Hematology 1 Module and Laboratory Mannual

Normel C.
Adarve, RMT, MPH, IMT(ASCP)

Clinical Hematology 1 Module & Laboratory manual 1
COURSE TITLE: CLINICAL HEMATOLOGY 1
COURSE DESCRIPTION: The course is intended for students who are taking up BS Medical
Technology/Laboratory Science. This course will provide the students the definite knowledge about the
normal development and physiology of the formed elements of blood, as well as the different diseases
involving blood. The different tests and techniques under a Hematology laboratory and its clinical
significance are also discussed.
STUDENT LEARNING OUTCOMES:

At the end of the course, the learner shall:
1. discuss the normal development and physiology of the formed elements of the blood.
2. discuss the pathophysiology of the different diseases associated with blood and its formed
elements.
3. explain the facts and principles of hematological determinations.
4. differentiate microscopically the normal and abnormal blood cells.
5. discuss the principle of the different hematological laboratory tests.
6. discuss the principle of the different cell counting machines used in hematology laboratory.
7. perform hematological tests with precision, accuracy and reliability.
8. correlate hematological tests to pathologic conditions.
9. assume responsibility in handling blood specimens, including examination and interpretation of
test results.
10. manifest the following values and skills: independence, dependability, honesty, critical analysis,
empathy and worth for life.
This material is downloaded for Ezra T. Estorninos (20190113901)

at FEU Dr. Nicanor Reyes Medical Foundation.
For personal use only. No other uses without permission. All rights reserved.
Table of Contents
LESSON I: Hematopoiesis (Blood cell production) ...............................................................................................6
Erythropoiesis ......................................................................................................................................................8
Granulocytopoiesis ........................................................................................................................................... 11
Monocytopoiesis............................................................................................................................................... 12
Lymphopoiesis .................................................................................................................................................. 13
Megakaryopoiesis ............................................................................................................................................. 14
LESSON II: Bone Marrow Examination and the Lymphoid Organs ............................................................... 17
LESSON III: Red blood cell Physiology ............................................................................................................. 26
Lesson VI: Leukocyte Kinetics and Function .................................................................................................. 39
The Adaptive immunity .................................................................................................................................... 45
White blood cell Kinetics .................................................................................................................................. 48
Lesson V: Complete blood count and other routine RBC procedures ......................................................... 52
Red cell Count ................................................................................................................................................... 52
White Blood Cell Count..................................................................................................................................... 55
Platelet count .................................................................................................................................................... 57
Sources of Errors in Manual Cell Count ............................................................................................................ 59
Hematocrit ........................................................................................................................................................ 61
Factors affecting the microhematocrit value................................................................................................... 62
Hemoglobin determination .............................................................................................................................. 63
Rule of three ..................................................................................................................................................... 66
Red cell indices .................................................................................................................................................. 67
White Blood cell differential Count (Indirect Relative Cell Count) ................................................................. 67
Other Routine RBC procedure .......................................................................................................................... 70
Reticulocyte count ........................................................................................................................................ 70
Erythrocyte Sedimentation Rate .................................................................................................................. 73
LESSON VI: Special Tests in Hematology ......................................................................................................... 78
Special test for diagnosis of certain RBC membrane defects .......................................................................... 78
Erythrocyte Osmotic Fragility Test (EOFT) ................................................................................................... 78
Ham’s test or Acidified Serum Test .............................................................................................................. 79
Special Test for assessing certain Hemoglobin disorders ................................................................................ 80
Dithionite solubility test ............................................................................................................................... 80
Sodium Metabisulfite test ............................................................................................................................ 81
Hgb Electrophoresis ...................................................................................................................................... 81
Acid Elution test ............................................................................................................................................ 82

Alkali denaturation test ................................................................................................................................ 83

Screening Test for unstable hemoglobin ......................................................................................................... 84
Isopropanol precipitation test ...................................................................................................................... 84
Heat denaturation test ................................................................................................................................. 84
Heinz Bodies inclusion test ........................................................................................................................... 84
Hemoglobin H preparation and staining ...................................................................................................... 85
Test for red cell energy and metabolic defect ................................................................................................. 85
Florescent spot test ...................................................................................................................................... 85
Glutathione reduction test ........................................................................................................................... 86
Auto Hemolysis test ...................................................................................................................................... 87
Ascorbate cyanide test ................................................................................................................................. 87
Special test for diagnosis of Autoimmune red cell disorder ........................................................................... 88
Donath-Landsteiner test ............................................................................................................................... 88
Direct Antihuman Globulin Test (DAT)......................................................................................................... 88
Lesson VII: Peripheral Blood Smear Examination .......................................................................................... 91
Lesson VIII: RBC Disorder ............................................................................................................................... 100
Intracellular Red Cell Disorders ...................................................................................................................... 104
Abnormal Iron Kinetics ............................................................................................................................... 104
Abnormal Nuclear Development................................................................................................................ 109
Red cell Membrane defects ........................................................................................................................ 111
Abnormal Hemoglobin................................................................................................................................ 115
Red cell Enzymopathy ................................................................................................................................. 121
Congenital Bone Marrow disorders ........................................................................................................... 123
Extrinsic Red cell Disorders......................................................................................................................... 123
Lesson IX: White Blood Cell Disorder .......................................................................................................... 128
Non-Malignant WBC disorder ........................................................................................................................ 128
Quantitative ................................................................................................................................................ 128
Qualitative (Non-Genetic) .......................................................................................................................... 130
Qualitative (Genetic)................................................................................................................................... 130
Other disorder of Phagocytes......................................................................................................................... 133
Malignant WBC disorder ................................................................................................................................ 134
Classification of Leukemia .............................................................................................................................. 134
Myeloid Leukemia or Myeloproliferative Leukemia ..................................................................................... 136
Acute myeloid Leukemia (AML) ................................................................................................................. 136
Myeloproliferative Neoplasm .................................................................................................................... 140

Myelodysplastic syndrome ......................................................................................................................... 141

Lymphoid Leukemia or Lymphoproliferative Leukemia ................................................................................ 142
Acute Lymphoblastic Leukemia .................................................................................................................. 142
Chronic Lymphocytic Leukemia (Mature Lymphoid Neoplasm) ............................................................... 144
Lymphoma....................................................................................................................................................... 145
Plasma Cell Neoplasm..................................................................................................................................... 145
Lesson X: Automated Hematology............................................................................................................... 147
Coulter Principle ............................................................................................................................................. 148
Radiofrequency ............................................................................................................................................... 151
Light Scatter .................................................................................................................................................... 151

LESSON MODULE
LESSON I
LESSON TITLE: Hematopoiesis (Blood cell production)
LESSON INTENDED LEARNING OUTCOMES (LILO)

At the end of this topic, the students are expected to:
1. discuss the hematopoietic phases.
2. discuss the theories of blood development.
3. discuss the different hematopoietic series.
4. identify the different stages of development of each formed elements of the blood.
5. enumerate the normal characteristics of the formed elements of the blood.
LESSON GUIDE:
I. Learning Guide Time Learning Resources
allotment
1. Discuss the Hematopoietic phases or periods. 3-hour lecture Learning Content:

1.1 Mesoblastic period - Lesson 1 module
1.2 Hepatic period page/s: 3-12.
1.3 Myeloid period - Rodak’s Hematology
2. Discuss the theories of blood development. pages: 76 – 80.
2.1 Monophyletic theory - Clinical Hematology
2.2 Dualistic theory principles, procedures,
2.3 Polyphyletic theory correlation pages: 46-
3. Discuss the different hematopoietic series. 57.
3.1 Erythropoiesis
3.2 Leukopoiesis
3.3 Megakaryopoiesis
4. Identify the different stages of development of each formed
elements of the blood.
4.1 Correct identification of the different progenitor cells of
Red blood cells, White blood cells and Megakaryocytes
as to nuclear and cytoplasmic changes and size.
5. Enumerate the normal characteristics of the formed
elements of the blood.
5.1 Correct identification of the normal structure of the
mature forms of red blood cell, white blood cell, and
thrombocytes in the blood.
6. Self-Directed Activity: Laboratory workbook
- Illustrate the stages of development of progenitor cell of red
blood cells, white blood cells, and platelets.
- Illustrate all the normal appearance and characteristic of
mature red cell, white blood cells, and platelets
7. For Submission: self- directed activity must be submitted 48 Moodle
hours after the lecture on hematopoiesis. e-mail account
8. Pre-test and Post-test Assessment Quizzes using the Moodle
platform

LESSON I: Hematopoiesis (Blood cell production)

There are three different periods or phases of blood production from the embryonic life to adult
life. According to the references, there are three periods of hematopoiesis, the first stage is the
mesoblastic period, followed by the hepatic period then the last is the myeloid period. In the graph
below it shows that the mesoblastic period usually starts at the 2 nd week of pregnancy and will end
before the 3rd month. The main source of blood production on this stage is the yolk sac. The type of
blood cell that is produced here is only red blood cell, which is of a primitive type due to its inability to
remove its nucleus. During this period there are three different forms of hemoglobin molecule, and
these are known as Gower I, Gower II, and Portland hemoglobin. As the yolk sac declines it blood cell
production capability, another organ now assumes that responsibility, and that organ is the liver. If the
liver now is the main source of blood cells then this period is called the hepatic phase. The graph below
shows that this phase usually starts during the 5th or 6th week of pregnancy, then the peak of it will be
between the 5th and 6th month of pregnancy, and commonly ends on the 1st or 2nd week after birth. The
majority of the hemoglobin type produced on this stage is known as the fetal hemoglobin or also known
as Hemoglobin F. The graph also demonstrates that there are secondary organs, such as spleen, lymph
nodes and thymus, which helps in the further differentiation and maturation of lymphocytes. Finally,
whenever the liver ends its blood-producing capability, the red marrow now assumes the responsibility
of producing blood cells, and this stage is known as the myeloid period of hematopoiesis. This last phase
commonly starts between the 4th and 5th month of gestation and will last until a person died. The red
marrow, where the major site of blood production, is widely distributed to the different parts of the
skeletal system such as, vertebra, pelvis, sternum, tibia and femur. A new type of hemoglobin is
developed during the myeloid period, and this is known as the adult hemoglobin called as hemoglobin
A1 and hemoglobin A2. As a person fully matures the concentration of Hgb A1 suddenly increases and
reaches around 97-98 % of the total Hgb type while the Hgb A2 will make up the remaining 2-3% of it.

Types of Normal Globin chains Period present

Hemoglobin
Gower I ζ2 , ε2 Mesoblastic
period
Gower II α2 , ε2 Mesoblastic
period
Portland ζ2 , γ2 Mesoblastic
period
Hgb F α2 , γ2 Hepatic period
and early
Myeloid period
Hgb A1 α2 , β2 Myeloid Period
Hgb A2 α2 , δ2 Myeloid Period
Table 1.1
Table 1.1 shows the different forms of normal hemoglobin variants that developed from
embryonic life to adult life, as well as the type of globin chains present on each specific type of it. Aside
from Hgb A1 and Hgb A2, the hemoglobin F may also be present during adult stage but it only covers
around less than 1 % of the total Hgb concentration during adult life. This low level of hemoglobin F
should be maintained in an adult individual to allow hemoglobin A1 to be the dominant type of
hemoglobin which is more efficient in delivering oxygen to the body. However, if an adult person
exhibits an increased concentration of hemoglobin F, like for example if it reaches more than 3%, then
this person may experience low oxygenation of his body, which might lead to cyanosis. This condition
is commonly seen on patient, that suffers from Beta thalassemia. Beta thalassemia, is a type of red cell
disorder that involves problem in hemoglobin synthesis, or development.
There are three different theories on how the blood cells are produced, and these are the
Monophyletic theory, Dualistic theory and the Polyphyletic theory. The most accepted among these
theories is the Monophyletic theory (or also known as Unitarian theory). This theory was first
introduced by a Russian histologist whose name is A.A. Maksimov. He suggested that there is a common
parent cell of all forming elements of blood-indifferent mesanchymal cells of myeloid, erythroblast and
lymphoid lineage. This common parent cell is also called as the multipotent hematopoietic stem cell or
pluripotent hematopoietic stem cell. The dualistic theory, which is proposed by several histologists
namely Erlich, Schridd, and O. Naegelli, suggest that there are two different sources of stem cells and
these are Myeloid stem cell and Lymphoid stem cell. The Polyphyletic theory or the Trialistic theory,
which is proposed by L. Aschoff, suggests that each blood cell lineage derived or originate from their
own unique stem cells namely myeloid stem cell, lymphoid stem cell and reticuloendothelial.
According to the monophyletic theory of hematopoiesis, all blood cells come from one single
stem cell known as the multi potent hematopoietic cell. The illustration below shows that there are
different growth factors needed to stimulate and dictate the continuous maturation of the human

blood cells. The multi potent stem cell requires the stimulation form several cytokines, such as
Interleukin 1, 3 and 6, to mature, and become a common myeloid progenitor cell. While Interleukin 1
and 6 are needed for the multi potent stem cell to development into the common lymphoid progenitor
cell. Further stimulation of the myeloid progenitor from the different growth factors, will eventually
transform it to another form of blood cell. The growth factors involved for platelet production is
thrombopoietin or TPO, and colony stimulating factor megakaryoblast or CSF Meg. For red cells, it
requires stimulation form erythropoietin or EPO, while for the granulocyte and monocyte development,
is the colony stimulating factor granulocyte monocyte or also known as CSF-GM. The lymphoid
progenitor stem cell requires various stimulation form the different cytokines, to complete its
differentiation, and eventually transform to either B lymphocyte or T lymphocyte. The thymus gland
plays a vital role on this process.
Erythropoiesis
In erythropoiesis the main growth factor necessary for the complete maturation of red cell is
erythropoietin. The EPO is produced by the kidney. The main stimulant of EPO release by the kidney is
hypoxia. Hypoxia is a condition where the human body is experiencing low oxygen pressure in the
blood, that makes the body react by stimulating the red marrow to immediately release red cell in the
circulation, to alleviate the hypoxic event. Once the stem cell is stimulated by the EPO, it will then
mature into various stages of development, the first cell developed is called the burst forming unit
erythroblast (BFU-E), followed by colony forming unit erythroblast (CFU-E), then after that, as what is
shown here, the progenitor red cell will eventually divide and mature further to these next six stages in
chronological order, which are, rubriblast, prorubricyte, rubricyte, metarubricyte, reticulocyte then
eventually mature red cell. If one uses the term “rubri” in giving names of this maturing red cell, this is
known as the Rubri nomenclature. There are other terms used in giving names on the different stages
of development of red cell, this term is known as Erlich and Normo nomenclature. The Erlich

nomenclature uses the term “erythroblast” or “erythrocyte” while the Normo nomenclature uses the
term “nomoblast” or “normocyte”. Thus, Rubriblast can also be called as, Proerythroblast or Pronormo
blast. The prorubricyte, is also known as basophilic erythroblast or normoblast. The rubricyte, is also
called as polychromatophilic erythroblast or normoblast. The metarubricyte, is also known as
orthochromatophilic erythroblast or normoblast. The reticulocyte, is also called as polychromatophilic
erythrocyte or normocyte, and lastly the mature red cell can be called as erythrocyte or normocyte.
The number of days it would take for the red cell to mature from rubriblast to red cell is approximately
between 3-5 days. Hypoxia is the key element that dictates on how short or long the day it would take
for a progenitor red cell to mature. There are morphological changes that generally take place during
the maturation of red cells. First is the cytoplasmic color. As the progenitor red cell matures the
basophilia decreases, the nuclear chromatin begins to tightly compact, the nuclear:cytoplasmic ratio
decreases, and as well as the cell size. Let us now discuss about the individual characteristics of this
maturing red cell relevant to the different changes that was mentioned. The rubriblast and the
prorubricyte has a basophilic cytoplasm, while the rubricyte has a lilac cytoplasmic color, the
metarubricyte has red to blue grayish color, the reticulocyte has diffusely basophilic cytoplasmic hue,
and the mature red cell has pinkish red or reddish orange cytoplasmic color. The change in cytoplasmic
color is related to the presence of hemoglobin in the cytoplasm. This is observable in the rubricyte’s
cytoplasm as well as in the succeeding progenitor cell after it. The chromatin material of the rubriblast
is described as having a fine lacy appearance which is diffused in the entire nucleoplasm, but as the cell
matures the chromatin material begins to aggregate. In the prorubricyte the chromatin material is
described as having a slightly compact chromatin material, moderate compactness of this is evident in
rubricyte, and tight or heavy compactness is seen in the metarubricyte. This process is known as nuclear
degeneration or karyorrhexis. That is why nuclear removal takes place on the metarubricyte due to the
complete degradation of the nucleus on this stage. The NC ratio for each cell is as follows, 8 is to 1 for
rubriblast, 6 is to 1 for prorubricyte, 4 is to 1 for rubricyte and 1 is to 2 for metarubricyte. There is no
NC ratio for reticulocyte and mature red cell because it doesn’t have any nucleus at all. The size of the
rubricyte is around 17 to 25 micrometers in diameter, the prorubricyte cell size is 15 to 17 micrometers,
rubricyte is 12 to 15 micrometers, metarubricyte is 8 to 12 micrometers, the reticulocyte is around 8 to
12 micrometer and lastly the red cell has 6 to 8 micrometers in diameter. The hemoglobin is one of the
major components of the red cell, therefore as it matures, the red cell has an obligation to synthesize
this unique protein and it usually begins as early as the rubriblast stage. The hemoglobin will
continuously form as the red cell progenitor matures up to the reticulocyte. Therefore, the last
immature cell that can still manufacture the hemoglobin is the reticulocyte, while the red cell has no
capability to synthesize it anymore. There are four mitotic divisions that take place during the
development of the red cell starting from the rubriblast going to rubricyte. Among these six stages of
red cell development, only the rubriblast, prorubricyte and rubricyte has the capability to undergo
nuclear division or mitotic division. According to one of the hematology books, entitled Hematology
principles and procedures by Rodak, the rubriblast usually divides once, which produces two daughter
prorubricyte. Then, the two produced prorubricyte each of them will divide twice, thus producing eight
daughter prorubricytes. After which, all of these eight rubricytes will eventually divides once, which

then produces 16 metarubricytes. The metarubricyte does not divide or it is now incapable to do further
mitosis due to the degenerative nucleus it has, it will just continuously mature to reticulocyte. The
reticulocyte if it is already formed, normally it resides for two days in the red marrow. This event is due
to the presence of a sticky protein that helps the reticulocyte to stay in the marrow for 48 hours. This
protein is known as fibronectin. This fibronectin gradually lost in the reticulocyte’s membrane as it
reaches its full maturation. And if this fibronectin is removed, eventually the reticulocyte will escape
and egress from the marrow and goes to the circulation. In the circulation, the reticulocyte needs
additional 24 hours to completely mature to red cells. This 24-hour period is enough for the spleen to
remove all the reticulocyte’s remnant, which is made up of ribosomes and mitochondria. This remnant
is also called as the RNA remnants. In hypoxic event, the increased level of EPO, stimulates the early
release of the immature reticulocyte from the red marrow. This immature reticulocyte is also called as
“shift cell”. That is why, if the person has a severe case of anemia, an increase number of this “shift cell”
is observable in his peripheral blood. This condition is called as “high polychromasia”. In a normal state,
the full length of days that a red cell has in the circulation is approximately 120 days. A red cell should
withstand the tremendous pressure and turbulence within the blood circulation for almost 3 months.
This is possible because of the unique appearance of red cells. The absence of nucleus and the presence
of the central pallor are one of the contributing factors for this. Usually the appearance and size of the
central pallor is vital in diagnosis of certain red cell disorder. The normal size of the central pallor should
not be more than 3 micrometers in diameter, or should only occupy at least one third of the total
diameter of the red cell. If the central pallor size increases, it only means that the hemoglobin content
of the red cell is decrease. This condition is known as “hypochromia”, and if the central pallor size
decreases, this is called as “hyperchromic”. As what has mentioned earlier, the size of a mature red cell
must be ranging between 6 to 8 micrometers in diameter, with an average diameter of around 7.5. If
the size of the red cell is lower than 6 micrometers, this red cell is called “microcytic”, and if it has more
than 8 micrometer size, it is called “macrocytic”. A microcytic red cell is seen in certain cases of anemia
such as, iron deficiency anemia, thalassemia, sideroblastic anemia, and anemia of chronic disease or
inflammation. While a macrocytic anemia is seen in megaloblastic anemia, which is a nutrient deficiency
anemia.
RBC nomenclature types

Rubri nomenclature Erlich nomenclature Normo Nomenclature
Rubriblast Proerythroblast Pronormoblast
Prorubricyte Basophilic erythroblast Basophilic normoblast
Rubricyte Polychromatophilic Polychromatophilic normoblast
erythroblast
Metarubricyte Orthochromatophilic Orthochromatophilic
erythroblast normoblast
Reticulocyte Diffusely basophilic erythrocyte Diffusely basophilic normocyte
Polychromatophilic erythrocyte Polychromatophilic normocyte
Mature red blood cell Erythrocyte Normocyte

Rubriblast Prorubricyte Rubricyte
Metarubricyte Reticulocyte Mature red cell
Figure 1.1
In leukopoiesis, the growth factors needed for the complete maturation of the granulocyte is
CSF-granulocyte while the monocyte development requires CSF-monocyte. Aside from this growth
factor, other cytokines are needed for the development of these cells. For neutrophil, basophil and
monocyte IL-3 and 6 are needed, while eosinophil requires IL-3 and IL-5.
Granulocytopoiesis
There are six stages of development in granulocyte development or also known as
granulocytopoiesis. The orders of granulocyte maturation after deriving it from the myeloid stem cells
are as follows: myeloblast, promyelocyte, myelocyte, metamyelocyte, band or stab cell, and mature
granulocytes known as neutrophil, eosinophil and basophil. According to some references, around 4 to
5 mitotic division took place during the development of the granulocytes. Within the red marrow, the
white blood cell progenitor is divided into two different pools namely the mitotic pool and maturation
pool. The mitotic pool can undergo nuclear division which the first three stages of progenitor cells are
included, while the maturation pool cannot undergo any nuclear division but they will just continuously
mature until they reach the final stage, in which the last three stages are part of this. Granulation is one
of the striking changes that happen in granulocyte maturation. The cytoplasmic granules increase in
number as the cell develop. The first granule may be seen in the promyelocyte stage. This granule is
called as primary granule, non-specific granule or azurophilic granule because it imparts a blue staining
reaction on Wright and Giemsa stain. During the myelocytic stage, a secondary granule is now
synthesized, this granule is also called as specific granules which imparts a reddish hue color on its
cytoplasm, reflecting a lilac appearance of its cytoplasm. As the granulocyte develop, they carry within
their cytoplasm the primary and secondary granules and additional granules are then develop as they
differentiate into either neutrophil, eosinophil or basophil. Another visible change occurs as this cell
matures is the nuclear indentation. The first three stages such as myeloblast, promyelocyte and
myelocyte do not exhibit any indentation. Nuclear indentation is observable only until the

metamyelocyte stage which resembles a “kidney” or “bean” shape nuclear pattern. And because of
this, the metamyelocyte is also called as “juvenile” cell. As it matures towards the band cell, the nuclear
indentation is becoming deeper which exhibit an “S” or “C” or “U” pattern. The mature granulocyte can
be differentiated by the nuclear lobulation they exhibit, and the type and color of their granules. For
neutrophil it has around 3-5 lobulation with lilac color of granules, the eosinophil has 2 to 3 lobulations
with striking bright red orange granules, while the basophil has 2 - 3 lobulations with dark blue color of
its granules. These mature granulocytes exit the bone marrow and go to the circulatory system. Around
50% of these cells adheres the walls of the blood vessels and the other 50% are circulating with the
blood. The one that adheres in the vessel wall is known as marginal pool while the one that circulates
is known as circulating pool.
Myeloblast Promyelocyte Myelocyte
Nutrophil Eosinophil
Metamyelocyte Band cell/Stab cell
Basophil
Figure 1.2
Monocytopoiesis
In the maturation of monocyte, or also known as monopoiesis, there are only three stages of
development involved and these are monoblast, promonocyte, and monocyte. The cytoplasmic color
and nuclear pattern do not change significantly. The cytoplasmic color which is seen in all stages is
characterized as having a red-blue grayish color with a ground glass cytoplasm. The nuclear chromatin
pattern is fine-lacy and evenly distributed within the nucleoplasm. This is seen in all stages of the
monocyte series. Cytoplasmic vacuolations may be present on the different stages of these cells.
Nuclear indentation commonly starts during the promonocyte stage and greatly evident in the

monocyte stage. The monocyte can travel to different parts of our tissue as macrophages or histiocytes
and reside on it for several days or weeks. These macrophages play an important part of our acquired
or adaptive immunity as an antigen presenting cell. Several names are called on these macrophages
depending on where they are located. In the liver , they are called Kupffer cells, in the dermis it is known
as Langerhans’s cell, in CNS it is known as microglial cells, in synovial fluid it is called synovial A cells, in
lymph nodes it is the dendritic cells, in the nephrotic tissue it is called as mesangial cell, in the lungs it
is called as alveolar macrophages and in the blood it is simply called as monocyte.
Monoblast Promonocyte Monocyte
Figure 1.3
Lymphopoiesis
In lymphopoiesis, there are three different stages of lymphocyte progenitor cells, known as
lymphoblast, prolymphocyte and lymphocyte. Like the monocyte maturation there is no significant
change in cytoplasmic color and nuclear appearance as the progenitor lymphocyte matures. The
cytoplasmic color from lymphoblast to lymphocyte is blue and the chromatin patterns is dense and
compactly arrange which gives a blue-black color under Wright’s and Giemsa stain. As the lymphocyte
lineage matures, the visibility of the nucleoli decreases. The lymphoblast has around 2-4 visible nucleoli,
the prolymphocyte has 1-2 visible nucleoli, while the lymphocyte has no visible nucleoli. There are two
types of lymphocyte, the small lymphocyte and the large granular lymphocyte or also called as “LGL”.
The large granular lymphocyte is our first line of defense against cancer cell and it can destroy virally
infected cell within our body. There are different names given on this LGL’s, and these are natural killer
cell or NK cell, lymphokine activated cell or LAK, and killer cell or K cell.
Lymphoblast Prolymphocyte Small Lymphocyte
Large Granular Lymphocyte
Figure 1.4

Megakaryopoiesis
Platelet production or megakaryopoiesis is stimulated by several growth factor that was
mentioned earlier. Aside from these growth factors, certain cytokines are taking part in the continuous
maturation of these platelet producing cell, and these are interleukin 7 and 11. There are four stages
or phases of platelet production and these are Megakaryoblast, followed by promegakaryocyte, then
megakaryocyte and lastly metamegakaryocyte. In the Rodak’s hematology book, it stated 3 phases
rather than four phases of platelet development, and these are megakayoblast or MK I,
promegakaryocyte or MKII, and megakaryocyte or MKIII. There are general changes that occurs during
the development of these cells which takes around 5 to 7 days to complete. These changes occur in the
nuclear and cytoplasmic content of the cell. The size also changes from 15 um to 100 μm in full size.
The size of megakaryoblast may vary between 15-30 μm in diameter, the promegakaryocyte is around
30-80 um, then both megakaryocyte and metamegakaryocyte has an approximate diameter of 30-100
um. As these progenitor cells develop or mature, the nucleus divides while the cytoplasm do not, and
this is called “endomitotic” division. And because of these, the number of nucleus inside increases,
which is very evident in both megakaryocyte and metamegakaryocyte. The number of nucleus in
megakaryocyte may vary between 2 or 4, while the metamegakaryocyte has 4 or more. Megakaryoblast
has 1 and promegakaryocyte has 2 nuclei. The cytoplasmic granule is absent in megakaryoblast, but as
the cell matures the granule formation is now visible on promegakaryocyte but with only few to
occasional number, while moderate number and increased number or aggregated granules are seen in
megakaryocyte and metamegakaryocyte, respectively. Another developmental changes within the
cytoplasm of these maturing platelet producing cell is the formation of “demarcating membrane
system” or also known as “DMS”. These “DMS” is thought to be the future plasma membrane of every
platelet that will be produced. Formation of this demarcating membrane system usually starts at the
promegakaryocyte stage. The nuclear chromatin material also changes from fine-lacy in
megakaryoblast to highly compact in the metamegakaryocyte stage. The nucleoli also becoming less
distinct as the cell mature. It was stated in several textbooks that only megakaryoblast shows distinct
or visible nucleoli. The N:C ratio decreases from 1:1 on megakaryoblast, 1:2 on promegakaryocyte and
1:12 on both megakaryocyte and metamegakaryocyte. Another part of change that happen as this
progenitor platelet producing cell matures, is the diminishing number of cytoplasmic protrusions or
“tags”. These tags are seen on both megakaryoblast and promegakaryocyte but it is absent on both
megakaryocyte and metamegakaryocyte. As the cell reaches either megakaryocyte with four nucleus
or metamegakaryocyte, the platelets are then released from their cytoplasm. The platelets are derived
from the fragmentation of the cytoplasm of either megakaryocyte or metamegakaryocyte, therefore,
whatever can be found in the cytoplasm of the platelet that is also found or present within the
cytoplasm of these progenitor cells. Normally around 2,000 to 4,000 platelets are produced by the
metamegakaryocyte and will have a life span of 7-10 days in the circulation. The approximate volume
of a single platelet is around 7-10 fL, while the diameter of it is between 2-4 um. Around 70 % of
platelets produced are in the circulation while 30% of it is sequestered in other organs primarily the
spleen. The main function of the platelets is to promote normal hemostasis or blood clotting process.

The estimated normal platelet count of an individual is between 150,000 cells/uL to 400,000 cells/uL. If
the platelet count decreases below the reference value it can cause severe consequence like
hemorrhage or bleeding, but if it is increased way above the reference value it may cause thrombus
formation within the blood circulation. Morphologically platelets are divided into three different zones,
the outer portion is called the peripheral zone, the middle portion is called sol-gel zone, and the
innermost part is the organelle zone. The peripheral zone is composed of the plasma membrane and
the different glycoproteins or receptors which is very useful for normal functioning of the platelets. The
HLA-antigens are also located on this region of the platelet which plays a vital role in immune activation.
An opening known as the “open canalicular system” is also located on this part. The sol-gel zone is
composed of microtubules, that supports and maintain the discoid shape of the platelet, and a
contractile protein known as thrombostenin that composed of actin-myosin protein complex. The
organelle zone of the platelet is made up of organelles such as mitochondria and ribosomes, and also
with four different forms of granules namely dense granule, alpha granule, lysosomal granule and
glycogen granule. The content of these granules is very vital to the normal functioning of the activated
platelets.
Megakaryoblast Promegakaryocyte Megakaryocyte Metamegakaryocyte
Figure 1.4

LESSON MODULE
LESSON II
LESSON TITLE: Bone Marrow Examination and the Lymphoid organs

1. discuss the anatomy, histology and physiology of bone marrow and lymphoid organs.
2. enumerate the sites for bone marrow collection.
3. discuss the sampling and processing of the bone marrow specimen.
4. describe the role of the Med. Tech. during bone marrow collection.
LESSON GUIDE:
II. Learning Guide Time Learning Resources
allotment
1. Discuss the anatomy, histology and physiology of the bone Learning Content:
3-hour lecture
marrow and lymphoid organs. - Lesson 1 module
1.1 Review of the anatomy and physiology of the Primary page/s: 14 – 20
and secondary lymphoid organs. - Rodak’s Hematology
2. Enumerate the sites for bone marrow collection. pages: 253 - 267.
2.1 Bone Marrow collection equipment and procedures. - Clinical Hematology
3. Discuss the sampling and processing of the bone marrow principles, procedures,
specimen. correlation pages: 366
3.1 Preparation of bone marrow films for evaluation. - 378.
3.2 Application and clinical use of routine and special stains
used for bone marrow samples.
3.3 Bone marrow evaluation report and interpretation.
4. Describe the role of Medical technologist/ Laboratory
Scientist during bone marrow collection.
4.1 The preparation of materials and equipment used for
bone marrow collection and processing.
- Answer the questions on the laboratory workbook related to
bone marrow evaluation and examination.
hours after the lecture on hematopoiesis.
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LESSON II: Bone Marrow Examination and the Lymphoid Organs

Bone Marrow and other Lymphoid organs
The adult hematopoietic tissue is situated in the primary and secondary lymphoid organs. The
primary lymphoid organ consists of the bone marrow and thymus, while the secondary lymphoid organ
consists of spleen, lymph nodes, and liver. The bone marrow is divided into two types and these are the
red and the yellow marrow. The yellow marrow is made up of adipose tissues while the red marrow is
where the hematopoietic cells are located. These hematopoietic cells are composed of maturing
erythroid, myeloid, megakaryocytic and lymphoid cells. The thymus is the location of further
differentiation of the lymphocyte into either T- cell, B cell or Killer cells, and then they will be distributed
to the different secondary lymphoid organs. Within the secondary lymphoid organs are the site of
lymphocyte activation in response to foreign antigens for functional adaptive immunity.
The bone marrow is enclosed within the cavities of the cortical bones and also within the
projection of calcified bone called the trabeculae. These trabeculae provide structural support to the
developing hematopoietic cells. In adults, these hematopoietic cells within the red marrow is widely
distributed in the flat bones such as sternum, pelvis, ribs, scapulae, skull and proximal portions of the
long bones, however during infancy and early childhood most of the cavity of the long bone is composed
of entirely red marrow. The replacement of red marrow to yellow marrow within the long bone from
infancy to adult age is known as retrogression. This process limits the red marrow only to the proximal
ends of the long bones in adults. However, the yellow marrow may revert back to red marrow in severe
hypoxic event that an individual may experience, like in hemolysis or severe blood loss, this is to
compensate the poor oxygenation of the body. Aside from the hematopoietic cells, the bone marrow
is also composed of other components such as stromal cells and blood vessels. Stromal cells include
adipocytes, macrophages, lymphocytes, endothelial cells, osteoblast, osteoclast and reticular
adventitial cells or fibroblast. Adipocytes are large cell with a fat vacuole that helps in the regulation of
the volume of the marrow in active hematopoiesis. The macrophage function as phagocytes and it also
“nursing” the developing progenitor red cells with iron. It also releases cytokines that regulates the
development of blood cells, in which, this function is also seen on lymphocyte residing within the
marrow. Other cells involved in cytokine release or production are the endothelial cells and reticular
adventitial cells. Moreover, the reticular adventitial cell forms an incomplete layer of cell that extends
to the vascular sinuses within the bone tissue. These reticular fibers are also responsible for providing
support to the developing hematopoietic cells. Osteoblast are involved in bone matrix formation and
remodeling while the osteoclast on the contrary is responsible for bone resorption or destruction. The
stromal cells secrete a semifluid extracellular matrix that anchors the hematopoietic cells. This matrix
contains substances such as fibronectin, laminin, collagen, thrombospondin, tenascin and
proteoglycans. Collectively, the stromal cells are very vital in the regulation of hematopoietic stem cell
and progenitor cells survival and differentiation. The red marrow within the bone is arranged in cords
known as extravascular cords. These cords are located in spaces between the vascular sinuses which
are supported by the trabeculae. The interaction of the cords to the vascular sinuses helps the
successful egress or exit of developed blood cell to the blood circulation. There are two arteries that
supplies oxygen and nutrition in the bone marrow and the bone matrix and these are the nutrient and
periosteal arteries. These arteries enter the bone through an opening known as bone foramina. The

nutrient artery supplies blood to the bone marrow only while the periosteal artery supplies blood to
both the osseous bone and the bone marrow. The vein that collects blood from the bone tissue to be
delivered back to the circulation is called the central longitudinal vein.
During the fetal development the liver is the major site of blood production which occurs during
the second trimester of pregnancy. In adult the liver cells known as hepatocytes had various functions
such as, protein and clotting factor synthesis, carbohydrate and lipid metabolism, drugs and toxin
removal, iron storage and recycling. The liver is also responsible for hemoglobin degradation that
produces conjugated bilirubin which eventually excreted out through the intestine and kidney. The liver
is composed of two lobes which is situated beneath the diaphragm of the abdominal cavity.
Anatomically, the hepatocytes are arranged in radiating plates originating from a central vein. Adjacent
to these plates are sinusoidal endothelial cell. There is a noncellular space that separates the
longitudinal hepatocyte plates and the sinusoidal endothelial cells. This spatial arrangement helps the
plasma to has straight contact to the hepatocytes for two directional flow of fluids and solutes. The
liver macrophage known as “Kupffer cells” are responsible for removal of senescent cells, cellular
fragments or debris from the blood that circulated within the liver tissue. It is also known that the
Kupffer cell helps in protein synthesis regulation within the liver cells by releasing certain mediators.
In the hepatic period of hematopoiesis, spleen contributes in the development and
differentiation of the lymphocytes. In adult the spleen filters the circulating blood to remove the
abnormal inclusion within the red cell and damaged red cell membrane. This function is known as the
“pitting” function. The spleen is also known as the graveyard of senescent cell or abnormal cells.
Histologically the spleen is enclosed by a capsule that projects inwardly forming trabeculae that divides
the spleen into regions. Within these regions there are three different splenic tissues namely, white
pulp, red pulp and marginal zone. White pulps are scattered follicles with germinal centers that contains
various cells such as lymphocytes, activated B-cell, macrophages and dendritic cells. There is an artery
that passes through these germinal centers where T lymphocyte aggregates and surround this artery
forming a region called periarterial lymphatic sheath or also known as PALS. Along the periphery of the
PALS are lymphoid nodules containing B-cells. The marginal zone that surrounds the white pulp forms
a reticular meshwork that contains blood vessel, macrophages, B memory cell and T helper cell. The
red pulp is made up of vascular sinuses separated by cords of reticular cell meshwork known as cords
of Billroth, this comprises of loosely connected special-activated macrophages. The cords of Billroth
creates a sponge-like matrix that produces tremendous pressure and stress on red cell. As the blood
enters on these vascular sinuses the blood flow decreases and temporarily makes the red cell stagnant
on this point. This resulted to the depletion of red cell’s glucose supply. Old red cell or senescent red
cells cannot withstand the harsh environment on this area which eventually leads to its destruction and
degradation. This function is known as “culling”. The blood supply of the spleen comes from the central
splenic artery and then the blood is withdrawn and collected in the splenic veins to be delivered back
to the blood circulation.
Lymph nodes play a very important role in the proliferation of lymphocytes for the activation of
the adaptive immunity. Lymph nodes are bean shaped organ that is divided into two regions, the cortex
and the medulla. The cortex is located on the outer portion of the lymph tissue where it is enclosed by
a capsule that forms trabeculae that penetrates within the cortex. The trabeculae forms divisions

known as follicles within the cortex. There are two types of follicles which are the primary and
secondary follicles in which the latter has germinal centers on it comprised of activated B-cell. The
medullary, on the other hand, is composed of chords containing plasma cells and B-cells. Another
structural part of the lymph node lies between the cortex and the medulla is the para cortex, where the
T-cells and macrophages are located.
Thymus is important for the development and differentiation of the lymphocytes produced by
the bone marrow known as the pre-thymic lymphocyte. The pre thymic lymphocyte that doesn’t have
yet a specified CD marker known as double negative cell goes to the outer cortical region of the thymus
and wait for cytokine stimulation. As these double negative lymphocytes travels inwardly the cortex, it
transforms into double positive T-cells with both CD4 and CD8 markers. Going deeply within the inner
part of the thymus, which is the medulla, the T cell transforms into either CD4 positive or CD8 positive
cell depending on the nature of the cytokines that stimulates their differentiation. B-cells on the other
hand, carries different CD markers such as CD19, 20, and 22 while large granular Lymphocytes has CD
16 and 56. Then eventually these different types of lymphocyte goes out from the thymus and widely
distributed to the different lymphoid tissues, waiting for activation and stimulation of foreign antigen
derived from the pathogenic organisms. The thymus is where self-antigen recognition by the T-cell
occurs and those T-cells that do not carry these self-antigens, called reactive T cells are eliminated via
apoptosis or self-destruction.
Bone Marrow Examination
The bone marrow examination is indicated to support the diagnosis of certain hematological
disease such as leukemia and other myeloproliferative disorders. It is also used to assess bone marrow
function, measure iron storage in the bone marrow samples and rule out or diagnose certain diseases
that cannot be detected by using only the peripheral blood smear. However, there are diseases that
bone marrow collection is contraindicated, and these are platelet dysfunction diseases, like Bernard-
Soulier syndrome and platelet storage pool defects, and blood clotting protein deficiency like
Hemophilia A and B. Person who has Vitamin K deficiency is also contraindicated to undergo bone
marrow collection, this is due to the poor activity of the vitamin K dependent prothrombin group
clotting factors. These different contraindications should be treated or corrected first before bone
marrow collection should be considered, because excessive bleeding or hemorrhage might occur
whenever the bone is punctured, considering that the bone tissue is highly vascularized. Moreover,
according to the Rodak’s hematology book, person who undergo Coumadin and heparin (blood
thinners) treatment should undergo bridging therapy to prevent the uncontrolled bleeding caused by
these blood thinners.
Bone marrow collection should be done aseptically to prevent introduction of infection. The
materials and equipment used for this procedure should be sterilized or autoclaved. The following
equipment used are no. 11 scalpel blade for skin incision, surgical gloves, shaving equipment, 10- or 20-
mL syringes, drape materials, disposable Jamshidi or Waserman-Jensen biopsy needle, and a 14-18
gauge aspiration needles. Materials used are microscopic slides or coverslip washed with 70% alcohol,
petri dish, vials or test tubes with stoppers, Wintrobe-hematocrit tubes, K2EDTA anticoagulant, Zenker’s
or B5 fixative and gauzes. There are two different specimens collected in bone marrow sampling, one

is the core biopsy and the other one is the aspirated bone marrow fluid. The core biopsy is collected
first to preserve the architectural structure of the bone marrow followed by aspiration of the bone
marrow fluid. The common site for adult bone marrow collection is the anterior and posterior iliac crest,
while for patient who is under 2 years of age the tibia is used. Other sites used for bone marrow
collection are sternum, spinous processes of the lumbar vertebrae and the distal and proximal ends of
the long bones. After the patients are prepared and positioned to supine or fetal position, using the
Jamshidi or Waserman biopsy needle, the physician punctures the cortex of the bone and penetrates
the medullary cavity of the bone where the hematological cells are located. The biopsy collected are
placed in the fixative to preserve it and used for histological technique.
Ideally, there are three different fixative used for bone marrow biopsy and these are Zenker’s
and B5 fixative which both contains mercury and another is the 5-10% buffered neutral formalin. The
aspirated bone marrow fluid is collected on a separate location from the biopsy. Around 1-1.5 mL of
this fluid is aspirated using a syringe with a 14-18-gauge needle inserted to the marrow. The collected
bone marrow fluid is passed to a medical technologist to prepare several bone marrows slides or films
from it, such as “direct aspirate marrow film” and “crush bone marrow film”. The “direct” film is
prepared by immediately transferring drops of the marrow specimen into around six to eight ethanol
washed microscopic slides and spread by using a spreader slide to create a wedge-shaped smear (see
figure 2.1. The “crush” film is prepared from the bone marrow aspirate containing spicules expelled to
a petri dish with K2EDTA anticoagulant. The spicules are collected and placed on several ethanol-
washed glass slides then gently pressed to crush it using another glass slide. A drop of 22% albumin is
directly added to the crushed bone marrow film if prolymphocytes or lymphoblast is suspected. The
albumin reduces the occurrence of “smudge” cell or “basket” cells often seen in chronic lymphocytic
leukemia or CLL. These bone marrow slides are used for evaluating the morphology of the
hematopoietic cells, detection of cancer cells or tumor cells, differential count of the bone marrow cells,
cellularity studies and myeloid erythroid ratio or M:E ratio. The difficulty in obtaining bone marrow fluid
is called “dry tap”, which usually occur in certain bone marrow disorders such as myelofibrosis, severe
aplastic anemia and in leukemia packed with abnormal cells. In this case, a biopsy is necessary, and the
observation of cell morphology is done by using a slide imprint or touch preparation. The slide imprints
are done by pressing the surface of the slide to the biopsy collected, that allows the cells to adhere or

transfer to the surface of the slide. Then the slide imprints will be appropriately stained and submitted
to the physician for microscopic evaluation. If a special examination is requested, another syringe with
aspirated bone marrow fluid containing heparin is used for special examination such as cytogenetic
studies, molecular diagnosis and immunophenotyping which is used for flow cytometry. A “buffy coat”
smear is prepared in a suspected case of aplastic anemia or bone marrow failure where small numbers
of nucleated cells are expected. The buffy coat preparation is used to concentrate the cells by
transferring the fluid into a Wintrobe hematocrit tube and centrifuge it at 2,500 gravitational force for
10 minutes. The centrifuged bone marrow fluid will produce four different layers. The uppermost layer
is the fat layer which comprises around 1-3 % of the fluid, followed by the plasma layer, the third layer
is the M/E layer or buffy coat layer which make up around 5 – 8% of it, and the last is the packed red
cell layer. After this the buffy coat is aspirated using a pipette and transferred into the microscopic
slides and spread like a wedge type smear preparation.
Figue 2.1
The bone marrow films submitted in the laboratory are routinely stained with Wright’s and
Giemsa stain for morphological and cytological examination of the hematopoietic cells. The stained
bone marrow slides are initially observed microscopically under the low power objective or 100x
magnification. Under this magnification the bone marrow films are observed for megakaryocyte
estimation, assessment of bony spicules, detection of tumor cells, detection of fat to marrow ratio and
cellularity studies. Other evaluation such as the differential counting of the bone marrow cells, M:E
ratio computation, identifying abnormal distribution and maturation gaps of the hematopoietic cells,
and observation of myelocytic and erythrocytic maturation are made using the high-power objective
which is around 1000x magnification. Special stains are also applied for several bone marrow film
prepared that may help to diagnose some hematological disorders. Several of these special stains are
myeloperoxidase stain, Sudan Black B stain and tdT stain. These set of stains are used to differentiate
acute myelogenous leukemia or AML, from acute lymphoblastic leukemia or ALL. The AML has a positive
staining reaction to both myeloperoxidase stain and Sudan black B but negative for tdT stain, while ALL
gives an opposite staining reaction with these stains. ALL gives a negative reaction on both
myeloperoxidase stain and Sudan Black B but positive with tdT stain. Other stains such as esterase stain
is used to differentiate granulocytic from non-granulocytic leukemia, the nitro-blue tetrazolium stain is

used to diagnose chronic granulomatous diseases or CGD, leukocyte alkaline phosphatase stain or LAP
stain is used to differentiate leukemoid reaction from chronic myelogenous leukemia or CML, tartrate
resistant acid phosphatase or TRAP is used to diagnose “hairy” cell leukemia, the pearl’s stain or
Prussian blue stain is used to stain ferritin granules or stored iron in the bone marrow, periodic acid
shift stain or PAS is used to diagnose erythroleukemia or AML M6, and factor 8 stain is used to confirm
the diagnosis of megakaryoblastic leukemia or AML M7. Several dyes are also used for evaluating bone
marrow condition such as Hematoxylin and Eosin (H&E) dyes, used to evaluate cellularity and
hematopoietic cell distribution and locate abnormal cell clusters; the reticulin and trichrome dyes are
used to examine bone marrow fibrosis, Gram stain or acid- fast stain is used to identify acid-fast bacilli,
fungi and bacteria in granulomatous disease, and immunocytochemical dyes are used to establish the
identity of malignant cell with dye-tagged monoclonal antibodies specific for tumor surface markers.
Bone marrow cellularity examination is one of the important parts of a bone marrow report.
This is done through macroscopic or microscopic examination. The Macroscopic cellularity is executed
by identifying the amount or percent volume that the different layers of centrifuged bone marrow fluid
has. According to what had mentioned earlier, the bone marrow fluid with K 3EDTA, is placed in a
wintrobe hematocrit tube and centrifuge for 10 mins at heavy spin. The reference value of the fat layer
should be 1-3 %, and the M/E layer or the buffy coat layer should be at least 5 – 8%, while the plasma
and the packed red cell layer is both variable. If this is the result obtained after centrifugation of the
bone marrow fluid, then it is term “normocellular” bone marrow, which means that the distribution of
fat and hematopoietic cell is normal, but if the fat layer is high and the buffy coat layer is below the
reference value, it is interpreted as “hypocellular” bone marrow, and if the fat layer is low and the buffy
coat is above the reference value, then it is interpreted as “hypercellular” bone marrow. Certain blood
disorders are expected to have a hypocellular bone marrow which includes, aplastic anemia, Fanconi’s
anemia, and myelofibrosis, while hypercellular bone marrow is expected in leukemia, different types of
myeloproliferative disorder, polycythemia vera and leukemoid reaction.

Normocellular Hypocellular Hypercellular
The microscopic cellularity study is evaluated by examining the bone marrow film that is
processed in the laboratory. A normocellular bone marrow is reported if the ratio of fat to marrow cell
is 1:1 or 2:1 which means it has an equal distribution of fat and marrow cells. It is expected that in a
normal adult individual the cellularity of his bone marrow should be 50%, but it declines as the person
aged. Using a simple mathematical formula, which is 100 – age = cellularity % plus or minus 10%, may
help physician to estimate the bone cellularity of an individual. For example, if the patient is 65 years
of age, the expected cellularity of his bone marrow is between 25% to 45 %. If the bone marrow exhibits
more fat over bone marrow cell or high fat to marrow ratio it is interpreted as hypocellular bone
marrow, and if the bone marrow cell is abundant over the fat cells then it is reported as hypercellular
bone marrow. Another part of the bone marrow examination is the differential count report. The
physician should observe around 300 to 1000 cells using the bone marrow films prepared and
submitted to the laboratory. The physician may use several slides to reach the desired number of cells
that should be observed. All the immature cells or progenitor cells of all blood lineage and mature white
blood cell in the bone marrow should be reported. This differential report may help assess or classify
leukemia, for example, if it is a granulocytic or a non-granulocytic leukemia and or if it is acute or a
chronic leukemia. The differential count result is also used to evaluate the myeloid: erythroid ratio or
M:E ratio. The myeloid represent all of the progenitor cells of granulocyte and mature granulocyte,
while the erythroid represents the progenitor or immature red cells. The normal M:E ratio is 4:1 and
2:1. Other references, provides another normal M:E ratios and these are, 1.3:1, 1.2:1 and 5:1. The M:E
report is used to help evaluate the status of the bone marrow in relation with the number of
granulocytic cells from the erythroid cells. An increased M:E report for example, 6:1, 10:1 or 11:4 may
indicate that the person has chronic myelogenous leukemia, while a reverse M:E ratio like 1:4 may
indicate normoblastic hyperplasia, which is suggestive of erythroleukemia or AML M6.
In the bone marrow microscopic examination, it is likely to observe other cell which is not part
of the hematopoietic cells. These cells are called non-hematological cell which are collected from either
the biopsy or aspiration of bone marrow fluid. These non-hematopoietic cells may be part of the bone
matrix or cortex and this are the osteoblast and osteoclast. Osteoblast plays an important role in bone
formation while the osteoclast is responsible for bone resorption or destruction. These non-
hematological cells can be mistaken as hematological cell because of the almost the same appearance
of between these cells. The metamegakaryocyte or megakaryocyte can be mistaken as osteoclast,

because both of these cells are of the same size and has multiple number of nucleus within the cell. To
differentiate the metamegakaryocyte from osteoclast, the microscopist should evaluate the cytoplasm
and the nuclear appearance of these cells. The metamegakaryocyte has cytoplasmic granulation while
the osteoclast does not have, and the multiple nucleus of the megakaryocyte is fused together while
the osteoclast’s multiple nucleus is not connected together. Other cells mistaken together are
osteoblast and plasma cell due to their similar appearance which is both exhibiting eccentrically located
nucleus, that makes the cytoplasm streams out behind the nucleus, this is called as “comet” or “water
bug” shaped appearance. To differentiate osteoblast from plasma cell, one must locate the presence
of nuclear halo, which is exhibited by plasma cell while the osteoblast does not have this. The nuclear
halo in the plasma cell is also called as the zone of “hof”. There are normal marrow cells observed in
the bone marrow tissue in addition to the developing progenitor cells, which includes macrophages and
mast cells. Normally only 1% of the total cell comprises the bone marrow tissue is macrophage, however
increase number of it is seen in certain disease. These diseases exhibit increase cell turnover such as
hemolytic anemia, idiopathic thrombocytopenic purpura and solid tumors. Morphologically abnormal
macrophages in the bone marrow is commonly associated with storage diseases such as Gaucher
disease and Nieman-Pick disorder. On the other hand, the origin of mast cell or tissue basophils is
undetermined. This cell participates in inflammatory and hypersensitivity reactions like basophils. Mast
cells are rarely seen in the bone marrow but increase number of this may be associated with refractory
anemia, chronic renal failure, Waldenstrӧm macroglobulinemia, and systemic mastocytosis.
The role of medical technologist or laboratory scientist who are trained in bone marrow
collection is to assist the person performing the procedure. As an assistant, the medical technologist
should act as an encouraging influence reliant on the response of the patient to the procedure.
Although the physician has usually explained the test to the patient and gained consent to carry out it,
the assistant medical technologist may have to review and explain the technique briefly. The assistant
medical technologist must be watchful for any problems that may arise regarding the procedure while
assisting in the technical part of the marrow procedure and preparing the specimen collected.

LESSON 3
LESSON TITLE: Red Blood Cell Physiology

1. enumerate the normal characteristics of red blood cell.
2. discuss the function of RBC.
3. differentiate the metabolic pathways of RBC.
4. discuss the normal red cell turnover.
5. discuss the differences between normal Hgb from abnormal Hgb derivatives.
LESSON GUIDE:
III. Learning Guide Time Learning Resources
allotment
Learning Content:
1. Enumerate the normal characteristics of red blood cell. 3-hour lecture
- Lesson 1 module
1.1 The properties and compositions of red blood cell
page/s: 22-34.
membrane and hemoglobin.
- Rodak’s Hematology
1.2 The formation or synthesis of Hemoglobin.
pages: 112 – 135.
- Clinical Hematology
2. Discuss the function of RBC.
principles, procedures,
2.1 The gas exchange and buffering effect of the red blood
correlation pages: 72-
cell.
87.
2.2 The oxygen dissociation curve and CO2 curve.
2.3 Comparison between the hemoglobin and myoglobin.
3. Differentiate the metabolic pathways of RBC.

3.1 Energy production of the red cells using the EMP and
HMP metabolic pathways.
4. Discuss the normal red cell turnover.
4.1 Hemoglobin destruction and recycling.
5. Discuss the differences between normal Hgb from abnormal

Hgb derivatives.
5.1 Methemoglobin, Carboxyhemoglobin and
Sulfhemoglobin formation and characteristics.
RBC physiology and characteristic.
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LESSON III: Red blood cell Physiology

The red blood cell is normally a biconcave disc, with central pallor and no cytoplasmic inclusion.
The diameter of the red cell is between 6 to 8 μm while its average volume is around 90 fL. The normal
thickness of the red cell is approximately between 1.5 to 2.5 μm and an average surface area of 140
μm. The normal life span of the red blood cell in the blood circulation is approximately between 90 to
120 days. In order for the red cell to reach this, the red blood cell should withstand the sheer pressure
innate within the blood circulation, and must have the ability to squeeze itself to small vasculature
without damage or hemolysis. This is made possible because of the red cell unique ability and that is
“deformability”. Deformability is the capability of the red cell to flex or stretched without losing its
normal discoid shaped as it bumps to the heart valves, in the blood vessel walls or entering small
capillaries. There are three important components of the red cell that contributes to the success of the
deformability of red cell, and these are red cell’s membrane, hemoglobin status, and energy production.
Any defect on these three components caused by either genetic or non-genetic reasons will affect the
deformability of the red cell which then eventually shorten the life span of it. This early or increased
destruction of the red cell is called hemolytic anemia. Hemolytic anemia may occur into two different
location and these are, extravascular and intravascular. Extravascular hemolysis is a type of hemolysis
that occurs outside the blood vessels but usually occurs within the reticuloendothelial system, such as
spleen and liver. The intra-vascular hemolysis, on the other hand, is a type of red cell destruction that
occurs within the blood vessels.
The membrane of the red cell is composed of lipids and proteins. The lipids are composed of
phospholipid bi-layer and cholesterol. The lipid bi-layer is made up of polar heads and a non-polar tail.
The polar heads of the phospholipid bi-layer is hydrophobic while the non-polar tail is hydrophilic. The
cholesterol, which provides additional cushion in the red cell membrane, is situated within the lipid
layer. In certain disorder that affect the metabolism of fats or cholesterol may lead to the abnormal
function and appearance of the red cell. Example of these abnormal rbc shapes are acanthocytes and
codocytes or “target cell”. Acanthocytes is associated with abetalipoproteinemia, while codocyte
formation is caused by the accumulation of cholesterol in the red cell membrane due to several factors
that affecting the level of cholesterol in the blood, such as chronic alcoholism and certain liver diseases.
There are two major membrane proteins found in the red cell and these are the peripheral protein and
transmembrane or integral proteins. The peripheral protein is located within the periphery of the
internal part of the red cell’s membrane while the transmembrane protein traverses the lipid bi-layer.
The transmembrane protein is connected or interlaced to the peripheral protein on several points that
helps provide stability on the entire red cell membrane. The molecular interaction of these two
membrane proteins keep and maintain the red cell’s discoid shape. Aside from providing membrane
stability, the transmembrane protein has other functions such as, serving as the gateway or channels
for the in and out of substances and ions within the red cells and it also provides attachment for
different antigens that helps dictate the blood group system of the red cell. Some example of these of
transmembrane proteins are glycophorins A, B and C and Rh proteins. The glycophorin carries blood
group system known as the MNSs antigens. Deficiency on this glycophorins may lead to disease related
to hemolysis. Rh protein is another important transmembrane protein, in which deficiency of this may
lead to early destruction of red cell and may cause abnormal appearance of red cell known as

stomatocytosis or “mouth cell”. Other blood group antigens are also found on red cell membrane like
the ABO blood group system. The ABO blood groups are attached to the red cell membrane as
glycolipids or glycosphingolipid. Another kind of transmembrane protein is the guanidine-phosphatidyl
inositol linked proteins or gpi-proteins. The formation of this protein is dictated by a gene known as the
PIGA gene. This protein is very essential to protect the red cell into a complement mediated hemolysis.
Red cell that lacks the formation of the gpi-linked protein may cause transient red cell hemolysis known
as “Paroxysmal Nocturnal Hemoglobinuria”. The expression of some CD markers such as CD 55 and CD
59 is greatly affected on this disorder. The loss of these CD markers will allow the red cell to be
susceptible to acid dependent - complement mediated lysis. These CD markers prevents the activity of
the complement convertase thus neutralizing the cytolytic capability of the complement proteins. The
CD 55 is also known as “decaying accelerating factor” which regulates the activity of C3 convertase by
preventing the formation and interaction of the C4b and C2b complement fragments. The CD 59 is also
known as the “membrane inhibitor of reactive lysis”, MAC- inhibitor protein, or protectin. The CD 59
prevents the complete formation of the “membrane attack complex”, (known also as C5b6789), of the
complement system by preventing the attachment of the C9 protein on it. The main function of the
peripheral protein is to provide a stable foundation of the red cell membrane. Examples of these
proteins are spectrin, ankyrin, protein band 4.2, protein band 3, and band 4.1. The spectrin protein is
made up of two chain namely the alpha and beta chain. This protein widely placed in the entire red cell
membrane from where the transmembrane protein is attached on it, as well as other peripheral protein
mentioned above. This interaction is very vital for the survival of the red cell within the circulation.
Deficiency or defective molecular structure of the spectrin protein is associated with hemolytic disease
known as hereditary spherocytosis. In Hereditary spherocytosis, as the red cell travels within the blood
circulation, the red cell membrane becomes loose and forms a membrane protrusion known as “blebs”.
These blebs formed on the red cell membrane will be remove eventually by the macrophages of the
spleen as the red cell enter on this organ. This event may lead to gradual loss of red cell membrane that
later decreases the size of the red cell and making the red cell fragile. The increase fragility of the red
cell membrane may lead to the early destruction of the red cell within the circulation which leads to
intravascular hemolysis. Patient who suffers on this disorder may have anemic symptoms during certain
viral infection like parvovirus B19. Hereditary spherocytosis is also associated with defective ankyrin,
protein band 4.2 and band 3. Defective protein band 3 may also associated with hereditary ovalocytosis
also known as Southeast Asia ovalocytosis. Defective protein band 4.1 is commonly associated with
hereditary elliptocytosis, hereditary pyropoikilocytosis, and Leach phenotype. Over all, intact membrane
is important to the normal red cell survival within the blood circulation.
The red cell’s cytoplasm is made up of entirely with hemoglobin. Molecularly, the hemoglobin
should be always in a reduced state and not in oxidized state. Oxidization of the hemoglobin make its
unstable and possibly precipitates within the red cell which forms visible inclusion bodies. One example
of this inclusion formed is known as Heinz bodies. This unstable hemoglobin may affect the normal
functioning of the red cell and its appearance, that may eventually lead to hemolytic anemia. The
hemoglobin synthesis begins in rubriblast stage and ends in the reticulocyte stage. The hemoglobin is
made up of two components, which are the “heme” and “globin”. The Heme is composed of a heme
ring known as protophorphyrin IX and ferrous iron (Fe+2). The globin is the protein part of the
hemoglobin and has different types of protein chains depending on the type of hemoglobin. In Hgb A1,

the type of globin chains are alpha and beta chains, while in Hgb A2 it is alpha and beta globin chains.
One molecule of hemoglobin is made up of 2 pairs of globin chains, four heme with four ferrous iron in
it, and a molecule of 2,3 DPG or BPG. The 2,3 DPG is very vital part of the hemoglobin molecule because
it helps in the normal and efficient release of oxygen from the hemoglobin to the tissue. This unique
component is synthesized within the Embden Meyerhof metabolic pathway of the red cell which is
known as the Rapaport-Leubering pathway. Therefore, an increase or decrease synthesis of the 2,3 DPG
may affect the normal oxygenation of the human tissue. The heme is initially formed within the
mitochondria of the progenitor red cell. A complex biochemical process occurs within the mitochondria
to produce a heme molecule. This starts with the action of ala-synthetase enzyme and co-enzyme A to
glycine which eventually produces phorphobilinogen. Further enzymatic activity will lead to production
of various products such as phorphobilinogen, uroporphyrinogen III, coprophorphyrinogen III,
protophorphyrinogen III and eventually protophorphyrin IX. Each of these products formed during
heme ring formation is collectively called as phorphyrins. (Please refer to the illustration below on how
the heme is completely formed). A defective heme formation due to several enzyme deficiency needed
for its formation may lead to a disease known as phorphyria. Phorphyria is a heme kinetic problem that
may lead to severe anemia and photophobia. The ferrous iron is inserted to the heme ring or also known
as P IX, with the help of an enzyme known as ferrochelatase. The iron is stored in various organs such
as liver, skeletal muscles, and bone marrow as ferritin. This ferritin is made up of a unique protein
known as apoferritin and a ferric iron. The apoferritin is a special kind of protein present on some
human cells that received iron from the blood-ferritin transport protein known as transferrin. The
transferrin collects and transports the iron absorbed in the small intestine or from the recycled iron of
the destroyed senescent red cells. As the heme is being synthesized in the mitochondria, the globin is
synchronize formed along with it. Globin chain formation is dictated by genes located on chromosome
number 11 and 16. The chromosome number 16 carries the alpha and zeta genes while the
chromosome number 11 has the beta, delta, gamma and epsilon chain. The cell uses the two processes
known as the translation and transcription process to promote protein synthesis. Transcription occurs
in the nucleus of the progenitor red cell while the translation occurs in the ribosomes. The number of
amino acids for the alpha and zeta chain is 141, while the other globin chains has 146. The molecular
structure of the globin chains forms a quarter nary appearance forming loops and folds within it, and
these are called helices. These helices formed was named as A, B, C, D, E and F helices. Normally, after
the globin chain was formed, they are bound together forming 2 pairs of it in the cytoplasm of the
maturing progenitor red cell. For Hgb A1 it is composed of 2 alpha and 2 beta chains, in Hgb A2 it has 2
alpha and 2 delta chains, and Hgb F has 2 alpha and 2 beta chains. The heme will then later be
incorporated in the F and E helices of the globin molecule completely and this occurs in the rubriblast
stage of maturing red cell.

The red cell has two functions and these are gas transport and blood buffering. When the blood
is pumped by the left ventricle of the heart to the different part of the tissue, the red cell delivers the
oxygen into the tissue and collects carbon dioxide from it, which later released as gas expelled in the
lungs. After the red cell delivers oxygen to the tissue there is a residual oxygen left within the red cell
and the partial pressure of this residual oxygen left is approximately 40 mm Hg. In the lungs the partial
pressure of oxygen within it is as the same as with our environment at sea level, which is around 100
to 120 mm Hg pressure. As the blood circulated back to the lung tissue, the high pressure of oxygen in
the lungs helps the oxygen to simply diffuse in to the red cell due to the low oxygen pressure within the
red cell. The movement of the molecules to a high concentration to low concentration to reach the
equilibrium of solutes to any parts of the solution is known as simple diffusion. This event effectively
took place between the lung and the red cell, thus after the red cell enters the lung’s environment, the
partial pressure of oxygen within the red cell is the same now with the lungs which is 100 mm Hg. If the
red cell reaches this oxygen pressure, it means that the Hgb molecule is 95.0%-97.5% or fully saturated
with oxygen. These fully oxygenated red cell will deliver the oxygen to the various tissue and
exchangeably collect carbon dioxide from the tissue. There are different biochemical processes that
occur during the exchange of gases between the red cell’s Hgb and the tissue and these processes also
provide buffering effect in the blood. First, as the red cell reaches the lungs, the Hgb is fully saturated
with oxygen meaning each of the hemoglobin molecule now carry four atoms of oxygen. The presence
of water molecule also plays a vital role on this complex process. Biochemically, this event resulted to
the formation of Hgb(O2)4 + H2O within the red cell’s cytoplasm. As the red cell goes to the tissue,
normally the oxygen is released from the Hgb to the tissue because the pressure of oxygen in the tissue
is lower than that of the red cells. This event makes the Hgb carry a negative charge due to the release
of a cation, which is the oxygen, thus giving the formation of Hgb- + H2O. On this same occasion, the
carbon dioxide or CO2 in the tissue is high which then binds with the water within the red cells and in
the plasma. The binding of CO2 and water will eventually produce carbonic acid or H2CO3 with the action
of an enzyme called carbonic anhydrase. This biochemical process resulted to the formation of Hgb - +
H2CO3 within the red cells. The negativity charge of the hemoglobin molecule attracts one hydrogen
atom from the carbonic acid resulting to the slight acidity of the Hgb. The loss of one hydrogen atom in
the carbonic acid resulted to the formation of a base which is called bicarbonate or HCO3- . The

biochemical process produced on this is the formation of HgH+ + HCO3- within the red cell. The release
of oxygen from the Hgb to the tissue and the binding of the H+ atom to the hemoglobin is known as
Bohr effect. This event is very useful if an individual is experiencing low blood pH or acidosis. One factor
that may lead to acidosis is hypoventilation. Whenever a person experience hypoventilation the CO 2
build up in the body, which eventually binds with H2O in the plasma forming carbonic acid or H2CO3.
The Hgb helps lower down the acidity of the blood due to high levels of H2CO3, by releasing more oxygen
than the usual amount to the tissue to accommodate the H+ atom from the accumulating H2CO3 in the
blood, and transforming it to HCO3- . The HCO3- or bicarbonate formed within the red cell will diffuse
out, and goes to the plasma which helps neutralize the acidity of the blood because of its basic property.
The release of HCO3- from the red cell to the plasma may cause an imbalance of negatively charge
molecule or anion within the red cell. To prevent this, the negatively charge chloride or Cl- in the plasma
diffuses in to the red cell in exchange to HCO3-. This is known as chloride shifting. On contrary, if a person
experience hyperventilation or high rate of breathing, the CO2 level in the blood drops which resulted
to alkalosis. Using the Bohr effect, the red cell’s Hgb this time holds more to the oxygen molecule
preventing the release of it to the tissue. This help prevent further formation of bicarbonate and allow
carbonic acid to form in the blood to neutralize the alkalinity of the blood. Now, as the blood circulates
back to the lungs, with a biochemical reaction of HgbH + HCO3-, the Hemoglobin binds with O2 more
than the H+ atom, which releases the Hydrogen atom that previously bound into it. The free H+ atom
will eventually bind back to the HCO3- to form H2CO3 or carbonic acid. This biochemical process resulted
to Hgb(O2)4 + H2CO3 formation. With the action of the carbonic acid anhydrase enzyme in the lung
tissue, it breaks down the H2CO3 into H2O and CO2 gas which resulted to the formation of Hgb(O2)4 +
H2O and CO2 gas. The carbon dioxide gas formed on this reaction eventually expelled from the lungs
through expiration.
The formation of the bicarbonates or HCO3- , is the process that the blood uses to remove the
CO2 in the tissue. About 70 % of carbon dioxide that is carried in the lungs is in the plasma bicarbonate
while 20% is in erythrocyte bicarbonate. This reaction method is not only removing the carbon dioxide
from the body but also help buffers the blood. The remaining 10 % of carbon dioxide is transported into
two other ways. Around 5% of the transported CO2 is dissolved and binds with the N-terminal amino
acid of the globin chains in non-oxygenated form Hgb to form carbaminohemoglobin, and the other 5%

is transported in other solution. The non-oxygenated Hgb (which is 0% O2 Hgb) binds more CO2 than
the oxygenated for of Hgb (which is 97.5% O2 Hgb). Therefore, the partial pressure of CO2 or pCO2 is
relatively high with non-oxygenated Hgb than the oxygenated Hgb. The difference of their pCO2
pressure in mm Hg is known as Haldane effect. The Haldane effect facilitates the binding of the Hgb in
the tissue, where the pCO2 is high, and the release of the carbon dioxide in the lungs, where the pCO2
is low. The Bohr Effect and the Haldane effect is an effective process that our blood use to buffer the
blood pH, or helps in maintaining the normal blood pH which is between 7.35 to 7.45.
The Adult hemoglobin known as Hgb A1, which is composed of 2 alpha and 2 beta globin chain,
may enter to either “T” form or “R” form. The “T” form or also known as the tense form is the kind of
hemoglobin where there is no oxygen atom on its heme. On the other hand, the “R’” form or also known
as relax form is where the hemoglobin has oxygen molecule in the heme. During the binding of oxygen
to the heme, the globin chains usually twist in a 15O angle. This twisting facilitates the successful
oxygenation of the hemoglobin molecule by opening its oxygen binding sites and pushing out the 2,3-
DPG molecule away from the beta globin chain. But when the hemoglobin is on its “T” form, the globin
chain twist back to a 0O angle to close and which in turn allows the binding of the 2,3-DPG molecule to
the Beta globin chain. The binding of the 2,3-DPG on the beta globin chain competes with the oxygen
binding with the hemoglobin molecule, thus promoting the efficient release of the oxygen to the tissue.
Therefore, a decrease synthesis of the 2,3-DPG will decrease the red cell’s capability to deliver oxygen
efficiently to the tissue, which usually occur in senile or senescent red cells and on red blood cell
products stored in Blood Bank refrigerator that are nearly expiring.
The Hemoglobin oxygen dissociation curve is very vital in assessing the oxygenation status of an
individual. According to what had mentioned earlier, the blood should be fully saturated or 97.5% to
100% saturated with oxygen. Lower oxygen saturation, like for example if it reaches 80%, the individual
may experience difficulty in breathing and may lead to abnormal level of blood pH. And if this is not
corrected the individual may eventually expire due to the complications of low oxygenation of the
blood. Normally, the blood should reach the 50% oxygen saturation or p50 at 26 to 27 mm Hg pO2 or
also known as the partial pressure of oxygen, and 100% oxygenation saturation or p100, must occur
when the pO2 reaches between 60-80 mm Hg. A further increase in partial pressure of oxygen will not
increase anymore the saturation of hemoglobin with oxygen because it already reaches the full capacity

or 100%, which allows no more space for oxygen binding within the hemoglobin molecule. As what is
shown in the figure below (fig.3.1). The normal oxygen curve shows a sigmoid curve and a plateau as it
reaches it full oxygen saturation. However, several factors that might shift the curve to the right or left.
Shifting the oxygen curve to the right signifies that the hemoglobin had an increased release of oxygen
or a decreased oxygen binding to the hemoglobin, while shifting of the curve to the left is vice versa.
The following factors that might shift the oxygen curve to the right due to increase release of oxygen
from the hemoglobin to the tissue are hypoventilation, hypercapnia, low blood pH or acidosis,
hyperthermia, high 2,3 DPG level, and hyperthyroidism. The shifting of the curve to the right due to
decrease binding of oxygen to the hemoglobin is due to some abnormal hemoglobin variants such as
methemoglobin, sulfhemoglobin, hemoglobin S and hemoglobin Kansas. These abnormal hemoglobin
derivatives are characterized by poor binding to the oxygen molecule. While the factors that may cause
shift of the oxygen curve to the left due to decrease release of oxygen from the hemoglobin to the
tissue are, hyperventilation, hypocapnia, high blood pH or alkalosis, hypothermia, low 2,3 DPG level
and hypothyroidism. The abnormal hemoglobin that causes the oxygen curve shift to the left due to
increase binding to an oxygen molecule are Hgb F, carboxyhemoglobin and hemoglobin Chesapeake.
Figure 3.1
Various forms of abnormal hemoglobin variants as what had mentioned above may be caused
by either genetic abnormality or sometimes it is acquired. Examples of these are methemoglobin,
sulfhemoglobin and carboxyhemoglobin. Normally the predominant Hgb in an adult individual should
be Hgb A1 which is around 97-98 % and the Hgb A2 should be 2-3 %. However, if any of the three
abnormal hemoglobin variants mentioned is present in an adult blood, normally, it should only
comprise around less than 1 % of the total Hgb concentration. High levels of these abnormal variants
may cause hypoxia and cyanosis. Methemoglobin is caused by the oxidization of the ferrous iron of the
heme then converted into ferric iron. Heme with ferric iron will never bind with oxygen thus leading to
poor oxygenation of the body or cyanosis. The allowable concentration of methemoglobin is only
around 1-3 %. More than 3% of it in the blood is called methemoglobinemia. This can be cause by a

genetic problem that leads to the deficiency of an enzyme necessary for reducing the ferric iron to
ferrous iron within the heme, and this enzyme is called methemoglobin reductase enzyme. The
methemoglobin reduction occurs in a special pathway under the EMP metabolic pathway and this is
known as the methemoglobin reductase pathway. This problem may be corrected by administering a
reducing substance such as ascorbic acid or methylene blue. However, if there is a genetic abnormality
that cause mutation in the globin chains known as Hgb M methemoglobinemia, it cannot be corrected
by administering these reducing substances. Acquired methemoglobinemia is caused by accidental
exposure to toxic substances like nitrate or nitrite, chlorates, quinine, quinolone, and benzocaine.
Increased level of this in the blood may cause an abnormal brownish hue on the blood specimen. The
sulfhemoglobin formation is caused by exposure to toxic substances that binds sulfur to the hydroxyl
groups of the hemoglobin molecule. these toxic substances are, sulfur containing drugs, hydrogen
sulfide, aromatic amine and nitrite or nitrate. Clostridium perfringens infection is also associated with
the formation of sulfhemoglobin. This abnormal hemoglobin variant may impart a mauve-lavander
color in the blood specimen. High levels of carboxyhemoglobin are associated with cigarette smoking
and carbon monoxide poisoning. Person who regularly smoke may increase the level of the
carboxyhemoglobin up to more than 5%. Carboxyhemoglobin binds and had a very high affinity to
oxygen, which is 200 times more than the normal oxyhemoglobin. This may cause the ineffective
release of oxygen to the body because the carboxyhemoglobin is not ready to let go of the oxygen. This
compromises the oxygenation of the body which may lead to hypoxia. High levels of this in the blood
specimen may impart a cherry red color especially in carbon monoxide poisoning.
The myoglobin is a heme pigment that is found in the skeletal muscles and cardiac muscle. The
myoglobin is composed of a single chain of 154 amino acid that forms eight alpha helices and one heme
or Protophorphyrin IX. The molecular weight of myoglobin is around 17,000 daltons which is obviously
lower than that of the hemoglobin which is 64,000 daltons. The myoglobin may have two different
forms and these are deoxymyoglobin which is free from oxygen, and the other is oxymyoglobin, which
is bound with oxygen. Unlike with hemoglobin, myoglobin doesn’t immediately release the oxygen
rather it binds strongly to it, which strongly suggests that its main function is to store oxygen within the
muscles. This unique property of the myoglobin produces a hyperbolic curve in the oxygen dissociation
curve rather than a sigmoid curve for hemoglobin (as shown in Fig.3.2). The myoglobin releases the
oxygen from its heme ring only if the oxygen tension in the muscle is severely decrease. This property
also helps the divers to hold their breath under water for longer period of time while keeping the muscle
oxygenated. Presence of myoglobin in the blood or plasma signifies an abnormal condition known as
“rhabdomyolysis”. Rhabdomyolisis is caused by damage to heart muscle due to myocardial infarction,
trauma or crush injury to the skeletal muscles, and viral, bacterial or parasitic infection that affects the
skeletal muscles. Urine specimen should not contain myoglobin because the kidney readily filters it, but
this may be present in urine in renal failure and extreme muscle injuries.

Figure 3.2
There are two metabolic pathways that the red cell uses to maintain its energy production and
stability of the hemoglobin molecule, and these are the Embden Meyerhof Pathway (EMP) and Hexose
Monophosphate pathway (HMP) or also known as Pentose Phosphate pathway (see figure 3.4). The
EMP is a glycolytic pathway that is responsible for producing enough energy anaerobically while the
HMP, an aerobic process, is responsible in protecting the hemoglobin molecule to any oxidation process
that may occur due to exposure from oxidants derived from drugs and toxic substances. Around 90-
95% the red cell enters the EMP pathway while 5-10% for the HMP pathway. There may be different
sugars used as the source of energy, but glucose is the main sugar used by the red cells. The glucose is
catabolized in the EMP pathway into three different phases. Each of these phases are consists of
complex biochemical and enzymatic reactions which produces 4 ATPs and consumes 2 ATPs. Therefore,
in every one molecule of glucose that enters in this pathway it produces 2 net ATPs in the process. The
pyruvate kinase is one of the major enzyme that is required to complete the production of ATP within
the red cells, therefore, deficiency of this enzyme may cause low energy production in the red cell and
that leads to the instability in the red cell’s membrane and early destruction of it or hemolysis. In
pyruvate kinase deficiency, the normal apprearance of the red cell may also be affected and shows
abnormal shapes such as burr cell or acanthocyte. There are two diversion pathways under the EMP
pathway, and these are Rapoport-Luebering and the Methemoglobin reductase pathway. The
Rapoport-Leubering pathway is responsible for the synthesis of the 2,3 biphosphoglycerate or
diphosphoglycerate, which is an essential part of the hgb molecule. The presence of the 2,3 BPG or DPG
in the hemoglobin molecule helps the efficient release of oxygen from the hemoglobin to the tissue.
Decrease synthesis of the 2,3 DPG may cause the poor release of oxygen to the tissue which then
eventually leads to low tissue oxygenation. This event is seen usually on senile red cells or senescent
red cells, and stored red cells in blood bank refrigerator. The methemoglobin reductase pathway is
responsible for reducing the ferric iron into ferrous iron within the heme. This process uses an enzyme
known as methemoglobin reductase or also known as cytochrome b5 reductase that controls the level
of methemoglobin into less than 1% in the blood. The Hexose Monophosphate pathway an oxidative
glycolysis consists of several enzyme reactions that prevents the sulfhydryl group of the globin molecule
from oxidation caused by the formation of peroxides derived from the metabolized administered drugs.

Example of drugs are antibiotics like streptomycin, sulfonamides, nitrofurantoin, and dilantins and
certain anti-malarial drugs. One of the major enzyme under the HMP is the glucose-6-phosphate
dehydrogenase enzyme or G-6-PD. As shown in figure 3.4, the G-6-PD with the help of reduced NADP
or NADPH, catabolizes the formed glucose-6-phosphate to 6-phosphogluconate. On the said process
reduced gluthathione (GSH) is produced which helps in the reduction of the oxidized sulfhydryl group
of the globin molecule. The oxidation of the sulfhydryl group is brought about by the action of the
formed peroxides (H2O2) from the metabolized drugs that were administered. Deficency of the the g-
6-PD enzyme may decrease the production of the reduced gluthathione within the red cell and may
cause instability or oxidation of the hemoglobin molecule. This event may cause the hemoglobin to
precipitate or crystalize within the red cell and forms an abnormal inclusion known as Heinz bodies. This
inclusion may appear as round bodies of different sizes and distributed within the red cells. The Heinz
bodies are removed by the macrophages of the spleen as the red cell enters into this organ,
unfortunately the red cell membrane is also damaged in the process resulting to hemolytic anemia. The
damaged red cell caused by this abnormality is called the “bite cells”(Fig 3.3).
Figure 3.3
Enzymes involved in the catabolism of glucose
First phase Second Phase Third phase
- Hexokinase - Glycerldehayde-3- - Phosphoglycerate mutase

- Glucose-6-phosphate phosphate dehydrogenase - Enolase
isomerase - Phosphoglycerate kinase - Pyruvate kinase
- 6-phosphofructokinase - Biphosphoglycerate
- Aldolase mutase
- Biphosphoglycerate
phosphatease

Embden- Meyerhof
Pathway H2O2
glutathione
GSH
Glucose glutathione
reductase peroxidase
ATP NADP+ H2O
+ GSSG
hexokinase
ADP glucose 6-phosphate NADPH
dehydrogenase Hexose Monophosphate
Glucose 6 Phosphate
Pathway
gluconate phosphate
6-Phosphogluconate
isomerase
NADP+ 6-phosgluconate
Fructose 6-Phosphate
NADPH dehydrogenase
ATP
phosphofructokinase
ADP CO2
Ribulose-5-Phosphate
Fructose 1 6-Diphosphate trans-
aldolase
aldolase
Methemoglobin
Dihydroxyacetone Glyceraldehyde 3 Reductase Pathway
Phosphate Phosphate
triose phosphate Methemoglobin
NAD Methemoglobin
isomerase NAD+ glyceraldehyde 3- reductase H+
phosphate dehydrogenase NADH Hemoglobin
Methemoglobin
NADPH + H+
reductase
1 3-Diphosphoglycerate 2-3-diphosphoglycerate
mutase
ADP
Phosphoglycerate
kinase RLP 2-3 Diphosphoglycerate
ATP
3-Phosphoglycerate 2-3-diphosphoglycerate
phosphatase
3-phosphoglycerate
mutase
2-Phosphoglycerate
enolase H2O
Phosphoenolpyruvate
ADP
Pyruvate kinase
ATP
Pyruvate
Lactate
dehydrogenase
Lactate
Figure 3.4

Whenever the red cell reaches the 120th day life span, this senile red cell will eventually come
to its destruction known also as “eryptosis”. There are several changes that occurs within the red cell
as it becomes senile or senescent and these are low energy or ATP production, increase Na+ and
decrease K+ inside the red cell, increase calcium ion influx within the cell, and changes in the molecular
structure of some proteins in the red cell membrane. The low ATP production is caused by the failing
glycolytic process and the consequence of this will be oxidation of membrane lipids and proteins. The
decrease on red cell’s energy production is further enhanced by the low glucose level within the
environment of the spleen. The increase intracellular Na+ will cause water influx within the red cell
which causes it to become spherical rather that discoid shape. All of these changes are further
enhanced by the stressful environment of the spleen as the senile red cell enters into this organ. Red
cells should be flexible to escape the splenic sieve in the basement membrane of the spleen, however
those senile red cells that becomes spherical cannot escape from it and will be trapped within it. These
trapped spherical red cells eventually will be phagocytized and destroyed by the macrophages of the
spleen. There are other factors that signals the macrophages to engulf or phagocytized this senile red
cells and these are, abnormal cluster of protein band 3 with attached auto-immunoglobulin IgG,
exposure of phosphatidylserine exteriorly from the red cell membrane, and the change in the molecular
structure of the integrin-associated protein or also known as CD47. The abnormal CD47 on senile red
cells binds with the thrombospondin-1 molecule that provides an “eat me” signal to the splenic
macrophages. As the macrophages destroys the red cell within it, the hemoglobin is released from the
destroyed red cell and degraded to heme and globin. The globin chains will be broken down into amino
acid which is returned back to the body’s amino acid pool. The iron is removed from the heme and
binds with transferrin to be transported and stored to different organs such as liver and bone marrow
which is then recycled and used up again by new maturing red cells. The remaining Heme or
Protophorphyrin IX inside the macrophages is degraded into biliverdin by the heme oxygenase enzyme.
The biliverdin is released to the plasma and reduced to unconjugated bilirubin by an enzyme known as
biliverdin reductase. The unconjugated bilirubin will enter the liver to form a conjugated bilirubin using
the enzyme known as glucoronyl transferase. This water-soluble conjugated bilirubin exits the liver via
the hepatic duct and goes to the common bile duct which eventually enters the intestine. The
conjugated bilirubin is oxidized by the intestinal bacteria forming a water-soluble urobilinogen. Most of
the urobilinogen form is further oxidized within the gut area transforming it to stercobilin that imparts
brown color to stool. Some of this water-soluble conjugated bilirubin and urobilinogen that is not
oxidized to become stercobilin, is absorbed in the intestine and goes to the blood which then
transported to the liver and recycled back in the bile or filter out and excreted by the kidney. The
excreted urobilinogen is further oxidized to form urobilin in the urine which imparts the yellowish color
in it. Most of the heme catabolism occurs in the macrophage of the spleen but this may also occur in
macrophages of the other organs known as reticuloendothelial system which includes the bone marrow
and lymph nodes, and circulating monocytes.
Normally most of the senile red cells are removed by the macrophage mediated lysis which is
also known as extravascular hemolysis, as what had been stated earlier. However, normally around 10
– 20% of this fragile senescent red cell are lysed inside the vasculature due to trauma resulted from the
turbulence created within the blood vessels. This event is known as intravascular hemolysis. The
damage red cells within the blood vessels releases the free hemoglobin in the plasma which is readily

filtered by the kidney. But the hemoglobin is toxic to the kidney therefore it should undergo a process
that will make it nontoxic substance, and this process is known as the “haptoglobulin-hemopexin-
methalbumin” system. The free hemoglobin on the plasma binds with the haptoglobulin forming the
hemoglobin-haptoglobulin complex. This complex cannot be filtered out by the kidney due to its large
molecular structure. As the hemoglobin-haptoglobulin complex travels in the blood and in tissues, it
will be taken up by the macrophages of the tissues using the haptoglobulin scavenger receptor on their
membranes known as CD136. This complex will be degraded within the cell’s lysosomes releasing the
globin, iron and protophorphyrin IX. The globin is catabolized, the iron will be recycled and the
protophorphyrin will be converted into unconjugated bilirubin. The unconjugated bilirubin will be
converted into conjugated bilirubin by the liver and further converted into urobilinogen. Some of the
free hemoglobin in the plasma will be oxidized forming methemoglobin. The methemoglobin formed
will be dissociated in the plasma to metheme or hemin and globin. The metheme will bind with
hemopexin forming hemopexin-metheme complex. This complex enters the liver through the use of a
receptor called CD91 which is part of a lipoprotein receptor-related protein (LRP1). Inside the liver cell
this complex is further degraded into bilirubin and iron which the latter is reused again. The metheme
can also bind with the albumin in the plasma forming metheme-albumin complex. This complex is just
temporary because as this complex stay in the plasma the metheme dissociates from the albumin and
binds with hemopexin, this is because the metheme is more attracted with binding to the hemopexin.
In severe hemolytic anemia, where there is high number of red cells destroyed, the level of free
hemoglobin in the plasma is relatively high which eventually exhaust the concentration of the proteins
needed for clearing all of this free hemoglobin. This resulted to lowering the levels of haptoglobulin and
hemopexin but may cause an increased methemealbumin concentration in the blood. Aside from this,
the high level of unconjugated bilirubin in the blood and an increased level of urobilinogen in the urine
may also signify that an individual is suffering from hemolytic anemia.

LESSON 4
LESSON TITLE: Leukocyte Function and Kinetics
1. classify the different types of WBC according to each characteristic.
2. discuss the properties and functions of each WBC.
3. discuss the leukocyte kinetics.
LESSON GUIDE:
IV. Learning Guide Time Learning Resources
allotment
Learning Content:
1. Classify the different types of WBC according to each characteristic. 3-hour lecture
- Lesson 1 module
1.1 Granulocytes and non-granulocytes.
page/s: 35-46.
1.2 Phagocytes and Non-phagocytes
1.3 Polymorphonuclear and Mononuclear cells
pages:149-163 .
2. Discuss the properties and functions of each WBC.
2.1 The role of Phagocytes in Natural immunity.
principles, procedures,
2.2 The role of Immune cells in the Adaptive immunity.
correlation pages: 292-
3. Discuss the leukocyte kinetics.
303.
3.1 The Egress and life span of the White blood cells.
RBC physiology and characteristic.
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Lesson VI: Leukocyte Kinetics and Function

The human immune system has two different types and these are the Innate or Natural
immunity and the Adaptive or acquired immunity. The innate immunity, which is already present at
birth, is providing the human body with protection against various pathogenic organism using the
natural physical and biochemical barrier, as well as certain neurological responses such as sneezing and
coughing. Examples of the physical barriers are intact skin and skin appendages while for biochemical
barriers are the body secretions like sweat, tears, mucus, and oil droplets on our skin. Most of these
secretions are provided with lysozyme enzyme which is very effective in digesting the walls of certain
susceptible bacteria. All of the said protection is part of the body’s first line of defense. The first line of
defense, even though it is effective, can be evaded by certain microorganism and successfully enter the
human body. Example of these are the Hookworm species (Necator americanus and Ancylostoma
duodenale) and Blood flukes (like Schistosoma japonicum) can penetrate an intact skin and can invade
various tissues and organs. On this cases, the human body fights back by using the provided second line

of defense in which the white blood cell is part of it. The white blood cell’s phagocytic function can keep
the human body free from any foreign particles that accidentally entered it, and these WBC’s are the
neutrophil, eosinophil, basophil and monocyte or macrophage. The phagocytes are effective in killing
and digesting organism that harbors and colonized tissues, however they are not that effective in
eradicating organism that already lives and multiply inside cells like the viruses. Viruses that evaded the
phagocytes and already lived inside the cell will be eradicated by the human body’s third line of defense
in which the lymphocytes are part of it, and these lymphocytes are the T-cells and B cells. The third line
of defense is providing the human body the acquired type of immunity because it has the ability to
recognized foreign organism that already entered the body by developing memory T-cells and memory
B-cells. This mechanism is effective on giving the human body a lifetime immunity against various forms
of viruses. Summary of other components of body defenses is listed on table below.
Body defenses
Natural or Innate Immunity Acquired or Adaptive Immunity
First line Second line Third line
▪ Physical barriers ▪ Phagocytosis ▪ Antigen presenting cells
- Intact skin - Nuetrohpils - Macrophages
- Skin appendages - Eosinophils - B-cells
- Beating of the cilia of - Basophils - Dendritic cells
respiratory lining - Monocytes/ macrophages ▪ Immunocytes
▪ Biochemical barriers ▪ LGL’s cytotoxicity - T-cell
- Body secretions (lysozyme) - NK cell - B-cell
- Acidity of the stomach and - K cell - Plasma cells
vagina - LAK cell Complement system
▪ Neurologic response ▪ Inflammatory response - Classical pathway
- Sneezing ▪ Complement systems - γ interferon
- Coughing - Alternative pathway - interleukins
▪ Urination - Lectin pathway
▪ Diarrhea Plasma proteins
- Α and β interferons
- interleukins
The white blood cells can be classified according to its nuclear lobulation, cytoplasmic
granulation and function. See table 4.2. The phagocytosis or also known as “cell eating” is commonly
part of the inflammatory response that the body experience. Inflammatory response is a body’s
reaction in any injury or infection that the human body experienced. There are three phases of
inflammation, the first phase is vascular response, the next is the cellular response and the last phase
is tissue repair. Whenever microorganism colonized and multiply on the tissues the blood vessel
responded by dilating its lumen to increase the blood flow on the area to recruit phagocytes on the site
of colonization, then later it constricts to allow the steps of phagocytosis to occur. The action of the
phagocytes is part of the cellular response. The recruited phagocytes on the site will eventually roll,
activated, adhere or arrest and do trans endothelial migration on blood vessel walls using their adhesive
molecules such as mucin-like cellular adhesion molecules (CAM), chemoattractant receptor, integrins
and heparan sulfate receptors. The mucin-like CAM binds with the E-selectin of the blood vessel wall,

the chemoattractant receptor binds with chemokine IL-6, while the integrin binds with the integrin-
superfamily CAM of the vessel wall. See figure 4.1.
Figure 4.1
After the trans endothelial migration or also known as “diapedesis”, the phagocyte will prepare
its membrane receptors and move towards where colonization of bacteria is located, this movement is
called “chemotaxis”. This positive movement is influenced by the chemotactic factors or signals which
directs the phagocyte in the location site. There are several chemotactic signals and these are bacterial
or viral proteins, complement fragments, immune complexes, and antigen-antibody reactions.
Phagocytes that is not stimulated by these chemotactic signals has a defective movement or motility
and these are the Lazy leukocyte syndrome and the Job’s syndrome or also known as the hyper IgE
syndrome. Individual with these WBC disorders are susceptible to recurrent infection or chronic
infection. As the phagocyte nears the foreign bodies it will open its cytoplasm and engulf it. As the
microorganism or foreign bodies are ingested it will be enclosed within a cytoplasmic vacuole forming
a structure known as phagosome. The cytoplasmic lysosomal granules of the phagocytes will adhere on
this phagosome forming a structure known as phagolysosome. The lysosome that adheres to the
phagosome will burst its vesicle releasing a hydrolytic enzyme that could kill the microorganism and
this process is known as “digestion”. The debris formed after digestion will eventually release out from
the cell and this process is known as secretion. The phagocytic power of the phagocytes is measured in
the laboratory using the phagocytic index.
In acute infection or inflammation, the predominating white blood cell is the neutrophil while
in chronic infection it is the monocyte. Aside from the released of hydrolytic enzymes within the

phagolysosome formed, another way that the neutrophils and monocytes or macrophages kill the
engulfed bacteria is using a biochemical reaction within it known as “respiratory burst” or oxidative
burst. The objective of this reaction is to initially form a reactive oxygen species (ROS) or singlet oxygen.
The biochemical reaction involved is the NADPH oxidase (NOX2) bound to the membrane of the
phagosomes will react with its substrate NADPH to allow the transfer of one electron to Oxygen to
create a superoxide free radical known as singlet oxygen (O2●). The superoxide form will be acted upon
by either the superoxide dismutase (SOD) or myeloperoxidase (MPO) producing Hydrogen peroxide
(H2O2). The H2O2 itself has an antibacterial property, however spontaneous reactions such as Haber-
Weiss reaction (also known as Fenton reaction) and Halide reaction may occur to further convert H2O2
to another substance. In the Fenton reaction the H2O2 plus iron Fe+3 will be catabolized into hydroxyl
compounds (●OH) that can induce damage to bacteria or other pathogenic organisms. In Halide reaction
the H2O2 plus the Cl- ion will be converted to hypochlorous acid with the action of myeloperoxidase
enzyme. The acid formed can disrupt the activity and multiplication of the bacteria which eventually
die within the phagosome. The H2O2 is also toxic to human cells therefore the white blood cell should
convert it to nontoxic substance by using the two enzymes which are the catalase enzyme that forms
water and oxygen, and the glutathione oxidase under the Pentose Phosphate pathway or HMP. The
Superoxide or singlet oxygen (O2●) can also react with the Nitrous Oxide (NO●) forming peroxynitrite
anion which can destroy bacterial proteins as well as the DNA. See figure 4.2. In Chronic Granulomatous
disease (CGD) the oxidative burst described above is defective which causes the inability of the
phagocyte to kill the ingested organism effectively, that may lead to serious recurrent infection.
Figure 4.2
Large organism such as parasitic larva or helminths that cannot be totally engulfed by
neutrophils are destroyed by the eosinophil. Eosinophil is effective in killing larvae and helminths

because it has the ability to release its granules outwardly from its cytoplasm towards the extracellular
space and adheres to the body of the larva. There are several processes on how the granular contents
of the eosinophil was released and these are classical exocytosis, compound exocytosis, and “piecemeal
degranulation”. The main components of the eosinophil’s granule that can digest the integument of
worms and larva are the “Major Basic Protein” (MBP) and eosinophil cationic protein. The eosinophil
also produces the reactive oxygen species, as what had been described above, that helps in eradicating
parasitic organisms. This WBC also plays a major role in immune regulation by removing double-positive
developing lymphocyte or thymocytes within the thymus tissue. The eosinophil regulates the allergic
reaction by releasing histaminase enzyme that neutralizes the action of histamine which causes the
symptoms of allergy. Increased number of eosinophil or eosinophilia is associated with certain allergic
disorders such as asthma, intestinal food allergy, allergic colitis, ulcerative colitis and Chron’s disease.
It can also act as antigen presenting cell and promotes the development of effector cells during adaptive
immunity. The eosinophil also influences the tissue survival and activity of the mast cells by releasing
the nerve growth factor. Additionally, mast cell degranulation is regulated by the release of MBP and
other cytokines from the eosinophil.
Basophils are previously thought only to be responsible for allergic reaction because of its close
relation with mast cell’s antigen crosslinking IgE mediated histamine release. But later they have found
out that the basophils also aid in the regulation of the adaptive immunity by releasing cytokines such
as IL-4 and IL-13 which activate the T helper 2 (Th2) effector cell. The Th2 is responsible for the
activation and differentiation of B-cell to promote Humoral immune response. Other functions of
basophils are inducing B-cell to synthesized IgE immunoglobulin, releasing of mediator of allergic
reaction known as granzyme B, taking part in angiogenesis (blood vessel formation) by helping the
expression of vascular endothelium (VEGF) and its receptors on endothelial cells, and promoting
eosinophilia and macrophages activation that help eradicate lung worm infection.
Monocyte or macrophage as a “professional phagocyte” has the capability to recognized wide

range of bacterial organisms using its toll-like receptors. This activity can induce cytokine release which
promotes inflammatory responses especially during chronic infections. Macrophages use several
components of its cytoplasm to effectively kill pathogenic organism as how the neutrophil does it. This
also acts as a “scavenger cell” which makes it effective in removing debris resulted from tissue damage
caused by trauma or infection. Macrophages located in the bone marrow is taking part in the nurturing
of developing red cells by giving off the stored iron within its cytoplasm for hemoglobin formation,
while splenic macrophages destroy senile and abnormal red cells. Aside from these functions, another
vital role of the macrophages is its capability to disintegrate antigens of pathogenic organisms and
present it to the T helper cell for the activation of the Adaptive immunity. As an antigen presenting cell,
the macrophages will engulf and destroy the pathogenic organisms, such as bacteria or viruses, and
capturing the degraded molecules of it. These degraded molecules are collectively called as small
antigenic peptides. These small antigenic peptides will migrate from the cytoplasm to the macrophages’
cell membrane and binds with the MHC class type II molecule located on the surface of the cell (figure
30. After which, the macrophages will release IL-1 to activate the T helper cell and finally present these
antigenic peptides by binding it to the T cell receptor site of the activated Th cell. Then the activated
Th cell will release IL-2 to initially trigger the Adaptive or Acquired immunity. See figure 4.4.

Figure 4.3
Cytoplasmic contents of Granulocytes and Monocyte

Neutrophil Eosinophil Basophil Monocyte
▪ nitric oxides ▪ Major Basic Protein ▪ Histamine ▪ hydrologic enzymes
▪ Antibiotic proteins ▪ Arysulfatase ▪ Prostaglandin ▪ Reactive oxygen
▪ Azurophilc granules ▪ Histaminase ▪ Thromboxane ▪ Metabolites
- Peroxidase ▪ Leukotrines ▪ Hydrogen peroxide
- lysozyme ▪ Heparin ▪ Singlet oxygen
- acid hydrolase ▪ Eosinophil chemotactic ▪ Hydroxyl radicals
▪ Specific granules factor
- Lactoferrin
- Lysozyme
▪ Tertiary granules
- Alkaline
phosphatase
Classification of White Blood Cells

According to Function According to Granulation According to Lobulation
Phagocyte Granulocyte Polymorphonuclear
Neutrophil Neutrophil Neutrophil
Eosinophil Eosinophil Eosinophil
Basophil Basophil Basophil
Monocyte
Non-phagocyte Non-granulocyte Mononuclear
Lymphocyte Lymphocyte Lymphocyte
Monocyte Monocyte
Table 4.2

The Adaptive immunity
The Adaptive or Acquired immunity is a series of cell activation influenced by the release of
different cytokines known as interleukins. Interleukins are chemical released by immunologic cells
which is responsible for growth, development and differentiation of the targeted cells. Interleukins can
also influence the maturation of hematopoietic cells that directs it to the proper order of development.
Different types of interleukins and specific functions of each is listed on table 4.4. As what is mentioned
earlier, the Antigen Presenting Cell or APC releases the IL-1 to activate the T helper (Thp). The activated
Thp spontaneously releases another IL-2 to activate the naïve T helper (THo) and will be transformed
to either T helper 1 (TH1) or T helper 2 (TH2) effector cell. The Th1 effector cell formation needs the
activation of IL-2, IL-12, and γ-IFN, while to become a Th2 effector cell a collection of interleukins is
needed and these are IL-2, IL-4, IL-10 and IL-13. These effector cells assume their own different function.
The Th 1 is responsible for the activation of the cellular mediated immune response while the Th2
triggers the humoral immune response. Both of this activated effector cell has memory on the specific
antigen that the APC presented to them. This is the reason why the adaptive immunity is unique from
the innate immunity due to its ability to recognized foreign pathogens thru its entire antigenic makeup.
Figure 4.4
The Cell mediated immune response is activated by the Th1 by releasing γ-Interferon that will
activate the T cytotoxic cell (Tc). The Tc cell is responsible for “cell-to-cell combat” that causes
cytotoxicity on a targeted cell by releasing its cytoplasmic contents known as perforin and granzymes.
This cell is responsible in killing virally infected cell and cancer cell or tumor cell. In humoral Immune

response the Th2 releases IL-4,5,6 and 2 to activate the B-cell to transform into either B-memory cell
or plasma cell. The plasma cell produces immunoglobulins that binds specifically on the antigen that
was initially presented to the THp via the APC’s. There are five immunoglobulin types that is synthesized
by the plasma cell and each of them has their different functions. See Table 4.3. The immunoglobulin
synthesized binds with the pathogenic organism and will cause immobilization of it, induce cytotoxicity
on viruses or bacteria by activating the complement system, enhances phagocytosis of this pathogenic
organisms through opsonization process, and neutralizes certain toxins that is produces by certain
pathogenic bacteria or fungi. The human immune response has two phases, first it is called the Primary
immune response in which the IgM immunoglobulin is the predominating antibody produced, and the
second phase is the Secondary immune response or also known as where the IgG antibody is the most
predominant one. The primary immune response is the state where the body first encountered the
invading organism which makes the antibody production takes a longer period of time due to the initial
activation of the immune cells. On the other hand, the secondary immune response is the second
encounter of the same organism which leads to rapid stimulation of antibody production due to the
stored memory cells already formed. The B memory cell will also react specifically with the recognized
antigen by the adaptive immunity. The mechanism of these two immune responses is very effective in
providing an individual with lifetime immunity against a pathogenic organism that had already
encountered by the body.
*The Activation of the Adaptive immunity

Immunoglobulin Types
IgM IgG IgA IgE IgD
Heavy chains μ γ α ε δ
No. of antigenic Five Two Four Two Two
binding sites
Molecular weight 900,000 daltons 150,000 daltons 385,000 daltons 200,000 daltons 185,000 daltons
Percentage of total 6% 80% 13% 0.002% 1%
Antibody in serum
Fixes complement Yes Yes No No No
Crosses placenta No Yes No No No
Fc portion binds to Phagocytes Mast cell and
Basophils
Function The major The major Found on body Main antibody Found on B-cells
antibody in antibody in secretions like associated with membrane as one of
primary immune Secondary mucus to allergy and anti- the immunoglobulin
response. immune neutralize toxins. parasitic action. markers.
Monomeric response. Present also on
forms are found Neutralizes toxin colostrum.
on B-cell and enhances
membrane. phagocytosis via
opsonization.
A special kind of lymphocyte known as the Large granular lymphocyte (LGL) has the ability to kill
virally infected cell, tumor cell, cancer cell and senile cells. There are three different forms of these
LGL’s and these are Natural Killer Cell (NK), Killer cell (K cell) and Lymphokine Activated Killer Cell (LAK).
These cells recognized abnormal cell nonspecifically and without memory that is why it is part of the
human body’s natural immunity. The virally infected cells are recognized by NK cells through the
expression of stressed associated molecule on the surface of those infected cells. The NK cell binds on
these molecules using its receptor known as Killer Associated Receptor (KAR) and then releases the
contents of its cytoplasmic granules which are called perforin and granzyme. The released perforin
attaches to the cell membrane of the infected cell and cause perforation on the cell wall which leads to
the osmotic imbalance of the cell. Then the granzyme enter on these holes on the membrane and goes
directly to the nucleus of the virally infected cell to inhibit the DNA synthesis of the cell which kills it
along with the virus within it. Cancer cells are recognized by the LGL by the downregulation of the MHC
molecule of these abnormal cells. The NK cell uses another receptor known as Killer Inhibition Receptor
(KIR) to bind on these abnormal MHC molecules located on the surface of the cancer or tumor cells and
eventually kills it the same way as what has been described above.

White blood cell Kinetics
NEUTROPHIL
The production of the neutrophil is approximately between 0.9 and 1.0 x 109 cells/kg per day.
Within the bone marrow the proliferative pool is composed of around 2.1 x 109 cells/kg while the
maturation pool is 5.6 x 109 cells/kg. The estimated transit time from myeloblast through myelocyte is
about 6 days while the transit time through the maturation pool is approximately 4 to 6 days. The
granulocyte is released from the bone marrow through the stimulation of the G-CSF. The Neutrophils
in peripheral blood is divided into two different pools known as the Circulating neutrophil pool (CNP)
and Marginal neutrophil pool (MNP). Normally, the distribution of the neutrophils on these pools are
50% for each.
EOSINOPHILS
Around 1 to 3% of nucleated cells in the bone marrow is eosinophil and 1/3 of it are mature and a
quarter of it are eosinophilic metamyelocytes. The eosinophils are comprising the 1 – 3% of the
peripheral blood leukocytes with an absolute number of 0.4 x 109/L. The length of mitotic division of
eosinophil in the bone marrow is approximately 3.5 days with a mean cell turnover of 2.2 x 108 cells/kg
per day. The eosinophil and its precursor cells are made up of around 9 to 14 x 108 cells/kg in the
marrow. The half-life of this cell in the circulation is around 18 hours but prolonged half – life when
eosinophilia occurs due to parasitic infection. Eosinophil that migrated to tissue may survive for about
2 to 5 days.
BASOPHILS and MAST cells

The basophil has similar function and properties to mast cell. This cell is considered to be least
numerous of the WBCs making up between 0 – 2% of circulating leukocytes and less than 1% of
nucleated cells in the bone marrow. The approximated life span of Basophil is 60 hours. Mast cells are
considered as tissue effector cells of allergic responses and inflammatory reactions. The origin of most
of its progenitor are from spleen and marrow. The Mast cells can also function as antigen-presenting
cells that may cause the differentiation of TH2 cells which can influence in both innate and adaptive
immunity. In addition, mast cells may enhance or suppress the immune responses due to its anti-
inflammatory and immunosuppressive functions.
MONOCYTE/MACROPHAGE
The macrophages size can be as large as 40 to 50 μm in diameter. Normally promonocytes go
through two mitotic divisions in 60 hours to produce a total of four monocytes, but in conditions when
there is an increased demand for monocytes, the promonocytes undergo four mitotic divisions to yield
a total of 16 monocytes in 60 hours. Unlike the neutrophils, there is no storage pool of mature
monocytes in the bone marrow and the monocytes are released instantly into the circulation upon
maturation. The monocytes remain in the circulation for approximately 3 days but it is longer whenever
it is residing in the tissues. The life span of macrophages in the tissues depends on whether they are
responding to inflammation or infection, or are “resident” macrophages such as Kupffer cells or alveolar
macrophages. Resident macrophages survive far longer than tissue neutrophils. The monocytes make
up around 18% to 42% of circulating leukocytes with an absolute number of 0.8 to 4.8x 109/L.

LYMPHOCYTE
The development and differentiation of the lymphocyte may occur in two ways and these are
Antigen dependent and antigen independent. In the Antigen independent the lymphocyte
development occurs in the bone marrow and thymus which are the central or primary lymphoid organs,
whereas the antigen-dependent lymphocyte development occurs in the secondary or peripheral
lymphoid organs such as spleen, lymph nodes, tonsils, mucosa-associated lymphoid tissue (MALT) like
the Peyer’s patches located in the intestinal wall, and bucosa associated lymphoid tissue (BALT).
Approximately around 3% to 21% of circulating lymphocytes are B – cells and the resting B lymphocytes
cannot be differentiated morphologically from resting T lymphocytes. One way to differentiate B- cell
from T-cell is by identifying of the CD markers, where CD19,20,21 is seen on B cells while CD 2,3,4,5,7,
and 8 is present on T- cells. Resting lymphocytes are small (around 9 mm in diameter) with an N:C ratio
ranging from 2:1 or 5:1. T cells comprise the 51% to 88% of the lymphocytes in the circulation. Lymphoid
progenitor cells travel from the bone marrow to the thymic cortex, where, under the influence or
regulation of cytokines produced by thymic epithelial cells. These cells develop through three stages
known as pro-T, pre-T, and immature T cells. Throughout these phases they undergo antigen receptor
gene rearrangement to produce T cell receptors that are exclusive or distinctive to each T cell. T cells
whose receptors react with self-antigens are allowed to undergo Apoptosis or spontaneous cell death.
T cells are subdivided into two major groups, depending on whether or not they have CD4 or CD8
antigen on their surfaces. The T helper cell is positive with CD4 marker while the T cytotoxic cell is CD8
positive. The Immature T cells then advances to the thymic medulla, where further apoptosis of self-
reactive T cells happens. The elimination of the self-reactive T cell is very vital to prevent the
development of autoimmune disorders. The remaining immature T cells (or antigen-naïve T cells) then
leave the thymus and travel to secondary lymphatic organs, where they will reside in specific zones like
the paracortical areas. T – cells in secondary lymphatic organs or in circulating blood eventually come
in contact with antigens resulting in cell activation and the creation of either memory cells or effector
T cells. The immune cells are regulated by a CD4+CD25+ T cell known as T reg cell. The T reg cell is
responsible for check and balance of the immune activity to prevent over reaction of the activated T
cells which may lead to autoimmune disorder, this mechanism is known as “self-tolerance” or “immune
tolerance”. NK Cells, on the other hand, are special kind of lymphocytes that make up the 4% to 29% of
circulating lymphocytes and they carry with them their surface antigens CD 56, 16, 3 and 7. The NK cell’s
cytoplasm contains azurophilic granules that are peroxidase negative.
Cytokine Main cell source Main Targeted cell Biological function

IL-2 T helper cell T cells ▪ Moderator of immune tolerance
NK cell B cells ▪ Controls T regulator cells
B cell NK cells ▪ Activation and Growth of T helper and
Monocytes T cytotoxic cells
IL-3 Activated T-cell Hematopoietic cells and ▪ Production or proliferation of
NK cell progenitor cells Hematopoietic cells (Myeloid cells)
IL-6 Macrophages T cell ▪ Growth and development of T cells and
T cell B cell B cells
Fibroblast Liver cell ▪ Helps in megakaryocyte maturation
▪ An acute phase reactant
IL-10 Th2 effector cell T cell ▪ Used as negative feedback to Inhibit
T helper Macrophages macrophages and cytokine production

T cytotoxic cells
Monocytes
Macrophages
IL-12 Macrophages T cells ▪ T h1 effector cell differentiation
IL-15 Activated T helper cell T helper cell ▪ T helper and T cytotoxic cell
T cytotoxic cell proliferation
NK cell ▪ Activates NK and Tc cell cytotoxicity
γ-IFN Dendritic cell Macrophages ▪ Antiviral activity
NK cell NK cells ▪ Allows increase MHC molecules
B cell expression
T cell
Macrophages
Fibroblast
Endothelial cells
Osteoclast

LESSON MODULE
LESSON V
LESSON TITLE: Complete Blood Count and other Routine RBC Procedure

1. discuss the principle and clinical significance of each parameters included in the CBC.
2. perform the manual procedures included in the CBC parameters.
3. analyze the factors affecting each test included in CBC.
4. memorize the reference value for each test with regards to age and gender of the individual.
5. discuss the clinical significance and principle of ESR and zetacrit.
6. discuss the clinical significance and principle of parameters that evaluate reticulocyte.
7. associate the interpreted results with diseases involving hematological disorders.
LESSON GUIDE:
V. Learning Guide Time Learning Resources
allotment
Learning Content:
1. Discuss the principle and clinical significance of each 4-hour lecture
- Lesson 1 module
parameters included in the CBC.
page/s: 47 - 73
1.1 Parameters of the complete blood count.
1.2 The clinical use of CBC.
pages: 187-205,235-
2. Perform the manual procedures included in the CBC
parameters. 251.
2.1 Performing the manual blood counts. - Clinical Hematology
2.2 WBC parameters: WBC differential count, WBC principles, procedures,
absolute count and Corrected WBC count. correlation pages: 108-
2.3 Methods of Hemoglobin determination. 122.
2.4 Methods of Hematocrit determination.
2.5 The use of the “rule of Three”.
2.6 The different red cell indices and their clinical use.
3. Analyze the factors affecting each test included in CBC.
3.1 The physiologic and technical factors that influence the
result of the CBC parameters.
3.2 The possible source of errors and its remedy.
4. Memorize the reference value for each test with regards to
age and gender of the individual.
5. Discuss the clinical significance and principle of ESR and
zetacrit.
5.1 Methods of ESR determination.
6. Discuss the clinical significance and principle of parameters

that evaluate reticulocyte.
6.1 Different methods of reticulocyte count determination.
6.2 Determination of Corrected reticulocyte count and
reticulocyte production index.

7. Associate the interpreted results with diseases involving

hematological disorders.
7.1 Parameters that differentiate Polycythemia form
anemia.
7.2 Difference between relative and absolute polycythemia
and anemia.
7.3 Classification of anemia using reticulocyte count and
red cell indices.
7.4 High WBC count in leukemia and leukemoid reaction.
7.5 Type of infections related to WBC differential count
report.
7.6 Platelet estimates in assessing Thrombocytosis and
thrombocytopenia.

CBC, ESR, and reticulocyte examination.
hours after the lecture on CBC and other routine RBC
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Lesson V: Complete blood count and other routine RBC procedures
Red cell Count

Complete blood count is the test routinely performed in a Hematology laboratory. This test is
very helpful in assessing blood disorders and also aids in the diagnosis of other underlying illness of
non-blood origin such as infections and other inflammatory diseases. One example of this is HIV
infection. Patients with progressive HIV infection usually is reflected with lowering white blood cell
count and decreasing number of the lymphocyte number. In cholera, one of the results obtained in the
CBC is the decrease of the neutrophil count which may be use as one of the tools to give a differential
diagnosis on enteric type of infection. A decreasing platelet count and high hematocrit value in dengue
hemorrhagic fever is another example where the CBC is a vital part in the diagnosis of this diseases. The
complete blood count includes the following parameters such as red cell count, white blood cell count,
platelet count or estimate, white blood cell differential count, and the red cell indices which includes
mean cell volume or MCV, mean cell hemoglobin or MCH, and mean cell hemoglobin concentration or
MCHC. The anticoagulant of choice for CBC is EDTA or ethylenediamine tetra-acetic acid, because of
the advantages of it over other blood anticoagulant, and these are, preserving the morphology of the
blood cells, preventing platelet clumping, and it does not alter blood counts. There are two types of
EDTA anticoagulant available in the laboratory and these are K2EDTA and K3EDTA which are also known
as versene and sequestrene, respectively. Between the two types of EDTA, the K2EDTA is more ideal for
manual hematocrit determination than the K3EDTA because the latter causes minimal shrinkage of red
cells. This shrinkage may cause a decrease of hematocrit value of approximately 2-3% from the original

hematocrit of the blood specimen submitted to the laboratory. The ideal concentration of the EDTA
anticoagulant that is used for CBC should be around 1.5 mg/dL ± 0.25; an increase concentration of it
may cause the red cell to shrink which falsely decrease the hematocrit value, while a low concentration
of EDTA will cause blood to clot which may decrease the blood counts. Blood withdrawn for CBC should
be process within two to three hours from the time of collection because any delay may elevate the
hematocrit and MCV and decreases the MCHC value. Therefore, proper handling of specimen should
be taken into consideration prior to executing the test.
The principle of the manual red blood cell count is that the whole blood is diluted into a specified
or desired dilution using a Thoma pipette (fig. 5.2), and the cells are counted with the use of the
hemocytometer which then calculated using a mathematical formula to get the number of red cells per
cubic milliliters/microliters or per liter of blood. The clinical significance of the red blood cell count is to
determine if an individual is suffering from either anemia or polycythemia, where the previous has a
decrease red cell number while the latter has an increased number of red cells. The characteristic of
the diluting fluid used for red cell count should be an isotonic fluid in able not to shrink or swell the red
cell while counting the cells. The available diluting fluid for manual RBC count are Dacie’s, Hayem’s,
Gower’s, Bethel’s, Toison’s and normal saline solution or NSS. Dacie’s is the most ideal diluting fluid
among the other diluting fluids because of its advantage of preserving well the morphology of the cells
and preventing fungal and yeast growth during its storage. However, in the presence of
autoagglutinating red cell, Dacie’s diluting fluid should not be used because it will cause more clumping
of the red cells. On this case, it is suggested that the 3.8% sodium citrate should be used instead. The
Hayem’s diluting fluid should not be use if hyperproteinemia is suspected in the blood specimen of an
individual because it may enhance further the clumping and rouleaux formation of the red cells. The
NSS or normal saline solution should be considered as the alternative on this situation. The
Hemocytometer has a ruled grids and lines (when viewed under the microscope) designed specifically
for manually counting cells of the blood and other body fluids such as CSF and pleural fluids, etc. This
ruled area is known as the Neubauer rulings, which is composed of a center RBC square and four corner
WBC squares. (Refer to figure 5.1) Please refer to steps for RBC manual counting.
WBC WBC
RBC
WBC WBC
Figure 5.1

Figure 5.2

RBC Count Reference value

Age Normal Reference Value
(x1012/L)
Children 6-11 months 3.60 – 5.20
1-3 yr. old 3.40 – 5.20
4-7 yr. old 4.00 – 5.20
8-13 yr. old 4.00 – 5.40
Male Adult 4.20 – 6.00
>50 yr. old 4.90 – 5.20
Female Adult 3.80 – 5.20
>50 yr. old 4.30 – 4.80
White Blood Cell Count

The principle of the manual white blood cell count is that the whole blood is diluted into a
specified or desired dilution using a Thoma pipette (fig. 5.2), and the cells are counted with the use of
the hemocytometer which then calculated using a mathematical formula to get the number of white
cells per cubic milliliters/microliters or per liter of blood. The characteristic of the diluting fluid used for
this procedure is hypotonic solution which lyses the red cell and retain the nucleated cells in the
solution including the nucleated red cells. The presence of nucleated red cell in the specimen may cause
a false increase in the overall WBC count, therefore, correction should be done on this situation by
using a mathematical formula known as the corrected WBC count (please see the table of sources of
error to see the formula). There are several conditions that caused the presence of this nucleated red
cells in the blood and these are thalassemia major, severe case of anemia and leucoerythroblastic
reaction. The diluting fluid used are 2% glacial acetic acid, 1% HCl and Turk’s fluid. The area of the
hemocytometer chamber used for counting the white cells are the four corner squares as shown in
figure 5.1. An increase of the white cell number in a given specimen is termed as leukocytosis which

usually seen in certain pathologic conditions such as bacterial or viral infections, inflammatory reaction,
leukemoid reaction, and leukemia. High counts are also common in normal physiologic responses of
the body such as strenuous physical activities, anxiety and stress. Certain drugs such as epinephrine
administration and cortisol may also give high value of the white cell count. Leukopenia, on the other
hand, is the term used for having a decrease white cell count in the blood which may be caused by
certain viral infections like HIV and bone marrow failure or fibrosis. Please refer to steps for manual
WBC count.

WBC Count Reference value

(x109/L)
Children 6-11 months 6.0-18.0
1-3 yr. old 5.50-17.5
4-7 yr. old 5.0-17.0
8-13 yr. old 4.5-13.5
Male Adult 4.0-11.0
>50 yr. old 7.10-9.20
Female Adult 4.0-11.0
>50 yr. old 7.10-9.20
Platelet count
The principle of the manual platelet count is that the whole blood is diluted into a specified or
desired dilution using a Thoma pipette (figure 5.2), and the cells are counted with the use of the
hemocytometer which then calculated using a mathematical formula to get the number of white cells
per cubic milliliters/microliters or per liter of blood. There are two procedures available in the
laboratory for manual platelet count and these are the Rees and Ecker or “Tocantin” method, and the
use of disposable blood cell count dilution system, in which one of it is known as the Unopette system.
In the Rees and Ecker method the blood is diluted using either the WBC pipette or the RBC pipette.
Routinely the blood is aspirated at 0.5 mark of either RBC or WBC pipette, which creates a 1:200 or 1:20
dilution of blood respectively. The Unopette system (figure 5.3) has a reservoir containing a preferred
volume of 1% ammonium oxalate from where a 20 microliter of blood is placed that eventually gives a
1:100 dilution. In the Rees and Ecker method the medical technologist can use either the RBC squares

or the WBC squares in counting the platelets. If the RBC square is used for counting the platelet the
technologist should use the RBC count formula in determining the platelet number, but if it is counted
in the WBC squares, the WBC count formula should be used. According to several references, the
minimum number of medium RBC square used is 10, as shown in figure 5.5, this is done to minimize
the error committed and to make an accurate count if one uses the RBC square for counting the
platelets. On the other hand, the Unopette system uses the 25 medium squares of RBC square which
therefore uses the RBC count formula for computing the number of platelets. Due to its small size, the
platelets are counted using a phase-contrast microscope as a reference method for easy visibility as
what is described by Brecher and Cronkite, but in absence of this type, the common light microscope
can be used. (Please refer to procedure for manual platelet count.)
The determination of the platelet count is the first step done in investigating Hemostasis
problem. Patient with low platelet count which is also known as thrombocytopenia, has tendency to
bleed, while those who has high counts or with thrombocytosis may form thrombosis within the blood
vessels. Thrombocytopenia is caused by several conditions such as bone marrow failure or Fanconi
anemia, megaloblastic anemia, Bernard-Soulier syndrome and other white blood cell anomaly like May-
Heglin, Wiskot-Aldrich syndrome, and etc. Decrease number of the platelets are also observed due to
increase platelet sequestration in splenomegaly and on tumors like giant hemangiomas. The production
of autoantibodies against platelets may also cause an increase destruction of it which leads to severe
thrombocytopenia, and example of these conditions are the Idiopathic Thrombocytopenic purpura or
ITP, Neonatal Isoimmune thrombocytopenia, and Post Transfusion purpura or PTP. Platelets are also
decreased in consumptive coagulopathy like disseminated intravascular coagulopathy (DIC) and
thrombotic thrombocytopenic purpura (TTP). While thrombocytosis is usually encountered in
myeloproliferative disorder like polycythemia vera (PV) and essential thrombocythemia (ET).
Steps of manual Platelet Count (Rees and Ecker)

Materials:
Red blood cell pipette
Rees and Ecker diluting fluid
Petri dish
Filter paper
Hemocytometer
Procedure:
1. Fill red cell pipette with blood up to mark 0.5.
2. Wipe-off blood on the pipette tip without affecting the column of
blood in the pipette stem.
3. Suck Rees-Ecker diluting fluid to mark 101.
4. Shake pipette thoroughly for 3 minutes.
5. Charge Hemocytometer.
6. Place a water-moistened filter paper inside the petri dish.
7. Place Hemocytometer atop filter paper and cover petri dish.
8. Let stand for 5 minutes.
9. Focus the white blood cells counting area under low power.
10. Shift to high power. Count the platelets in 4 white cell squares.
11. Calculate platelet count using the formula below:
𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕 𝒄𝒐𝒖𝒏𝒕
𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓 𝒙 𝑫𝒆𝒑𝒕𝒉 𝑭𝒂𝒄𝒕𝒐𝒓
=
𝑨𝒓𝒆𝒂 𝑭𝒂𝒄𝒕𝒐𝒓

Plt. Count Reference value

(x109/L)
Children 6-11 months 150-450
1-3 yr. old 150-450
4-7 yr. old 150-450
8-13 yr. old 150-450
Male Adult 150-450
84-98 yr. old 256-298
Female Adult 150-450
84-98 yr. old 256-298
Steps of manual Platelet count (Unopette system)

Materials:
Blood collection apparatus
Hemacytometer and coverslip
Microscope
Counter
Unopette system
Petri dish with moist filter paper
Procedure:
1. Using the protective shield on the capillary pipette, puncture the diaphragm of the
reservoir.
2. Remove the shield from the pipette assembly by twisting.
3. Holding the pipette horizontally, touch the tip of the pipette to the blood. Pipette will
fill by capillary action. Filling will cease automatically when the blood reached the
capillary bore in the neck of the pipette.
4. Wipe the outside of the capillary pipette to remove excess blood, which could
interfere with the dilution.
5. Squeeze reservoir slightly to apply negative pressure thus removing some air inside.
6. Cover the opening of the overflow chamber of the pipette with index finger and seat
the pipette securely in the reservoir neck.
7. Release pressure on the reservoir. Then remove finger from the pipette opening
allowing blood to draw into the reservoir.
8. Squeeze the reservoir several times to allow adequate mixing of blood and diluent.
9. Place index finger over the upper opening and gently invert several times to
thoroughly mix the diluent.
10. Let the mixture stand for 10 minutes.
11. Convert the system to dropper assembly.
12. Discard several drops (3-4) then charge properly the hemocytometer.
13. Place the hemacyometer in a moist Petri dish for 10 minutes.
14. Mount the hemacytometer on the microscope and locate the RBC square.
15. Count the platelets on all 25 tertiary square or the entire RBC square.
16. Calculate Platelet count using the formula below:
𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕 𝒄𝒐𝒖𝒏𝒕 = # 𝒐𝒇 𝑷𝒍𝒕. 𝑪𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟏𝒙 𝟏𝟎 𝒙 𝟏𝟎𝟎
Sources of Errors in Manual Cell Count

The margin of error in manual cell counting may vary between 10 – 20 %, this is due to several
factors which makes it less accurate if compared with the automated cell counting machines. These
factors are “error of the specimen”, “error of the pipette”, “error of the chamber”, and “error of the

field”. Error of the specimen includes the improper handling of the specimen and poor collection
technique that may cause a significant discrepancy in the outcome of the cell counts which leads to
misdiagnosis of the illness. Hemolysis and clotted specimen are the two most common problems that
encountered in specimen handling. Hemolysis may cause decrease in the result of the RBC count and
increase Platelet count, while clotted specimen may cause decrease RBC count, WBC count and Platelet
count. Error of the pipette includes poor technique in aspiration of the blood, bubbles formed during
blood and diluting fluid aspiration, and glass pipettes bulb size and bore size are not standard. Error of
the chamber includes the lines and grids of the Neubauer ruling is not with standard dimensions, and
difficulty of counting the cell due to low visibility of this ruling during cell counting. Error of the field is
the uneven distribution of the cells in the chamber which is observable whenever cells are grouping on
one area and less cells on some area, this maybe due to drying of the mixture during cell counting.
Physiologic factors may also put the reliability and accuracy of the count in jeopardy which includes
polycythemia vera or severe hypoplasia of the bone marrow. In polycythemia vera the severely high
number of the cell may cause the difficulty in locating the grids and lines of the chamber due to
overlapping of numerous cells. This can be corrected by making a higher dilution than the routine
dilution made for cell counting. In hypoplastic bone marrow on the other hand may cause low number
of observable white blood cells which may also lead to an inaccurate counting, to correct this, one
should make a lower dilution than the usual routine dilution. Other source of error for each count is
described in the table below.
Source of Error
RBC count WBC count Platelet count
Factor Remedy Factor Remedy Factor Remedy
1. Polycythemia Make a 1:301 Extremely high Make a 1:200 Extremely high Make a 1:200
dilution leukocyte number dilution platelet dilution
>100 x 10 9/L
2. Severe anemia Make a 1:101 Extremely low Make a 1:10 Presence of platelet Collect new
dilution leukocyte number dilution clumps specimen
<3.1 x 10 9/L
3. Dirt, lint and dried Use clean Moderately high Make a 1:100 Presence of Platelet Collect citrated
blood on pipettes, equipment prior to leukocyte number dilution satellitism sample
hemocytometer cell counting >30 x 109/L
4. Presence of NRBC *Do corrected <80 platelets Count the

WBC count counted on 10 platelets on 25
medium RBC medium squares
squares of both chamber
Repeat the count
< 50 platelets and make a 1:20
counted on both dilution
sides of the chamber
*Corrected WBC count should be done if the medical technologist observed >5 nRBC per 100 white blood cells
observed in the differential count. The formula for corrected WBC count is stated below:
𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑡. 𝑥 100

𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑅𝐵𝐶 + 100

Hematocrit
The microhematocrit is the measurement of the packed cell volume on a given specimen after
the application of centrifugation. The microhematocrit centrifuge used for this test should be calibrated
to maintain its standard RPM of 10,000-12,000 and RCF of 10,000-15,000 gravitational force. The clinical
use of this parameter is to determine if the patient is suffering from anemia or polycythemia. A decrease
value of the hematocrit is seen in anemic individual while high levels of it is seen on cases of
polycythemia. Relatively the level of hematocrit value is higher in children than adults and higher on
males than female. The hematocrit value is also influenced by the geographical location of the individual
where higher values are seen on persons who lives at high altitude places, where there is low oxygen
pressure, than living at sea level. The low oxygen level on this place may cause hypoxia driven
erythropoietin release by the kidney which triggers the bone marrow to produce more red cells as a
compensatory mechanism. Hypoxic event may also experience by individual who suffers from heart or
lung problems like COPD which causes the HCT level above the normal value. Smokers also has higher
value of HCT than non-smokers because of the presence of carboxyhemoglobin on smoker’s blood. As
what had mentioned previously, the carboxyhemoglobin has higher affinity of oxygen than the normal
oxyhemoglobin which causes hypoxia on the body. To compensate, the kidney releases the EPO to
increase the production of red cell in the marrow.
The microhematocrit determination uses capillary glass tubes with a length of 70-75 mm and a
bore diameter of 1.2 mm. There are two types of capillary tubes one of it has a heparin and the other
does not have any anticoagulant or additive. Blood collected directly from the fingerstick should use
the heparinized capillary tube which has a red ring on one of its ends, while the capillary tube without
anticoagulant should be used if the blood is already mixed with K2EDTA. Refer to procedure 5.4 for
microhematocrit. Compared to the use of automated cell counter, the HCT value on spun blood is
approximately 1-3% higher than the HCT result obtained from the automated cell counters, this is due
to the presence of trapped plasma in between the packed red cell that contributes to the said increase.
The trapped plasma is great in certain hematological disorders that extremely affecting the size or
shape of the red cells like in sickle cell anemia and with severe case of thalassemia.
Hematocrit Reference value

(%)
Children 6-11 months 36-51
1-3 yr. old 38-48
4-7 yr. old 36-46
8-13 yr. old 35-49
Male Adult 40-54
84-98 yr. old 40-47.1
Female Adult 35-49
84-98 yr. old 37-43

Factors affecting the microhematocrit value

There are several factors that may cause a false increase or decrease of the microhematocrit
result on a given blood specimen. These are classified into either Physiologic or Technical factors.
Physiologic factors are uncontrollable causes, while the technical factors are controllable actions made
by the medical technologist that influences the outcome of the test. Examples of this technical factors
are poor blood specimen collection and handling, and improper technique in executing the HCT
procedure. The responsibility of the technologist is to identify these factors and do some corrections if
possible, especially on the technical ones. The list of these factors is stated in following the table below.
Physiologic factor
Increase Cause Decrease Cause
Red cell disorders These red cell disorders change the size Hypernatremia High plasma sodium
• Macrocytosis and appearance of the red cell which allows the efflux of fluid
• Spherocytosis increases the tapped plasma in between from the red cell causing
• Thalassemia packed red cells. crenation of the red cells.
• Hypochromic anemias
• Sickle cell anemia
Dehydration or loss of body fluid Loss of fluid may cause
• Severe Diarrhea hemoconcentration of the blood.
• Hyperemesis
• Burnt patients

Hyponatremia Low plasma sodium increases the entry

of fluid in the red cell causing it to swell
increase the trapped plasma in between
packed red cells.
Technical factor
Increase Cause Decrease Cause
Poor Blood collection Practices: Poor blood collection practices:
• Prolong tourniquet This action may result to • Puncture done above
application hemoconcentration of the the IV infusion line IV fluid contamination
• Allowing the patient to do blood. • Did not stopped the IV may cause hemodilution
Fist pumping during line for 2 minutes prior of the blood specimen.
venipuncture to blood collection
Improper handling of specimen: Improper handling of the

• Use of old EDTA samples or This resulted to swelling the specimen:
more than 6-hour old blood red cells that increases trapped • Use of high This factor may cause
sample stored in room temp. plasma in between packed red concentration of EDTA crenation on red cells.
cells. • Use of underfilled EDTA
tubes
• Filling the tubes more
than the desired This may trigger clot
amount of blood formation.
Common problem on specimen used: Common problem on specimen
• Use of non-heparinized The blood will clot inside the used:
capillary tubes on blood capillary tube preventing it to • Clotted specimen Trapped red cells on
collected directly on completely pack the red cells fibrin clots causing low
fingerstick after centrifugation. number of free red cells.
• Hemolyzed specimen Red cells are destroyed

leads to low number of it.
Poor Technique on microhematocrit Poor Technique on
determination: microhematocrit determination:
• Under centrifugation of • TheLeaking
red cellor
is washing
not completely
out of blood specimen during centrifugation Improper sealing of the
capillary tubes packed. capillary tubes prior to
centrifugation. The
• Placing the spun capillary Packed red cells may tend to sealant made should
tubes on horizontal position loosen resulting to high HCT. occupy at least 4-6 mm
on table tops instead of of the capillary tube in
vertically. one of its ends.
• Reading above the buffy coat Buffy coat is composed of

layer. nucleated cells like WBC,
therefore reading should be
done below the buffy coat
layer.
Hemoglobin determination
Measuring the level of hemoglobin concentration determines the oxygen carrying capacity of
the person’s blood. The clinical use of this parameter is the same that of the hematocrit determination
which is to establish or diagnose anemia or polycythemia. Low values of hemoglobin are seen in anemia
while high levels of it is common in polycythemia vera. There are tests or methods done previously in

detecting the level of hemoglobin on patient’s blood and this may fall in either colorimetric,
gasometrical or chemical principles. The most ideal and acceptable method used nowadays is the
chemical principle known as the cyanmethemoglobin determination. The cyanmethemoglobin uses the
Drabkin’s Reagent which is composed of two active ingredients known as potassium ferricyanide
K3Fe(CN)6 and potassium cyanide KCN. This reagent should be stored on dark place or brown bottle due
to its photosensitive property. Exposure to light may cause the deterioration of the Drabkin’s reagent
which will cause false decrease on the hemoglobin values obtained. This is a toxic reagent due to the
cyanide on it, therefore proper use, handling and disposal of this should be done properly. There are
two types of this reagent and these are the modified Drabkin which contains dihydrogen potassium
phosphate as its buffer solution and the original Drabkin which has sodium bicarbonate instead. The
advantages of using the modified one are shorter incubation time needed after the blood is mixed with
the reagent, and it is not affected by abnormal precipitating proteins in the plasma. The Drabkin
solution should be prepared fresh. The procedure includes placing a 20 uL of blood to either 5 ml or 6
mL of the said reagent, making a 1:251 or 1:301 dilution, respectively. Upon the blood is placed into
the Drabkin reagent the red cells are lysed and the hemoglobin is released and reacted with the active
components of the reagent. The chemical reaction involved after the blood is mixed with the reagent
is that the hemoglobin is oxidized by the K3Fe(CN)6 into methemoglobin (Hi) which then acted upon by
the KCN to convert it to the cyanmethemoglobin (HiCN) which has a light absorbance at 540 nm
wavelength (fig 5.6). This chemical reaction may take 10-15 minutes to complete if on uses the original
Drabkin, while it takes 3-5 minutes incubation for the modified one. The concentration of the
hemoglobin is computed using a formula devised by the Beer’s law of spectrometric analysis relative to
the concentration of the standard used, as what is shown below.
All forms of hemoglobin can be converted to cyanmenthemoglobin and measured by this

method except for sulfhemoglobin. On the other hand, the carboxyhemoglobin takes approximately 60
minutes for complete conversion which may cause erroneous result on blood specimen of heavy
smokers which contains high level of this hemoglobin type. The presence of abnormal Hgb derivative
like Hgb SS or Hgb SC may cause sickling of the red cell which prevents the lysis of these red cells. This
may result to decrease hemoglobin value obtained due to the confinement of the Hgb within these
sickle red cells thus conversion to cyanmethemglobin will not occur. Therefore, correction should be
made on this situation. (Please refer to procedure below for Cyanmethemoglobin determination).

Steps of Cyanmethemoglobin method

Materials:
Drabkin’s reagent
Sahli-Hellige pipette (20ul)
Test tube
Spectrophotometer
Hemoglobin Standard reagent
Procedure:
1. Label 2 test tubes with “T” (test) and “S” (standard), Sample computation:
respectively. A test = 0.231
2. Place 5 ml of Drabkin’s reagent to each tube to create a
A standard = 0.450
1:251 dilution.
3. Draw blood up to mark of the Sahli-Hellige pipette. C standard = 80mg/dL
4. Deliver blood into tube “T” and rinse pipette 3x with the Supply the data using the formula
solution.
below:
5. Cover tube with parafilm and mix by inversion 2-3 times and
stand for 10-15 minutes and place the tube in a dark place. 0.251 ( 0.231 𝑥 80)
𝐶 𝑡𝑒𝑠𝑡 (𝑔/𝑑𝐿) =
6. Fill the Sahli-Hellige pipette, up to mark, with the standard. 0.290
7. Deliver the standard into tube “S” and mix by inversion, 2-3
times and stand for 10-15 minutes and place the tube in a C test = 15.9 g/dL
dark place.
8. Set spectrophotometer at 540 nm wavelengths.
9. Place a small volume of the mixture in tube “S” in a cuvette,
read the absorbance in the spectrophotometer, and record
as “As”.
10. Drain the content of the cuvette and drain-dry the mouth
with tissue paper. Using the same cuvette, read the
absorbance of tube “T” mixture and record as “At”.
11. Using the derived formula from the Beer’s Law below,
calculate for the hemoglobin value. (C = concentration; A =
Absorbance).
0.251 ( 𝐴 𝑡𝑒𝑠𝑡 𝑥 𝐶 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 )

𝐶 𝑡𝑒𝑠𝑡 =
𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
Each laboratory may create its own hemoglobin standard curve for every new slot or set of
reagents to be used. The concentration of the standard can be predetermined by mixing the standard
reagent with an initial concentration of 80mg/dL into different volumes of the Drabkin’s reagent as
what is shown in the table below. The absorbance of each predetermined standard Hgb concentration
will be determined and plotted to a semilogarithmic paper as what is shown below.
Hgb conc. Blank 5 10 15 20

g/dL
Standard rgt. 0 1.5 3 4.5 6
Drabkin’s rgt. 6 4.5 3 1.5 0

The Hgb concentration of the patient can be determined using this standard curve by plotting
the absorbance made of the patient’s samples after the spectrometric analysis.
Poor specimen collection that causes hemoconcentration or hemodilution of the blood
specimen prior to test may elevate or decrease the result respectively. Improper handling of the
specimen that leads to clotting of the sample may cause decrease values, but hemolysis will not affect
the hemoglobin value. There are physiologic factors that may falsely elevate or decrease the Hgb
concentration of the patient’s sample. The role of the medical technologist is to identify these problems
and apply a specific remedy to correct it.
Factors Hgb value Remedy

Lipemia Increased Do plasma blanking by adding 20 ul of plasma to 5 ml or
6 ml of the Drabkin’s rgt. This is to correct the
absorbance created by the lipids in the plasma.
Icteric sample Increased Do the same as in lipemic samples.
High WBC ct. (>30,000/uL) Increased After mixing and incubating the Drabkin solution and the
blood, centrifuge it at heavy spin to allow the WBC’s at
the bottom of the tube then determine the absorbance
of the supernatant. This action eliminates the additional
absorbance created by the WBC in the mixture.
Severely high platelet count Increased Do the same as in high WBC count.
Presence of abnormal plasma Increased Use the modified Drabkin solution instead of the original
precipitating proteins one.
Presence of non lysing RBC such Decreased Dilute the blood using a 1:2 dilution using distilled water.
as Sickle cells and Target cell This will completely lyse the red cell and allows the
release of the Hgb in the solution to completely convert
r Codocyte it to cyanmethemoglobin. However, the computed
c red cells concentration of the hemoglobin should be multiplied
first to a factor 2 before reporting.
Rule of three
The “rule of three” is a simple mathematical formula that can be used by multiplying each of the
red cell parameters which are the RBC ct., Hgb and Hct to a factor 3. According to the rule of three, if
the RBC ct. is multiplied by 3 the result is the Hgb ± 1.5 and when the Hgb value is multiplied by 3 the
result is the Hct ± 3%. This can be done if the blood picture of the peripheral blood shows a normocytic
and normochromic red cell. The rule of three can also be used to validate the results of the RBC ct.,
Hgb, and Hct obtained from the automated cell counting analyzers. The results of these parameters
should conform the “rule of three” before releasing the result, but if it is not then an investigation
should be done to identify the cause and a specific remedy should be applied to correct it. Refer to the
examples below.
a. RBC ct. of 4.0 x 1012/L x 3 = Hgb 12 g/dL ± 1.5 (the acceptable value is between 10.5 – 13.5 g/dL)
b. Hgb of 12 g/dL x 3 = 36% ± 3 (the acceptable value is between 33-39%)

Red cell indices
The parameters include in the red cell indices are Mean Cell Volume (MCV), Mean Cell
Hemoglobin (MCH), and Mean Cell Hemoglobin Concentration (MCHC). This can be determined by
using a mathematical formula using the three red cell parameters which are the RBC ct., Hgb and Hct
values, as what is shown below.
𝐻𝑐𝑡
𝑀𝐶𝑉 = 𝑥 10
𝑅𝐵𝐶 𝑐𝑡.
𝐻𝑔𝑏
𝑀𝐶𝐻 = 𝑥 10
𝑅𝐵𝐶 𝑐𝑡.
𝐻𝑔𝑏
𝑀𝐶𝐻𝐶 = 𝑥 100
𝐻𝑐𝑡
The MCV is used to measure the average volume of the red cells in a given specimen and the
reference value of it is 80-100 fL. If the computed MCV is below the reference value it indicates that
the red cells are microcytic, and if it falls within the reference value the red cells are normocytic, while
if it is above the range the red cells are macrocytic. There are anemias that exhibit a microcytic red cell
and these are iron deficiency anemia, thalassemia, sideroblastic anemia, and anemia of chronic disease
or inflammation. Normocytic red cell is found on certain disease such as bone marrow failure or aplastic
anemia, Fanconi syndrome, kidney failure, uremia, myelofibrosis, leukemia and myelopthisic anemia.
Macrocytic red cells are seen on macrocytic anemia caused by either cobalamin or folate deficiency.
Cobalamin deficiency includes pernicious anemia, Zollinger-Elison syndrome, Chron’s disease,
Immerlund-Grasbeck syndrome, transcobalamin deficiency, Tapeworm infection, blind-loop syndrome,
etc. While Folate deficiency commonly caused by drug related absorption inhibition and chronic
alcoholism. The MCH is used to measure the average weight of the hemoglobin in the red cells and the
normal reference range of it is 26-32 pg. Low values of it signifies that the amount of hemoglobin on
red cells are decreased and these can be termed as hypochromic red cells. If the computed MCH is
within the reference value the red cells are normochromic while if it is above the range it is termed as
hyperchromic red cells. Hypochromic red cells are usually seen on conditions that shows microcytic red
cell which are mentioned above. Normochromic red cells are seen in certain disease under the
normocytic red cells as what is stated earlier, while the hyperchromic red cells are seen on macrocytic
anemias. The MCHC is measuring the average concentration of the hemoglobin in a given specimen and
the reference value of it is 31-37 g/dL or g%. the clinical application of the MCHC is the same with MCH,
therefore low value of it the red cell can be evaluated as hypochromic red cells, normochromic if it falls
within the reference value while increase value is hyperchromic red cell.
White Blood cell differential Count (Indirect Relative Cell Count)

The principle of this test is to observe a total of 100 white blood cells in the peripheral blood
smear and get the percentage distribution of each types of it. Normally mature WBC’s are populating

the blood, but in certain disorders of bone marrow like in leukemia and myelopthisic anemia the
immature WBC may be seen in the peripheral blood. The medical technologist should be well trained
in the correct identification of both mature and immature white blood cells. In thalassemia major the
immature red cell or nucleated red cell may be seen in the peripheral blood smear and can be mistaken
as lymphocyte due to their almost same size and appearance. That is why a technologist should know
how to differentiate nRBC from lymphocytes to make a proper correction. The presence of increase
number of nRBC may cause the elevation of WBC ct., therefore a corrected WBC ct. should be done.
The differential count also gives the initial diagnosis of what possible infection that the patient has. In
acute bacterial infection the predominating type of white cell in the blood is the neutrophil, while if it
is a chronic infection the monocyte is more likely to be seen. In viral infection the lymphocyte will
increase in number and may show reactive changes on its cytoplasmic appearance and size. This
reactive lymphocyte is also known as atypical lymphocyte or variant lymphocyte. Eosinophil number is
increased during parasitic or larval infections as well as in allergic reaction. The increase number of
eosinophils in allergic reaction is to keep the symptoms controlled to prevent allergic shock or
anaphylaxis. The cell responsible in developing symptoms of allergy is the increased number of
basophils in the peripheral blood. Initially, the medical technologist should prepare a stained wedge
blood smear in order to observe and evaluate each white cell. Refer to procedure for wedge smear
preparation and for white blood cell differential count.

Neutrophil Eosinophil Basophil Band cell Monocyte Lymphocyte
WBC Differential Count

WBC type Normal Reference Value (in %)
Neutrophil 47 – 79
Lymphocyte 14 – 47
Monocyte 2 – 11
Eosinophil 0–7
Basophil 0–2
Stab or Band cell 0–7
Routinely, a total of 100 white cell should be observed in executing the indirect cell differential
count, however, it is suggested that if the total WBC count is more than 35.0 x 109/L the medical
technologist should observe 200 cells instead. In severe leukopenia, where the total WBC ct. is less than
2.0 x 109/L, fewer than 100 cells can be observed due to the possibility of not seeing enough number
of white cells in the blood smear. On this situation the technologist may observe only a total of 50 or
25 white cells and then multiplied by 2 and 4, respectively to convert it to 100%.
The absolute count of each type of WBC can also be reported as part of the CBC to offer more
accurate assessment of the actual or definite number of the white blood cell. The formula is shown
below:
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 = 𝑖𝑛𝑑𝑖𝑟𝑒𝑐𝑡 𝑐𝑒𝑙𝑙 𝑑𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑡𝑖𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝑥 𝑡𝑜𝑡𝑎𝑙 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡

Example:
Total WBC ct.: 5.0 x 10 9/L
WBC Differential count x total WBC ct. = Absolute count of each cell type Normal Reference value
Neutrophil 70% 3.5 x 109/L 1.8 – 7.7 x 109/L
Lymphocyte 27% 1.35 x 109/L 1.0 – 4.8 x 109/L
Monocyte 8% 0.40 x 109/L 0.0 – 0.8 x 109/L
Eosinophil 5% 0.05 x 109/L 0.0 – 0.45 x 109/L
Basophil 0% 0.00 x 109/L 0.0 – 0.30 x 109/L
Other Routine RBC procedure

Reticulocyte count
The assessment of the reticulocyte number is not only used to evaluate the efficiency of the bone
marrow in red cell production as a response to anemia, but it is also used to categorized anemia into
either hemolytic or non-hemolytic anemia. High reticulocyte count or reticulocytosis is commonly found
in cases of hemolytic anemia and decrease count or reticulocytopenia is seen in non-hemolytic types.
Example of these hemolytic anemias are thalassemia, microangiophatic syndromes, autoimmune
hemolysis, RBC enzymopathy like G6PD deficiency, and RBC membrane disorders such as hereditary
spherocytosis and elliptocytosis, etc. Non-hemolytic anemia includes iron deficiency anemia,
megaloblastic anemia, aplastic anemia and kidney failure. Reticulocyte number can be reported as
relative count or absolute count. The relative reticulocyte count is done by getting the estimated
percentage number of retics per 1000 red cells observed in a wedge blood smear, while the absolute
count is determining the actual number of it in a given EDTA sample. Reticulocytes are stained by
supravital stains such as new methylene blue or brilliant cresol blue. These stains are used to see the
cytoplasmic inclusion bodies of the reticulocyte which is known as the RNA remnants. However, there
are also other red cell inclusion bodies that can be stained by this supravital stains and these are Heinz
bodies, Pappenheimer, Howell-Joly, and Hgb H. Red cells with these inclusion bodies can be mistaken
as reticulocytes, therefore, use of different reagents is suggested to differentiate this inclusion bodies
from that of the reticulocyte’s inclusions. Heinz bodies can be confirmed by using a special reagent
known as acetyl phenyl hydrazine. The Pappenheimer bodies, which contains ferritin, shows positive
reaction with Prussian blue stain or pearl’s stain. The Howell-jolly, which is a nuclear DNA remnant,
gives a positive reaction to Fuelgen reaction. The Hgb H, which is seen on α-thalassemia major, can be
differentiated with reticulocyte remnants using a modified brilliant cresol blue. The Hgb H gives a blue
to greenish color reaction while the reticulocyte remnant imparts blue to purplish hue. There are two
approaches in performing the relative reticulocyte count, one of these is counting the reticulocyte using
the common ocular eyepiece of the light microscope and the other one is using a special type of ocular
eyepiece known as the Miller Disc method. The use of miller disc allows the medical technologist to
observe around 4500 red cells in getting the relative number of reticulocyte than the usual 1000 red
cells. Theoretically, observing more red cells while performing the relative retic count gives a more
accurate result, therefore the use of the Miller disc is the more ideal one. Please see the procedure for
relative reticulocyte count.

Steps of Relative Reticulocyte Count Steps of Relative Reticulocyte Count (Miller Disc)
Materials: Materials:
Slides Slides
Test tube Test tube
Capillary tube Capillary tube
New Methylene Blue stain New Methylene Blue stain
Formula 0.5 gm. NMB powder Procedure:
1.6 gm. Potassium oxalate 1. Place equal amounts of anticoagulated blood
100 ml distilled water (to dissolve) and NMB stain in a tube. Gently tap the tube’s
Procedure: side to mix.
1. Place equal amounts of anticoagulated blood 2. Charge a capillary tube with blood and stain
and NMB stain in a tube. Gently tap the tube’s mixture.
side to mix. 3. Stand for exactly 10 minutes at room
2. Charge a capillary tube with blood and stain temperature.
mixture. 4. Place a drop of the mixture on a slide and
3. Stand for exactly 10 minutes at room make a smear. Air dry.
temperature. 5. Scan smear under oil immersion. Using the
4. Place a drop of the mixture on a slide and miller disc, count the reticulocytes in Square
make a smear. Air dry. “A” and observe the red cells on Square “B”
5. Scan smear under oil immersion. Enumerate using 9 fields. Approximately around 500 red
1000 red cells while counting the number of cells in square “B” are observed per field used.
reticulocytes therein. 6. Calculate retic (reticulocytes) count using the
6. Calculate retic (reticulocytes) count using the following formula below:
following formula:
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑖𝑐 𝑐𝑜𝑢𝑛𝑡
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑥 100 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐴 𝑥100
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑡 = =
1000 𝑅𝐵𝐶 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵 𝑥 9

Parameters associated with Reticulocyte count

Parameter Formula Comment
Absolute 𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 This is to determine the
Reticulocyte 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑅𝑒𝑡𝑖𝑐 𝑐𝑜𝑢𝑛𝑡 𝑥 𝑅𝐵𝐶 𝑐𝑡. true value of the
= 𝑥 1000 reticulocyte in a given
Count 100 𝑅𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
EDTA blood sample.
Normal Reference range:

25 x 109/L– 75 x 109/L
Corrected 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 The reticulocyte count
Reticulocyte 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 may falsely elevated if the
= 𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑖𝑐 𝑐𝑜𝑢𝑛𝑡 𝑥 red cell number decreases,
Count 0.45
therefore correction
should be made to resolve
this relative to the
hematocrit of the person.
Reticulocyte 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝐼𝑛𝑑𝑒𝑥 This parameter is used to
Production 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 assess the capability or
= efficiency of the bone
Index # 𝑜𝑓 𝑑𝑎𝑦𝑠 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑡ℎ𝑒 𝑐𝑖𝑟𝑐𝑢𝑙𝑎𝑡𝑖𝑜𝑛
marrow to compensate to
correct the anemia of the
HCT value # of days of reticulocyte person. If the computed
maturation RPI is <2 it means that the
0.40 – 0.50 1 day bone marrow can not
0.30 – 0.39 1.5 day anymore compensate but
0.20 – 0.29 2 days if it is >3 it means that the
0.10 – 0.19 2.5 days marrow still can.
Red cell inclusions that can be stained using supravital stains

Inclusions Microscopic exam Associated Disorder
- High levels are seen in
Hemolytic anemia
Reticulocyte remnants - Decrease level is seen in
None-Hemolytic anemia
- Glucose 6 Phosphate
dehydrogenase deficiency
Heinz bodies
- Unstable Hemoglobin

- Sideroblastic anemia
Pappenheimer bodies
- Megaloblastic anemia
Howell-Jolly bodies - Thalassemia
- Alpha thalassemia with 3

α genes deleted
Hemoglobin H
Relative retic count

Reference value
(%)
Children 0-1 day 2-6
2-4 days 1.5-4.7
5-30 days 0.2-1.0
3 mos. – 13 0.5-1.5
years old
Male Adult 0.5-1.5
Female Adult 0.5-1.5
Erythrocyte Sedimentation Rate

The clinical use of the erythrocyte sedimentation rate is to determine if the person is
experiencing an acute or chronic inflammatory reaction. This test measures the length of fall in
millimeter of the red cell in a blood column placed in a special type of tube after one hour standing.
There are two methods available to measure the ESR and these are the Wintrobe and the Westergren
method. The table 5.1 shows the differences between the two methods.

Methods Wintrobe Westergren

Anticoagulant used EDTA or ammonium Original method: 3.8% sodium
potassium oxalate citrate
Modified method: 2.0 mL EDTA

blood diluted into either 0.5 mL
of 0.85% NaCl or 3.8% sodium
citrate.
Total length of the tube 110-115 mm 300 mm
Length of the ruled area 0-100 mm 0-200 mm
Bore diameter of the tube 3 mm 2.65±.15 mm
Table 5.1
There are three phases that occurs during the standing of the anticoagulated blood in a glass
column after one hour. The first phase is that the red cells will tend to stack together which is called
rouleaux formation that takes around 15 minutes to complete. The second phase is called fast settling
of the stacked red cells forced by the pull of earth’s gravity and usually completed around 40 minutes.
The third phase is called the final packing of the red cells as they find their way to settle at the bottom
of the tube and this usually takes around 15 minutes to complete. This is the reason why 60 minutes is
the waiting time for this procedure. Reading the result less than an hour may cause a decrease reading
of the ESR value because the phases are not yet complete, while reading it beyond 1 hour may cause
an increase reading of it due to the excessive packing of red cell within the blood column. The rouleaux
formation of red cells are enhanced by hyperproteinemia, therefore, high ESR values are obtained on
this condition. Hyperproteinemia may be due to certain plasma cell dyscrasia like Waldenstrӧm’s
macroglobulinemia, multiple myeloma, and etc. High number of red cells cause a decrease reading of
the ESR value and this is a common result on patients with Polycythemia vera. Therefore, the value of
the ESR is directly proportional to the protein level in the plasma while it is inversely proportional to
the number of red cells in the blood. The value of the ESR is also influence by age and gender of the
individual. High ESR values are common on elderly while low values are seen on children, on the other
hand, male has lower ESR value compared to female. This is related to the number of red cells in the
blood, in which children has higher red cells in their blood compared to elderly, and men has higher red
cell number than that of female. There are also other factors that might influence the ESR result and
these are listed on table 5.3 and 5.4.
Phases Duration
Rouleaux Formation 10 minutes
Fast settling of red cells 40 minutes
Final Packing of red cell 10 minutes

Reference value
Wintrobe method Westergren method
Male: 0-10 mm/hr Male <50 yrs. old: 0 – 15 mm/hr
Female: 0-20 mm/hr > 50 yrs. old: 0 – 20 mm/hr
Female <50 yrs. old: 0 – 20 mm/hr
>50 yrs. old: 0 – 30 mm/hr
Children: 0-10 mm/hr

Factors Affecting ESR value
Physiologic Factors Technical Factors
Increase Decrease Increase Decrease
▪ Hyperproteinemia ▪ Hyperalbuminemia or ▪ Hot or humid room ▪ Low or cold room

▪ Hyperfibrinogenemia hypoproteinemia temperature temperature
▪ hypergammaglobuline ▪ Hyperglycemia ▪ Slanted or tilted ▪ Bubbles within
mia ▪ High phospholipids tube the glass column
▪ Hypoalbuminemia ▪ High bile salts ▪ Vibration or ▪ Narrow ESR tube

unstable surface diameter
▪ Hypercholesterolemia ▪ Lecithin
▪ Reading the result ▪ Use of mixture of
▪ Macrocytosis ▪ Microcytosis
beyond 60 minutes Heparin and
▪ Menstruation ▪ Sickle cells oxalate as
▪ Anemia ▪ Echinocytes anticoagulant
▪ Leukemia ▪ Spherocytosis ▪ Reading the result
▪ rd
After 3 month of ▪ Acanthocytosis before 60 minutes
pregnancy ▪ Marked anisocytosis ▪ Use of more than
2-hour old EDTA
▪ Presence of Hgb C
samples
▪ Polycythemia
▪ Marked Leukocytosis
Table 5.3
Clinical Conditions Drugs affecting the ESR

affecting ESR value values
Increase Decrease increase Decrease
▪ Diabetes ▪ Cachexia ▪ Dextran ▪ Cortisol
▪ Gout ▪ Congestive Heart ▪ Heparin ▪ Cortisone
▪ Malignancy failure ▪ Vitamin A ▪ Quinine
▪ Multiple myeloma ▪ Polycythemia vera ▪ Procainamide ▪ Salicylates
▪ Myocardial ▪ Theophylline ▪ Ethambutol
infarction ▪ Penicillin amide ▪ ACTH administration
▪ Collagen vascular ▪ Corticotropin
disorders
▪ Rheumatoid arthritis
▪ Syphilis
▪ Rheumatic fever
▪ End stage renal
diseases
▪ Acute bacterial
infections
▪ Leukemia
▪ Anemia
Table 5.4

LESSON MODULE
LESSON 6
LESSON TITLE: Special Test in Hematology

1. enumerate the different special laboratory parameters.

2. discuss the principle of the test and sources of errors.
3. associate the test results with a hematological disorder.
4. perform the different test with confidence and reliability.
LESSON GUIDE:
VI. Learning Guide Time allotment Learning Resources
1. Enumerate the different special laboratory parameters for 3-hour lecture Learning Content:
RBC. - Lesson 1 module
1.1 Special Tests for assessing different red cell disorders. page/s: 75-86.
- Hematology: Principles
2. Discuss the principle of the test and sources of errors. and Procedure. Page/s
2.1 Special laboratory tests used to screen and confirm 127-201.
different RBC disorders characterized as to:
- Membrane defect
- Hgb defect
- Energy defect
- Autoimmune cause
3. Associate the test results with a hematological disorder.

3.1 Appropriate interpretation and assessment of red cell
disorders using laboratory data.
4. Perform the different test with confidence and reliability.

- Answer related questions for Special test

platform

LESSON VI: Special Tests in Hematology
Special test for diagnosis of certain RBC membrane defects

Erythrocyte Osmotic Fragility Test (EOFT)
The Erythrocyte Osmotic Fragility test is used to diagnose hereditary spherocytosis. Hereditary
spherocytosis is a genetic membrane defect associated with deficiency or abnormal molecular structure
of either the α or β chain of spectrin. Other defective peripheral proteins are also associated with this
disorder and these proteins are ankyrin, protein band 3 and protein band 4.2. In hereditary
spherocytosis the red cell membrane becomes loose and fragile which cause the easy destruction of
red cells as it travels within the vasculature. Red cells with this defect commonly form “blebs” on its
membrane which eventually removed by the macrophages of the spleen every time the red cells enter
into this organ. This event decreases the red cell membrane which eventually forms a small and sphere-
shaped red cell instead of a disc shaped. These red cells are small that it cannot contain the volume of
HGB inside it, that is why it is likened to a “bag full of things”. Spherocytes has a low surface to area
volume ratio and usually measures <5 um in diameter resulting to low MCV but with high MCHC value
due to the loss of its central pallor. The principle of the EOFT is subjecting the red cells into different
concentration of salt starting from hypertonic solution (1.0% NaCl) or isotonic solution (0.90-0.85%
NaCl) to hypotonic solution (0.0% NaCl) and then identify at what salt concentration shows the initial
and complete hemolysis. Normally, red cell shrinks at hypertonic solution and will not lyse in isotonic
solution, but swells and eventually lyse at hypotonic solution. A normal red cell without any defect on
its membrane can withstand up to 0.5 % NaCl hypotonic solution without lysis, and partial hemolysis
will only happen if it is placed at 0.40-0.45% NaCl and complete hemolysis occurs at 0.30-0.35% NaCl.
Partial hemolysis means that some red cells in the solution are lysed but some still are intact, while
complete hemolysis means that all the red cells in the solution are totally lysed. In hereditary
spherocytosis the red cells are lysed higher than 0.5% NaCl, as shown in figure 6.1. This is due to the
increase fragility and less resistance of the spherocytes membrane.
Fig.6.1

There are other abnormal red cells that shares the same result with spherocytes if these are also
subjected to the EOFT studies and these are macrocytes, elliptocytes, and ovalocytes because of the
increase fragility and decrease resistance of their membrane in osmosis. On the other hand, non-lysing
red cells such as hypochromic red cells, target cells or codocytes, and sickle cells has high membrane
resistance and decrease membrane fragility and shows delay lysis if these are placed to a hypotonic
solution.
Ham’s test or Acidified Serum Test
The Ham test or acidified serum test is used to confirm the diagnosis of Paroxysmal Nocturnal
Hemoglobinuria or PNH. This is done immediately after the blood specimen shows a positive result with
the screening tests for PNH which is the sugar water hemolysis test. PNH is a red cell membrane defect
associated with the genetic deficiency of guanidine phosphate inositol linked protein. Loss of this
protein also leads to decrease to absence expressions of both the CD55 and CD59 on red cells as well
as in other white cells. The use of flow cytometrical analysis on white cells is also used to evaluate the
extent of loss of these CD markers which helps establish the type of PNH that the person has. There are
three types of PNH and these are Type 1, Type 2 and Type 3. The PNH type 1 shows mild loss of the gpi
proteins, while type 2 and 3 exhibits moderate and complete absence of this protein, respectively. Red
cells without the CD 55 and CD 59 markers are prone to spontaneous complement mediated hemolysis
specially if the blood pH becomes slightly acidic. This blood characteristic in PNH was used as the basis
to develop the Ham’s test. In this test three different type of serum is prepared and these are normal
serum, patient’s serum and inactivated serum. Inactivated serum is prepared by heating the serum at
56OC for 30 minutes to deactivate certain complement proteins such as C4 and C2. After which, 0.2N
HCL is place on each serum type to acidify it which will make the red cells to be tested susceptible to
complement lysis. Exactly 20 μL of defibrinated blood is placed on each serum preparations and
incubated for one hour at water bath temperature or 37OC. After the incubation period, it is expected
that in PNH the tubes with patient’s serum and normal serum will show hemolysis while the tube with
inactivated serum will show no hemolysis leaving the supernatant clear. Other hematological disorder
may show positive result with Ham’s test and these are CDA type II or also known as “HEMPAS” and
Hereditary spherocytosis. In CDA type II hemolysis of red cells may occur in normal serum and
inactivated serum but not with patient’s own serum, while in Hereditary spherocytosis it may show
mild hemolysis in all of the serum preparations made. See figure 6.2.
Fig. 6.2

The Sugar Water test as mentioned earlier is the screening test used for the assessment of PNH.
This test is known to be sensitive but not specific to PNH, therefore if the result shows a negative result
then there is no need to proceed to any confirmatory test for PNH, however if it shows a positive result
it is necessary to do a confirmatory test such as sucrose hemolysis or Ham test. The principle of this test
is that the patient’s whole blood will be mixed with sugar water solution at room temperature and
allow it to stand for 10 minutes, then after which check the mixture for hemolysis. The presence of
hemolysis in the mixture is positive for PNH while no hemolysis is negative for the said disorder. On
the other hand, the sucrose hemolysis test uses an isotonic sucrose solution containing normal ABO
compatible serum from where the patient’s washed red blood cells will be placed. The PNH red cell is
sensitive under this condition than the normal red cells in which the previous will lyse but the latter will
not. Other conditions may also show positive result in sucrose hemolysis test and these are leukemia
and myelosclerosis, that is why it is suggested that there should be a good correlation between the
results of the sucrose hemolysis test with that of the Ham or acidified serum test.
Special Test for assessing certain Hemoglobin disorders

Dithionite solubility test
The Dithionite solubility test is one of the screening tests to evaluate the presence of Hgb S in
the patient’s blood. Hgb S is a kind of unstable hemoglobin where point mutation of a single amino acid
occurs in the beta globin chain. This genetic defect causes the substitution of glutamic acid with valine
on the 6th position of amino acid in either one or two beta globin chains of the hemoglobin. This
abnormality could result to the instability of the Hgb molecule causing it to precipitate within the red
cells that changes the appearance of red cells from disc shape to sickle shape (see figure 6.3). This shape
change is triggered by several factors such as hypoxia, acidosis and dehydration. There are three
different forms of this hemoglobin defect and these are sickle cell anemia known as Hgb SS, sickle cell
trait known as Hgb AS and sickle cell diseases such as Hgb SC, SG, SD, SE, S-β thalassemia, etc. The
principle of the dithionite solubility test is that the red cells are placed in a solution containing saponin
and sodium dithionite reagent. The red cells are lysed by the saponin reagent liberating the hemoglobin
in the solution, then if Hgb S is present, it will be oxidized by the sodium dithionite forming hemoglobin
tactoids or precipitates. Therefore, the positive result of this test is the presence of precipitates causing
turbidity of the solution. See figure 6.4. Commonly those individuals with either Hgb SS or Hgb AS shows
a positive result on this test, on the contrary, sickle cell diseases show otherwise.
Figure 6.4

Sodium Metabisulfite test

The sodium metabisulfite test or also known as the sickling test is used as a screening test for
Hgb S. Red blood cells containing either Hgb SS or AS will cause a significant change on its shape forming
either a sickle or a “holly leaf” appearance after these cells are incubated with the sodium metabisulfite
reagent. This test requires microscopic visualization of the blood to rule out this disorder. See figure
below.
Holly leaf appearance Sickle cell
Figure 6.3
Hgb Electrophoresis
The Hemoglobin electrophoresis is used to identify specifically the type of hemoglobin present
on an individual’s blood. That is why this special test is used to confirm several blood disorders such as
thalassemia and other hemoglobinopathy. There are two types of Hgb electrophoresis depending on
the what type of medium used and the pH environment of the test. One of these is the use of Cellulose
acetate Hgb electrophoresis with a pH environment of 8.0 and the other one is the Citrate Hgb
electrophoresis that utilizes a pH of 6.0-6.2. Proteins are known to migrate at varying points on a certain
medium after it was subjected into an electric current. Hemoglobin types migrate differently at
isoelectric point in a medium forming band at different regions. These bands formed are tag with a
special type of stain for easy visibility of it. There are Hgb standards used for this test and these are the
Hgb A1, Hgb F, Hgb S and Hgb C. In cellulose acetate medium after an electric current run through this
medium, the Hgb A1 migrates the fastest towards the anode (positive charge) which is then followed
by Hgb F, then Hgb S and the slowest among these is the Hgb C as what is shown in figure 6.6. The
pattern of migration will be different on citrate agar which is this time the Hgb C migrates nearest
towards the anode area followed by Hgb S, while both Hgb F and A1 migrates towards the cathode
(negative charge) area where the previous is nearer into it than the latter.

Other Hemoglobin type migrates differently on a specific isoelectric point using the cellulose
acetate medium, like Hgb D and G migrates the same point where Hgb S is, while Hgb E, O-Arab and
Hgb A2 migrate at the same point with Hgb C. The Bart’s Hgb, Hgb I and Hgb H migrates faster towards
the anode area bypassing the point where Hgb A1 is located. See figure 6.7. This migration pattern of
the said hemoglobin will significantly change if they will be subjected to citrate medium. The Hgb A2,
D, G, E, and O-Arab will migrate at the same point where Hgb A1 is located at this medium.
Acid Elution test

The Acid Elution test is intended to determine the degree of distribution of the red cell
containing Hgb F in a given specimen. This is useful in the diagnosis of certain disorders where the level
of Hgb F is high such as hereditary persistence of fetal hemoglobin (HPFH) and the presence of fetal red
cells in maternal circulation. The principle of this test is the blood smears made, either directly from a
finger stick or from a less than 6-hour old EDTA sample, is fixed with ethyl alcohol and then incubated
in a citric-acid solution. Those red cells that contains Hgb F or fetal Hgb, will not lysed or remain intact
even it is subjected to an acid solution, this is due to the fact that Hgb F is not eluted by the action of
an acid, but red cells containing other Hgb type is lysed and destroyed. The blood smears made then
are stained with hemotoxylin to stain the white cell nuclei and erythrosin B to stain the red cells (see

figure 6.9). After which, the medical technologist will view the smears microscopically using around 20
– 25 field to determine the number of intact red cells and assess the percentage of red cells that
contains the Hgb F using the two formulas below.
# 𝑜𝑓 𝑖𝑛𝑡𝑎𝑐𝑡 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
# 𝑜𝑓 𝐻𝑔𝑏 𝐹 𝑅𝐵𝐶/𝑓𝑖𝑒𝑙𝑑 =
# 𝑜𝑓 𝑓𝑖𝑒𝑙𝑑 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
# 𝑜𝑓 𝐻𝑔𝑏 𝐹 𝑅𝐵𝐶/𝑓𝑖𝑒𝑙𝑑
% 𝑜𝑓 𝑅𝐵𝐶 𝑤𝑖𝑡ℎ 𝐻𝑔𝑏 𝐹 = 𝑥100
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 # 𝑅𝐵𝐶/𝑓𝑖𝑒𝑙𝑑
*Intact red cell containing Hgb F is completely stained showing an intense reddish color.
In Hereditary persistent fetal hemoglobin, the amount of Hgb F is even in all red cells which
makes it stained consistently. Varying staining intensity may be found in certain disorders where the
levels of Hgb F is variable, such as thalassemia, acquired aplastic anemia, sickle cell and other forms of
hemoglobinopathies. Reticulocytes may resist elution by the acid which therefore causes confusions
and discrepancy in the laboratory report.
Alkali denaturation test

This test is used to quantify the concentration of Hgb F in a given specimen. Normally the
concentration of Hgb F is high in newborn which is approximately 60-90 %, but should constantly
decline at the age of 4 months reaching around only 10%. By the age of 6-12 months the Hgb F
concentration should be less than 2%. In certain blood disorders the levels of Hgb F are high above 15%
in hereditary persistent fetal hemoglobin and normal to 20% in sickle cell anemia. In β– thalassemia
minor the levels of Hgb F may reach around 2-5%, whereas in β-thalassemia major it may reach from
15% up to 95%. High levels of Hgb F are also seen on other conditions such as acquired aplastic anemia,
megaloblastic anemia, PNH, myelofibrosis, leukemia, refractory anemia and during pregnancy. There
are two methods of alkali denaturation test and these are the Betke and the Singer method. The Betke
method uses sodium hydroxide as the alkali reagent while the Singer method uses the potassium
hydroxide. The principle of this test is the red cell hemolysate will be incubated with an alkali solution
which allows the denaturation of all forms of hemoglobin except the Hgb F. The denatured hemoglobin
will precipitate after mixing it with a precipitating reagent such as ammonium sulfate which leaves the

Hgb F suspended in the solution. After which the precipitates are filtered out, and the filtrate containing
the Hgb F will be subjected to cyanmethemoglobin test to determine the optical density (O.D.) of the
said filtrate using the spectrometric analysis. The optical density of the total Hgb is also determined
which is used to calculate the percent concertation of the Hgb F using the formula below.
𝑂. 𝐷. 𝑜𝑓 𝐻𝑔𝑏 𝐹
% 𝐻𝑔𝑏 𝐹 = 𝑥 100
𝑂. 𝐷. 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑥 10
Screening Test for unstable hemoglobin
Isopropanol precipitation test

The red blood cell hemolysate prepared from a blood specimen suspected with unstable
hemoglobin is incubated with 15% or 17% isopropanol and incubated at 37 OC. The mixture is checked
for precipitation or flocculation at 5 minutes and 20 minutes. Unstable hemoglobin shows a faster rate
of precipitation formation than the normal hemoglobin because their bonds are easily weakened by
isopropanol. A normal hemoglobin usually starts to precipitate at 45 minutes while unstable
hemoglobin shows heavy flocculation at 5 minutes and 20 minutes.
Heat denaturation test

The hemolysate prepared mixed with tris buffer solution with a pH of 7.4 is incubated at 50 OC
for 2 hours and another duplicate sample is incubated at refrigerated temperature. Usually the unstable
hemoglobin will precipitate easily at high temperature than the normal hemoglobin. Using the
spectrometric analysis, the absorbance of the hemoglobin concentration of the supernatant of both
the heated and refrigerated mixture will be determined using the cyanmethemglobin method. The
concentration of the unstable Hgb is calculated using the formula below.
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑟𝑒𝑓𝑟𝑖𝑔𝑒𝑟𝑎𝑡𝑒𝑑 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐻𝑒𝑎𝑡𝑒𝑑
% 𝑜𝑓 𝑢𝑛𝑠𝑡𝑎𝑏𝑙𝑒 𝐻𝑔𝑏 = 𝑥 100
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑟𝑒𝑓𝑟𝑖𝑔𝑒𝑟𝑎𝑡𝑒𝑑
*If the calculated concentration of unstable Hgb is high additional test should be performed to confirm it and
these tests are isopropanol precipitation test and tests for inclusion bodies.
Heinz Bodies inclusion test

Red cells with unstable hemoglobin will form Heinz inclusion bodies after the cells are incubated
with a reducing substance known as acetylphenylhydrazine. The blood is stained with crystal violet and
observed microscopically. Red cells with faulty reducing systems will show more than 32% of red cells
with 5 or more inclusions but normal cells will show less than 32% with 5 or more inclusions (See figure
6.10). Heinz bodies are also common in certain enzymopathies like G-6-PD deficiency, glutathione
reductase deficiency, 6-phosphogluconate dehydrogenase deficiency, glutathione peroxidase
deficiency, and triosephosphate isomerase deficiency.

Hemoglobin H preparation and staining

The presence of Hgb H is seen in α- thalassemia major where three alpha genes are absent or
deleted. The loss of the 3 α genes causes the excessive production of β globin chain in the cytoplasm of
the red cells forming abnormal inclusions which is rapidly removed by the macrophages of the bone
marrow or the spleen. This event causes early destruction of the red cell which leads to hemolytic
anemia. Commonly Hgb H is not detected by the hemoglobin electrophoresis due to the small number
of red cells that survived the hemolytic function of the bone marrow and splenic macrophages. To
increase the yield of positive detection of the presence of Hgb H in the patient suspected with
homozygous alpha thalassemia, the blood specimen is incubated with an oxidizing substance such as
1% brilliant cresyl blue. Microscopically, the red cells with Hgb H or excessive β globin chains will form
a multiple, evenly distributed bluish-green colored bodies in the red cell that seem like a golf ball
pattern. See figure 6.11. On the other hand, reticulocyte inclusion is also stained by this reagent but
shows a blue to purple color inclusion instead.
Figure 6.11
Test for red cell energy and metabolic defect
Florescent spot test

The florescent spot test is the screening test used to determine the presence of normal activity
of the G-6-PD and Pyruvate kinase in the blood. G-6PD deficiency is the most common enzyme
deficiency which may cause acute hemolytic anemia after the patient is exposed to an oxidizing
substance produced from the metabolized administered drugs. Example of these drugs are certain
antibiotics and anti-malarial drugs. These drugs form peroxide which oxidizes the hemoglobin in the
absence of G-6PD enzyme forming Heinz bodies. To screen the blood for G-6PD, approximately 1 ml
drop of anticoagulated whole blood using either EDTA, Heparin or ACD anticoagulant, is placed to a
special filter paper which is then mixed with a drop of a test reagent containing glucose-6-phosphate
and NADP. If the red cell has enough level of G-6-PD a chemical reaction will occur which converts the
NADP to reduced NADP or also known as NADPH, as shown below. After which, the blood is subjected
into a UV light, and due to the presence of the NADPH formed, the blood drop is expected to fluoresce
or glow. If the result shows no fluorescence, it means that there is a deficiency of this enzyme.

𝐺−6−𝑃𝐷𝐻
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 − 6 − 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝑁𝐴𝐷𝑃 → 6 − 𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑢𝑐𝑜𝑛𝑎𝑡𝑒 + 𝑁𝐴𝐷𝑃𝐻
Pyruvate kinase is one of the major enzymes under the EMP pathway which is responsible for
the production of enough ATP. Deficiency of this enzyme may cause the instability of the red cell
membrane due to the insufficient production of energy that eventually leads to hemolytic anemia. The
pyruvate kinase activity in the blood can be assessed by using the Fluorescent spot test as what was
described earlier for G-6PD test. A drop of blood is placed in a filter paper and mixed with a test reagent
containing phosphoenol pyruvate, ADP, and reduced nicotinamide-adenine dinucleotide or NADH. If
there is enough pyruvate kinase in the blood a chemical reaction will occur that leads to the conversion
of NADH to NAD, as what is shown below. The presence of NAD (or oxidized form of nicotinamide-
adenine dinucleotide) in the blood will hinder the formation of fluorescence of the blood under UV
light. Therefore, blood samples that fluoresces is negative for pyruvate kinase enzyme.
𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 𝑘𝑖𝑛𝑎𝑠𝑒
𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑒𝑛𝑜𝑙 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐴𝐷𝑃 → 𝐴𝑇𝑃 + 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒
𝐿𝑎𝑐𝑡𝑎𝑡𝑒 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒
𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 → 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷
There are factors that may cause a false normal result on both tests, and these are high WBC
count of more than 20,000/μL and high platelet count. This is due to the fact that white cells and
platelets has high levels of G-6PD enzyme and pyruvate kinase. Reticulocyte has high levels of G-6-PD
which may also cause false normal result. On the other hand, plasma has a significant level of pyruvate
kinase which may lead to false positive result; therefore, washed red cells is commonly used in assessing
the level of this enzymes on red cells. Hemoglobin has a quenching effect on fluorescence and may
cause unreliable result. To prevent this, if the hematocrit of the person is more than 50% it is
recommended that only half of the required amount of specimen is used, but if the hematocrit is less
than 20% the amount of specimen should be doubled.
Glutathione reduction test

The principle of this test is also employing the same principle used in the fluorescent spot test.
A drop of blood specimen is placed in a filter paper and mixed with a test reagent containing oxidized
glutathione (GSSG) and reduced nicotinamide-adenine dinucleotide phosphate (NADPH). If the blood is
containing normal levels of Glutathione reductase (GSSG-R) the NADPH will be converted to NADP. (See
the reaction below). This conversion causes the blood to fluoresce for 20 minutes under long-wave UV
rays and apparently disappear after, but in glutathione reductase deficiency the fluorescence will
persist and continue for more than 1 hour.
𝐺𝑆𝑆𝐺−𝑅
𝐺𝑆𝑆𝐺 + 𝑁𝐴𝐷𝑃𝐻 → 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 𝐺𝑙𝑢𝑡𝑎𝑡ℎ𝑖𝑜𝑛𝑒 + 𝑁𝐴𝐷𝑃

Auto Hemolysis test

This test is used to screen the two most common red cell enzymopathy and these are the G-6-
PD deficiency and pyruvate kinase deficiency. Blood specimen is subjected into warm temperature
ranging from 37OC to 40OC for 48 hours. If the red cells are deficient with either of these two enzymes,
these red cells will be lysed and will cause the plasma reddish after incubation. To correct the
occurrences of hemolysis a glucose reagent or an ATP reagent is mixed on two separate tubes with
blood specimens, respectively. If the hemolysis is corrected by adding glucose reagent but not with ATP
reagent, then the patient has G-6PD deficiency. But if the hemolysis is not corrected by the glucose
reagent but corrected by the ATP reagent, then the patient has pyruvate kinase deficiency. If both the
glucose and ATP reagent corrected the hemolysis, then the patient is suffering from hereditary
spherocytosis. The result obtained in G-6PD deficiency is known as Auto hemolysis type I, for pyruvate
kinase deficiency it is Auto hemolysis type II, while for hereditary spherocytosis it is known as Auto
hemolysis type III.
Ascorbate cyanide test

The ascorbate cyanide test is used to evaluate the activity of the Hexose monophosphate
pathway or Pentose phosphate pathway. One of the known methods used is the Jacob and Jandl. This
method uses an aerated whole blood (oxyhemoglobin) that is mixed with sodium cyanide and sodium
ascorbate. The mixture of sodium ascorbate and oxyhemoglobin produces a toxic substance hydrogen
peroxide, which is acted upon by either the catalase enzyme, normally found in red cells, or the
glutathione peroxidase, generated from the metabolism of pentose phosphate pathway, to convert it
to non-toxic substance or inactivating it. The action of the sodium cyanide is to inhibit the activity of
the catalase enzyme, which is not part of the pentose phosphate pathway, thus allowing only the action
of the glutathione peroxidase in deactivating the hydrogen peroxide produced, hence making the test
to specifically assess the said metabolic pathway. If the activity of the pentose phosphate pathway is
normal the blood sample will remain red, which means that the hydrogen peroxide is reduced and
deactivated, but if it is defective the blood specimen will turn into brown color. The methemoglobin is
one of the oxidized products produced by the action of hydrogen peroxide which causes the formation
of this brown pigment on blood samples.

Special test for diagnosis of Autoimmune red cell disorder
Donath-Landsteiner test
The Donath-Landsteiner test is used to diagnose an autoimmune disorder known as Paroxysmal
Cold hemoglobinuria (PCH). PCH is a condition where a person produces an anti-P autoantibody (also
known as Donath-Landsteiner Antibody) secondary to other diseases such as viral infection or bacterial
infection. One of the known infections that triggers the production of this autoantibody is syphilis which
is caused by a spirochete known as Treponema pallidum. The auto anti-P is an IgG antibody that has a
biphasic characteristic. This autoantibody usually binds with the red cell membrane at cold temperature
and triggers red cell hemolysis at 37OC by activating the complement system. The principle of the
Donath-Landsteiner test is it uses two separate tube filled with whole blood. One of the tubes is
incubated at 37OC for 60 minutes which is the control tube and the other tube is incubated first at 0-
4OC for 30 minutes then later at 37OC for 60 minutes. After which check the serum of both tubes for
hemolysis. If the patient has PCH, a positive test should show hemolysis only on tube that is incubated
first at 0-4OC and later at 37OC, and no hemolysis should be observed on control tube. A negative result
is if there is no hemolysis on both tubes after incubations made. If both tubes showed hemolysis after
incubation this is also interpreted as negative for PCH.
Direct Antihuman Globulin Test (DAT)

The Direct Antihuman Globulin or DAT test is used to determine the in vivo attachment of an
auto or allo antibody and complement fragments on red cell membrane. This in vivo attachment occurs
in several conditions such as autoimmune hemolytic anemia, hemolytic transfusion reactions,
hemolytic disease of the newborn and PCH. Autoimmune Hemolytic anemia includes several diseases
such as cold hemagglutinin syndrome, warm agglutinin syndrome and drug induced autoimmunity. Cold
hemagglutinin syndrome is caused by the production of a pathologic auto anti-I after Mycoplasma
pneumoniae infection, and anti-i after Epstein-Barr virus infection that causes infectious
mononucleosis. Warm hemagglutinin syndrome is usually caused by the production of antibody against

the RH blood group system and this is usually the auto anti-e. The DAT test uses a reagent called as
Antihuman globulin (AHG) or Coomb’s reagent which is known to contain an antibody against human
immunoglobulin and complement fragments. The collected blood sample from persons suspected with
cases mentioned above will be placed in an EDTA tube. On a separate tube several drops of the washed
EDTA anticoagulated blood with red cells that is sensitized with IgG antibody or with attached
complement fragments in vivo, is mixed with the Coomb’s reagent and eventually creates a visible
clumping of these red cells or agglutination. The agglutination is caused by the cross bridging of the
sensitized red cells with immunoglobulins as shown in figure the figure below.

LESSON MODULE
LESSON 7
LESSON TITLE: Peripheral Blood Smear Examination

1. enumerate the procedure of blood smear procedure.

2. discuss the properties of a well-made blood smear for microscopic examination.
3. perform the peripheral blood smear examination.
4. associate the diseases related to red cell morphology abnormalities.
VII. Learning Guide Time allotment Learning Resources
1. Enumerate the procedure of blood smear procedure. 3-hour lecture Learning Content:
1.1 The different types of blood smear preparation. - Lesson 1 module
2. Discuss the properties of a well-made blood smear for page/s: 88-95.
microscopic examination. - Rodak’s Hematology
2.1 Characteristics of a good quality Wedge smear. pages: 235 - 252.
2.2 Factors that caused the poor smear preparation.
3. Perform the peripheral blood smear examination.
3.1 Microscopic evaluation of red blood cell as to size, shape,
hemoglobin concentration, inclusion and distribution.
3.2 White blood cell and platelet number estimation.
4. Associate the diseases related to red cell morphology
abnormalities.
4.1 Anisocytosis and Poikilocytosis.
4.2 Polychromatophilia.
4.3 Red cell inclusions.
- Answer questions related to peripheral blood smear
platform

Lesson VII: Peripheral Blood Smear Examination

In investigating blood diseases one of the reliable laboratory tools that is used is the examination
of the peripheral blood. The peripheral blood smear examination includes the microscopic evaluation
of the morphology of the red blood cell, white blood cell, and platelets, as well as the estimated number
of the white blood cells and platelets is reported. The medical technologist should know how to prepare
a good quality of blood smears. There are different kinds of smear preparation and these are the
coverslip method, coverslip-slide method, and wedge method, in which the latter is the most ideal and
commonly used. The wedge method is also called as the double slide technique or the slide spreader
technique. Even though an automated smear preparation is available, like the spinner or centrifugal
method and the miniprep technique, not all laboratory is equipped with this machine. This is the reason
why most laboratories rely on the skill of the technologists on manual smear preparations. The coverslip
method is ideal for bone marrow preparation and for white cell differential count but it cannot be used
for evaluating the morphology of the red cell as well as in getting the estimate number of the white cell
and platelets, in which the wedge smear is more appropriately used for these activities. The list of
comparison of advantages and disadvantages between the coverslip method and wedge method is
listed on table 7.1.
Table 7.1
The coverslip method is prepared by placing a drop of blood or bone marrow fluid at the center
of one of the coverslips and putting another coverslip on top of it making an eight-pointed star like
appearance, as what is shown in figure 7.1. After which, the two coverslips will be pulled apart to make
a blood smear. The two smears made will be appropriately stained and mounted to a glass slide for
microscopic evaluation.

Figure. 7.1
The wedge smear is prepared by placing a 2-3 mm drop of blood around 1-2 cm from the edge
of the slide. Hold each end of the slide with the thumb and the middle finger of one end, respectively.
On the other hand, hold another slide in similar way, this will serve as the spreader slide. Place the
lateral side of the spreader slide against the slide with blood, such that the slide and the spreader
slide will form around 30-40-degree angle. Pull back the spreader slide until the edge of it touches the
blood drop, and allow the blood to spread laterally on the entire edge of the spreader slide. Keeping
the angle constant, push the spreader slide toward the other end of the slide to spread the blood.
Note that the blood smear prepared should show a thicker part at the starting point and becoming
thinner towards the end and producing the so-called “feathery edge”. See figure 7.2. Then carefully
lay slide on a flat surface to dry immediately and fixed with 10 % vol/vol of methanol. Delay in the
drying of the smears made may cause crenation of the red cell or also known as echinocytes. See
figure 7.3.
Figure.7.2
Figure. 7.3

A well-made wedge smear should meet the following criteria to make it acceptable for
microscopic evaluation and these are; 1. The length of the smear should occupy at least ½ to ¾ of the
slide, 2. The smear should have a transition from thick portion to thin portion with fine feathered edge
at the end, 3. It should occupy the entire width of the slide, 4. The smear should be free from any spaces
and foreign particles, 5. The edge should show a rainbow appearance if it placed against a white light.
The smears prepared will be stained with the available routine stains used in a Hematology laboratory
such as Wright’s and Giemsa, Lieshman, May-Grunwald, Jenner-Giemsa and Romnowsky stain.
Generally, these stains are composed of two main staining reagent which are methylene blue and eosin
Y. The methylene blue which is the basic component of the stain, commonly stains the acid portion of
the cell which is the nucleus; while the eosin Y, which is the acid component of the stain, stains the
basic portion of the cell which is the cytoplasm. The steps for staining the smears made are as follows;
1. Fixation, 2. Buffer solution, 3. Eosin Y, 4. Methylene Blue, 5. Washing, 6. Drying. Ideally the buffer
solution used should maintain the pH of the stains to 6.8. However, if one wishes to see the malarial
stippling within the infected red cells, the pH of the stain can be adjusted to 7.2. The well stained smear
should impart a purple hue color macroscopically to be acceptable for microscopic evaluation.
However, there are certain factors that cause excessive blueness or redness coloration of the blood
smears making it unacceptable for microscopic evaluation. The factors that may cause too blue or too
red staining of the blood smear is listed on the table below.
Ideally, blood smears should have a transition from thicker to thinner portions, but if the smears
made is thick all throughout it can cause too blue appearance. A very thick blood smear is not used
because it might obscure the microscopic observation of the red cell due to the too much overlapping
of the cells, and it may cause falsely increase estimated count of the white blood cells and platelets.
There are several reasons why smears made are thick and these are, too big drop of blood used, high
angle of smears made during spreading, and fast spreading of the blood. On the contrary, thinner blood
smear created is due to either using small amount of blood, low angle of spreading, and slow spreading
of blood. It was mentioned earlier that the ideal angle made should be around 30-45-degree angle,
however if the blood specimen is derived from a polycythemia patient, the medical technologist should
lower the angle to 20-25 degree. This is done to prevent thicker blood smear that possibly might be
created due to the high viscosity nature of a polycythemic blood. The laboratory personnel should
prevent the contamination of water on every reagent used for staining the smear because this may

induce hole formation in the cytoplasm of red cells or also called as “moth-eaten red cell”. See Figure
7.3. This water artifact may cause confusion especially in investigating red cell anomalies.
Figure. 7.3
In the peripheral blood smear examination, the entire characteristics of the red cell should be
evaluated and reported. The medical technologist should assess the red cell size, shape, hemoglobin
concentration, cytoplasmic inclusion and even the red cell distribution. Normally the red cell size is
around 6-8 micrometer in diameter and should have almost the same size with small lymphocytes,
however if the red cell’s size is deviated from its normal size then it is called as anisocytosis. The extent
of anisocytosis varies from slight, moderate to severe depending on the severity of anemia that an
individual has. Medical technologist can also report terms such as microcytic red cell if the red cell size
is small, normocytic red cell if the size falls within the normal range and macrocytic red cells if it has
large size. Microcytic red cells are commonly associated with certain anemia such as iron deficiency
anemia, thalassemia, sideroblastic anemia, lead toxicity and anemia of chronic disease. Normocytic red
cell is found in bone marrow and kidney failure, while macrocytic red cells are seen on patients with
megaloblastic anemia. This report should be correlated with the MCV result of the patient. On the other
hand, red cell shape should be discoid but if it is deviated from its normal shape it is termed as
poikilocytosis. The severity of the poikilocytosis can be reported from slight, to moderate to severe,
however medical technologist may use a grading chart to assess how extent is the shape and even size
variation of red cells present on patient’s blood. (Refer to table 7.2).
Grading scale for Red cell morphology

Anisocytosis/Poikilocytosis
Normal 5%
Slight 5 – 10 %
+1 10 – 25%
+2 25 – 50%
+3 50 – 75%
+4 >75%
Grade +4
Table 7.2

Specific cell types or poikilocytes are reported using either of the suggested grading charts
below.
+1 +2 +3
Dacrocytes
Acanthocytes
1-5/hpf 6-10/hpf >10/hpf
Schistocytes
Spherocytes
*polychromatophilia
Burr cells
Ovalocytes
3-10/hpf 11-20/hpf >20/hpf
Stomatocytes
Bizarre RBC shapes
Poikilocytosis
Target cells
Cell type Grading (# of cell type per hpf)

Occasional +1 +2 +3 +4
Target cells <5 5 - 10 10 – 30 30 – 60 >60
Sickle cells
Acanthocytes
Stomatocytes
Tear drop cells <1 1–3 3–6 6 – 12 >12
Schistocytes
Spherocytes
Elliptocytes <6 6 – 20 20 – 50 50 – 75 >75
Ovalocytes
Echinocytes <10 10 -25 25 – 50 50 – 75 >75
Blister cells <1 1–5 5 – 10 10 – 15 >15
The hemoglobin concentration is evaluated by determining the central pallor size of the red
cells. Normally, the size of the central pallor should occupy approximately 1/3 of the total diameter of
the red cell or should not exceed 3 μm. The central pallor size may increase in certain anemia such as
iron deficiency anemia, thalassemia, sideroblastic anemia and anemia of chronic disease. On the
contrary, decreased size of it is commonly associated with megaloblastic anemia, spherocytosis and in
other hemoglobinopathies such as Hgb S and Hgb SC. The Medical technologist reported the Hgb
content using terms such as normochromic, hypochromic and hyperchromic if the central pallor size is
normal, increased and decreased, respectively. Keep in mind that the report should be correlated with
MCH and MCHC result to prevent discrepancy that may lead to misinterpretation of the patient’s red
cell status. The hypochromicity of the red cells can be graded depending on how big is the central pallor
size. This grading chart will be very helpful in assessing on how extensive was the decreased hemoglobin
content that the blood specimen has.

Grade +1 +2 +3 +4
Central pallor size 1/2 2/3 3/4 Thin rim of

hemoglobin
Red cells normally should not have any cytoplasmic inclusion; however, certain anomalies
may cause the formation of this and could affect the normal functioning of the red cell. One example
of this is Heinz bodies which is commonly associated with g-6-PD deficiency and unstable hemoglobin.
The list of other inclusions with their associated disorders is stated on the table below.
Abnormal Red cell Microscopic appearance Disease associated

inclusion
Heinz bodies ▪ Enzymopathy
- G-6-PD deficiency
- glutathione reductase
deficiency
- 6-phosphogluconate
dehydrogenase
deficiency
- glutathione peroxidase
deficiency
- triosephosphate
isomerase deficiency
▪ Presence of unstable
Hgb
Pappenheimer bodies ▪ Sideroblastic anemia
▪ Refractory anemia
Basophilic stippling’s ▪ Sideroblastic anemia

▪ Thalassemia
▪ Arsenic poisoning
▪ Lead poisoning
▪ Pyrimidine 5’-
ribonuclueotidase
deficiency
▪ Megaloblastic anemia

Howell-jolly bodies ▪ Megaloblastic anemia

▪ Thalassemia
▪ splenectomy
Cabot rings ▪ Megaloblastic anemia

▪ thalassemia
Hgb H ▪ alpha thalassemia major
Malarial stippling ▪ Plasmodium sp. infection
Babesia ▪ Babesia microti infection
The red blood cells are evenly distributed with no overlapping in the thin area of a well-made
smear. This is due to the repulsion made by red cell towards each other because of the electron cloud
on its membrane which is known as the zeta potential. Abnormal distribution of red cell occurs on
certain blood problems and these are the rouleaux formation and agglutination or clumping of red cells.
Rouleaux formation is caused by increased plasma protein such as hyperfibrinogenemia and also seen
in cases of plasma cell dyscrasia like multiple myeloma, Waldenstrӧm’s macroglobulinemia and heavy
chain disorders. While agglutination is caused by the presence of an autoantibody or alloantibody

directed against red cell antigens that cause aggregation or clumping of the RBC. These antibodies may
either be IgG or IgM types. If the responsible antibody causing agglutination is IgM this is referred to as
cold agglutinin while if it is IgG antibody it is referred as warm agglutinins. The extent of red cell’s
rouleaux formation can be reported using the suggested grading chart below.
Grading for Rouleaux

formation
+1 3 – 4 Rouleaux formation
+2 5 – 10 Rouleaux formation
+3 >10 Rouleaux formation
White blood cell and Platelet number estimate

Part of the blood smear examination is reporting of the estimated number of the WBC and
platelet. Using the smear, the medical technologist can give a relative count of the WBC and platelets
using the formula below.
WBC count
a. 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝐵𝐶 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑥 2000

b. 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝐵𝐶 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑥 3000
Note: If the total magnification of the microscope is x40 use the factor 2,000 but if it is x50 use the
factor 3,000.
Platelet count
a. 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑥 20,000
𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑥 𝑅𝐵𝐶 𝑐𝑡.

b. 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑝𝑙𝑎𝑡𝑒𝑙𝑒 𝑐𝑜𝑢𝑛𝑡 = 200 𝑅𝐵𝐶

LESSON MODULE
LESSON 8
LESSON TITLE: Red cell Disorders

1. classify red cell disorders
2. discuss the mechanisms involved in anemia and polycythemia
3. discuss the different types of polycythemia.
4. discuss the different forms of anemia.
5. correlate the lab result on different red cell disorders
VIII. Learning Guide Time allotment Learning Resources
1. Classify red cell disorders. 6-hour lecture Learning Content:

1.1 Relative and Absolute Polycythemia - Lesson 1 module
1.2 Relative and Absolute Anemia page/s: 97-122.
1.3 Absolute anemia classification. - Rodak’s Hematology
pages:284 - 473.
2. Discuss the mechanisms involved in anemia and
polycythemia. principles, procedures,
3. discuss the different types of Polycythemia. correlation pages: 126
3.1 Primary and Secondary Absolute Polycythemia. – 285.
4. Discuss the different types of absolute anemia.
4.1 Intrinsic red cell disorders.
4.2 Extrinsic red cell disorders.
5. correlate the lab result on different red cell disorders
5.1 Special Laboratory results, PBS exam and
hematological parameters.

- Answer questions related to RBC disorder
platform

Lesson VIII: RBC Disorder

Red cell anomalies are initially diagnosed by taking note of the signs and symptoms that the
patient exhibits and with the use of Hematological parameters like the CBC, PBS exam and special
hematological examination. The RBC ct., Hgb and HCT are parameters that is requested to know if the
patient is suffering from either Polycythemia or Anemia. High result of these three parameters is
commonly associated with polycythemia while decrease level of those is seen in anemia. However,
there are factors that may cause false decrease or increase of the said parameters which may lead to
false interpretation known as Relative Polycythemia or Relative Anemia. Relative Polycythemia is
caused by the efflux of fluid from the blood vessel to the interstitial fluid which causes the
hemoconcentration of the blood within the vasculature. Relative anemia, on the other hand, is caused
by the influx of fluid from the interstitial tissue or other source to the blood vessel which causes the
hemodilution of the blood. Dehydration is an example of a condition that may lead to false high HCT
and Hgb value due to loss of fluids or relative polycythemia. Blood collected contaminated with I.V
fluids may cause hemodilution which leads to false decrease level of the Hgb and HCT or relative
anemia. Therefore, Medical laboratory scientist should keep in mind that any abnormal laboratory
findings there is a necessity to do further investigation before releasing laboratory results, this is to
prevent the possible misdiagnosis on part of the physician who will interpret the report. List of factors
that causes relative polycythemia or relative anemia is listed on table 8.1.
Relative Polycythemia Relative Anemia
▪ Dehydration ▪ Poor blood specimen handling

-
Prolong diarrhea - Using hemolyzed samples
-
Hyperemesis - Using clotted EDTA samples
▪ Stress/Anxiety ▪ Poor blood collection technique
▪ Tobacco smoking - Blood collected above the I.V infusion site
▪ Poor blood collection technique - I.V lines are not stopped for 2 to 3 min. before
- Prolong tourniquet application blood collection
- Advised the patient do hand pumping during - Blood collected on edematous arm
venipuncture
Table 8.1
An increase or decrease of blood production due to pathological disorder causing a true increase
or decrease of Hgb and HCT value is known as Absolute Polycythemia or Absolute Anemia, respectively.
These pathological disorders that affects the production or survival of the red cell may either due to
genetic or acquired reasons. Absolute Polycythemia is classified into primary or secondary. Primary
absolute polycythemia is a genetic problem which causes the uncontrolled or over production of the
blood cells especially the red cell that causes serious health problems if intervention is not applied. This
disorder is known as “Polycythemia vera”. The gene defective in Polycythemia vera is the mutated JAK
2 V617F gene which leads to spontaneous kinase production that causes overstimulation of the
hematopoietic cell to multiply unceasingly. The consequence of this is making the blood so viscous that

it may cause decrease blood tissue perfusion and cyanosis. Due to the high viscosity of the blood it may
stagnate within the vessel and eventually may clot that leads to thrombosis or infarctions. The EPO
level on the patient is severely decrease due to the overproduction of the red cells by the bone marrow.
The laboratory findings on this condition includes pancytosis (high RBC count, WBC count and Platelet
count), low ferritin, high LDH, high vitamin B12, hypercellular bone marrow and the LAP score is high
reaching more than 200. The ESR value on this condition is decreased due to the excessive number of
red cells in the given sample. Clinical manifestations include headaches, dizziness, weakness, shortness
of breathing, abdominal fullness due to splenomegaly, excessive reddish tone of skin, itching during
warm shower, pain or numbness of hands and feet, blurred visions, and bone pain. One of the common
remedies to alleviate the symptoms is the therapeutic phlebotomy in which approximately 500 mL of
blood is removed from the patient. However, there are therapeutic drugs available to treat this
condition.
The Secondary Polycythemia is triggered by high EPO stimulation on the bone marrow to
promote red cell production which increases the Hgb and Hct value. The secondary polycythemia is
divided in to either hypoxia driven EPO release (also known as appropriate secondary polycythemia) or
non-hypoxia driven (also known as inappropriate secondary polycythemia). Hypoxia driven includes
patients with heart and lung problems such as COPD, cigarette smoking, living or going up on high
altitudes. While non hypoxia driven are kidney disorders that accidentally release the EPO triggering
red cell production, and example of these are polycystic kidney and kidney tumors.
Absolute anemia is caused by a pathological disorder that leads to the decreased value of the
Hgb and Hct accompanied by general signs and symptoms such as fever, weakness, extreme fatigue,
pale skin, chest pain due to increase heartbeat, shortness of breath, headaches, dizziness or
lightheadedness, and coldness of palm and feet. Absolute anemia can be classified into various ways
depending on what is the nature or source of disease and characteristic of the blood picture that it
exhibits. See the table below. Acute blood loss or chronic blood loss can also lead to anemia.

Red cell Size

Microcytic Anemia Normocytic anemia Macrocytic anemia
(MCV <80fL) (MCV 80-100fL) (MCV >100fL)
▪ Iron Deficiency anemia (IDA) ▪ Bone Marrow disorders ▪ Megaloblastic anemia
▪ Thalassemia - Aplastic anemia - Vitamin B12 deficiency
▪ Sideroblastic Anemia - Fanconi anemia • Pernicious anemia
▪ Lead poisoning - Shwachman-Bodian-diamond • Immerslund-
▪ Anemia of Chronic syndrome Grasbeck syndrome
Disease/Inflammation - Diamond- Blackfan anemia • Transcobalamin
▪ Chronic hemorrhage - Myelopthisic anemia deficiency
- Myelofibrosis • Zollinger Ellison
- Leukemia syndrome
- CDA • D. latum infection
▪ Kidney Failure • Blind loop
▪ Uremia syndrome
▪ Acute hemorrhage • Chron’s disease
• Decrease Intrinsic
factor secretion
- Folate deficiency
• Chronic alcoholism
• Therapeutic drug
administration
Table 8.2
Reticulocyte Number
Hemolytic anemia Non- Hemolytic anemia
(with reticulocytosis/ high retic count) (with Low to normal reticulocyte count)
▪ Sideroblastic anemia (Lead Poisoning) ▪ Iron Deficiency anemia
▪ Autoimmune Hemolytic anemia ▪ Megaloblastic anemia
- Cold agglutinin syndrome ▪ Bone marrow failure or Aplastic anemia
- Warm agglutinin syndrome ▪ Leukemia
- Drug induced ▪ Kidney failure
- HDFN
- PCH
▪ Hemoglobin disorders
- Thalassemia (α and β)
- Hemoglobinopathy
• Sickle cell anemia
• Hgb CC or SC and other unstable
hemoglobins
▪ Red cell membrane disorders
- Hereditary spherocytosis
- Hereditary elliptocytosis
- Hereditary ovalocytosis
- RH null phenotype
- PNH
▪ Enzymopathy
- G6PD deficiency

- Pyruvate Kinase deficiency

- Glutathione oxidase deficiency
- P-5’-Nucleotidase deficiency
▪ Microangiopathic Hemolytic anemia
- Disseminated Hemolytic anemia
- TTP
- HUS
- HELLP syndrome
- Eclampsia/Pre-eclampsia
- Parasitic infection
• Malaria
• Babesia
- Mechanical damage of red cell due to toxic
substances, burns, infections, etc.
Table 8.3
Source of Disease
Intracellular Red cell disorders Extracellular red cell disorders
▪ Abnormal Iron Kinetics ▪ Immune cause
- Iron Deficiency anemia - Cold agglutinin syndrome
- Anemia of chronic disease/inflammation - Warm agglutinin syndrome
- Sideroblastic anemia - Drug induced
- Hemochromatosis - HDFN
- PCH
▪ Abnormal Heme Kinetics ▪ Non immune
- Porphyria - Microangiopathic Hemolytic anemia
- Sideroblastic anemia • Disseminated Hemolytic anemia
- Lead poisoning • TTP
▪ Abnormal Nuclear development • HUS
- Megaloblastic anemia • HELLP syndrome
- CDA • Eclampsia/Pre-eclampsia
▪ Abnormal Globin synthesis - Parasitic infection
- Thalassemia (α and β) • Malaria
- Hemoglobinopathy • Babesia
▪ Abnormal Membrane disorder - Mechanical damage of red cell due to toxic
- Hereditary spherocytosis substances, burns, infections, etc.
- Hereditary elliptocytosis
- Hereditary ovalocytosis
- Hereditary stomatocytosis
- Acanthocytosis
- PNH
▪ Congenital Bone Marrow disorders
- Fanconi anemia
- Shwachman-Bodian-diamond syndrome
- Diamond- Blackfan anemia
▪ Enzymopathy
- G6PD deficiency
- Pyruvate kinase deficiency
Table 8.4

Intracellular Red Cell Disorders
Abnormal Iron Kinetics
Iron deficiency anemia

Iron deficiency anemia is the most common cause of anemia especially in women. There are
several cause of iron deficiencies and these are inadequate intake, increase demand, impaired
absorption, and chronic blood loss. There are three phases of iron deficiency anemia and these are Iron
Replete, Iron Deplete, and Iron deficiency. In Iron replete the patient does not yet experience any signs
and symptoms of anemia because on this stage both stored iron (Serum Ferritin) and functional iron
(Serum Iron) are still in normal level. The normal serum ferritin for male is 12 to 300 ng/L while for
female it is 12 to 150 ng/L, while serum iron is 60-170μg/dL (10-30 μmol/L). The Hgb and Hct is normal
as well as other parameters such as Total iron binding capacity (TIBC), and the percentage of Transferrin
saturation or also known as Transferrin saturation index (TSI). TIBC is one of the blood parameters that
can determine indirectly the level of iron in the blood and the normal reference range of it is 240 to
450 microgram/dL or 42.9 to 80.55 μmol/L. The percentage of transferrin saturation is measured by
dividing the serum iron concentration by the TIBC then the factor is multiplied by 100. The TSI is used
by clinicians in determining how much iron is bound to transferrin (transport protein for iron). The
average TSI is 25%, for male the maximum value is 45% and for female it is 30%, however, an increase
value of more than 50% may possibly cause organ failure or damage as what is seen on
hemochromatosis. In Iron Deplete the individual still doesn’t exhibit any signs and symptoms of anemia
because almost all parameters is within the reference range like the Hgb, Hct, Serum Iron, TIBC and
Transferrin saturation, however, due to several factors such as increase demand or inadequate intake
on growing child or in pregnant woman, the stored iron or ferritin is starting to deplete. As the stored
ferritin is continuously depleting and not replenished, this event may lead to the exhaustion of all of
this stored iron which then cause decrease production and abnormal development of red cells within
the marrow. This stage is the Iron deficiency, where patient is experiencing the general signs and
symptoms of anemia with additional manifestation such as paresthesia, angular stomatitis and other
unique features for IDA known as PICA and koilonychia. PICA is a neurological disturbance where the
patient has these craving to chew on or eat non-food items such as soil, clay pots and ice cracks.
Koilonychia or also known as “spoon-shaped nail” is the formation of depression on the nail bed due to
low oxygenation on fingers or digits. On this stage the Hgb and Hct is decreased and the TIBC is
increased due to low serum iron while the transferrin saturation is decrease. The red cell produced on
this anemia is microcytic (low MCV), hypochromic (low MCHC and MCH) red cells due to the
asynchronous development of the nucleus and cytoplasm of it within the marrow. The maturation of
the cytoplasm lags behind the nucleus which causes the red cell membrane looks irregular or described
as shaggy. Other abnormal shape on red cells are occasionally found on the peripheral blood such as
target cell or elliptocytes. The diagnosis of IDA should be appropriate because other anemia also exhibit
a blood picture of microcytic, hypochromic red cells such as thalassemia minor, sideroblastic anemia,
and anemia of chronic disease or inflammation. Therefore, complete set of parameters should be used
to distinguished all of these microcytic hypochromic anemias to confirm the diagnosis and these are

Serum iron, serum Ferritin, TIBC and Transferrin Saturation. Comparison of different Microcytic anemia
along with lab result is listed on table 8.5.
Microcytic, Hypochromic anemia

Serum iron Serum Ferritin Transferrin TIBC FEP
Saturation
Iron Deficiency
anemia
Sideroblastic anemia
Thalassemia Minor
N to N to N to N
Anemia of Chronic
Disease/Inflammation
Lead Poisoning Variable N N
Table 8.5
If the cause of iron deficiency anemia is due to nutritional deficiency or increased need, it can
be treated by oral administration of 3 tablets per day of ferrous sulfate containing 65 mg of elemental
iron. Other conditions that lead to iron deficiency anemia such as hookworm infection, ulcers and
tumors, should be corrected first before giving oral iron supplements on these patients. Red cell
transfusion is not necessary as part of treating iron deficiency anemia unless the Hgb of the patient
reaches the low critical value.
Sideroblastic anemia
There are two forms of sideroblastic anemia and these are congenital or acquired type. The
congenital type is due to the genetic defect that causes the deficiency of ALA-synthetase which is a vital
enzyme on the initial formation of heme. The hereditary inheritance of this congenital type includes
either x-linked or autosomal diversities. Because the Heme is not fully synthesized the stored iron
accumulates in the mitochondria that surrounds the nucleus of a developing normoblast. This
mitochondria with accumulated iron can be visualized by staining it with a Prussian blue stain that
shows a blue color that circles the nucleus which is known as ringed sideroblast, which is the hallmark
of sideroblastic anemia. See figure. 8.2.
The acquired type is divided into either primary or secondary cause. The primary is associated
with refractory anemias while the secondary is caused by toxic substance or drugs that affects the
formation of heme and the incorporation of the iron in the heme. Drugs associated that may cause
sideroblastic anemia includes chloramphenicol, and anti-TB drugs like isoniazid. Toxic substances like
alcohol may also induce sideroblastic anemia that is why chronic alcoholism can be associated with this
disorder. Heavy metals such as in lead toxicity affects the formation of the heme by inhibiting several

enzymes such as ALA dehydratase or also known as PBG synthase causing the inability to form
porphobilinogen (PBG) from aminolevulinic acid (ALA) in which the latter accumulates within the
normoblast. Lead also inhibits the ferrochelatase or heme synthase, in which this enzyme allows the
integration of Ferrous iron into the Protoporphyrin IX to form the Heme. Another enzyme that is
affected by lead toxicity is the P-5’-ribonucleotidase, an enzyme responsible for degrading ribosomal
RNA to prevent its aggregation that will promote the formation of coarse basophilic stippling within the
red cells. Lead also inhibits the Pentose phosphate pathway that enables the red cells to be sensitive to
oxidative substance which can lead to hemolytic anemia. The use of lead chelating substance like salts
of EDTA is used to treat this condition which allows the excretion of lead in the urine. Lead poisoning
does not only cause acquired sideroblastic anemia but also cause acquired porphyria. *Porphyria is a
condition where porphyrins accumulate in the body which is reflected by high levels of it in the blood,
urine and fecal samples of an individual. Porphyrins are the different products produced from the
biochemical enzymatic reactions needed for complete formation of heme ring. Hereditary porphyria is
an impaired heme production due to genetic defect that causes the deficiency of an enzyme which is
vital for heme synthesis leading to anemia and photosensitivity. Refer to table 8.6 for the list of
erythropoietic porphyria.
Erythropoietic Porphyria
Congenital Erythropoietic X-Linked Erythropoietic
Erythropoietic Protoporphyria (EPP) Protoporphyria (XLEPP)
Porphyria (CEP)
Enzyme affected Uro III synthase Ferrochelatase ALA-synthase 2
Inheritance Autosomal Recessive Autosomal Dominant X- lined
Affected gene UROS FECH ALAS2
Photosensitivity Yes Yes Yes
Anemia Yes Yes Yes
Increased porphyrins in red Uroporphyrin Protoporphyrin Protoporphyrin
blood cells Coproporphyrin
Increased porphyrins in Uroporphyrin
urine Coproporphyrin ----- -----
Increased porphyrins in Coproporphyrin Protoporphyrin Protoporphyrin
feces
Table 8.6
*Note: Porphyria is not associated with Sideroblastic anemia.
The peripheral blood smear of individual suffering from sideroblastic anemia shows a
combination of microcytic and normocytic red cells. This blood picture is known as dimorphic blood
picture or dimorphism. Acquired dimorphism, however, is observed on patients that underwent bone
marrow transplant and blood transfusion. Inclusion bodies such as Pappenheimer bodies and basophilic
stippling are commonly observed. Mature red cells stained with Prussian blue may show blue remnants
of ferritin granules in which these red cells are called “siderocytes”.

Anemia of Chronic Disease/Inflammation

Anemia may develop in some systemic disorders such as malignancy or cancer, prolonged
infection such as Tuberculosis or HIV, and chronic inflammatory conditions like rheumatoid arthritis. In
chronic infection the body produces cytokines that promotes the development of anemia and these are
IL-1, α-TNF, and γ-IFN. During chronic infection the macrophages continuously releases these cytokines
that eventually prevents the release of stored iron from the macrophages to be used up by the
developing red cells within the bone marrow. This event caused the accumulation of stored iron in the
body which is reflected by increased serum ferritin and decreased level of functional serum iron in the
blood. In Chronic inflammation the body releases acute phase reactants on which some of it may cause
anemia and these are Hepcidin, ferritin and lactoferrin. During prolonged inflammatory response the
macrophages releases IL-6 that influences the liver cells to release volumes of hepcidin in the blood and
prevented the release of iron in the plasma, which causes the accumulation of iron in various organ like
liver and bone marrow. This event resulted to decrease serum iron and increased serum ferritin in the
blood. Red cells hemoglobin synthesis is also affected by this process which causes poor cytoplasmic
maturation of the red cell leading to microcytic, hypochromic anemia. Hepcidin is a hormone
manufacture by the liver which is responsible for regulating iron release by the macrophages and
hepatocytes, as well as controlling the intestinal absorption of iron. If the body has low iron level, the
hepcidin release is decreased to allow increase absorption of iron in the intestine and to allow
exportation of iron form the liver cells and enterocytes or macrophages. The liver cells or enterocytes
uses the transmembrane protein known as ferroportin to export iron from them and releases to the
plasma or to the progenitor red cell for hemoglobin formation. Hepcidin interacts with the ferroportin
and degrades it thus preventing the release of iron from the hepatocytes and macrophages or
enterocytes. Lactoferrin is an iron-binding protein that is derived from neutrophil granules that has high
affinity with iron than the transport protein for iron which is the transferrin. Lactoferrin is used by the
phagocytes to prevent the engulfed bacteria or organism to use the cytoplasmic iron for their own
metabolic used. In chronic inflammation neutrophils released the lactoferrin and competes with
transferrin on binding with iron which leads to the formation of lactoferrin-ferritin complex. The
lactoferrin-ferritin complex will eventually bind with macrophages membrane and internalized it and
degrades the iron on it, which deprived the developing red cell with iron to be utilized for heme
synthesis. The bound iron to lactoferrin also is not delivered to the bone marrow tissue due to the
absence of the receptors for it. Finally, high levels of ferritin binds with significant number of iron and
will not delivered to the developing red cell because it lacks receptor for ferritin.
Hemochromatosis
Hereditary hemochromatoses are collection of disorders that affects the regulatory mechanism
for iron absorption and release to and from the cell, resulting to the increased level of iron within the
cell. This disorder has mutated gene which is inherited as autosomal recessive. There are different
forms of mutated gene detected on this disorder, known as Hemochromatosis phenotypes, but one of
most common is the defective HFE gene (a.k.a. Classic Hereditary Hemochromatosis). The HFE gene,
which is located on the short arm of chromosome number 6, encodes for the synthesis of the HFE
protein or also known as “hereditary hemochromatosis protein”. The function of the HFE protein in

regulating iron absorption within the cell is it binds with the β2-microglobulin that allows it to travel to
the membrane of the cell. As the HFE protein is already on the surface of the cell it then interacts with
the Transferrin receptor 1 (TfR1) to receive the transferrin (transport protein for iron) coupled with
iron. As the Transferrin with iron binds with the TfR1 receptor the HFE protein is eventually released
which then binds with Transferrin receptor2 (TfR2). This interaction between HFE protein and TfR2
produces a signal for Hepcidin release by the liver to prevent further absorption of iron in the intestine
as well as iron intake of the cell. A mutated HFE gene produces an abnormal HFE molecule which doesn’t
interact with β2-microglobulin that subsequently impaired the release of the HFE molecule to bind with
TfR2. Without this interaction the Hepcidin released from the liver will not occur or decreases that leads
to continuous absorption of iron in the intestine as well as cell intake of iron unceasingly. This may
result to over accumulation of iron in various organ that may cause organ failure due to toxicity.
Normally iron absorbed by the parenchymal cells of various organs transform it first to a non-metabolic
iron known as ferritin and the excess iron to hemosiderin. These two types of stored iron within the cell
is not toxic to the cell, however, if the iron absorption is not controlled the capability of the cell to
convert it to these non-toxic substances will wear-off. On this event the cell will absorb the iron as plain
ferrous iron. The ferrous iron within the cell’s cytoplasm will interact with oxygen that initiate the
formation of free radicals and superoxide that can cause destruction of the lipid containing membrane
of the cell’s nucleus, lysosomes, mitochondria and the cytoplasmic membrane. The damaged lysosome
will release its enzymes digesting the cell internally which enhances the destruction of the cell. The cell
death occurs on various tissue is irreversible which may lead to organ failure. Vitamin E and C help
prevent the action of these free radical form by coating it to neutralize it, however in hereditary
hemochromatosis, this protective action may exhaust or deplete. The common organs affected are
heart, liver, pancreas and skin. The deposition of excess iron on skin causes a brownish coloration of it
which is called “bronzed diabetes”. The affected liver may lead to cirrhosis or hepatocellular cancer
resulted from the mutation of p53 gene by these free radicals. On the other hand, hemochromatosis
can be acquired by individual who underwent massive blood transfusion or on those with severe
thalassemia that regularly receiving red blood cell component. Several treatment or remedy are
proposed to reduce the level of iron in the body one of this is the removal of around 500 mL of blood
though therapeutic phlebotomy and another is the administration of iron chelating substance known
as desferrioxamine. Thalassemic patient who regularly received red cell component is suggested to
received young red cells using neocytopheresis technique, because it is believed that young red cells
containing less amount of iron than the older ones.
Laboratory findings of Hemochromatosis includes high serum ferritin, transferrin saturation and
serum iron. Elevated liver enzyme such as alanine transaminase or ALT is seen on patient with affected
liver and Troponin I in serum is present if the heart muscle is damaged. Diabetes or high glucose level
in the blood may develop due to the reduce insulin synthesis if the pancreatic tissue is affected. Urine
may show sloughed cells containing hemosiderin as one of the evidences of impaired regulatory
mechanism for iron.

Abnormal Nuclear Development
Megaloblastic anemia
Megaloblastic anemia is a nutrient deficient type of red cell disorder that resulted to the
production of large sized red cell from the bone marrow known as macrocytes. The nutrient deficiency
on this disorder is either vitamin B12 (cobalamin) or folate (pteroyl-glutamate). The interaction of these
two nutrients are very important for DNA synthesis within the nucleus. Absorption of the Vitamin B12
requires HCL and intrinsic factor (IF) which are normally secreted by the parietal cells of the stomach,
while Folate doesn’t require any of those for normal absorption of it in the intestine. Deficiency of either
of these two will cause asynchronous nuclear to cytoplasmic maturation of the red cell, where the
nucleus had shunted development while the cytoplasm continuously develops causing enlarge size of
the cell. There are several factors associated with Vitamin B 12 deficiency and these are inadequate
intake, increased need, impaired absorption, errors in absorption or transport and competition for
vitamins. Impaired absorption includes affected release or lack of Intrinsic Factor which can occur in
pernicious anemia, Intrinsic factor deficiency, H.pylori infection, and gastrectomy. As what was
mentioned earlier the Intrinsic Factor is very vital for normal absorption of vitamin B12, therefore
evaluating it should be part of the investigation. One of the diagnostic tools used to assess the ability
of the stomach to release IF is the Schillings test. In Schillings test the patient is first administered intra-
muscularly with a preferred volume of vitamin B12 and this is known as the flushing dose. The objective
of the flushing dose is to saturate all the entire individual’s cell receptor site with Vitamin B12. After
which, the patient will be given a radioactive Vit B12 pill orally. If the IF is enough the Vitamin B12 will be
absorbed in the intestine and goes to the blood but it will not be absorbed by the body because the
body is already full of Vit B12 due to the flushing dose given earlier. The radioactive vitamin B12 in the
blood will just be excreted out in the urine. A 24-hour urine is collected to measure the amount of
excreted radioactive vitamin B12 using a gamma scintillation counter. The normal excretion should be
>7% of the administered radioactive vitamin B12 should be in the urine, lesser value means that there
is a decrease or lack of IF in the stomach. Errors in absorption includes Imerslund-Grasbeck syndrome
where the intestine lacks the receptor site for IF-Cobalamin complex resulted to inability to absorbed
the vitamin B12 from the intestine to the blood. Error in transport is due to the lack of transcobalamin
II, an important protein that delivers or transports cobalamin to different parts of the body. Vitamin B12
may also occur if the Individual is infected with fish tapeworm known as Dyphyllobothrium latum
because this parasitic worm competes for the vitamin B12 within the host’s body. Normal flora of the
intestine may also compete for this nutrient which occurs in “blind loop syndrome”.
Folate deficiency may occur due to inadequate intake, increased need, impaired absorption,
impaired used of drugs and loss due to dialysis. Certain drugs related to folate deficiency includes
pentamidine, phenytoin, trimethoprim, cholestyramine, and metformin. Chronic alcoholism is also
associated with impaired absorption of folate in the intestine. Combined Vitamin B12 and Folate
deficiency are seen on individuals with tropical sprue and gluten sensitive enteropathy.
There are sequential events that usually took place before developing anemia and this starts
with low vitamin levels in the blood, followed by abnormal morphology on white blood cell known as

hyper-segmented neutrophils (with >5 nuclear lobulations), then the bone marrow will show
megalobastosis (larger immature hematopoietic cell than the usual), which then eventually affected
the size and shape of the released red cell showing macrocytosis and ovalocytosis in the peripheral
blood smear. Aside from having the general signs and symptoms of anemia additional features on this
anemia includes, angular stomatitis, glossitis, and megaloblastic madness. The megaloblastic madness
is a psychosis hallucination due to the degeneration of neurons of the central nervous system. The
laboratory findings of this anemia include decreased cell counts knowns as pancytopenia, low
reticulocyte count, high MCV and MCH but normal MCHC. The peripheral blood shows macrocytic,
normochromic to hyperchromic red cells, with macro-ovalocyte and giant platelets. Red cell inclusions
such as Howell-Jolly bodies, Cabot rings and basophilic stippling are observable. The blood chemistry
findings are high LDH isoenzyme 1, high iron level, and low serum vitamin levels. Urine chemical analysis
shows an increase level of homocysteine and FiGlu for both Vitamin B12 deficiency and folate, while the
presence of methylmalonic acid in urine occurs only in cobalamin deficiency. Additional laboratory test
is used to diagnose this anemia and these are microbiological assay, deoxyrudine suppression test,
serum antibody detection against intrinsic factor, serum gastrin analysis, gastric analysis,
holotranscobalamin assay and stool analysis for parasitic infection. See the table below for the
differential diagnosis between vitamin B12 and folate deficiency.
Specific diagnostic test Vitamin B12 deficiency Folate deficiency
Serum Vit. B12 Decrease Normal
Serum folate Normal to increase Decrease
RBC folate Normal to decrease Decrease
Serum Methylmalonic acid Increase Normal
Serum Homocysteine Increased Increased
Serum gastrin Elevated in pernicious anemia Normal
Gastric analysis Decrease or achlorhydria in Normal

pernicious anemia
Halotranscobalamin assay Decrease Normal
IF antibodies Positive in pernicious anemia Negative
Fecal analysis for parasites Positive for D. latum egg Negative
Microbiological studies: Serum inoculated with Euglenia Serum inoculated with

gracilis or Lactobacilus leichmani Lactobacillus casei shows no
shows no turbidity. turbidity.
Doxyrudine suppression test Low suppression of labeled Low suppression of labeled

thymidine into the DNA is thymidine into the DNA is
corrected by adding Vitamin B12 corrected by adding folate
Table 8.7

There are several conditions that shows macrocytic red cell in the peripheral blood or with high
MCV but they are not caused by megaloblastic anemia and these conditions are collectively called as
“megaloblastoid reaction”. These macrocytic non-megaloblastic anemias include reticulocytosis,
acquired bone marrow failure, liver diseases and chronic alcoholism. Macrocytosis is normally found
on newborns.
Congenital Dyserythropoietic Anemia (CDA)

This abnormal nuclear development of the red cell showing binuclearity or multinuclearity of
and characterized by ineffective erythropoiesis that resulted to decrease Hct and Hgb. There are four
types of CDA in which the most common type is type II. The cause of this abnormality is due to the
genetic mutations that causes abnormal appearance of developing red cell or dyserythopoiesis. The
defective gene for CDA type I is the CDAN1, for CDA type II is SEC23B, CDA type III is the KIF23, and CDA
type IV is KLF1 gene. The most common type of among the CDA type is the Type II where the SEC23B
gene is mutated which affects the instructions for protein formation that is responsible for transport of
protein within the cell. The comparison between CDA types is listed on the table below.
Red cell Membrane defects
Hereditary spherocytosis
Hereditary spherocytosis (HS) is a genetic defect that promotes the poor development of the
red cell’s membrane peripheral proteins which resulted to the impaired interaction of it to the
transmembrane or integral membrane proteins. This disorder increases the fragility of the red cell that
resulted to early red cell destruction which occurs either extravascular or intravascularly. According to
some studies, 2/3 or 75% of the said cases is genetically inherited as autosomal dominant, while 1/3 or
25% of it is inherited as recessive or nondominant. There are several mutated gene associated with HS,
and these are ANK1 gene that instruct the formation of Ankyrin, SPTA1 gene for α chain of spectrin,

SPTB1 gene for β chain of spectrin, EPB42 gene for protein Band 4.2, and SLC4A1 gene that encodes the
formation of protein band 3. As the red cell travels within the cardiovascular system, due to the loose
protein cytoskeleton structure, the RBC membrane eventually forms “bleb” like structure caused by
dislocated membrane lipid from the membrane proteins. These loose blebs or small vesicles formed
spontaneously detaches as the red cells circulates, or are removed or eaten up by the macrophages of
the spleen. This gradual loss of red cell membrane will make the red cell size small or spherocytic with
tightly confined Hgb within it which leads to decreased surface area to volume ratio. Additionally, the
inability of the spherocytes to deform or squeeze, these abnormal cells will sequentially be trapped
within the tight vasculature of the spleen that increases the chance of hemolysis within this organ. The
abnormality of the membrane may also cause the excessive activity of the Na+-K+ ATPase that may
bring about to increase efflux of sodium ion along with water from the red cell that makes it dehydrated.
The peripheral blood smear of the patient suffering from HS will show variable grade of
anisocytosis and poikilocytosis with increased polychromasia or reticulocytosis. Presence of
spherocytes paralleled with a history of childhood hemolytic anemia and familial anemia with the same
condition is greatly suggestive of this disorder. Aside from anemia, symptomatic patients with
moderate to severe case of HS also exhibit signs such a splenomegaly and jaundice caused by increased
level of serum bilirubin. The levels of bilirubin may vary between 2 – 3mg/dL for moderate HS while
more than 3 mg/dL on severe cases. The MCV is low and the MCHC is high which gives the blood picture
of microcytic, hyperchromic red cells (small red cells without central pallor). Aside from spherocytes,
which is considered as the hallmark of HS, the PBS exam on some case of HS may show other
poikilocytes such as acanthocytes if the abnormal protein involve is the β-spectrin, and bizarre shape
like pinched or mushroom shaped red cell on splenectomized patient with defective red cell membrane
protein band 3. The blood chemistry gives a low result on serum haptoglobin and increased LDH
isoenzyme 1 which is highly suggestive of increased hemolysis of red cell. In mild HS the patient is
asymptomatic with normal levels of Hgb because of good compensatory mechanism of the bone
marrow, however, the bilirubin level is slightly increased with values between 1 to 2 mg/dL, with high
reticulocyte count and the PBS shows few spherocytes. Asymptomatic patient may develop
complications known as hemolytic crises, aplastic, and megaloblastic crisis. Hemolytic crisis may
develop after viral infection, aplastic crises is associated with Parvovirus B19 infection resulting to
severe decreased on reticulocyte number and hemoglobin that frequently necessitate for blood
transfusion, and megaloblastic crisis occurs when folate level drops in the patient’s body due to
increased need specially during pregnancy. Splenectomy is one of the recommended treatments to
reduce the destruction of the red cell, however, the consequence of this is high risk of infection caused
by encapsulated bacteria like Hemophilus influenzae type b that may pose a great danger in developing
cardiovascular diseases. Therefore, vaccination practice against this organism is recommended prior to
splenectomy. Other consequences of splenectomy include increased number of target cell, red cell with
pappenheimer bodies and Howell-Jolly inclusion bodies, leukocytosis and thrombocytosis. On the other
hand, spherocytes are observable in the PBS of some individual with no genetic abnormality mentioned
above associated for HS and this is known as acquired spherocytosis. Acquired spherocytosis occurs in
other conditions such as immune mediated hemolytic anemia, HDFN, and hemolytic transfusion
reaction, this may also find on patient that underwent red cell transfusion, as well as on traumatized
red cell due to external and mechanical damage caused by toxins, severely burnt patients and in

disseminated intravascular coagulopathy (DIC). Differential diagnostic test should be done to

differentiate HS from acquired one like DAT test, Eosin-5’-maleimide binding test and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The use of the EOFT studies is not a
confirmatory test for HS because it may also give a positive result on other conditions which shows
spherocytic red cells in the blood; however, it is a vital part as one of the laboratory tests used for
investigating this disorder.
Hereditary Elliptocytosis
Hereditary Elliptocytosis (HE) is caused by genetic defect that causes the poor parallel contact
of the red cell membrane proteins. This abnormality deprived the red cells the ability to recoil its
membrane after experiencing absolute pressure within the cardiovascular system and also through
squeezing to small blood vessels resulted to an elongated shaped red cell called elliptocytes. This is
inherited as autosomal dominant with a defective SPTA1 gene (codes for α-spectrin), SPTB gene (codes
for β spectrin) and EPB41 gene (codes for protein band 4.1). This mutated gene resulted to the
abnormal molecular interaction between α-spectrin, β-spectrin and protein band 4.1 which makes the
red cell prone to early hemolysis as it travels within the circulatory system. Normally red cell can
withstand temperature up to 49℃ without causing it to lyse, but the RBC in HE is easily degraded after
exposing it to 41 to 45 ℃ , this condition is called hereditary pyropoikilocytosis. Protein Band 4.1 is also
affected by the deficiency of Gerbich membrane protein known as the Leach phenotype as well as the
deficiency of glycophorin C due to defect on GPC gene. The peripheral blood smear of patient suffering
from hereditary elliptocytosis may show few to 100% elliptocytes (cigar shaped red cell) depending on
the severity of the disorder. However, other conditions may also exhibit this abnormal RBC shape like
myelodysplastic syndrome, megaloblastic anemia, iron deficiency anemia and in myelofibrosis of
primary cause. Mostly patient with this disorder are asymptomatic but around 10 % of the cases may
show moderate to severe anemia showing high polychromasia or reticulocytosis in the blood.
Hereditary ovalocytosis
Hereditary ovalocytosis or also known as Southeast Asian ovalocytosis is caused by the defective
membrane protein band 3 due to the occurrence mutation on SLC4A1 gene. These mutations resulted
to the deletion of 27 base pairs that causes the highly fitted binding of protein band 3 with ankyrin
causing stiffness on red cell membrane. This stiffness on red cell membrane will affect the flexibility of
the red cell as it squeezes within the small blood vessels which may lead to hemolysis. Patients with
this abnormality shows oval shaped red cell with one or two cross ridges.
Hereditary Stomatocytosis
This disorder occurs in two different forms and these are overhydrated hereditary
stomatocytosis and dehydrated hereditary stomatocytosis. In overhydrated HS, the Na+ ion influx while
K+ ion efflux to and from red cell increases which resulted to high volume of water inside the red cell.

This occurs in RH null phenotype due to defective RHAG gene. The dehydrated HS or hereditary
xerocytosis is caused by the low K+ ion inside the red cell that cause cation imbalance of proportion
causing the water out from the red cells. The defective gene on this condition is the PIEZO1 gene that
is responsible for synthesis of a protein known as Piezo-type mechanosensitive ion channel component
1 protein (PIEZO1) that is part of the regulating mechanism for cationic transport. The peripheral blood
smear on the overhydrated HS shows stomatocytes and high MCV with approximate value of 110-150
fL and low MCHC, while in dehydrated HS, the PBS shows stomatocytes, with other RBC forms like burr
cells and target cells. Other red cell membrane disorder with stomatocytosis includes Familial
Pseudohyperkalemia, Cryohydorcytosis, and RH null syndrome.
Acanthocytosis
The presence of acanthocyte in the peripheral blood with neurological disorder is associated
with a group of disorder called “neuroacanthocytosis”, which includes abetalipoproteinemia, McLeod
phenotype and Chorea. Acanthocyte is characterized by the presence of irregular shaped and spaced
pointed spicules which usually mistaken with other poikilocytes like burr cells or echinocytes. The burr
cell spicules differ from that of the acanthocytes because the latter has regular shape and spaced blunt
spicules. Burr cells are associated with certain conditions such as uremia, renal insufficiency and
pyruvate kinase deficiency. In abetalipoprotenemia the abnormality comes from the defective MTP
gene that is responsible for the synthesis of MTP protein which is needed for lipid transport and lipid
formation onto apolipoprotein B resulting to decrease production of lipid products such as
chylomicrons, vLDL and LDL. The absence of these lipids will cause the diminished level of triglycerides
and cholesterol but with increased sphingomyelin level in the plasma. The consequence of this is low
fluidity within the lipid component of the red cell membrane that changes the normal shape of the red
cell. Patients with this disorder may experience episodes of mild hemolytic anemia but with normal
MCV, MCH and MCHC that gives a blood picture of normocytic, normochromic red cells. Other clinical
manifestation includes ataxia, fat malabsorption, retinitis pigmentosa and neuropathy due to myelin
degeneration. Macleod phenotype is an X-linked inherited disorder that affects the formation of the
Kell antigen on red cell membrane because of gene mutation on Kx gene and Kell gene. Clinical
manifestation on this disorder includes few acanthocytes on blood, myopathy, neuropathy and
psychotic episodes. Lastly, Chorea acanthocytosis is caused by mutated VPS13A gene resulting from
deficiency of Chorein, in which this protein helps in the trafficking of membrane protein. The deficiency
of this protein may cause abnormal protein formation and acanthocytosis. Other clinical manifestation
of this disorder includes hyperkinesia, poor cognitive and neuropsychiatric episodes.
Paroxysmal Nocturnal Hemoglobinuria (PNH)

Paroxysmal Nocturnal Hemoglobinuria is an X-linked genetic abnormality that causes a chronic
type of intravascular hemolytic anemia with a defect on PIGA gene that encode for the synthesis of the
GPI anchor proteins which consists of phosphatidylinositol and a glycan core. Red cell that lacks this
anchor protein leads to the absence of the GPI linked protein in the red cell membrane that resulted to

the diminished to no CD 55 and CD 59 markers. This CD markers are very important for the inhibition
of red cell lysis mediated by the activation of the complement system. The CD 55 which is also known
as the decaying accelerating factor (DAF) protects the red cell form complement cytotoxicity by
deactivating the C3 convertase formed while the CD 59 which is known as the membrane inhibitor
reactive lysis (MIRL) prevents the incorporation of the C9 in the membrane attack complex which lowers
the activity of it to cause cell lysis. The red cells in PNH is highly susceptible on cell cytotoxicity during
night time where slight acidity of the blood occurs with evidence of hemoglobinuria on urine specimen
submitted using the fresh morning samples. Other cells such as white blood cells also lack this CD
markers which can be confirmed using the flow cytometrical analysis. There are three types of PNH,
the type I is characterized by normal expression of the CD markers mentioned above with slight to no
complement associated hemolysis, the type II has moderate loss of the CD markers which is unaffected
by complement associated lysis, and type III with no CD markers at all is highly susceptible to
complement associated hemolysis. Complications of PNH includes anemia, chronic renal failure due to
toxic level of hemoglobin in renal tissues, cholestasis and esophageal spasm and erectile dysfunction.
Esophageal spasm is caused by decrease level of nitric oxide resulted from increased elimination of it
during degradation of free hemoglobin. Patient with this disorder has low platelet count but with high
activity that may lead to thrombosis. Patient with thrombotic tendencies may suffer to various
complications associated with cardiovascular occlusion such as stroke, myocardial infarction, deep vein
thrombosis or DVP, portal hypertension, Budd-Chari syndrome or hepatic venous thrombosis and
cerebral infarction. Laboratory diagnosis in PNH includes hemoglobinemia, hemoglobinuria,
hemosiderinuria high retic count, initially slightly increased MCV due to reticulocytosis but later may
decrease the MCV due to gradual loss of iron which leads to microcytosis, and DAT negative test. If
folate is deficient because of erythroid hyperplasia megaloblastic anemia may develop. Acquired
aplastic anemia is also associated with PNH as secondary to a disease process. Screening test for PNH
includes sugar water test and confirmatory tests includes Sucrose hemolysis test, HAM’s test, Flow
cytometry with Fluorescein labeled proaerolysin variant or FLAER. The use of FLAER flow cytometry is
used to detect GPi linked proteins on monocytes and macrophages by detecting the presence or
absence of several CD markers such as CD 55, 59, 24, 16, 66b, and 14.
Abnormal Hemoglobin
Thalassemia
Thalassemia is a quantitative hemoglobin defect caused by various causes such as either
abnormal gene deletion, transcription error or point mutation. There are two general forms of
thalassemia and these are α thalassemia and β thalassemia, where the latter is the most common type.
Beta thalassemia has six different types and these are β thalassemia silent, β thalassemia minor, β
thalassemia major, β thalassemia intermedia, hemoglobin Lepore, and Hereditary persistent Fetal
Hemoglobin (HPFH). Most of the types of β thalassemia manifest signs and symptoms of anemia except
for the silent carrier and minor types due to the normal levels of both Hgb and Hct. A normal adult
individual is expected to have normal concentration of Hgb A1 which is 97 - 98%, Hgb A2 which is 2-3%,

while Hgb F is <1 %; however, in Beta thalassemia the levels or concentration of these three types of
hemoglobin may vary depending on its type. See table below.
Genotype Clinical Findings Hgb A1 Hgb A2 Hgb F Hgb Lepore

β/β Normal Hematological Normal Normal Normal Absent
parameters
ΒSilent/β Asymptomatic Normal Normal Normal Absent
Normal Hematological
parameters
β Thalassemia
minor
Β+/β Slight Decrease Slight Increase Normal to Slight Absent

Asymptomatic to mild Increase
Β0/β hemolytic anemia with Slight Decrease Slight Increase Normal to Slight Absent
microcytic, hypochromic red Increase
δβ0/β cells. Slight Decrease Slight Increase Approximately 5- Absent
25%
δβLepore/β Slight Decrease Slight Increase Slight Increase 5 – 15%
β Thalassemia
intermedia
βsilent/βsilent Mild to moderate Hemolytic Slight Decrease Slight Increase Slight Increase Absent
Β+/βsilent or anemia with microcytic Slight Decrease Slight Increase Slight Increase Absent
β0/βsilent hypochromic red cells. May
not require blood transfusion
δβ0/δβ0 Absent Absent 100% Absent
but warranted in pregnancy or
δβ0/β0 infection.
Absent Normal Moderate Absent
Increase
β Thalassemia
major
β+/β+ Moderate Variable Moderate Absent
Severe Hemolytic anemia with decrease increase
β0/β0 microcytic, hypochromic red Absent Variable Moderate Absent
cells. Requires regular blood increase
β+/β0 transfusion. Severely Variable Moderate Absent
decrease increase
δβLepore/δβLepore Absent Absent 80% 20%
Table 8.8

Features of Types of β Thalassemia

Genotype Clinical manifestation and Blood picture Hgb/Hct Poikilocyte Other Name
Characteristic value present in
PBS exam
ΒSilent/β Asymptomatic Slight Normal Normal Silent Carrier
microcytosis State
β Thalassemia One beta gene is affected while Microcytic, Hgb 12 – 14 Target cell β-thalassemia
minor the other beta gene is normal. hypochromic g/dL for male Elliptocytes trait or
Most patient is asymptomatic with and 12.8 heterozygous
but some may show polychromatic g/dL for
hepatosplenomegaly red cells female
The bone marrow shows
moderate erythroid hyperplasia
β Thalassemia Untreated case may show Microcytic, (MCV 3-4 g/dL Target cells β thalassemia
major skeletal bone deformation and 50-70fL) Teardrop cells homozygous ,
expansion showing a “hair on hypochromic Elliptocytes Cooley’s
end” on skull x-rays. with high anemia
Typical facial features include polychromasia.
frontal bossing, prominent Severe
cheekbones and upper jaw. anisocytosis and
Neutropenia and poikilocytosis.
thrombocytopenia are present. Red cell inclusion
Jaundice due to high levels of is present such
bilirubin. In children it may show as Howell-jolly,
hepatosplenomegaly, basophilic
retardation, and decrease sexual stippling, and
maturity. pappenheimer
Iron overload resulted from bodies.
regular transfusion of red cell
component or decrease
Hepcidin level in the blood.
β Thalassemia Varying degree of anemia and Microcytic, 7 g/dL Target cell Dominantly
intermedia jaundice. Splenomegaly is hypochromic elliptocytes inherited β
present. Low neutrophil and with high thalassemia
platelet count. high iron polychromasia.
absorption. Heart, liver and
endocrine problems are
possible.
Table 8.9
The types of β thalassemia where the β gene cluster are affected are the Hereditary Persistent
Fetal Hemoglobin (HPFH), δβ0 – thalassemia and Hgb Lepore. HPFH and δβ0 – thalassemia both are
characterized by the deletion of the δβ gene on chromosome number 11 that leads to the increase
production of the Hgb F. The increase of Hgb F is due to the boost production of the γ globin gene to
compensate for the lack of normal β gene activity. The concentration of Hgb F for both homozygous
HPFH and δβ0 – thalassemia (δβ0/ δβ0) is 100%. However, Non-deletional type may occur on HPFH and

δβ0 – thalassemia. The non-deletional type is characterized by the increase γ gene stimulation that
overtakes the synthesis of the other non-α genes. Both the deletional and non-deletional HPFH are
asymptomatic with slightly normal levels of Hemoglobin, this is because even though the Hgb F has high
affinity with oxygen it can somehow still deliver oxygen to the tissue but not very effective as compared
to Hgb A1. The concentration of Hgb F in heterozygous deletional type HPFH is around 10-30% and
usually has normal MCV. For the heterozygous δβ0 – thalassemia the concentration of Hgb F varies
between 5 – 20%, and with normal levels of Hgb A2, decreased level of Hgb A1 and low MCV. The Hgb
Lepore thalassemia is characterized by the fusion of the δ and β globin genes which is known as Lepore
gene. The Lepore gene is dictated more by the δ gene than the β gene which resulted to decrease
production of the β globin chains, however in anti-Lepore gene, the β gene is more active than the δ
gene which resulted to normal β globin chain synthesis. Hgb Lepore may be inherited as homozygous
(δβLepore/δβLepore) or heterozygous form (δβLepore/β). The Homozygous form has no δ and β globin chain
production that leads to severe anemia and with 80% Hgb F, 20% Hgb Lepore and no Hgb A1 and A2,
while the heterozygous has decreased level of Hgb A1 and A2 and increased level of Hgb F and with
about 5 -15% of Hgb Lepore.
The α thalassemia is the decreased production of the alpha globin chain that affects the
concentration of all the Hgb types that contains the said chain, like Hgb A1, HgA2 and Hgb F. This may
result to over synthesis of the other non-α globin chain such as the β and γ globin chain that may
aggregate within the cytoplasm of the developing cell or mature red cell. This red cell with aggregates
of globin chain is subsequently removed by the macrophages of the bone marrow which therefore
cause the early destruction of the red cell. Those red cells that escaped form the bone marrow will
eventually removed by the spleen. There are four different type of α thalassemia and these are silent
carrier, α- thalassemia minor, Hgb H disease, and Hgb Bart Hydrops fetalis. The silent carrier has three
functional α gene, the α thalassemia minor has two functional α gene and two deleted α gene, Hgb H
disease has one normal α gene and three deleted α genes and produces Hgb H with four γ globin chain,
the Hgb Bart diseases has four deleted α genes and produces Hgb Bart with four β globin chain. The
genotypes and the characteristics of the α thalassemia is shown on the table below.
α Thalassemia Genotypes Clinical manifestation Hematological PBS exam Type of Hgb
types Parameters produced
Silent carrier -α/αα Asymptomatic Normal Shows normal size and Hgb A: normal
αCSα/αα shape of red cell with Hgb Constant
αTα/αα no other abnormality. Spring: < 1% (for
αCSα/αα)
Newborn:
Hgb Bart 1 -2 %
α thalassemia -α/αα Asymptomatic to mild Hgb falls Microcytic , Hgb A: slight
minor -α/-α hemolytic anemia. between 12 – hypochromic red cells decrease.
αCSααCSα Some may have 13g /dL with Target cells Hgb constant
jaundice and Spring: <6% (for
enlargement of spleen αCSααCSα)
and liver.
Newborn:
Hgb Bart: 5 -15%

Hgb H --/-α Mild to moderate Hgb value is 7 Microcytic, Hgb A: decrease

--/αCSα hemolytic anemia. -10 g/dL hypochromic red cell, Hgb Constant
spleen is enlarged and Retic count is with target cell and Spring: <1 % (for --
the bone marrow 5 – 10 % marked poikilocytosis /αCSα)
shows red cell and bizarre RBC
hyperplasia. shapes. nRBC is Newborn
present. Hgb Bart: 10-40%
Red cell with inclusions
characterized as “golf Adult:
balls” or “raspberries” Hgb H(β4): 1-40%
on BCB.
Hgb Bart Hydrops --/-- Severe anemia. Hgb value: 3-8 Microcytic, Hgb A1: absent
Fetalis syndrome Still birth or infants g/dL hypochromic red cells Newborn
died after delivery. Retic count: with target cells and Bart Hgb(γ4): 80-
Fetus or infants high marked poikilocytosis. 90%
shows, ascites, nRBC is present. Hgb Portland
cardiomegaly, Bizarre RBC shape is
hepatosplenomegaly present. Marked
and edema. Bone Polychromasia is
marrow shows present.
normoblastic
hyperplasia
Table 8.10
Thalassemia with Hgb variants

Thalassemia combined with abnormal Hgb variants is characterized by a genetic deletion of
either α or β gene mixed with a point mutation or amino acid substitution. Examples of these are
Hemoglobin S-thalassemia, Hgb C- thalassemia, and Hgb E- thalassemia. Generally, all of these types
have moderate decreased to absence of Hgb A1, and increased concentration of both Hgb A2 and F.
other Hgb Is present like Hgb S, Hgb C, and Hgb E, relative to the type of Hemoglobin abnormality.
Presence of Target cell and microcytosis are consistent with the said abnormal conditions.
Hemoglobinopathy
Hemoglobinopathies are collection of disease where there is a point mutation or amino acid
substitutions within the globin molecule. This is a qualitative hemoglobin defect that affects the stability
of the Hemoglobin molecule which causes it to precipitate within the red cell forming crystals or rigid
forms which in turn changes the normal appearance of the red cells. One of the common clinically
important hemoglobinopathies and well-studied is the Sickle cell anemia. Sickle cell anemia is
characterized by the defective beta globin chain where the glutamic acid is replaced by valine on the
6th position. This hemoglobin abnormality is called Hgb S, and there are three forms of Hgb S and these
are Homozygous (Hgb SS) or sickle cell anemia, heterozygous (Hgb AS) or sickle cell trait, and other
sickle cell disease (i.e. Hgb SC, Hgb SE, Hgb SD etc.). The presence of Hgb S causes the polymerization
of the globin molecule which is triggered by several factors such as low oxygen tension, dehydration

and acidosis. This polymerization elongates the hemoglobin molecule and causes a significant change
on RBC shape from discoid to elongated with pointed structure called “sickle cells” or drepanocytes.
These sickle cell formed may be trapped and clogged within the minute vasculature especially of the
kidney and spleen and cause occluded within it. This condition is called Vaso-occlusive crisis. The said
crisis event could affect various tissues and organs aside from kidney or spleen like bone, lungs, liver
eyes, and CNS.
Other complication of sickle cell disease includes bacterial infection that advances to sepsis
including S. aureus, S. pneumoniae and H. influenzae, cardiac anomalies, stunted growth, high risk
pregnancy and priapism on males. Special screening and confirmatory test are available to diagnose
sickle cell anemia and these are Sodium dithionite solubility test and Hgb electrophoresis, respectively.
The sickle cells formation affects the Psickle cell channels that allows the influx of Ca+ ion within the red
cells and the Gardos channel which allows the efflux of K+ and the influx of Cl- on the red cells. The in
and out of these ions on red cells allows the exit of water from the red cell which in turn dehydrating it
and further contributes to the polymerization of the Hgb S molecule. Deficiency of G-6PD enzyme is
common with sickle cell disorders that apparently contributes to the stress related hemolysis, but this
keeps the malarial like P. falciparum to infect red cells leading to its resistant to this parasitic infection.
The peripheral blood smear of sickle cell anemia shows normocytic, hypochromic red cells, increased
anisocytosis and poikilocytosis, increase reticulocyte count, positive for nRBC, presence of abnormal
red cell inclusions like pappenheimer bodies, Howell-Jolly bodies with few spherocytic red cells. The
presence of codocyte or Target cell and sickle cell is considered to be diagnostic for Sickle cell disorders.
Other lab result includes high RDW, mild shift to the left differential count result with neutrophilia, high
LAP score, and serum IgA is increased. Elevated bilirubin is possible in chronic cases that is reflected
with jaundice.

Other forms of Hemoglobinopathy
Abnormal Hemoglobin Defective structure

Hgb SC α2 β6Glu-Valβ6Glu-Lys
Hgb C α2β6Glu-Lys
Hgb D α2β121Glu-Gln
Hgb SD α2 β6Glu-Val β121Gln-Glu
Hgb E α2β26Glu-Lys
Hgb O-Arab α2 β121Glu-Lys
Hgb C- Harlem or Georgetown Two amino acid substitution
α2β6Glu-Val;73Asp-An
Hgb G-Philidelphia Alpha chain is affected
α268ASn-Lys β2
Hgb Gun Hill Deletion of five amino acid on
β chain 91-95
Hgb Constant Spring CS Elongation of the alpha chain
Additional of 31 amino acid
Table 8.11
Hgb CC and Hgb SC forms a unique appearance of Hgb polymerization forming red cell inclusion
that causes shape change in red cell. The Hgb CC forms a hexagonal blunt crystal while Hgb SC forms
rigid crystal with pointed end described as “Washington monument” crystal or sometimes with blunt
ends are observable.
Red cell Enzymopathy
Glucose-6-Phosphate Dehydrogenase deficiency

The G6PD enzyme is part of the pentose phosphate pathway that enables the production of
NADPH from the action of it in converting glucose-6-Phosphate to 6-phosphogluconate. The NADPH
form is utilized by the glutathione reductase to reduce the oxidized glutathione forming reduced
glutathione. The reduced glutathione is used by the glutathione reductase to convert the hydrogen
peroxide to water and oxygen. The hydrogen peroxide if not reduced will cause the formation of free
radicals within the red cell and oxidizes the sulfhydryl group of the hemoglobin leading to the instability
of its molecule and eventually precipitates and adhere to the red cell membrane. The precipitated Hgb
is also called as Heinz inclusion bodies. In G6PD deficiency this protective mechanism is impaired due
to the low to lack of NADPH synthesis. This is the most common enzymopathy on red cell which is
characterized by an X-linked inheritance of defective G6PD gene. The Heinz inclusion bodies form within
the red cell membrane is spontaneously removed by the macrophages of the spleen that subsequently
damage the red cell membrane leading to acute hemolytic anemia. The acute Hemolytic anemia is
associated with oxidative stress brought about by the administration of certain drugs or chemicals that
lead to the formation of peroxides such as hydrogen peroxide. Example of these drugs are Sulfonamide,

streptomycin, nitrofurantoin, Dilantin, dapsone and antimalarial drugs such as hydro chloroquine,
quinolone, etc. Favism is another form of acute hemolytic anemia caused by eating fava beans on
certain susceptible individuals who also lacks the said enzyme. Exposure to naphthalene mothballs also
induces the same effect. Infection may also cause the acute hemolytic anemia related to G6PD enzyme
deficiency due to the formation of hydrogen peroxides when phagocytes uses the respiratory burst in
killing ingested organism. Other clinical syndromes related to G6PD enzyme deficiency are neonatal
hyperbilirubinemia and chronic hereditary non-spherocytic hemolytic anemia (HNSHA). Patient
suffering with acute hemolytic episodes may show general symptoms of anemia, along with jaundice
and dark colored urine. The laboratory findings include abrupt decrease in Hgb and Hct, high
reticulocyte count or polychromasia. The PBS shows normocytic normochromic red cells, normal to
marked anisocytosis, poikilocytosis, spherocytosis and even schistocytes in severe cases and red cells
shows Heinz inclusion bodies after using supravital stains. Presence of bite cells is another red cell
abnormality that may be observable in the PBS exam which resulted from membrane eating made by
the macrophages to remove the Heinz bodies embedded on it. The Blood chemistry shows evidence of
hemolytic episodes such as decrease haptoglobin and hemopexin, increased LDH enzyme, presence of
free hemoglobin. Additional laboratory test like the DAT test shows negative result and urine samples
is positive with hemoglobin.
Pyruvate kinase Deficiency

The gene defective on this enzymopathy is the PKLR gene which is inherited as autosomal
recessive trait. The PKLR gene synthesized the formation of pyruvate kinase enzyme on red cell to
promote ATP production for energy use. Lack of ATP due to PK deficiency will cause depletion of ATP
production and increase in 2,3-DPG synthesis within the red cell. The low ATP production may cause
membrane instability and early red cell destruction in either extravascular space or intravascularly.
Patient with PK deficiency may show chronic hemolysis with general signs of anemia, jaundice,
splenomegaly, gallstone, bone marrow aplasia, and skin ulcerations sometimes may occur. PBS exam
shows burr cells, anisocytosis and poikilocytosis, in certain case Howell-Jolly bodies and pappenheimer
bodies and target cell is also present. The WBC and platelet count are normal to increase and the serum
haptoglobin is decrease and the urine is positive with urobilinogen.
Pyrimidine-5’-nucleotidase deficiency
The reticulocytes ribosomal RNA inclusion is removed by the action of pyrimidine-5’-
nucleotidase type 1 enzyme. This enzyme removes the phosphate from the pyrimidine-5’-
ribonucleotodase monophosphate forming ribonucleoside and inorganic phosphate which eventually
diffuse out the red cell as it matures. Lack of the said enzyme cause the accumulation of the precipitated
ribosomal RNA within the reticulocyte causing the formation of large coarse basophilic stippling. The
consequence of this is early red cell lysis which usually occurs in the spleen related to the pitting
function of this organ. Patient with this condition may experience mild to moderate anemia.
Reticulocytosis, jaundice and in some extent mental retardation may be seen on some patients.

Congenital Bone Marrow disorders

The etiology of bone marrow failure or aplastic anemia is caused by either genetic abnormality
or acquired. Acquired bone marrow failure is the most common cause of aplastic bone marrow caused
by exposure to toxic agents like benzene and lead. Certain drugs if given above its recommended
therapeutic dose may also affect the development of the marrow cells, example of this drugs is
chloramphenicol. Patient who are expose significantly on radioactive materials and hazardous
biochemicals are another possible cause of bone marrow failure. Infections to viral infection may also
alter the normal production of hematopoietic cell within the marrow. Genetic causes on the other
hand, includes diseases such as Fanconi anemia and Schwachman-Bodian syndrome. Fanconi anemia is
an X- linked chromosomal instability disorder with mutation and deletions and breaks of several genes
such as FANCA, FANCB, FANCC, and FANCD1. These abnormality affects the ability of all hematopoietic
cell to do mitotic division and maturation which lowers the production of all blood cell causing decrease
counts which is called pancytopenia. Patient with Fanconi syndrome shows physical abnormalities like
thumb malformation, microcephaly, mental retardation, scoliosis and hip dislocation. Brown
pigmentation is also observable on the surface of their skin. The Shwachman-Bodian-Diamond
syndrome is a genetic abnormality that triggers the increase apoptosis of the bone marrow cell that
leads to low cell counts. Patient with this condition may also exhibit abnormal pancreatic enzyme
secretion and a Bone marrow studies shows hypoplastic with low M:E ratio report. Diamond-Blackfan
is a pure red cell aplasia caused by mutation on nine different genes that is responsible for encoding
structural ribosomes. Diamond-Blackfan has low red cell count with normal white cell and platelet
count and with high Hgb F and adenosine deaminase. Patient with this condition shows physical
malformation of the thumb which is also similar with that of the Fanconi anemia. Stunted growth, poor
sexual organ development and mental retardation are among of the clinical manifestation it has.
Myelopthisic anemia is a condition where a space occupying lesion is present within the
marrow, like tumor cell and fibrous connective tissue, which replaces the normal hematopoietic cell.
This will cause decrease number of developing bone marrow progenitor cells resulting to low cell
counts. Anemia, bleeding and infection are the complications that occurs on this condition. The
peripheral blood smear may show immature white blood cell with nucleated red cells or also called as
“leukoerythroblastoid reaction”. Idiopathic myelofibrosis is a genetic disorder causing abnormal release
of the platelet derive growth factor (PDGF) from the abnormal platelets in the bone marrow. This PDGF
stimulates the fibroblast forming fibrous tissue within the red cell. The peripheral blood smear of
patient with myelofibrosis may show schistocytes and dacrocytes or tear drop cell resulted from the
trauma experienced by the red cell as they pass thru with the fibrous connective tissue within the
marrow.
Extrinsic Red cell Disorders

Hemolytic anemia due to external factors are divided into two groups and these are
immunologic and non-immunologic cause. Immunologic cause is due to the presence of antibody that
is directly binds with red cell antigens. The type of immunoglobulin usually associated with this are IgG
and IgM antibody. Under this condition includes autoimmune hemolytic anemia, Hemolytic disease of

the Fetus and Newborn (HDFN), and PCH. Autoimmune Hemoyltic anemia has three disorders under it
and these are Cold Hemagglutinin disease (CHAD), Warm Hemagglutinin Disease (WHAD) and Drug
induced Hemolytic anemia. In CHAD it is caused by the formation of autoantibodies known as anti-I and
anti-i. Autoanti-I production is associated with Mycoplasma pneumoniae infection while the
development of autoanti-i is attributed to EBV infection that cause infectious mononucleosis or IM.
These two autoantibodies are IgM antibody that causes clumping of red cell at cold temperature and
possibly may trigger the complement system in vivo which may result to hemolytic anemia. The WHAD
is caused by the development of IgG autoantibodies commonly reacting with the e Rh antigen of the
red cells. The IgG antibody mediated hemolysis usually occurs extravascular specifically within the
spleen. Certain Drugs like penicillin, certain antibiotics, Keflin, and alpha methyl dopa, are examples of
drugs that may trigger autoantibody production due to the drug metabolites produced after the body
metabolized the drugs. There are several mechanisms involved on how the autoantibody develop
against red cell depending on the drug administered and these are Hapten mechanism, red cell
membrane modification, and autoantibody development.
HDFN is a condition where the mother’s red blood cell antigen is not compatible with the fetal
red cells antigen. The common incompatibility associated with HDFN is the RH blood group system. On
this case, the mother usually is RH- while the developing fetus is Rh+. The pathophysiology of this
condition is that the mother’s immune system is triggered to produce anti-RH antibody after
sensitization from the fetal red cells. Due to the Nature of the IgG to cross the placenta the anti-RH
antibody that comes from the mother will go to the fetal circulation and destroys the red cell causing
lethal consequences. Fetus may suffer with complications such as anemia, kernicterus,
hepatosplenomegaly, and edema. Commonly fetus died if no intervention is done.
Paroxysmal Cold Hemoglobinuria (PCH) is a chronic hemolytic anemia which is caused by the
production of auto anti-P after the individual suffers from certain infection like syphilis and viruses. The
auto anti-P or also known as Donath-Landsteiner antibody, is an IgG antibody that binds with the red
cell when incubated at cold temperature and initiate red cell lysis if it is incubated at 37℃.
Non-Immunologic external red cell hemolysis is cause by mechanical damage on red cell without
the involvement of antibody or the immune cells. Example of the condition that may lead to early
destruction of red cell is microangiopathic hemolytic anemia, toxins, burns and prosthetic heart valve
transplant. The hallmark of this is seeing fragmented red cell on PBS exam or also called as
“schistocytes”. Other poikilocyte may also observed on this case such as teardrop cell or dacrocyte,
blister cells, and keratocytes. Microangiopathic hemolytic anemia is a collection of disorder that shares
almost the same etiology on haw the red cell is destroyed intravascularly and these are Disseminated
Intrvascular Coagulopathy (DIC), Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic Uremic
Syndrome (HUS), and HELLP syndrome. DIC is secondary to disease process that triggers the coagulation
of the blood within the vasculature that cause thrombus formation that blocks the blood flow on the
affected vessel. As the red cell passes through this fibrin mesh or clots or thrombus, the red cell
fragments due to trauma resulting to intravascular hemolysis. This event of hemolytic pattern is true
also with the other disorders mentioned earlier. The Laboratory test shows decreased Hgb, Hct, and
platelet count as well as low fibrinogen level. The Decrease platelet and fibrinogen is cause by
consumption of all components necessary to blood coagulation. The low fibrinogen level may cause the

PT and PTT test to be abnormal or prolonged. The PBS shows low platelet estimates and Schistocytes
with normocytic, normochromic blood picture.
Abnormal Red Blood cell shapes

Poikilocyte Appearance Synonyms Related disorders
Codocyte Tear drop, Mexican Hat Thalassemia,
cell, sombrero cells, Hemoglobinopathy, Obstructive
Leptocyte (thin) Liver diseases, Iron deficiency,
Abnormal Fat metabolism,
Chronic Alcoholism
Stomatocyte Mouth cell Rh null syndrome
H. stomatocytosis
Defective Na+-K+ ATPase
membrane enzymes
Burr cell Sea urchin cell, Crenated Uremia
red cells, Echinocyte Renal insufficiency
PK deficiency
Acanthocyte Spur cell Abetalipoproteinemia

PK deficiency
McLeod phenotype
Elliptocytes Rod shape cell, pencil H. elliptocytosis, Iron deficiency

form red cell anemia, thalassemia,
Ovalocyte Macro-ovalocyte Megaloblastic anemia, H.

ovalocytosis or SAO
Sickle cell Drepanocyte Sickle cell Disorders, Sickle cell

anemia
Anulocyte Punch out cells Severe hypochromic anemia

Dacrocyte Tear drop cells Myelofibrosis,

Macroangiopathic hemolytic
anemia
Schistocyte Schizocyte, helmet Macroangiopathic hemolytic

shaped cell, fragmented anemia, myelofibrosis
red cell
Degmacyte Bite cells G6PD deficiency
Table 8.12

LESSON MODULE
LESSON 9
LESSON TITLE: White Blood Cell Disorders

1. identify the Non-malignant from Malignant white blood cell disorders.
2. discuss the Non-malignant WBC disorder.
3. classify the different kinds of leukemia.
4. describe the characteristics and morphological features of each of leukemic cells.
5. discuss the principle of the special stain and its used in evaluating leukemia
6. discuss the characteristics of lymphoma.
7. discuss the different types of plasma cell neoplasms.
IX. Learning Guide Time Learning Resources

allotment
1. Identify the Non-malignant from Malignant white 6-hour Learning Content:

blood cell disorders. lecture - Lesson 1 module
1.1 Qualitative and Quantitative White cell page/s: 123-142.
disorder related to stress and genetic - Rodak’s Hematology
abnormalities. pages: 475 - 643.
1.2 Classification of Acute and Chronic Leukemia - Clinical Hematology
principles,
2. Discuss the Non-malignant WBC disorder.
procedures,
2.1 Qualitative and Quantitative White cell
correlation pages:
disorder related to stress and genetic 336 - 484.
abnormalities.
3. Classify the different kinds of leukemia.
3.1 Acute and Chronic under FAB classification
and WHO classification.
4. Describe the characteristics and morphological
features of each of leukemic cells.
5. Discuss the principle of the special stain and its
used in evaluating leukemia
5.1 Principles of special stains and its respective
use.
6. Discuss the characteristics of lymphoma.
6.1 Hodgkin and non-Hodgkin Lymphoma.
7. Discuss the different types of plasma cell
neoplasms.
1.1 Multiple myeloma and other plasma cell
neoplasm.
- Answer questions related to WBC disorder

12. For Submission: self- directed activity must be submitted Moodle

48 hours after the lecture on hematopoiesis. e-mail account
platform
Lesson IX: White Blood Cell Disorder
The diseases involving white blood cells are categorized into two general groups which are the
Non-malignant and Malignant WBC disorders. Non-Malignant is divided into either stress/reactive
disorder or Genetic disorder. The Stress or Reactive disorder is usually caused by infection, trauma,
injury, or inflammatory responses that could cause the quantitative or qualitative anomaly on the white
blood cells. The quantitative disorder is characterized by an absolute increase or decrease of a specific
white cell in response to an infection or other stressful event that a person’s body is experiencing, while
the qualitative disorder is pertaining to the morphological changes that occurs on white cells during
infections. The said abnormality is reversible especially after the stressful events mentioned above
subsides. The Genetic disorder of non-malignant type, causes an abnormal appearance of the white cell
which is nonreversible, in which, the lethality of this abnormality may vary from mild to severe case.
On the other hand, the malignant WBC disorders are myeloproliferative or lymphoproliferative
disorders that leads to the uncontrolled growth and proliferation of the hematopoietic cells which
causes severe illness to a person including infection, bleeding, and anemia. Example of this malignancy
are leukemia and lymphoma.
Non-Malignant WBC disorder
Quantitative
Whenever the human body is experiencing an infection the immediate response is to increase
the production of the WBC by the bone marrow to help eradicate the pathogenic organism that gain
entry to the body. The increase on white cell number is termed as leukophilia or leukocytosis. The body
responses to type of pathogens by releasing a specific white blood cell that is effective to kill it. Usually
acute bacterial infection will increase the number of Neutrophil in the blood but in chronic infection it
is dramatically replaced by monocyte or macrophages. Eosinophil number increases during parasitic
infection or larval invasion due to the major basic protein contained within its granules that can destroy
those organisms. Basophil may increase in certain immune responses and allergic reaction. In viral
infection, on the other hand, the lymphocyte number is increased to help the body attain the life time
immunity against a specific viral pathogen. However, there are several factors that may case the
decrease of the white blood cell number which is called as leukopenia. Example of conditions that may
decrease white cell number are bone marrow failure or aplastic bone marrow, certain viral infection,
etc. Each white blood cell may fall below with their respective reference number caused by different
factors or diseases. Physicians used to assess the absolute cell count is used to determine if there is a

significant increase or decrease of each white blood cell. Please refer to table 9.1(absolute count is
x109/L).
The summary that resulted to the absolute increase and decrease number of each white cell
type is listed in the table below.

Qualitative (Non-Genetic)
Morphological changes on neutrophil related to severe infection like septicemia or bacteremia

is the appearance of Toxic granulation and Dohle-Amato bodies on its cytoplasm. Toxic granules are
enlarged azurophilic or primary granules due to the overreaction of the white cell in eradicating the
invading bacteria. The presence of this granules is also found during leukemoid reaction. Dohle bodies
are blue to round inclusion bodies found on neutrophil’s cytoplasm during trauma, stress or infection.
This inclusion usually mistaken with May-Hegglin inclusion due to the similarity of their appearance,
however, the May-Hegglin inclusion usually measures > 5 μm in length and can be found on other white
cells’ cytoplasm like monocyte and lymphocytes. Decreased platelet count and the presence of
enlarged or giant platelets are another feature in May-Hegglin which differentiates it form dohle bodies.
Another cytoplasmic change that occur on Neutrophil caused by infection is the appearance of
vacuolation after the digestion of the engulfed organism via respiratory burst or oxidative mechanism,
though it may also observe whenever white cells are exposed too long with EDTA anticoagulant.
Cytoplasmic vacuolation may also observed in myelokathexis, a condition where there is a normal
production of granulocyte but defectively released to the circulation. Myelokathexis is part of the
“WHIM” syndrome where there is a hyperactivity of the CXR4 gene resulting to cellular imbalances,
poor regulatory and trafficking of the cell, low lymphocyte and neutrophil count and decrease antibody
synthesis. Conversely, nuclear change may occur on neutrophil in some nutrient deficiency anemia like
the presence of hypersegmented neutrophil in megaloblastic anemia.
Toxic Granules Dohle-Amato Bodies
Toxic vacuolation Toxic hypersegmentation
Figure 9.1
Qualitative (Genetic)
Certain genetic disorders can affect the appearance and function of the WBC which may lead to
serious illness like recurrent infection, poor blood clotting that may lead to bleeding tendencies and
low antibody development. Please refer to table 9.3 for the list of disorders that is associated with it.

Disorder Microscopic Genetic Characteristic

appearance abnormality
Pelger -Huet anomaly Lamin B receptor ▪ Impaired or halted nuclear
gene (Chr 1q41-43) lobulation resulting to bi-lobed
PMNs.
▪ associated with pince-nez
appearance (nuclei attached with
thin filament) that resembles a
mirror image appearance
▪ estimated number on differential
count showing > 80% of neutrophil
showing hyposegmentation
▪ Pseudo Pelger-Huet is established
if the count is <50% and occurs in
Myelogenous leukemia.
May-Hegglin anomaly MHY9 gene (Chr 22q ▪ Blue spindle inclusion body that
12-13) resembles Dohle bodies
▪ Characterized by low platelet
count, giant platelet and
dysfunctional platelet activity
▪ It affects the synthesis of myosin
heavy chain type II that cause
defect of megakaryocyte
development and platelet
fragmentation.
Alder-Reilly anomaly Inherited as ▪ Large and darkly stained
autosomal recessive metachromatic granules resulted
trait from partially digested
mucopolysaccharides that is
observable in the cytoplasm of
neutrophil, lymphocyte and
monocytes
▪ Resembles the toxic granules.
▪ Occurs on group of disorders called
mucopolysaccharidosis such as
Hurler and Hunter’s syndrome. It is
also seen in Maroteuax-Lamy
polydystrophic dwarfism and Tay-
Sach’s disease.
▪ WBC function is not affected.

Chediak-Higashi CHS1 LYST gene ▪ Giant lysosomal inclusion found on

syndrome all white cell.
▪ Patient exhibiting photophobia,
albinism, and immune deficiency.
▪ The genetic defect caused problem
in trafficking proteins within the
cells.
▪ Patient may bleed due to
dysfunctional platelet.
▪ Patient is prone to recurrent
pyogenic infection due to
abnormal WBC functions.
Table 9.3
Dysfunctional White blood cell disorders

Disorder Genetic abnormality Characteristic
Chronic Granulomatous Disease Mutated NADPH oxidase gene ▪ Defective phagocytic activity due
(CGD) to lack of oxidative or respiratory
burst.
▪ Patient may suffer with recurrent
bacterial and fungal infections.
▪ Affected individual forms
granuloma on tissue or organs
that causes obstruction on it.
Leukocyte adhesion disorders type 1 Mutated gene responsible for ▪ Person with this disorder is
encoding adhesion molecule β2- susceptible to recurrent bacterial
integrin and fungal infections.
▪ Other findings include skin
lesions, lymapadenopathy, low
neutrophil number and
splenomegaly.
Leukocyte adhesion disorders type 2 Mutated SCL35C1 gene responsible ▪ Person with this disorder is
for encoding adhesion molecule susceptible to recurrent bacterial
ligands and fungal infections.
▪ Individual with this condition may
have poor H antigen expression
on red cell, neurological problem
and stunted growth.
Leukocyte adhesion disorders type 3 Mutated Kindin-3 gene responsible for ▪ Person with this disorder is
activating β-integrin needed for susceptible to recurrent bacterial
neutrophil rolling on blood vessel for and fungal infections.
white cell attachment. ▪ Person with this condition is
susceptible to bleeding due to
defective platelet aggregation
function.
Myeloperoxidase enzyme deficiency Mutated MPO gene ▪ White blood cell can still kill
ingested organism but in a slow
rate.

▪ Individual doesn’t suffer

recurrent bacterial and fungal
infection due to other mechanism
of killing action used by the
phagocytes in killing it.
G6PD enzyme deficiency Mutated G6PD gene ▪ White cell has defective killing
action due to lack of available
reduced glutathione that
detoxifies the production of
peroxides within the phagocytes.
Table 9.4
Other disorder of Phagocytes
Lipidoses is one of the disorders associated with inability of the cell including the macrophages
to metabolize completely the lipids within it, resulting to the accumulation of a specific fat on its
cytoplasm depending on the enzyme deficiency. There are two known disorder associated with this
condition and these are Gaucher and Nieman-Pick disorder. In Gaucher disease the enzyme deficient is
called β-glucocerebrosidase enzyme that is responsible for metabolizing glucocerobroside, in which this
lipid accumulates within the cell resulted to abnormal appearance of the macrophage known as
Gaucher cell. The Gaucher cell is characterized with formation of striations in the cytoplasm which is
likened to a crumpled tissue appearance. Nieman-Pick disease on the other hand, is an autosomal
recessive abnormality where the deficient enzyme is the sphingomyelinase that resulted to the
accumulation of lipids such as sphingomyelin and cholesterol. The fats accumulated forms a bubble like
or foamy like appearance on the macrophage’s cytoplasm, that is why this cell is also called Foam cells.
Gaucher cell Nieman-Pick cell
Figure 9.2
Phagocytes with defective motility poorly response to any chemotactic signals which in turn
resulted to recurrent infection on those individual with this disorder. Normally phagocytes move in two
different ways and these are progressive or directional movement and random movement. In Lazy
Leukocyte syndrome both the directional and random movement are impaired causing poor
phagocytosis due to unresponsiveness to signals, while in Job’s syndrome the progressive movement is
the one impaired. The Job’s syndrome is also known as Hyper IgE syndrome that is why it is believed
that high levels of IgE in the blood affects the normal movement of the phagocyte.

Malignant WBC disorder
This is a group of white blood cell diseases characterized by development and uncontrolled
proliferation of the abnormal cells in the lymphoid tissues which is triggered by genetic abnormality.
The abnormal cells may invade tissues and organs like skin, bone marrow, lymph nodes, blood, etc. The
malignant white cell disorder can be classified into two types namely Leukemia and Lymphoma.
Leukemia is defined as the uncontrolled growth of neoplastic cell of the bone marrow that invades the
blood and sometimes other tissues, while Lymphoma is a clonal neoplastic proliferation of lymphoid
cell that mostly involving the lymphoid organs than the blood. One striking laboratory result that
commonly encountered on patient suffering from leukemia is the moderate to severely increase WBC
ct. However, the increased WBC ct. also occur on patient that has Leukemoid reaction, a condition
where the bone marrow is over stimulated to produce white blood cell in response to severe infection
like septicemia, therefore a differential diagnosis should be establish between the two disorders before
ruling out a disease. The use of the PBS exam and the LAP score are some of the laboratory tool that is
used to differentiate the two conditions. In Leukemia the PBS shows immature forms of the white blood
cells like the blast cells while in leukemoid reaction there is no blast form but immature cells may be
found like metamyelocytes and band cell, and Toxic granulation may be observed. The LAP score in
leukemia is decreased to no score but it is usually high in Leukemoid reaction reaching more than 100.
The comparison between Leukemia and Leukemoid reaction.
Comparison between Leukemia and Leukemoid reaction.
Classification of Leukemia
Leukemia can be classified into either Acute or Chronic type. The said classification depends on
the course of the disease process, how differentiated the hematopoietic cells were in the bone marrow
as well as in the blood, and the percentage number of the abnormal blast populating the marrow tissue.
In acute leukemia the course of illness is fast and usually patients die within three months from the
time it was detected, while in chronic leukemia the length of survival is from 1 year to 3 years. Although,
this characteristic is not always reliable because medical interventions are done like chemotherapy
which may prolong the life of the patient. In Acute leukemia the bone marrow is less differentiated
meaning more abnormal blast populate the bone marrow while in chronic type it is more differentiated

which means that the hematopoietic cells further mature resulting to seeing less blast. Lastly, using the
French-American-British (FAB) classification, in Acute leukemia the bone marrow or blood shows 30%
or more blast while in chronic leukemia it has around 10% or less blast. Although, in the W.H.O
classification, observing at least 20% abnormal blast is already considered as acute form of leukemia.
Acute or Chronic leukemia is further classified into myeloid or lymphoid type. In myeloid leukemias, the
involved abnormal cell is the progenitor cell derived from the myeloid stem cells, like the
metamegakaryocytes, monocytes, granulocytes and red cells, while in lymphoid leukemias the
abnormal progenitor cell is derived from the Lymphoid stem cells where the T-cell, B-cell, and NK cells
came from.
Myeloproliferative Disorder Lymphoproliferative disorder

▪ Acute myelogenous leukemia ▪ Acute lymphoblastic leukemia (FAB)
- AML M0 with minimal differentiation - L1
- AML M1 without maturation - L2
- AML M2 with maturation - L3
- AML M3 promyelocytic leukemia ▪ Acute lymphoblastic leukemia (WHO)
- AML M4 myelomonocytic leukemia - B lymphoblastic leukemia/Lymphoma
- AML M5 monocytic leukemia - T lymphoblastic leukemia/Lymphoma
- AML M6 erythroleukemia ▪ Chronic Lymphocytic leukemia
- AML M7 megakaryoblast leukemia - B-cell type
▪ Myeloproliferative neoplasm (MPN) • Hairy cell leukemia
- Chronic myelogenous leukemia • Burkit Lymphona
- Polycythemia vera - T-cell type
- Essential thrombocythemia • Adult T-cell leukemia
- Primary myelofibrosis • Mycoses fungodes
▪ Myelodysplastic syndrome (MDS)
- Refractory anemia
- Refractory anemia with ringed sideroblast
- Refractory anemia with excess blast
- MDS-U
- MDS-del(5q)
- RCMD
▪ MPN/MDS
- Chronic myelomonocytic
- Atypical chronic myeloid leukemia
- Juvenile myelomonocytic
- Unclassified MPN/MDS
Table 9.5

Myeloid Leukemia or Myeloproliferative Leukemia
Acute myeloid Leukemia (AML)
AML is the most common leukemia in children less than 1 y.o. but usually rare in children and
adolescents and most common type of leukemia in adults. Patients with this condition exhibit bone and
joint pain in 25% of cases, splenomegaly, hyperuricemia, hyperphosphatemia, hypokalemia, and
hypocalcemia. Splenomegaly and hyperuricemia are due to the increase cell turnover of because of
over production of it. There are eight types of AML which is listed on table 9.5. In the FAB classification,
the AML are categorized based on what predominating cell is seen on the bone marrow samples, the
morphology and type of blast present (refer to table 9.7), and the cytochemical reaction it exhibit on
special stains used such as peroxidase, Sudan black B, esterase, PAS stain, etc. On the other hand, The
WHO classify it by identifying the genetic abnormality for each type of leukemia, it also relies on the
use of flow cytometrical analysis to detect the CD markers present on the targeted abnormal cells to
determine the exact course of leukemia and type. The presence of Auer rod on the blast is one of the
unique features of AML. Auer rod is bar shaped blue cytoplasmic inclusion which is an abnormal
aggregation of azurophilic or primary granules that is peroxidase stain positive (see figure 9.3). Please
refer to table 9.6 for the characteristic of each type of AML using the FAB classification.
Table 9.6

Type of Blast
Table 9.7
The laboratory tests including bone marrow examination, peripheral blood examination and the
use cytochemical stains are very helpful in the differential diagnosis of leukemia, however modern
techniques are now available to confirm each type of it like, flow cytometry, immunocytochemistry,
genetic sequencing using PCR, etc. The principle and the clinical purpose of the special stain used is
discussed below.
Auer Rod
Figure 9.3
M1 M2 M3 M4 M5
M6 M7

CYTOCHEMISTRY is the microscopic study of chemical constituents within cells, useful for the
identification of malignant cell types and abnormal cell constituents.
I. Enzymatic Techniques
a. Peroxidases- catalyze the oxidation of substances by H2O2; used as marker for primary neutrophilic
granules; important for determining the presence or absence of a granulocytic component in acute leukemias;
routinely performed on all newly diagnosed leukemias; recommended for the demonstration of Auer Rods
b. Diamino Benzidine Method- uses 3,3 diaminobenzidine tetrahyrochloride; (+) dark brown granules;
samples should be stained within 2 weeks of collection because myeloperoxidase (MPO) deteriorates on
storage
c. Cyanide Resistant Peroxidase Stain- useful for the identification of an eosinophilic component of
leukemia or acute eosinophilic leukemia; due to the activity of eosinophilic peroxidase even in the presence of
cyanide; (+) brown
d. Esterase
i. Isoenzymes 1, 2, 7, 8, and 9- present in PMN; can be stained with chloroacetate as substrate;
specific esterase
ii. Isoenzymes 3, 4, 5, and 6- monocytes; either butyrate or acetate substrate; nonspecific
1. Naphthol AS-D Chloroacetate Esterase- marker for mature and immature PMN’s and
mast cells; less sensitive than peroxidase but more stable (useful even for paraffin blocked
specimens); (+) bright red cytoplasmic granules
2. Alpha Naphthyl Acetate Esterase (ANAE)- for isoenzymes 3, 4, 5, and 6; can be used
to demonstrate monocytes, megakaryocytes, plasma cells, and some lymphocytes; also, for
the T helper phenotype; (+) red brown for monocytes, focal or dotlike for lymphocytes
3. Alpha Naphthyl Butyrate Esterase- useful for mesoblasts, promonocytes, and
monocytes; for isoenzymes 2 and 4; (+) dark red precipitate in cytoplasm
4. Combination Alpha Naphthyl Butyrate- Chloroacetate Esterase Stains- distinction of
M4, M5a, and M5b from other ANLL, CMML, and DMPS; (+) dark red precipitate in monocytes
and histiocytes, blue granules in PMN and precursors
e. Phosphatases
i. Acid Phosphatase (ACP)- acid pH; 7 nonerythroid isoenzymes: 0, 1, 2, 3, 3b, 4, 5
ii. ALP- alkaline pH; present in PMN, osteoblasts, vascular endothelial cells, and sometimes
lymphocytes
iii. ACP- present in all hematopoietic cells; located in lysosomes; (+) discrete purplish to dark red
granules
iv. Tartrate Resistant Acid Phosphatase (TRAP)- for Hairy Cell Leukemia (Leukemic
Reticuloendotheliosis); isoenzyme band 5 on PAGE; (+) discrete purplish to dark red cytoplasmic
granules
v. Leukocyte Alkaline Phosphatase/ Neutrophil Alkaline Phosphatase (LAP/NAP)- only PMN have
various amounts; differentiates CGL from Leukemoid Reaction.
II. Nonenzymatic Techniques

a. Periodic Acid Schiff Stain (PAS)-reacts with glycogen and mucoproteins; (+) bright fuchsia pink
b. Sudan Black B- to demonstrate phospholipids and lipoproteins; (+) in granulocytes and monocytes and
macrophages but negative in lymphocytes
c. Toluidine Blue- binds acid mucopolysaccharide to form metachromatic complexes; useful for mast cell
disease, acute and chronic basophilic leukemia; (+) reddish clue metachromatic granules of basophils and mast
cells
d. Perl’s Prussian Blue Stain- for ferric iron; sideroblasts, siderocytes; (+) blue to blue green with red
nucleus.

Cytochemical staining reaction of AML and ALL
Cytochemical Staining reactions of AML types
AML with recurrent Genetic Abnormalities (2008 WHO classification)

AML with t(8;21)(q22;q22):RUNX1-RUNX1T1 ▪ Similar to FAB M2
▪ Myeloblast with dysplastic granular cytoplasm
▪ Positive with Eosinophilia, Pelger-huet, and
hypogranulation
AML with inv(16)(p13.1q22);CBFB-MYH11 ▪ Core Binding Protein
▪ PBS shows myeloblast, monoblast and
promyelocyte
APL with t(15;17)(q22;q12):PML-RARA ▪ Abnormal hypergranular promyelocyte
▪ May lead to DIC

▪ Presence of “Butterfly” or “coin-on-coin”

nuclues
AML with t(9;11)(p22;q23):MLLT3-MLL ▪ Presence of granules and vacuoles in blast
▪ Commonly occurs in children with gingival and
skin involvement
▪ DIC is possible
▪ Increase number of monoblast ans
promonocyte
AML with t(6;9)(p23;q34);DEK-NUP214
AML with inv(3)(q21;q26) or t(3:3)(q21;q26.2):RPN1-
EV11 ▪ Rare types of Leukemia
AML (Megakaryoblastic) with t(1:22)(p13;q13):RBM15-
MKL1
Myeloproliferative Neoplasm
The abnormal cell proliferates because of a mutated gene that constantly activates tyrosine
kinase release that activates growth factor-independent proliferation and survival of marrow
progenitor cells causing the expansion, excessive production and growth of red cells, granulocytes, and
platelets in the bone marrow and blood tissue. The Table 9.8 shows the different types of
myeloproliferative neoplasm with their respective genetic abnormality.
Table 9.8
In Chronic myelogenous leukemia around 20% of the case are seen on male than female. The
genetic abnormality of it is due to the translocation of BCR and ABL gene which is located at
chromosome number 9 and 22, respectively. The t(9:22) of BCR/ABL gene is called as the Philadelphia
chromosome. Around 90% of patient with CML is positive with Philadelphia chromosome and usually
shows a good prognosis. The bone marrow is hypercellular, with maturing granulocytic precursors
including elevated quantities of eosinophil and basophil as well as the megakaryocytes are increased
while the erythroid progenitors is normal to mildly decrease. The lipid loaded macrophages shows an
abundant wrinkled, green-blue cytoplasm known as the “sea-blue histiocytes”. The peripheral blood

smear exhibits a marked leukocytosis, that often surpasses 100,000 cells per μL. The differential count
shows several Blasts that usually makes up around < 10% of circulating cells which is composed of 5%
Promyelocyte and 1% Myeloblast. All stages of granulocytes are present such as neutrophils, band
forms, metamyelocytes, myelocytes, eosinophil and basophil and the platelets are usually increased.
In CML the LAP score is markedly decrease usually ranging between 0-13 (the normal LAP score is 20-
100).
Polycythemia Vera is a condition with an excessive proliferation of all myeloid cell lines in
marrow known as panmyelosis. The clinical features of this disorder include ruddy cyanosis
splenomegaly, thrombotic or hemorrhagic phenomenon, and gastrointestinal bleeding. Myelofibrosis
with myeloid metaplasia (MMM) is a chronic, progressive panmyelosis characterized by triads of
findings such as fibrosis of marrow tissue, splenomegaly, and leukoeryhtroblastotic anemia with
marked red cell abnormality, immature granulocytes, atypical platelets. Essential Thrombocythemia
(ET) is a condition where the platelet count reaches around 1,000 X 10 9/L with dysfunctional activity
and could cause blockage to the vessels. There are secondary cause of ET and these are splenectomy
and chronic infection.
Myelodysplastic syndrome
Myelodysplastic syndrome or MDS are collection of disorders caused by poorly formed or

dysfunctional blood cells or dyspoiesis in one or more cell lines called dyserythropoiesis,
dysmyelopoiesis or dysmegakaryopoiesis. Dyserythopoesis is characterized with a bone marrow cell
that has > 1 nucleus or abnormal nuclear shape, uneven cytoplasmic staining, and ringed sideroblast
while the blood shows oval macrocytes, hypochromic microcytes and dimorphic population. In
dysmyelopoiesis, it is characterized by the presence of nuclear-cytoplasmic asynchrony, uneven
cytoplasmic basophilic staining, abnormal granulation, absence of promyelocytic and myelocytic
granulation and with monocytopoiesis hyperplasia in the bone marrow, while the blood has
cytoplasmic basophilia of mature cells, abnormal granulation. And finally, in dysmegakaryopoiesis there
is a mononuclear megakaryocyte, micromegakaryocytes, and micromegakaryoblasts in the bone
marrow while the blood exhibits giant platelets with abnormal, fused, decreased or no granulation, and
presence of micro-megakaryocytes. Refer to table 9.9 for the list of different forms of MDS.

Table 9.9
Lymphoid Leukemia or Lymphoproliferative Leukemia
Acute Lymphoblastic Leukemia
Around 25% of childhood cancers and 75% of childhood leukemias is diagnosed as ALL.
Statistically, approximately 85% of the ALL are of B cell type, which typically manifests as most
childhood acute leukemias while the T cell ALL is common among adolescent males as thymic
lymphoma. In FAB classification ALL are classified as ALL L1, L-2 and L-3 which is dependent on the size
and morphology of the lymphoblast considering the cytoplasmic appearance and nuclear chromatin
abnormality. L1 type is common among children and shows good prognosis among patients, L2 is
usually mistaken as AML, while the L3 is considered as the Burkitt’s type with translocation of genes
cMYC and Ig loci gene located at chromosome number 8 and 14 and characterized with the worst
prognosis among the ALL types. In WHO, ALL is classified as B-ALL and T-ALL, with subclassifications for
B-ALL, as what is shown in table 9.10.

Table 9.10
Immunophenotyping of the CD markers and genetic analysis are the most reliable indicators of
cell origin for ALL types. See table 9.11.
Table 9.11

The prognosis of ALL is better in children than teens and infants, poor outcome usually
associated with the presence of myeloid surface marker CD66c, and worse outcome if the blood
lymphoblastic count reaches 20 – 30 x10 9/L with hepatosplenomegaly and lymphadenopathy.
ALL-L1 ALL-L2 ALL-L3
Chronic Lymphocytic Leukemia (Mature Lymphoid Neoplasm)
CLL is characterized by abnormal proliferation of dysfunctional mature lymphocyte which is

classified into either mature B-cell type or mature T cell and NK cell type. The B cell Type of CLL includes
different types in which the Hairy cell leukemia and Burkitt Lymphoma is the most common while for
T-cell CLL comprises of Adult T-cell Leukemia, Mycoses Fungoides and Sezary syndrome. Hodgkin and
Non-Hodgkin Lymphoma is also including under this category. In mature B-cell CLL there is > 5 x109/L
of monoclonal small lymphoid cells in the blood associated with indolent lymphoproliferative disorder
of the elderly (65yrs old). One of this B-cell CLL is the Hairy cell leukemia which is characterized with
small B-lymphocytes with abundant hair like projections that are found predominantly in BM and
spleen, and the cell affected has no specific chromosomal abnormality but positive with CD19,20,22
and TRAP stain. Another B-Cell CLL is the Burkitt’s type lymphoma which is characterized with medium
sized lymphoma cells with basophilic and vacuolation cytoplasm, round nuclei containing finely
clumped chromatin and small nucleoli and positive with CD19,20,10 and BCL-6 markers. The gene
affected with it is the t (8:14) which involves the cMYC and Ig loci that is commonly implicated with EBV
infection. For T-cell CLL one of the most common is the cutaneous lymphoma with small to medium
sized lymphoma cells with cerebriform nuclei that Widespread skin involvement causing Pautrier
micro-abscess, and Sezary syndrome including lymphadenopathy and the presence of circulating
lymphoma known as Sezary cells, which is positive with CD2,3,4,5 but negative for CD7 marker. Adult
T-cell Leukemia is associated with HTLV-1 virus infection that can be transmitted via blood transfusion
containing infected Lymphocytes with the said virus. On the other hand, a combined T-cell and B-cell
CLL is the prolymphocytic leukemia which is a rare disease of the elderly that contains both B-cell and
T-cell lymphoproliferation involving the lymph nodes. The WBC ct. may reach > 100,000/μL, and the B-
cell prolymphocyte is positive with CD19,20,22 and FMC7, while the T-cell prolymphocyte CD3,2, and 5
markers.

Lymphoma
There are several types of Lymphoma and these are Follicular lymphoma, Diffuse large B-cell
lymphoma, Mantle zone lymphoma, Burkitt lymphoma and Hodgkin lymphoma. Hodgkin Lymphoma
arises in a single node or chain of nodes and spreads first to anatomical contiguous lymphoid tissue
which is more often contained or restricted to single or solitary axial group of nodes such as cervical,
mediastinal, and para-aortic. Abnormal cells may spread from the nodes then to spleen and eventually
may spread to other lymphoid organs such as liver and bone marrow. The presence of the Reed-
Sternberg cell is the diagnostic cell for this type of lymphoma. The Reed-Sternberg cell is characterized
by binucleated cell with enlarged nucleoli that resembles an “owl’s eye appearance”. There are five
subtypes of Hodgkin Lymphoma under the WHO classification as what is shown on table 9.12.
Reed Sternberg cell
Table 9.12
Plasma Cell Neoplasm

Plasma cell neoplasm is categorized as terminally differentiated B-cell neoplastic cells which
may be localized or disseminated involving the BM and the bone. The affected cell has diverse
rearrangements of genes involving the IgH;13q deletions resulting to abnormal release or accumulation
of parts of the immunoglobulin molecules within the cell. There are several types under this leukemia
and these are Plasma cell myeloma or also known as multiple myeloma, Plasma cell leukemia,
Monoclonal gammopthy, Solitary plasmacytoma and Extraosseous plasmacytoma. In Multiple
myeloma, statistic shows that it usually seen on Elderly patient commonly around 70 years old. It is
characterized with overpopulation of plasma cell within the marrow tissue known as bone marrow
plasmacytosis. Patients with multiple myeloma may exhibit a collection of disorder known as CRAB
syndrome which includes Hypercalcemia, renal problems, anemia and bone lesions. The abnormal
plasma cell cannot fully manufacture a complete immunoglobulin but releases the light chains such as
the kappa κ and lambda λ chains (i.e. monoclonal proteins), which resulted to
hypogammaglobulinemia. This monoclonal protein is not only present in serum but also in urine
samples. The affected plasma cell shows abnormal cytoplasmic inclusion known as the Russel bodies
which is characterized with bubble like or Mott like inclusion bodies. Urine samples with this disorder

are positive with a unique protein known as Bence-Jones protein. On the other hand, Plasma cell
leukemia is a condition where there is a blood involvement of > 2.0 X 109/L or 20% circulating plasma
cells, while monoclonal gammopathy of undetermined significance has BM plasmacytosis of <10%
plasma cells and the production of <30 g/L of monoclonal gammopathy with no lytic bone lesions
contrary to multiple myeloma. The Solitary plasmacytoma there is a solitary bone mass of plasma cell
while the Extraosseous plasmacytoma involves the extraosseous (outside of the bone) mass of plasma
cell.
Bone Marrow Plasmacytoid in Russell Bodies/Mott/Morula cell

Multiple myeloma

LESSON MODULE
LESSON X
LESSON TITLE: Automation

1. enumerate the different automated cell analyzer available.
2. discuss the different principles used in hematological cell analyzers
3. identify the sources of errors affecting cell analyzer results.
4. apply the proper collection and handling of specimen used for automated cell analyzer.
X. Learning Guide Time Learning Resources

allotment
1. Enumerate the different automated cell analyzer available. 6-hour Learning Content:
1.1 Principles applies for Sysmex, Coulter, Abott lecture - Lesson 1 module
and Siemens cell counting machines. page/s: 144-149.
2. Discuss the different principles used in hematological cell - Rodak’s Hematology
analyzers pages: 208-234.
2.1 Coulter, Radiofrequency and Light Scatter. - Clinical Hematology
3. Identify the sources of errors affecting cell analyzer results principles,
3.1 Factors that affect cell counting analyzers. procedures,
4. Apply the proper collection and handling of specimen used correlation pages:
for automated cell analyzer. 486-530.
4.1 Proper handling of specimen for automated
hematology
- Answer questions related to Automation
6. For Submission: self- directed activity must be submitted Moodle
48 hours after the lecture on hematopoiesis. e-mail account
platform
Lesson X: Automated Hematology

There are three fundamental principles used by the different cell counting machine and these
are Coulter principle, Radiofrequency, and Light scattering technique. The Coulter Principle or also
known as the electrical impedance or electrical resistance assess the number of the cells by determining
its volume without considering the character of it, while the radiofrequency and the light scatter
evaluate the cell’s size as well as its cytoplasmic and nuclear characteristics. There are a lot of factors
that affects the result of the cell counting analyzers that a medical technologist should consider, and
one of those is the proper handling of the EDTA blood specimen. EDTA blood samples that will be run
on cell-counting analyzers should be processed within 2 to 3 hours from the time of collection. Using a

more than 4-hour old specimen will cause the swelling of the platelets which resulted to increase Mean
Platelet volume (MPV) and low platelet count report. Old EDTA sample also cause the development of
cytoplasmic vacuolation, nuclear fragmentation and washing out of the granules of the white cell that
resulted to variable differential count report. Running a 6-hour old or more EDTA sample on a cell
counting analyzers, will increase the Hematocrit and Mean Cell Volume (MCV) while decreases the
Mean Cell Hemoglobin concentration (MCHC) result, this is due to the swelling effect on red cells caused
by prolonged contact with EDTA anticoagulant. Other factors that affects the cell-counting analyzers’
report on hematological parameters are listed on table 10.4.
Coulter Principle
The Coulter machine, developed by Wallace Coulter during early 1940’s, determines cell number
by using electrical current, and the change in electrical voltage or pulses is proportional to the cell
volume of the cell. Cells are considered to be poor conductor of electricity which cause resistance to
the flow of electricity within a conducting fluid used. The cells volume is determined in a specific part
of the machine known as the Aperture. The aperture is composed of different parts and these are
external glass that hold the conducting fluid (isotonic fluid), an internal U-shaped glass with a bore
called aperture hole from where the cell will enter, and two electrodes (negative and positive).
As the cell enters the aperture hole the electric current flowing within the conducting fluid will
be cut-off causing the released of voltage pulses that is determined by the sensing zone within the
aperture. The voltage pulses are relative to the size or volume of the cell. The machine is calibrated
which in turn uses preferred volume for each cell type. The volume range calibrated for platelet is 2 –
20 fL, for red cells it is 36 – 360 fL, for the lymphocyte it is 35-90fL, for monocyte it is 90-160 fL and for
the granulocyte it is from 160 to 450 fL. The size or volume determination will be plotted to a graph
with y-axis that represent the cell number or concentration and an x-axis that represents the volume
or size of the detected cell. See Figure 10.1.

Figure 10.1
Specimens with abnormal cells or components are usually detected by the modern cell counting
analyzers which notify the medical technologists or hematologist by using the flagging system. For the
White blood cell histogram, there are five different flags registered and this are, Flag 1 that represents
between red cells and lymphocytes, Flag 2 is between lymphocyte and monocytes, Flag 3 is between
the monocyte and neutrophil zone, Flag 4 is for granulocytes and Flag 5 is for undetermined
abnormality. The list of the cause of these red flags are listed on table 10.1. The obligation of the
medical technologist is to investigate and confirm the said cause of the flags by examining the patient’s
blood smear and look for any abnormal cells if present before releasing the result.
Table 10.1
For the Red cell histogram normally the graph or wave produced is a bell curve and with its peak
should fall between 80-100 fL as what is shown in figure 10.1. If the Peak of the red cell curve skew to
the right it means that the volume of red cell increases or macrocytosis is present on the sample, and
this are seen in megaloblastic anemia and megaloblastoid reactions. The skewing to the right is relative
to the increase in MCV result. On the other hand, if the peak skew to the left it signifies that the red cell
volume is decreased or microcytic red cells are present in the patient’s sample. This is possible in cases
of microcytic anemias such as IDA, thalassemia, Sideroblastic anemia and Thalassemia (see figure 10.2).
It is also very important that the red cell size should be near the 80 -100 fL mark which makes the bell
curve narrow, but if the wave is broader than normal then it signifies that there is a significant variation

of the red cell size. The extent or degree of size variation is measured by using the red cell distribution
width or RDW. The RDW reference value should be around 10 – 15%, meaning that if there is variation
of red cell size in a given specimen it should only fall on that percentage or number. Commonly in
certain anemia the RDW is high due to the different sizes of the red cell as what is seen in thalassemia
or IDA. Please refer to table 10.3 for the list of diseases that gives high and normal RDW relative to the
MCV result.
Figure 10.2
Table 10.3
For the platelet histogram, normally it shows that the average volume of platelets is
approximately 7 fL and the bell curve should touch the base of the x-axis. However, there are factors
where the platelet curve doesn’t touch the x-axis and creates a gap in between. These factors include
presence of enlarged or giant platelets, aggregated platelets or platelet clumping, micro-red cells or
micro spherocytes, and using old EDTA samples. The technologist should investigate further if he
encounters such problem to make a reliable reporting of results. (see figure 10.3)

Figure 10.3
The cells that enter in the aperture should have a constant flow of cell known as laminar flow.
This is to prevent the entry of cells simultaneously in the aperture hole which causes error on cell
counting which is also known as passage loss or coincidence error. To prevent this, engineers employed
the principle of hydrodynamic focusing which uses a fluid sheath that forced the cells to form a single
line. Coincidence error cause a positive error that would falsely increase the size or volume of the cell
and decreases the blood counts.
Radiofrequency
Machines using the principle of radiofrequency uses two different parameters to determine the
character of the cell as well as the size of it. Most of the Sysmex machines incorporate this principle as
way to determine the cells. It uses the Low- voltage DC impedance in conjunction with RF resistance
(Conductivity). The DC signal measures the size of the cell while the RF signal determines the
cytoplasmic character of the cells.
Light Scatter
The light scattering technique determine the cell using allowing the cell to pass through a quartz
flow glass with laser focused on it and thus create a scattered light in various angle. The low angle

scattered light, ranging between 0 to 5-degree angle, measure the size of the cell, while the high angle
scattered light, which is between 10 to 90 degree, determines the cytoplasmic content of the cell.
(Figure 10.6). The use of this mechanism allows the machine to differentiate the white cells into five
different types which are called 5-part differential machines. Cytochemical stains are used to enhance
the cytoplasmic content of the white cells and reticulocyte remnant for correct detection of the cell.
Example of the stain used for granulocyte like neutrophil and basophil is the peroxidase stain, cyanide
resistant peroxidase stain for eosinophilic granules, and o-toluidine blue for basophilic granules. The
analyzed blood cell is plotted in a scatterplot form that can be viewed on the computer monitor as
shown in figure 10.7.
Figure 10.6
Figure 10.7

Factors Affecting Cell Analyzers
Table 10.4
Principles used by various cell counting analyzers

References:
1. Bennett S., C. Lehman and G. Rodgers. Laboratory Hemostasis: A Practical Guide for Pathologists. USA: Springer,
2007.
2. Brown, Barbara. Hematology: Principles and Procedures 6th ed. Philapdelphia: Lea and Febiger, 1993
3. Colman R. Hemostasis and Thrombosis: Basic Principles and Clinical Practice 5 th ed. USA: Lippincott Williams and
Wilkins, 2004.
4. Greer J., J. Foerster and J. Lukens. Wintrobe’s Clinical Hematology 11th ed. USA: Lippincott Williams and Wilkins,
2003.
5. Harmening D. Clinical Hematology and Fundamentals of Hemostasis 5 th ed. USA: F.A. Davis Co., 2008.
6. Hoffman R. et al. Hematology: Basic principles and Practice 4th ed. USA: Churchill Livingstone, 2004.
7. McClatchey, Kenneth. Clinical Laboratory Medicine 2nd ed. USA: Lippinott Williams and Wilkins, 2002.
8. Mcpherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods
21st ed. Philadelphia, Elsevier Inc., 2007.
9. Reddi, V., M. Marques and G. Fristma. Quick Guide to Hematology Testing. USA: AACC Press, 2007.
10. Rodak B., G. Fritsma and K. Doiq. Hematology: Clinical Principles and Applications 5th ed. Philadelphia: W.B.
Saunders, 2007.
11. Schmaier A. and L. Petruzelli. Hematology for the Medical Student. USA: Lippincott Williams and Wilkins, 2003.
12. Scott M., A. Gronowski and C. Eby. Tietz’s Applied Laboratory Medicine 2nd ed. USA: Wiley-Liss, 2007.
13. Steininger, Cheryl at al. Clinical Hematology: Principles, Procedures and Correlations. Philadelphia: J.B. Lippincott,
1992.
14. Turgeon, Mary Louise. Clinical Hematology: Theory and Procedures 4 th ed. USA: Lippincott Williams and Wilkins,
2004.
Electronic References:
1. http://themedicalbiochemistrypage.org/
2. http://www.bloodline.net/
3. http://www.emedicine.com/med/topic132.htm
4. http://www.lib.uiowa.edu/hardin/md/hem.html

Laboratory manual
Table of Contents
FAR EASTERNUNIVERSITY-NICANOR REYESMEDICALFOUNDATION:Philosophy, Vision,
Mission, and Goals ......................................................................157
Exercise 1: Blood Collection Instruments ...........................................159
Exercise 2: Capillary Puncture ........................................................161
Exercise 3a: VENIPUNCTURE (Syringe Method) .....................................163
Exercise 3b: VENIPUNCTURE (Vacutainer Method) .................................165
Practical 1: Capillary puncture .......................................................169
Practical 2: Venipuncture (Syringe Method) ...... Error! Bookmark not defined.
Practical 3: Venipuncture (Vacutainer method) ...................................173
Exercise 4: Red Cell Count (Hemocytometer) ......................................176
Practical 4- Red Cell Counting ........................................................179
Exercise 5a: Hematocrit (Macro-Method) ...........................................180
Exercise 5b: Hematocrit (Micro Method) ............................................181
Excersice 6a: Hemoglobinometry (Acid Hematin Method) ........................184
Exercise 6b: Hemoglobinometry (Cyanmethomoglobin Method) .................185
Exercise 7: Red Cell Indices (MCV, MCH, MCHC) ...................................188
Exercise 8a: Erythrocyte Sedimentation Rate (Wintrobe Method) ..............191
Exercise 8b: Erythrocyte Sedimentation Rate (Westergren Method)............192
Exercise 9: Osmotic Fragility Test (Sanford’s Method) ............................194
Exercise 10: Reticulocyte Count (New Methylene Blue Method) ................198
Exercise 11a: Blood Smear Preparation (Wedge Film) ............................201
Exercise 11b: Blood Smear Preparation (Cover Glass Method)Error! Bookmark not
defined.
Exercise 12: Staining of Blood Film .................................................203
Practical 5: Blood Smear Preparation (Wedge Method) ...........................204
Exercise 13: Stained Red Cells .......................................................205
Exercise 14: White Cell Count ........................................................207
Practical 6: WBC Counting ............................................................210
Exercise 15: Normal White Blood Cells (Stained) ..................................211
Exercise 16: Differential Count .......................................................212
Exercise 17: Immature White Blood Cells (Peripheral Blood Film) ..............213
Exercise 18: Leukemia .................................................................214

Exercise 19: White Cells With Morphologic Variation .............................215

Exercise 20: Buffy Coat Preparation .................................................217
Exercise 21a: Platelet Count – Direct (Rees And Ecker Method) .................218
Exercise 21b: Platelet Count (Unopette System) ..................................219
Practical 7 – Platelet Count – Direct .................................................222
Exercise 21c: Platelet Count – Indirect ( Fonio’s Method) ........................223
Exercise 22: Examination Of Peripheral Blood SmearError! Bookmark not defined.
Exercise 23: Acid Elution Test For Hgb F (Kleihauer-Betke ) .....................229
Exercise 24: Acidified Serum Test (Ham’s Test) ...................................231
Exercise 25: Sugar Water Test ........................................................234
Laboratory Activity Evaluation Sheets ………………………………………………………….. 239

FAR EASTERN UNIVERSITY-NICANOR REYES MEDICAL FOUNDATION
PHILOSOPHY
Excellence inmedical and paramedical education, quality health care services, and
relevant research.
VISION
FEU-NRMF envisions itself to be a world-class academic and training institution providing

excellent health care services.
MISSION
FEU-NRMF commits to develop competent and compassionate professionals adhering to the

highest level of global standards in healthcare, education and research.
GOALS
1. Attain local & international recognition as Center of Excellence in health care, education and research;
2. Participate actively in health advocacy and community services;
3. Provide opportunities to meet the needs of socio-economically disadvantaged sectors of society;
4. Provide accessible and comprehensive health care services;
5. Develop critical thinkers, leaders and life –long learners among students, faculty and staff;
6. Promote interprofessional collaboration;
7. Integrate modern technology in education, health service and research;
8. Manage efficiently and ethically the institution’s human, financial, physical and technological
resources;
9. Ensure continuous improvement through quality assurance activities;
10. Instill loyalty to the Foundation.
VALUES
Fidelity – devotion to duty, loyalty to profession and to the Foundation

Excellence – highest standards in Education, Health care and Research
Universality – to regard everyone with equity and respect
Nationalism – love of country
Responsibility –social and professional accountability
Morally Upright – adheres to moral and ethical principles
Family-oriented–cognizant of the role of the family in education, health care and research

Course Intended Learning Outcome for Hematology 1
Course Title : HEMATOLOGY 1
Course Description: This course deals with the study of blood as a tissue and the
pathophysiology of the cellular elements of the blood.
COURSE CREDIT: 4 UNITS

CONTACT HOURS: 3 HOURS LECTURE PER WEEK,
3 HOURS LABORATORY PER WEEK
PLACEMENT : THIRD YEAR, FIRST SEMESTER

PRE- REQUISITES: BIOCHEMISTRY AND HUMAN ANATOMY AND
PHYSIOLOGY
Course Intended Learning Outcomes (CILOs)

•At the end of this course, the student is able to:
11. explain the facts and principles of hematological determinations.
12. differentiate microscopically the normal and abnormal blood cells.
13. correlate hematological tests to pathologic conditions.
14. perform hematological tests with precision, accuracy and reliability (QUALITY
ASSURANCE).
15. assume responsibility in handling blood specimens, including examination and
interpretation of test results.
16. manifest the following values and skills: independence, integrity, honesty, critical
thinking, empathy and value for life.

Exercise 1: Blood Collection Instruments
Objectives:
At the end of the laboratory exercise the student shall:
1. identify the instruments used for blood collection
2. discuss the appropriate used of the different blood collection instruments
3. enumerate the different anticoagulant used for blood collection
4. discuss the appropriate use of each anticoagulant used for blood collection
Instruction: Illustrate, label the parts and state the uses of the following blood collection instruments.
A. Lancets B. Capillets
___________________________ ____________________________
___________________________ ____________________________
___________________________ ____________________________
Instruction: Illustrate, label the parts and state the uses of the following blood collection instruments.
C. Syringe
______________________________________________________________________
__
_________________________________________________________________

D. Vacutainer set
________________________________________________________________________
__________________________________________________________________
E. Vacutainer tubes
__________________________________________________________________
__________________________________________________________________

Exercise 2: Capillary Puncture
Objectives:
1. identify the appropriate materials used for capillary blood collection
2. locate the ideal sites used for capillary blood collection
3. perform the capillary blood collection properly
4. discuss the appropriate technique for handling capillary blood specimens
5. determine the technical factors affecting capillary blood collection procedure
Materials:
Ethyl Alcohol 70 %
Cotton Balls
Blood Lancet
Capillets
Procedure:
1. Disinfect the ventral side of the chosen finger using a cotton ball wet with 70
% ethyl alcohol. Allow spontaneous drying.
2. Using one hand, apply a small amount of pressure at the base of the subject’s
finger. The other free hand is for handling the blood lancet.
3. Make a standard wound puncture with one swift motion with the lancet blade
to be in line with the finger crease.
4. Allow a small drop of blood to form on the puncture site and using a dry
cotton ball, lightly wipe it off.
5. Allow another drop of blood to form and collect it into a capillet, without
allowing the capillet tip to touch the skin.
6. After collecting the desired amount of blood, place a dry cotton ball on the
punctured site and make the subject’s thumb hold the cotton ball in place not less
than 10 minutes.
Pointers:
• The finger for the puncture should not be callused, free of any dermatosis, or
any condition that will lessen the depth of wound.
• Choose the finger of the less used hand for the puncture.
• It is less painful to puncture the ventral part of the finger that right at the tip
• Warm clothed are sometimes placed on the intended puncture site to allow
good circulation of blood on the area.
• Too much pressure applied on the finger will result to hemoconcentration,
which will in turn results to inaccurate examination values.
• The first drop of blood that forms immediately after the puncture is likely to

• be mixed with tissue juices, which will alter results of blood examination.
• Skin puncture may be done on sites other than finger such as earlobe and, for
babies, the ball of the heel or big toe.

Exercise 3a: VENIPUNCTURE (Syringe Method)
Objectives:
1. identify the parts of syringe used for venipuncture
2. locate the ideal site for a venipuncture collection procedure
3. perform properly the venipuncture procedure using syringe method
4. list technical factors that affect blood collection procedure using syringe method
Materials:
Disposable syringe with 21gauge needle
Sterile cotton balls
70% ethyl alcohol
Tourniquet
Test tubes
Procedure:
1. Assemble equipment. Check needle attachment to syringe, such that it can
easily be removed but not to lose to allow air to enter while plunger is pulled
during collection.
2. Snugly apply the tourniquet 2-3 inches above the antecubital area.
3. Choose the vein through palpation using the index finger.
4. Loosen tourniquet and disinfect site with a cotton ball wet with 70% ethyl
alcohol, following a circular motion from in to outside of the chosen site.
Allow skin to dry spontaneously.
5. Re-apply tourniquet. Do not touch site of puncture anymore.
6. Using the thumb of one hand, tense the skin just below the intended puncture
site to fix the vein.
7. Hold syringe with the other hand, index finger of which is placed on the
barrel’s end near the needle while the thumb and other fingers firmly hold the
syringe’s barrel.
8. With the needle’s bevel directed upward, insert the needle into the skin near
the chosen vein. When whole bevel is already under the skin, aim to insert the
needle into the lumen of the vein.
9. Check entry of blood in the needle’s hub. If none appears on the hub, lightly
pull the plunger.
10. As soon as the blood appears on the hub, loosen the tourniquet and aspirate
the desired amount of blood without causing any to-and-fro movement of the
syringe.
11. Place a cotton ball directly on top of the needle’s entry point and gently press
the cotton as the needle is withdrawn. Press the cotton ball firmly after the
needle is fully drawn out and ask patient to raise the arm while applying

pressure on the site for not less than 5 minutes.

12. Remove the needle by putting on the cover followed with a gentle twist and
dispose it in sharp receptacle.
13. Carefully transfer the blood sample into a test tube by allowing it to flow onto
the inner side of the tube.

Exercise 3b: VENIPUNCTURE (Vacutainer Method)
Objectives:
1. identify the material used for a venipuncture procedure using vacutainer method
2. identify the different vacutainer tubes used for multiple blood collection
3. locate the ideal site for a venipuncture collection procedure
4. perform properly the venipuncture procedure using syringe method
5. list technical factors that affect blood collection procedure using vacutainer method
Materials:
Vacutainer and collecting tube
Sterile cotton balls
70% ethyl alcohol
Tourniquet
Procedure:
1. Attach the needle into the adapter. Do not remove needle’s cover until puncture is
done.
2. Choose the vein as stated in steps 2-5 in the syringe method.
3. Place collection tube into the adapter but do not puncture yet the stopper. Carefully
hold the vacutainer with one hand with palm supporting the collection tube in the
adapter.
4. Fix the vein by tensing the skin below the intended puncture site.
5. With precise motion, insert the needle into the skin first then into the vein, making
sure that the needle will be in the lumen of the vein.
6. Without causing any movement of the needle and adapter, press the collection tube to
allow inner needle to puncture the stopper.
7. When collection tube is filled with blood, loosen the tourniquet with the free hand.
8. Without moving the needle in the vein, carefully pull the collection tube and set it
aside on the rack.
9. Place a dry cotton ball directly on top of the puncture site and press it immediately
after the needle is withdrawn.
10. With the cotton ball still in place, ask the patient to flex arm and not to lower it until
after 10 minutes.

Pointers:
• Lowering the arm for a while or successive closing and opening of the hand while the
tourniquet is in place will engorge the vessels, which makes choosing the vein easier. The
process should not be too long, as it will cause hemoconcentration.
• To prevent hematoma formation, choose the most immovable vein, do not puncture
the vein through and through, making sure the whole bevel of the needle is inside the vein
throughout the procedure, and apply pressure on the punctured site for not less than 10
minutes.
• Choose the more or less straight veins so that, at least 1/3 rd of the needle length
maybe inserted in the lumen during the phlebotomy. This is quite useful in preventing
being out of vein, especially in uncooperative patients, and thus avoiding hematoma
formation.
• With the use of vacutainer, multiple collection tubes may be filled depending on the
number and the nature of test desired.
• Among patient with deep-seated veins, pull the plunger slightly to produce a small
amount of negative pressure in the syringe after inserting the needle into the skin and
prior to puncture of the vein.
• Veins of the limbs with intravenous fluids inserted are not good sites for venipunture.
• The sense of “give” is felt when the needle pass through the vein and its point is
already in the blood vessel’s lumen. This however, is appreciated better with more
practice.
• In the so-called “two step puncture technique,” the needle is inserted into the vein not
immediately after it is inserted in the skin, but having little millimeter distance between
the skin’s entry point from that of the vein. This is a good technique to prevent hematoma
formation.
• Do not re-insert a needle, either that of syringe or vacutainer, even though blood is not
drawn.

Related Question:
1. List all the indications for Capillary puncture.
2. Give the contraindications for venipuncture blood collection.
3. What is the meaning of following the principle of “Order of Draw” in a Vacutainer

blood collection procedure?
4. What is the maximum allowable time for venipuncture blood collection and what will
happen if it was collected beyond the allowable time?

5. List all the technical errors that may cause hemo-dilution and hemo-concentration of
the blood specimens collected.
6. What are the expected changes if the blood mixed with EDTA was left standing for
more than 4 hours at room temperature before processing it?
7. List the concentration of each anticoagulant commonly used Hematology Lab.
A. EDTA
B. Citrate
C. Heparin
D. Oxalate

Practical 1: Capillary puncture
PRINT ___________________________________________ Section ___________

Surname First name M.I. Score ___________
I. Pre-collection 8
A. Materials
1. Complete 2
2. Incomplete 1
B. Proper Patient Identification and Interaction (X 2)
1. done 2
2. not done 1
C. Proper Patient Positioning (x 2)
1. done 1
2. not done 0
II. Collection 27
A. Site Selection (x2)
1. best choice for the patient 3
2. second choice for the patient 2
3. last choice for the patient 1
B. Antiseptic Application
1. done properly 2
2. done improperly 1
3. omitted 0
C. Manner of Puncturing (x2)
1. appropriate 2
2. inappropriate 1
D. Manner of Blood collection ( x3)
1. applying enough pressure 2
2. excessive squeezing 1
E. Amount of Blood Withdrawn (x3)
1. sufficient 3
2. insufficient 2
3. none 1
III. Post-collection 5
A. Pressure Application (x2)
1. one firm pressure until the bleeding stops 2
2. not maintained until the bleeding stops 1
B. Labeling of specimen (x2)
1. proper labeling 2

2. incompletely labeled 1
C. Discarding of Used Materials
1. done as soon as finished and disposed
in the proper waste container 1
2. improperly disposed 0

Practical 2- Venipuncture (Syringe Method)
PRINT ___________________________________________ Section ___________

Surname First name M.I. Score ___________
I. Pre-collection 8
A. Materials
1. Complete 2
2. Incomplete 1
B. Proper Patient Identification and Interaction (X 2)
1. done 2
2. not done 1
C. Proper Patient Positioning (x 2)
1. done 1
2. not done 0
II. Collection 38
A. Vein Selection
B. Tourniquet Application (x 2)
1. proper distance from the puncture site
with enough tightness 3
2. either the distance from the 2
puncture site is improper
or too tight or loose
3.both were done incorrectly 1
C. Antiseptic Application (x2)
1. done properly 2
3. omitted 0
D. Needle Insertion
1. inserted bevel up with the appropriate angle 3
2. either it was not inserted bevel up
or the angle was inappropriate 2
3. the angle was inappropriate and was not
inserted bevel up 1
E. Blood Withdrawal (x3)
1. proper speed of pulling the plunger 2
2. either too fast or too slow 1

F. Amount of Blood Withdrawn (x3)

1. sufficient 3
2. insufficient 2
3. none 1
G. Tourniquet Removal (2)
1. done within 2 minutes from application,
before needle withdrawal 3
2. either removed beyond 2 minutes from
application or done after needle withdrawal 2
3. has exceeded 2 minutes from application
and was preceded by needle withdrawal 1
C.Needle Withdrawal
1. done in one swift motion after tourniquet removal 1
2. done ahead of tourniquet removal 0
1. done after needle withdrawal using a dry cotton, 3
with one firm pressure, with the arm extended
and elevated slightly above the heart, until the
bleeding stops
2. one to three of the appropriate actions (use 2
of dry cotton, one firm pressure, arm extension
and elevation slightly above the heart) was
omitted but done until the bleeding stops
B. Transfer of Blood (x2)
1. proper (fishing out during recapping was done,
appropriate speed, no bubbles, appropriate
position of syringe and test tube) 3
2. one to three of the appropriate actions
were done but fishing out was done 2
3. none of the appropriate actions
were done correctly 1
1. done as soon as finished and disposed
in the proper waste container 2

Practical 3: Venipuncture (Vacutainer method)
PRINT ___________________________________________ Section __________

Surname First name M.I. Score __________
Venipuncture: Evacuated Tube System Method (Multiple Draw)
I. Pre-collection 8
A. Materials
1. Complete 2
2. Incomplete 1
B. Proper Patient Identification and Interaction (x2)
1. done 2
2. not done 1
C. Proper Patient Positioning (x2)
1. done 1
2. not done 0
II. Collection 40
A. Vein Selection
B. Tourniquet Application (x2)
1. proper distance from the puncture site
with enough tightness 3
2. either the distance from the puncture site is
improper or too tight or lose 2
3. both were done incorrectly 1
C. Antiseptic Application (x2)
1. done properly 2
3. omitted 0
D. Needle Insertion
1. inserted bevel up with the
appropriate angle 3
2. either it was not inserted bevel up
or the angle was inappropriate 2
3. the angle was inappropriate and was not
inserted bevel up 1

E. Blood Withdrawal (x3)

1. proper speed of pulling the plunger 2
2. either too fast or too slow 1
F. Order of Draw
1. Correct 2
2. Incorrect 1
G. Amount of Blood Withdrawn (x3)
1. sufficient 3
2. insufficient 2
3. none 1
H. Tourniquet Removal (x2)
1. done within 2 minutes from application,
before needle withdrawal 3
2. either removed beyond 2 minutes from
application or done after needle withdrawal 2
3. has exceeded 2 minutes from application
and was preceded by needle withdrawal 1
I. Needle Withdrawal
1.done in one swift motion after
tourniquet removal 1
2. done ahead of tourniquet removal 0
1. done after needle withdrawal using 3
a dry cotton, with one firm pressure,
with the arm extended and elevated
slightly above the heart, until the
bleeding stops
2. one to three of the appropriate actions 2
(use of dry cotton, one firm pressure,
arm extension and elevation slightly
above the heart) was omitted but done
until the bleeding stops

B. Transfer of Blood (x2)

1. proper (fishing out during recapping 3
was done, appropriate speed, no
bubbles, appropriate position of
syringe and test tube)
2. one to three of the appropriate 2
actions were done but fishing out was done
3. none of the appropriate actions
were done correctly 1
1. done as soon as finished and 2
disposed in the proper waste container

Exercise 4: Red Cell Count (Hemocytometer)
Objectives:
1. discuss the principle of manual red cell counting
2. discuss the significance of performing the manual red cell counting
3. list all the materials and equipment used for manual red cell counting
4. identify the appropriate area in the Neubauer Hemocytometer used for counting red cells
5. lists all the different diluting fluid used for manual red cell counting
6. perform the steps for manual red cell counting properly
7. calculate the RBC count correctly
8. identify the technical errors affecting the manual red cell counting
Materials:
Red cell pipette
Red cell diluting fluid
Counting chamber
Microscope
Blood collection materials and equipment
Procedure:
1. Perform venipuncture on a patient and place blood with anticoagulant.
2. Fill pipette, with blood, up to the 0.5 marking. With a piece of clean tissue paper, wipe the
blood on the outside of pipette stem being careful not to reduce the amount of blood inside.
3. Without allowing the air to go in, fill pipette with diluting fluid up to 101 mark using the
sucking tube.
4. Remove the sucking tube and hold each end of the pipette in between the thumb and middle
finger.
5. Mix the contents by making a figure of “8” motion of the pipette.
6. Discard the fluid contained in the stem including a small amount from the bulb of the
pipette.
7. Charge the counting chamber and set it on flat surface for 1-2 minutes.
8. Set the chamber on a microscope and focus the ruled area under low power objective lens.
9. Bring the RBC counting area in the center of the field then shift to HPO lens.
10.Count the red cells in one chamber and list. Do the same in the other chamber. Get the
average count and calculate the red cell count using the formula below:
RBC count = # cells x 10 x 5 x 200

or
RBC count = # cells x 10,000

Related Question:
1. Illustrate the Improved Neubauer Counting Chamber rulings, indicating the area (mm2) of each
square used for RBC, WBC, and Platelet counting.
2. What will be the correct action of the medical technologist if he noticed that there is an
overcrowding of cells during counting of RBC?
3. List all the available diluting fluids used for RBC counting.

4. Give the possible source of error that can give high RBC count value.
5. Give the possible sources of error that can give Low RBC count value.
6. What are the Physiologic errors that could contribute to the discrepancy of reported RBC
count values?
7. What is the Coulter counter principle that is used by several automated machines for RBC
counting?

Practical 4- Red Cell Counting
PRINT ___________________________________________ Section __________

Surname First name M.I. Score __________
A. Manner of filling up of pipette with blood (x3) 9
a. excellent control 3
b. good control 2
c. poor control 1
B. Dilution of Blood (x2) 6
a. properly diluted blood with 3
no bubbles or spaces
b. improper dilution of blood with 2
minimal bubbles or spaces
c. improper dilution of blood with 1
many bubbles and spaces
C. Mixing of Blood and diluting fluid 2
a. correct manner of mixing 2
(following figure of “8”)
b. incorrect manner of mixing 1
(shaken vigorously)
D. Charging of hemocytometer (x2) 4
a. not overcharged or undercharged 2
b. overcharged or undercharged 1
E. Knowledge of the counting chamber (x2) 2
a. has located the desired square 1
for counting
b. has not located the desired area 0
for counting
F. Cell counting (x4) 12
a. has counted + or – 2 from the proctor’s actual count 3
b. has counted + or – 3 from the proctor’s actual count 2
c. have counted + or – 4 form the protor’s actual count 1

Exercise 5a: Hematocrit (Macro-Method)
Objectives:
1. discuss the principle of hematocrit determination using macro-method
2. explain the significance of hematocrit determination
3. identify the materials used for macro-hematocrit method
4. perform the hematocrit determination using macro-method properly
Materials:
Wintrobe tube
Whole blood with anticoagulant
Centrifuge
Canula
Procedure:
1. Gently but thoroughly mix the whole blood sample.
2. Using the syringe with a canula, fill-up the tube with blood up to “O” mark.
3. Centrifuge for 30 minutes at 3500 rpm speed.
4. Read the graduation mark for the pack red cell, i.e. excluding the buffy coat.
5. Calculate the hematocrit using the formula below
HCT% = Volume of packed red cell x 100

Volume of whole blood used
6. If the tube is not graduated, a ruler may be used to measure, in millimeters, the
column of packed red cell and that of the whole blood.
7. Record and report result.

Exercise 5b: Hematocrit (Micro Method)
Objectives:
At the end of the laboratory exercise the student shall be able to:
1. discuss the principle of hematocrit determination using micro method
2. explain the significance of hematocrit determination
3. identify the materials used for micro hematocrit method
4. perform the hematocrit determination using micro-method properly and
appropriately
5. discuss the advantage of using micro-hematocrit determination over micromethod
6. explain the physiologic and technical factors that affects the micro hematocrit
procedure
Materials:
Heparinized capillary tube
Micro-hematocrit centrifuge
Micro-hematocrit reader
Clay and paraffin sealant
Procedure:
1. Perform a standard finger stick.
2. Fill up ¾ of the length of the capillary tube with blood.
3. Hold mid-part of the tube between the thumb and middle finger while the index finger
of the same hand covers one end of the tube.
4. Press the other free end of the tube on top of the sealant clay then reinforce with
paraffin. Remove index finger while pressing.
5. Spin tube in Adam’s micro-hematocrit centrifuge for 5 min.
6. Place the tube in the reader. Bottom of blood column at 0 mark, top of the plasma
layer on the uppermost diagonal line of the reader.
7. Bring adjustable marker to the top of packed red cells column and read the hematocrit
reader.
8. Read and record the result.

Related Question:
1. Illustrate the end result of the exercise 5a (Macro Method) below, as well as write how the
resulting value is calculated.
2. Illustrate the Hematocrit reader with the tube place to show how the value for micro-
hematocrit determination is obtained.
3. State the principle of Micro-hematocrit determination.

4. Compare the Two methods of Hct determination by stating the advantages and disadvantages.
5. List the possible source of error that leads to false increase in Hct reading. And give
appropriate action to resolve each error.
6. List the possible source of error that leads to false decrease in Hct reading. And give
appropriate action to resolve each error.

Exercise 6a: Hemoglobinometry (Acid Hematin Method)
Objectives:
1. discuss the principle of hemoglobin determination using acid hematin method
2. explain the significance of hemoglobin determination
3. identify the materials used for acid hematin method
4. perform properly the hemoglobin determination using acid hematin procedure
Materials:
Hemoglobinometer set
Sahli-Hellige pipette
0.1N HCl
Distilled water
Procedure:
1. Place 0.1 N HCl to mark 2 of the scale of a graduated Sahli-Hellige hemoglobinometer
tube.
2. Draw blood up to mark of a Sahli-Hellige pipette.
3. Place blood sample into the hemoglobinometer tube and rinse pipette 3 times with the
solution inside the tube.
4. Stand for 10 minutes at room temperature.
5. Place hemoglobinometer tube in a comparator block and add distilled water, drop by
drop, until the color matches with that of the standard in the comparator block.
6. Read meniscus on scale of the tube. Numeric value is expressed in grams %
hemoglobin.
7. Record result.

Exercise 6b: Hemoglobinometry (Cyanmethemoglobin Method)
Objectives:
1. discuss the principle of hemoglobin determination using cyanmethemoglobin method
2. explain the significance of hemoglobin determination
3. identify the materials used for cyanmethemoglobin method
4. perform the hemoglobin determination using cyanmethemoglobin properly and
appropriately
5. calculate the concentration of hemoglobin of the sample using the formula used for
cyanmethemoglobin determination
6. discuss the physiologic and technical factors affecting the result of hemoglobin using the
cyanmethemoglobin method
Materials:
Drabkin’s reagent
Sahli-Hellige pipette (20ul)
Test tube
Spectrophotometer
Standard
Procedure:
1. Label 2 test tubes with “T” (test) and “S” (standard), respectively.
2. Place 5 ml of Drabkin’s reagent to each tube.
3. Draw blood up to mark of the Sahli-Hellige pipette.
4. Deliver blood into tube “T” and rinse pipette 3x with the solution.
5. Cover tube with parafilm and mix by inversion 2-3 times.
6. Fill the Sahli-Hellige pipette, up to mark, with the standard.
7. Deliver the standard into tube “S” and mix by inversion, 2-3 times.
8. Set spectrophotometer at 540 nm wavelengths.
9. Place a small volume of the mixture in tube “S” in a cuvette, read the absorbance in the
spectrophotometer, and record as “As”.
10. Drain the content of the cuvette and drain-dry the mouth with tissue paper. Using the
same cuvette, read the absorbance of tube “T” mixture and record as “At”.
11. Using the formula below, calculate for the hemoglobin value and write the result and
computation below. (C = concentration; A = Absorbance).
𝐶𝑇 𝐴𝑇
=
𝐶𝑆 𝐴𝑆

Related Question:
1. What is the clinical significance of the tests?
2. State the principle of Acid Hematin Determination.
3. State the principle of Cyanmethemoglobin Determination.
4. Illustrate the Cyanmethemoglobin standard curve.

5. What are the disadvantages of using Acid Hematin determination?
6. List all the technical errors relevant to Cyanmethemoglobin determination and state how to
resolve each error.
7. List all the Physiologic errors relevant to Cyanmethemoglobin determination and state how to
resolve each error.
8. State if the item will give false increase or false decrease in Hgb value.
A. Hemoconcentration
B. Hemodilution
C. Clotted blood specimen
D. Presence of carboxyhemoglobin
E. High WBC count
F. Lipemic sample

Exercise 7: Red Cell Indices (MCV, MCH, MCHC)
Objectives:
1. explain the significance of determining the red cell indices
2. memorize the formula used for computing red cell indices parameter
3. calculate the MCV, MCH and MCHC correctly
Procedure:
1. Determine the red cell count, hematocrit, and hemoglobin values of the patient’s
blood, either using freshly withdrawn blood with or without antocougulant.
2. Use the following formulas to calculate for the red cell indices:
𝐇𝐜𝐭 𝐱 𝟏𝟎
𝑀𝐶𝑉 =
𝐑𝐁𝐂 𝐜𝐭.
𝐇𝐠𝐛 𝐱 𝟏𝟎
𝑀𝐶𝐻 =
𝐑𝐁𝐂 𝐜𝐭.
𝐇𝐠𝐛 𝐱 𝟏𝟎𝟎
𝑀𝐶𝐻𝐶 =
𝐇𝐜𝐭
Data: RBC count: ___________ Hct: __________ Hgb: __________

Calculation:
1 Mean Corpuscular Volume (MCV)
2 Mean Corpuscular Hemoglobin (MCH)
3 Mean Corpuscular Hemoglobin Concentration (MCHC)

Related Question:
1. What is the significance of determining the red cell indices of certain hematologic patients?
2. List all the diseases that have Low and High MCV value.
3. List all the Diseases that have Low and High MCH value.

4. Give types of anemia that has Normocytic, Normochromic blood picture.
5. Give types of anemia that has Microcytic, Hypochomic blood picture.
6. Give types of anemia that has Macrocytic, normochromic blood picture.

Exercise 8a: Erythrocyte Sedimentation Rate (Wintrobe Method)
Objectives:
1. discuss the principle of ESR determination using wintrobe
2. explain the significance of ESR determination
3. identify the materials used for ESR method using Wintrobe method
4. perform the ESR determination using wintrobe method properly
5. discuss the factors affecting the ESR results
Materials:
Wintrobe tube
Canula
Syringe
Oxalated tube
Procedure:
1. Withdraw 5 ml of blood from the patient and place in tube with oxalate anticoagulant.
Gently mix thoroughly.
2. Attach a canula to a syringe.
3. Aspirate about 3 mL of the oxalated blood.
4. Without allowing blood to flow, insert canula into a wintrobe tube until the canula tip
is almost touching the end of the tube.
5. Gradually withdraw the canula as blood is slowly delivered into tube up to O mark.
6. Place the tube vertically in a perfectly set and balanced rack.
7. Let set-up stand at room temperature for 1 hour.
8. Note for the junction of the plasma column and the sedimented cells and read the
value directly at the left side of the tube, which is the ESR expressed in mm/hr.
9. Illustrate the end result of the test below and record the value obtained.

Exercise 8b: Erythrocyte Sedimentation Rate (Westergren Method)
Objectives:
1. discuss the principle of ESR determination using Westergren method
2. explain the significance of ESR determination
3. identify the materials used for ESR method using Westergren method
4. perform the ESR determination using Westergren method properly
5. discuss the technical and physiological factors that affects the ESR results
Materials:
Westergren tube and rack
Citrated tube (3.8% Sodium Citrate)
Canula
Syringe
Procedure:
1. Place patient’s blood in citrated tube and gently mix well.
2. Set Westergren tube in its rack and carefully fill it with patient’s blood.
3. Read result after 1 hour and after 2 hours.
4. Illustrate and record test result below.
Pointers:
• Blood not mix well prior to filling the tube, in either Wintrobe or Westergren
method, is likely to produce inaccurate result.
• Air bubble in the tube may produce faulty result. To remove air bubble, gently
whisk the tube once or twice.
• Incorrect blood-anticoagulant proportion, positioning of the tube, manner of
reading, timing of the test, and incorrect amount of blood delivered in the tube
are sources of technical errors that are likely to produce inaccurate results.
• Hot or Humid temperature may result erroneous values.

Related Question:
1. What is the clinical significance of the ESR determination?
2. What are the physiologic and technical factors that give an increase and decrease ESR
reading? Explain why these factors give such reading.
3. Give the normal reference range of the test as to gender and age of the patient.
4. Compare the two ESR methods used in clinical Hematology laboratory.
Wintrobe method Westergren method
a. length of tube
b. bore diameter of tube
c. anticoagulant used
d. manner of reporting

Exercise 9: Osmotic Fragility Test (Sanford’s Method)
Objectives:
1. discuss the principle of EOFT determination using Sanford’s method
2. explain the significance of EOFT determination
3. identify the materials used for EOFT method using Sanford’s method
4. perform the EOFT determination using Sanford’s method properly
5. discuss the factors affecting the EOFT results
6. discuss the different red cell abnormalities that resulted to a decrease or increase sensitivity
on EOFT studies
Materials:
12 Kahn or ordinary test tube
Test tube rack
0.5% NaC1
Capillary pipette or Sahli-Hellige pipette
Distilled Water
Heparinized tube
Procedure:
1. Place 4.5ml blood in a heparinized tube.
2. Label 12 Kahn tube 14 to 25, and place tubes in a rack.
3. With a capillary pipette, place in each tube number of drops of 0.5% NaC1 equal to the
number in the tube.
4. In each tube, add number of drops of distilled water that will bring the amount in each
test tube equals to 25 drops. Use the following table as guide.
Tube # 0.5% NaC1 (drops) Distilled H2O(drops)

25 25 0
24 24 1
23 23 2
22 22 3
21 21 4
20 20 5
19 19 6
18 18 7
17 17 8
16 16 9
15 15 10
14 14 11

5. Gently tap the side of each tube to mix contents.

6. Using a Sahli-Hellige pipette, deliver 0.02ml blood into each tube.
7. Cover all tubes with parafilm then mix all by inverting tubes once or twice. Stand for
two hours.
8. Examine each tube against a white background. Look for the first test tube that shows
initial hemolysis and for the first tube showing complete hemolysis.
9. Calculate for the salt concentration by multiplying the number of the tube that shows
initial and complete hemolysis by 0.02.
10. Illustrate test and record result (with computation) below.
Results:
Initial hemolysis Tube # ________ = _________ % NaCl
Complete hemolysis Tube # ________ = _________ % NaCl

Related Question:
1. State the principle of the test.
2. What is the clinical significance of the test?
3. What are the possible technical errors that a medical technologist should be aware of to
avoid discrepant result?
4. How does the surface area: volume ratio of red cell influence the EOFT result?

5. List the different poikilocyte with high resistance to osmosis.
6. List the different poikilocyte with low resistance to osmosis.

Exercise 10: Reticulocyte Count (New Methylene Blue Method)
Objectives:
1. discuss the principle of reticulocyte counting
2. explain the clinical significance of reticulocyte count
3. identify the different materials used for reticulocyte count
4. identify the reticulocyte in the blood smear stained with New methylene blue
5. perform the reticulocyte count properly
6. calculate the different formula used for reticulocyte determination
7. discuss the technical factors affecting the reticulocyte counting
Materials:
Slides
Test tube
Capillary tube
New Methylene Blue stain
Formula 0.5 gm. NMB powder
1.6 gm. Potassium oxalate
100 ml distilled water (to dissolve)
Procedure:
1. Place equal amounts of anticoagulated blood and NMB stain in a tube. Gently tap the
tube’s side to mix.
2. Charge a capillary tube with blood and stain mixture.
3. Stand for exactly 10 minutes at room temperature.
4. Place a drop of the mixture on a slide and make a smear. Air dry.
5. Scan smear under oil immersion. Enumerate 1000 red cells while counting the number
of reticulocytes therein.
6. Calculate retic (reticulocytes) count using the following formula:
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑥 100
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑡 =
1000 𝑅𝐵𝐶
7. Illustrate reticulocytes in the circle and record retic count below:
Retic count = %

Related Question:
1. What vital stains are used to demonstrate reticulocyte?
2. Give the clinical significance of determining the reticulocyte count on some patient.
3. List the following disease associated with increase reticulocyte count.
4. List the following disease associated with decrease reticulocyte count.
5. What are the other RBC inclusions that can be stained with vital stains which could be
mistaken as reticulocyte?

6. How will you differentiate reticulocyte inclusions from these other RBC inclusions?
7. Using your reported relative reticulocyte count, compute the following parameter:
Given: RBC count: 3.0 x 10 12 /L ;Hct: 30 vol %
a. Actual Reticulocyte Count (ARC)
b. Corrected Reticulocyte Count (CRC)
c. Reticulocyte Production Index (RPI)

Exercise 11a: Blood Smear Preparation (Wedge Film)
Objectives:
1. discuss the principle of blood smear preparation using Wedge method
2. explain the use of blood smears in the clinical diagnosis of various hematological disorders
3. perform the Blood smear preparation using Wedge method correctly
4. discuss the technical factors that affect the blood smear preparation
Materials:
2 glass slides
EDTA Blood sample
Procedure:
1. On a clean, scratch-free glass slide, place a drop of blood 1-2 cm from one end.
2. Hold each end of the slide with the thumb and the middle finger of one end,
respectively.
3. On the other hand, hold another clean slide in similar way.
4. Place the lateral side of the clean slide against the slide with blood, such that both
slides form 30-45 angle and the blood drop is behind the clean slide.
5. Pull back the clean slide until the backside touches the blood, which will spread
laterally.
6. Keeping the angle constant, push the clean slide toward the other end of the slide
with blood. The blood is spread then with a thicker part at the starting point
becoming thinner towards the end of the motion and producing the so-called
“feathery edge”.
7. Carefully lay slide on a flat surface to dry.
8. Practice making several blood films.
9. On the edge of the thick part of the smear, write the label with a lead pencil and
attach the slide in the box below.

Exercise 11b: Blood Smear Preparation (Cover Glass Method)
Objectives:
1. discuss the principle of blood smear preparation using cover glass method
2. explain the use of blood smears in the clinical diagnosis of various hematological disorders
3. perform the blood smear preparation using cove glass method correctly
4. discuss the technical factors that affect the blood smear preparation
5. lists the advantages and disadvantages of the cover glass method
Materials:
Cover glass – 2 pcs.
Blood sample
Procedure:
1. Hold two adjacent corners of a cover glass between thumb and index finger of one
hand.
2. Place a small amount of blood at the center of the cover glass.
3. Place another clean cover glass on the top of the one with blood such that they are
positioned as shown below.
4. When blood has spread between the contact areas of the cover glasses, pull them
apart with a smooth, lateral, parallel and sliding motion.
5. Lay smear, blood-side up, on a flat surface to air-dry.
6. Apply stain.

Exercise 12: Staining of Blood Film
Objectives:
1. list the different stain used for staining blood smear
2. perform the staining of blood film correctly
3. create an ideal stained blood film/smear
4. identify the technical and physiologic factors that affects the quality of a stained blood
smear
Materials:
Blood smears
Droppers
Wright’s stain
Distilled water
Staining Disk or Pan
Procedure:
1. Fix blood smear in methanol for 30 sec. – 1 min.
2. Air dry smear and place it on a stain rack.
3. Cover entire smear with Wright’s stain using a dropper and count the number of drops
of stain needed.
4. Let stand for 1 minute.
5. Add equal number of drops of buffer solution.
6. Gently blow on the side of the slide to mix stain and buffer solution until a metallic
sheen appears on the surface of the mixture.
7. Let stand for 3-5 minutes (depending on the thickness of the smear and age of the
stain).
8. Using a spouted bottle of water, flush the stain gently until no more color comes off
with the water.
9. Gently blot-dry the smear on a piece of tissue paper.
10. Air-dry and examine under oil immersion objective.
11. Clean slide with xylene after examination to remove the oil immersion oil.

Practical 5: Blood Smear Preparation (Wedge Method)
PRINT ______________________________________ Section __________

Surname First name M.I.
Score __________
A. Amount of drop of blood placed (x2) on the 2

glass slides
1. enough amount 2
2. too large or too little amount 1
B. Angle of spreader (x2) 4
1. proper angle 2
2. too high or too low angle 1
C. Manner of spreading (x3) 6
1. continuous smooth spreading 2
2. too fast or too slow spreading 1
D. Blood Smear appearance 4
1. with feathery edge, no spaces, 4
more than ½ the length of the slide
2. with feathery edge, with spaces, 3

more than ½ the length of the slide
3. with feathery edge, with spaces, not more than ½ the 3
length of the slide
4. w/o feathery edge, with spaces, not more than ½ 1

the length of the slide
E. Labeling of the Smear (x2) 4

1. Completely labeled 2
2. Incompletely labeled 1
3. Not labeled 0

Exercise 13: Stained Red Cells
Objectives:
1. identify correctly the different abnormal shape of red cell
2. illustrate the different abnormal shape of red cell
3. correlate each of the abnormal shape of red cell with various hematological disorder
Procedure:
1. Examine several stained blood films under oil immersion.
2. Look for the different morphologic variations of red blood cells.
3. Illustrate each cell below and label. Use next page if needed.

Exercise 13: Stained Red Cells (Continuation)

Exercise 14: White Cell Count
Objectives:
1. Discuss the principle of manual white cell counting
2. Discuss the clinical significance of determining the white cell count
3. list the diluting fluids and materials needed for manual white cell counting
4. identify correctly the area of the Neubauer Hemocytometer used for WBC counting
5. perform the manual white cell count properly
6. enumerate the technical factors that falsely elevate and decrease the white cell count
results
Materials:
White blood cell pipette
Hemocytometer
WBC diluting fluid
Procedure:
1. Perform standard finger stick on a patient. Wipe off the first drop of blood and allow a
good-sized drop of blood to form.
2. Suck blood up to mark 0.5 using the white cell pipette.
3. Wipe-clean the tip of the pipette with a clean tissue paper but not reducing the
amount of blood in the stem.
4. Carefully suck diluting fluid up to mark 11.
5. Cautiously remove the rubber tube and place each of the pipette between the thumb
and middle finger, respectively.
6. Mix the solution by lateral figure-8 motion
7. Discard the fluid that occupies the stem of the pipette and some few drops from the
bulb.
8. Charge the chamber and let stand for 30-60 seconds on a flat surface.
9. Place hemocytometer on a microscope and focus on the white cell counting area.
10. Count the cells. Calculate for the WBC count using the formula below.
# 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓 𝒙 𝑫𝒆𝒑𝒕𝒉 𝑭𝒂𝒄𝒕𝒐𝒓

𝑾𝑩𝑪 𝒄𝒕 =

Related Question:
1. Illustrate the WBC pipette.
2. State the principle of the test.
3. What is the clinical significance of the test?
4. What are the diluting fluids used for manual WBC count?

5. To have an accurate manual WBC count report; what is the proper action of the medical
technologist if the specimen is suspected to have leukocytosis? How about leukocytopenia?
6. Give the formula for Corrected WBC count.
7. When should the medical technologist will correct the performed manual WBC count?

Practical 6: WBC Counting
PRINT ___________________________________________ Section _________

Surname First name M.I. Score _________

b. good control 2
c. poor control 1
(shaken vigorously)
D. Charging of hemocytometer (x2) 4
a. have located the desired square 1
for counting
b. have not located the desired area 0
for counting
a. have counted + or – 2 from the 3
proctor’s actual count
b. have counted + or – 3 from the 2
c. have counted + or – 4 form the 1
protor’s actual count

Exercise 15: Normal White Blood Cells (Stained)
Objectives:
1. identify correctly the normal mature white blood cells
2. classify the different white cell according to its granulation and nuclear lobulation
3. illustrate the different white blood cell
Procedure:
1. Examine stained blood films under oil immersion objective.
2. Look for the listed white blood cells and illustrate below, with color, inside the circles.

Exercise 16: Differential Count

Objectives:
1. discuss the significance of evaluating the white cell differential counting

2. identify correctly the normal mature white blood cells
3. differentiate the mature from immature white blood cells
4. perform the white cell differential count correctly
5. illustrate the different normal mature white blood cells
Materials:
Stained blood film
Microscope
Counter
Procedure:
1. Scan smear under oil immersion. Enumerate the different white blood cells until a total
of 100 cells are identified.
2. Illustrate and label each cell enumerated and fill-up the form below with the count.
Differential Count:
Neutrophil: Monocytes: Basophils:
Lymphocyte: Eosinophils: Stab/Band cells:

Exercise 17: Immature White Blood Cells (Peripheral Blood Film)
Objectives:
1. identify correctly the different immature white blood cells
2. differentiate the mature from immature white blood cells
3. illustrate the different immature white blood cells
Procedure:
1. Secure stained blood film likely to contain immature white blood cells, such as from
patients with leukemia.
2. Examine under oil immersion and illustrate and give diagnostic characteristics of each
cell.
MYELOBLAST PROMYELOCYTE MYELOCYTE
METAMYELOCYTE BAND CELL

Exercise 18: Leukemia
Objectives:
1. differentiate leukemia from leukemoid reaction
2. identify types of leukemia using the blood smears
3. identify cells that confirms a leukemic process using blood smears
4. differentiate AML from CML using blood smear preparation
5. differentiate ALL form CML using blood smear preparation
Procedure:
1. Examine stained blood films under oil immersion objective.
2. Identify type of leukemia cells and illustrate below, with color, inside the circles.
AML CML
ALL CLL

Exercise 19: White Cells with Morphologic Variation
Objectives:
1. differentiate the normal white cell from white cell with morphologic variation
2. identify white cell with abnormal cytoplasmic inclusion and vacuolation
3. identify white cell with abnormal nuclear features
Procedure:
1. Secure stained blood films and examine under oil immersion.
2. Look for white cells with morphology other than the normal and illustrate and label
each cell below.
Pelger- Huet Toxic Granules
Dohle bodies May-Heglin

Exercise 19: White Cells with Morphologic Variation
Atypical Lyphocyte Hypersegmented PMN
Gaucher cell Nieman-pick cell
Flame cell Reed-Sternberg cell

Exercise 20: Buffy Coat Preparation
Objectives:
1. list the materials required for preparing a buffy coat smear
2. explain the use of evaluating the buffy coat of the patient
3. discuss the principle of buffy coat preparation
4. perform the buffy coat preparation correctly
Materials:
Patient’s blood in EDTA tube
Wintrobe tube
Pasteur pipette
Slides
Procedure:
1. Fill Wintrobe tube with well-mixed EDTA-anticoagulated blood.
2. Centrifuge for 6 minutes at 2000 rpm.
3. Remove plasma with a Pasteur pipette until a very small amount greater than the buffy
coat layer remains.
4. Aspirate remaining plasma, buffy coat layer, and the very top of the mature red cell
layer and place them onto a watch glass or clean glass slide.
5. Rinse Pasteur pipette several times with the mixture to remove all cells possibly
sticking to the glass.
6. Mix sample well with a glass rod.
7. Transfer a drop of the mixture on a clean glass slide (or cover glass) and make a smear.
8. Air-dry and stain.
9. Examine under oil immersion. Illustrate a portion of the smear (and label cells
observed) as seen under the microscope in the circle below.

Exercise 21a: Platelet Count – Direct (Rees And Ecker Method)
Objectives:
1. discuss the principle of manual platelet counting
2. discuss the significance of performing the manual platelet counting
3. list all the materials and equipment used for manual platelet counting
4. identify the appropriate area in the Neubauer Hemocytometer used for counting platelets
5. lists all the different diluting fluid used for manual platelet counting
6. perform the steps for manual platelet counting properly
7. calculate the formula for manual platelet count correctly
8. identify the technical errors affecting the manual platelet counting
Materials:
Red blood cell pipette
Rees and Ecker diluting fluid
Petri dish
Filter paper
Hemocytometer
Procedure:
1. Fill red cell pipette with blood up to mark 0.5.
2. Wipe-off blood on the pipette tip without affecting the column of blood in the pipette
stem.
3. Suck Rees-Ecker diluting fluid to mark 101.
4. Shake pipette thoroughly for 3 minutes.
5. Charge Hemocytometer.
6. Place a water-moistened filter paper inside the petri dish.
7. Place Hemocytometer atop filter paper and cover petri dish.
8. Let stand for 5 minutes.
9. Focus the white blood cells counting area under low power.
10. Shift to high power. Count the platelets in 4 white cell squares.
11. Calculate platelet count using the formula below:
𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓 𝒙 𝑫𝒆𝒑𝒕𝒉 𝑭𝒂𝒄𝒕𝒐𝒓

𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕 𝒄𝒐𝒖𝒏𝒕 =

Exercise 21b: Platelet Count (Unopette System)
Objectives:
2. discuss the significance of performing the manual platelet counting using unopette System
3. list all the materials and equipment used for manual platelet counting using unopette
system
4. identify the appropriate area in the Neubauer Hemocytometer used for counting platelets
5. lists all the different diluting fluid used for manual platelet counting using the unopette
system
6. perform the steps for manual platelet counting using the unopette system properly
Materials:
Hemacytometer and coverslip
Microscope
Counter
Unopette system
Petri dish with moist filter paper
Procedure:
1. Using the protective shield on the capillary pipette, puncture the diaphragm of the
reservoir.
2. Remove the shield from the pipette assembly by twisting.
3. Holding the pipette horizontally, touch the tip of the pipette to the blood. Pipette will
fill by capillary action. Filling will cease automatically when the blood reached the
capillary bore in the neck of the pipette.
4. Wipe the outside of the capillary pipette to remove excess blood, which could interfere
with the dilution.
5. Squeeze reservoir slightly to apply negative pressure thus removing some air inside.
6. Cover the opening of the overflow chamber of the pipette with index finger and seat
the pipette securely in the reservoir neck.
7. Release pressure on the reservoir. Then remove finger from the pipette opening
allowing blood to draw into the reservoir.
8. Squeeze the reservoir several times to allow adequate mixing of blood and diluent.
9. Place index finger over the upper opening and gently invert several times to thoroughly
mix the diluent.

10. Let the mixture stand for 10 minutes.

11. Convert the system to dropper assembly.
12. Discard several drops (3-4) then charge properly the hemocytometer.
13. Place the hemacyometer in a moist Petri dish for 10 minutes.
14. Mount the hemacytometer on the microscope and locate the RBC square.
15. Count the platelets on all 25 tertiary square or the entire RBC square.
16. Calculate Platelet count using the formula below:
𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕 𝒄𝒐𝒖𝒏𝒕 = # 𝒐𝒇 𝑷𝒍𝒕. 𝑪𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟏𝒙 𝟏𝟎 𝒙 𝟏𝟎𝟎

Related Question:
1. What is the purpose of placing moist filter paper inside the Petri dish in platelet counting?
2. What is the diluent present in the reservoir of the unopette system used for platelet count?
3. List some Technical errors that will influence the accuracy of manual platelet counting.
4. Illustrate and label parts of the unopette system.

Practical 7 – Platelet Count – Direct
PRINT ______________________________________________ Section _________

Surname First name M.I.
Score __________

b. good control 2
c. poor control 1
(shaken vigorously)
D. Charging of hemacytometer (x2) 4
a. have located the desired square 1
for counting
b. have not located the desired area 0
for counting
a. have counted + or – 2 from the 3
b. have counted + or – 3 from the 2
c. have counted + or – 4 form the 1

Exercise 21c: Platelet Count – Indirect (Fonio’s Method)
Objectives:
2. identify correctly the platelets in the peripheral blood smear
3. discuss the significance of performing the manual platelet counting indirectly using Fonio’s
method
4. perform the steps for indirect manual platelet counting properly
Materials:
Blood sample
Slides
Wright’s or Giemsa stain
Procedure:
1. Make a wedge smear of a finger-stick-obtained blood sample.
2. Air-dry and stain the smear.
3. Examine blood smear under oil immersion objective.
4. Observe 5 – 10 fields while counting the number of platelets in each oil immersion
field.
5. Calculate average number of platelets in the entire field examined.
6. Using the formula below, calculate platelet count.
Platelet count = average # of platelet x 20,000
7. Illustrate and label cells in the circle below a portion of the smear as seen in the oil
immersion field.

Related Question:
1. Compare relative platelet count which uses the PBS with Absolute platelet count that uses
dilution of blood and hemacytometer.
2. If the medical technologist notices platelet satellitism on PBS; which causes falsely decrease in
machine platelet count, what is his proper action to resolve the problem?
3. List some diseases associated with Thrombocytosis.
4. List some diseases associated with Thrombocytopenia.
5. What is the normal Mean Platelet volume?
6. What is the Clinical Significance of increase platelet distribution width?

Exercise 22: Examination of Peripheral Blood Smear
Objectives:
1. identify the normal features of the cell using the low power and high power objective
2. perform an estimated WBC count using the peripheral blood smear under high power
objective magnification
3. evaluate the red cell morphologic and shape alteration using the blood smear
4. evaluate the morphologic alterations of white blood cell and platelets
5. calculate the formula for corrected WBC count
Materials:
Blood Collection apparatus
Slides
Hematology Stain
Microscope
Procedure:
Three necessary steps:
A. Low Power Scan
1. Determine the overall staining quality of the smear.
2. Determine if there is good distribution of cells.
a. Scan the edge and center of smear to make sure no RBC, WBC, and
platelet clumping occurs.
b. Scan the edge for abnormal cells.
3.Find optimal area for examination and enumeration of cells.
a. The RBC should not touch each other.
b. There should be no large number of broken cells.
c. RBC should have gradual loss of stain towards the central pallor.
B. High Power Scan
1.Determine WBC estimate.
a. WBC estimate is done under High Power (40x) magnification
b. Use the table for WBC estimation
No./High Power Estimated Total WBC

Field Count / mm3
2-4 4000-7000
4-6 7000-10,000

6-10 10,000-13,000
10-20 13,000-18,000
2.Correlate the estimate with WBC Count.
C. Oil Immersion Scan

1. Perform WBC differential count.
2. Evaluate RBC anisocytosis, poikilocytosis, hypochromasia, polychromasia, and inclusion.
3. Evaluate Morphology of WBC, and report any abnormalities.
4. Perform platelet estimate (refer to Ex. #21 b) and evaluate platelet morphology.
5. Correct total WBC count if there are >5 -10 nRBC observed in the PBS per 100 WBC differential
count.

Related Question:
1. What is Rouleaux Formation? What is its significance if observed in the PBS?
2. What is Agglutination? What is its significance if observed in the PBS?
3. Why it is that large and abnormal cell are often observed in the edges of the blood smear?
4. What is the clinical significance of toxic granulation?

5. How will the medical technologist differentiate Leukemoid reaction from Leukemia by simply
using the PBS evaluation?
6. What is Leukoerythroblastoid reaction?
7. Give an example of PBS evaluation report.

Exercise 23: Acid Elution Test For Hgb F (Kleihauer-Betke )
Objectives:
1. discuss the principle of the Kleihauer-Betke test
2. explain the significance of determining the level of Hgb F on patient’s blood
3. perform the Kleihauer- Betke test properly
4. compute the formula used to determine the concentration of Hgb F using the Kleihauer
Betke test
Materials:
Reagents:
Red cell Fixing Solution: 80% Ethanol
Citrate/Buffer 0.2mol/L
Hemoglobin Staining Solution
Blood Collection Apparatus
Slides
Microscope
Coplin jar
Procedure:
1. Collect Maternal Blood Sample and place it in EDTA tube.
2. Mix the blood sample by gentle inversion.
3. Place 3 drops of 0.85% saline and 2 drops of blood into a test tube, and mix gently.
4. Prepare a blood smear (wedge), and air dry at room temperature.
5. Place the slide in a coplin jar containing sufficient amount of Red cell fixing solution to
cover the smear. Allow the slide to remain in the solution for 5 min.
6. Remove the slide from the fixing solution, rinse thoroughly with deionized water, and air
dry.
7. Place the slide in a coplin jar containing sufficient Citrate/Buffer solution to cover the
smear. Allow the slide remain in the solution for 10 min.
8. Remove the slide and wash it with deionized water and blot dry the edge.
9. Place the wet slide in a coplin jar with sufficient Hemoglobin Staining Solution to cover the
smear. Allow it to remain in the solution for 3 min.
10. Remove the slide and wash with deionized water and allow it to dry.
11. Examined the slide sunder oil immersion objective.
12. Calculate % Fetal Cells or Fetal/Adult Cell ratio.
a. % Fetal Cells = Total Fetal cells x 100
Total RBC counted
b. Fetal/Adult Ratio= Total Fetal Cells

Total RBC counted
Note: Fetal Cells will stain a dark reddish-pink
Adult Cells will stain a white to light-pink with a slightly darker center

Related Question:
1. What is the clinical significance of the test (Kleiheur-Betke)?
2. What is Rh immune globulin and explain its purpose?
3. List some diseases associated with increase Hgb F concentration in the patient’s blood.

Exercise 24: Acidified Serum Test (Ham’s Test)
Objectives:
1. discuss the principle of Ham’s Test
2. list all the material and sample used for Ham’s test
3. explain the clinical use of performing Ham’s test in the laboratory
4. perform the Ham’s test properly
5. identify the positive and negative reactions obtained from the test
6. correlate the results obtained with different hematological disorders
Materials:
Venous blood (Normal Control) (ABO compatible)
5 test tubes
1-mL serological pipette
2 Erlenmeyer flasks
Glass beads
0.2n HCl
Saline solution
Procedure:
1. Collect venous blood from patient.
2. In separate Erlenmeyer flask containing glass beads, defibrinate the venous blood of
patient and control by swirling.
3. Centrifuge the defibrinated blood and separate serum from cells. Save the normal
control, patient’s serum, and RBC’s.
4. Wash the RBC from the patient and control three times with saline, and dilute to a 50%
cell suspension.
5. Label test tubes 1 through 5, and add into it the reagents as shown below:
Tubes
Reagents 1 2 3 4 5
Patient 0.5 mL 0.5 mL
serum
Normal 0.5 mL 0.5 mL 0.5 mL
serum
0.2 N HCl 0.5 mL 0.5 mL 0.5 mL
50 % 1 drop 1 drop 1 drop 1 drop
Patient’s
RBC
50% 1 drop
Normal RBC
6. Cover paraffin film and incubate all tubes for 1 hour at 37 OC.
7. Centrifuge and examine supernatant for hemolysis.

Related Question:
1. What is the clinical significance of the Ham’s test?
2. How will you interpret the result obtained using this test?
3. What is the optimum pH of the test?
4. What diseases will possibly give false positive result?

5. Using Ham’s test; what is the expected result if the patient has “HEMPAS”?
6. What is “HEMPAS”?

Exercise 25: Sugar Water Test

Objectives:
1. discuss the principle of sugar water test
2. list all the material and sample used for sugar water test test
3. explain the clinical use of performing sugar water test in the laboratory
4. perform the sugar water test properly
5. identify the positive and negative reactions obtained from the test
6. correlate the results obtained with different hematological disorders
Materials:
Stock Sucrose solution:
a. Dissolve 486g sucrose and 5.1 g sodium barbital in 500 mL
distilled water.
b. Adjust the pH to 7.3 to 7.4 with HCl and water to reach 1 L.
c. Store at 4 O C.
Working Sucrose solution:

a. Mix 20 mL stock solution with 80 mL water.
0.1 M NH4OH
Test tubes
1 mL serologic pipettes
Spectrophotometer
Saline solution
Procedure:
1. Prepare a 50 % blood suspension from an EDTA anticoagulated whole blood.
Determine the ABO blood Group.
2. Get type compatible fresh normal serum or serum from an AB donor. (This can be
stored at -20 OC for up to 1 week).
3. Prepare the following mixture using test tubes as shown below:
Test 1 2 3 4
tubes
Sucrose, 0.90 0.95 0.95
mL
Cells, mL 0.05 0.05 0.05
Serum mL 0.05 0.05
0.01 M 0.05
Nh4OH

4. Incubate the mixture for 1 hour at room temperature.

5. After incubation, add 4 mL of 0.15 M NaCl and centrifuge to remove residual cells for 1
to 2 minutes at 3400 rpm.
6. Read the absorbance readings of the supernatant against water blank at 540 nm and
calculate the % lysis using the formula below.
OD tube 1 – (OD tube 2 + OD tube 3) x 100

% Lysis = (OD tube 4 – OD tube 2)

Related Question:
1. State the principle of the Sugar water test.
2. What is the importance of the complement proteins in the serum used for this test?
3. What should be the computed % Lysis to consider it diagnostic for PNH?
4. Discuss the patho-physiology of PNH?

5. How will you differentiate PNH from “HEMPAS”?
6. List some disease that possibly gives a false positive sugar water test result.

References:
1. Anderson S. and K. Poulsen. Anderson’s Atlas of Hematology. USA: Lippincott Williams and Wilkins, 2003.
2. Brown, Barbara. Hematology: Principles and Procedures 6th ed. Philadelphia: Lea and Febiger, 1993.
3. Carr, Jaqueline and Bernadette Rodak. Clinical Hematology Atlas 3rd ed. Philadelphia: W.B. Saunders, 2008.
4. Garza, Diana and Kathleen Becan-McBride. Phlebotomy Handbook: Blood Collection Essentials 7th ed. USA:
Prentice Hall, 2004.
5. Greer J., J. Foerster and J. Lukens. Wintrobe’s Clinical Hematology 11th ed. USA: Lippincott Williams and
Wilkins,2003.
6. Harmening D. Clinical Hematology and Fundamentals of Hemostasis. 5th ed. USA: F.A. Davis Co., 2008.
7. Phlebotomy: Principles and Procedures. USA: Delmar Learning, 2006.
8. Steininger, Cheryl et al. Clinical Hematology: Principles, Procedures and Correlations. Philadelphia: J.B.
Lippincot, 1992.
9. Theml, H. Color Atlas of Hematology: Practical, Microscopic and Clinical Diagnosis 2nd ed. USA: Thieme,
2004.
10. Turgeon, Mary Louise. Clinical Hematology: Theory and Procedures 4th ed. USA: Lippincott Williams and
Wilkins, 2004.

Laboratory Activity Evaluation Sheets
Name of Student: _________________________________ Sec: _______
Laboratory Activity Evaluation Sheet
Laboratory Activity: Capillary Puncture Properly Done

Steps: YES NO
1 Disinfect the ventral side of the chosen finger using a cotton ball wet with 70
% Ethyl alcohol. Allow spontaneous drying.
2 Using one hand, apply a small amount of pressure at the base of the subject’s
finger. The other free hand is for handling the blood lancet.
3 Make a standard wound puncture with one swift motion with the lancet blade
to be in line with the finger crease.
4 Allow a small drop of blood to form on the puncture site and using a dry
cotton ball, lightly wipe it off.
5 Allow another drop of blood to form and collect it into a capillet, without
allowing the capillet tip to touch the skin.
6 After collecting the desired amount of blood, place a dry cotton ball on the
punctured site and make the subject’s thumb hold the cotton ball in place not less
than 10 minutes.
Comments: ______________________________________________________
Evaluator: __________________________________________

Name of Student: _________________________________ Sec: ________
Laboratory Activity: Venipuncture (Syringe method) Properly Done

Steps: YES NO
1 Assemble equipment. Check needle attachment to syringe, such that it can
easily be removed but not toloose to allow air to enter while plunger is pulled
during collection.
2 Snugly apply the tourniquet 2-3 inches above the antecubital area.
3 Choose the vein through palpation using the index finger.
4 Loosen tourniquet and disinfect site with a cotton ball wet with 70% ethyl
alcohol, following a circular motion from in to outside of the chosen site.
Allow skin to dry spontaneously.
5 Re-apply tourniquet. Do not touch site of puncture anymore.
6 Using the thumb of one hand, tense the skin just below the intended puncture
site to fix the vein.
7 Hold syringe with the other hand, index finger of which is placed on the
barrel’s end near the needle while the thumb and other fingers firmly hold the
syringe’s barrel.
8 With the needle’s bevel directed upward, insert the needle into the skin near
the chosen vein. When whole bevel is already under the skin, aim to insert the
needle into the lumen of the vein.
9 Check entry of blood in the needle’s hub. If none appears on the hub, lightly
pull the plunger.
10 As soon as the blood appears on the hub, loosen the tourniquet and aspirate
the desired amount of blood without causing any to-and-fro movement of the
syringe.
11 Place a cotton ball directly on top of the needle’s entry point and gently press
the cotton as the needle is withdrawn. Press the cotton ball firmly after the
needle is fully drawn out and ask patient to raise the arm while applying
pressure on the site for not less than 5 minutes.
12 Remove the needle by putting on the cover followed with a gentle twist and
dispose it in sharp receptacle.
13 Carefully transfer the blood sample into a test tube by allowing it to flow onto
the inner side of the tube.
Comments: ______________________________________________________
Evaluator: __________________________________________

Name of Student: _________________________________ Sec: _________
Laboratory Activity: Venipuncture (Evacuated Tube method) Properly Done

Steps: YES NO
1 Attach the needle into the adapter. Do not remove needle’s cover until puncture is
done.
2 Choose the vein as stated in steps 2-5 in the syringe method.
3 Place collection tube into the adapter but do not puncture yet the stopper.
Carefully hold the vacutainer with one hand with palm supporting the collection
tube in the adapter.
4 Fix the vein by tensing the skin below the intended puncture site.
5 With precise motion, insert the needle into the skin first then into the vein, making
sure, that the needle will be in the lumen of the vein.
6 Without causing any movement of the needle and adapter, press the collection
tube to allow inner needle to puncture the stopper.
7 When collection tube is filled with blood, loosen the tourniquet with the free hand.
8 Without moving the needle in the vein, carefully pull the collection tube and set it
aside on the rack.
9 Place a dry cotton ball directly on top of the puncture site and press it immediately
after the needle is withdrawn.
10 With the cotton ball still in place, ask the patient to flex arm and not to lower it
until after 10 minutes.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Manual RBC count Properly Done

Steps: YES NO
1 Perform venipuncture on a patient and place blood with anticoagulant.
2 Fill pipette, with blood, up to the 0.5 marking.
3 With a piece of clean tissue paper, wipe the blood on the outside of pipette stem
being careful not to reduce the amount of blood inside.
4 Without allowing the air to go in, fill pipette with diluting fluid up to 101 mark
using the aspirator tube.
5 Remove the sucking tube and hold each end of the pipette in between
the thumb and middle finger.
6 Mix the contents by making a figure of “8” motion of the pipette.
7 Discard the fluid contained in the stem including a small amount
from the bulb of the pipette.
8 Charge the counting chamber and set it on flat surface for 1-2 minutes.
9 Set the chamber on a microscope and focus the ruled area under low power
objective lens.
10 Bring the RBC counting area in the center of the field then shift to HPO lens.
11 Count the red cells in one chamber and list. Do the same in the other chamber.
Get the average count and calculate the red cell count using the formula.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Hemoglobinometry (Acid Hematin) Properly Done

Steps: YES NO
1 Place 0.1 N HCl to mark 2 scale of the graduated Sahli-Hellige hemoglobinometer tube.
2 Draw blood up to mark of a Sahli-Hellige pipette.
3 Place blood sample into the hemoglobinometer tube and rinse pipette 3 times with
the solution inside the tube.
4 Stand for 10 minutes at room temperature.
5 Place hemoglobinometer tube in a comparator block and add distilled water, drop by
drop, until the color matches with that of the standard in the comparator block.
6 Read meniscus on scale of the tube. Numeric value is expressed in grams %
hemoglobin.
7 Record and report the result.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Cyanmethemoglobin Properly Done

Steps: YES NO
1 Label 2 test tubes with “T” (test) and “S” (standard), respectively.
2 Place 5 ml of Drabkin’s reagent to each tube.
3 Draw blood up to mark of the Sahli-Hellige pipette.
4 Deliver blood into tube “T” and rinse pipette 3x with the solution.
5 Cover tube with parafilm and mix by inversion 2-3 times.
6 Fill the Sahli-Hellige pipette, up to mark, with the standard.
7 Deliver the standard into tube “S” and mix by inversion, 2-3 times.
8 Set spectrophotometer at 540 nm wavelengths.
9 Place a small volume of the mixture in tube “S” in a cuvette,
read the absorbance in the spectrophotometer, and record as “As”.
10 Drain the content of the cuvette and drain-dry the mouth with tissue paper.
Using the same cuvette, read the absorbance of tube “T” mixture
and record as “At”.
11 Using the formula below, calculate for the hemoglobin value and
write the result and computation below. (C = concentration; A = Absorbance).
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: ESR (Wintrobe method) Properly Done

Steps: YES NO
1 Withdraw 5 ml of blood from the patient and place it in tube with citrate-oxalate.
2 Gently mix thoroughly.
3 Attach a canula to a syringe and aspirate about 3 mL of the oxalated blood.
4 Without allowing blood to flow, insert canula into a wintrobe tube until the canula
tip is almost touching the end of the tube.
5 Gradually withdraw the canula as blood is slowly delivered into tube up to O mark.
6 Place the tube vertically in a perfectly set and balanced rack.
Let set-up stand at room temperature for 1 hour.
7 Note for the junction of the plasma column and the sedimented cells and read the
8 Illustrate the end result of the test below and record the value obtained.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity Evaluation Form
Laboratory Activity: ESR (Westergren method) Properly Done

Steps: YES NO
1 Withdraw 5 ml of blood from the patient and place it in EDTA-Citrate tube.
2 Gently mix thoroughly.
3 After mixing. Remove the cap of the tube and insert the Westergren tube until fit.
4 Note that the blood should fill up the tube to 0-millimeter mark.
5 Set Westergren tube in its rack and carefully fill it with patient’s blood.
6 Read the result after 1 hour.
7 Note for the junction of the plasma column and the sedimented cells and read the
8 Record the value obtained.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: EOFT Properly Done

Steps: YES NO
1 Place 4.5ml blood in a heparinized tube.
2 Label 12 Kahn tube 14 to 25, and place tubes in a rack.
3 With a capillary pipette, place in each tube number of drops of 0.5% NaC1 equal
to the number in the tube.
4 In each tube, add number of drops of distilled water that will bring the amount in
each test tube equals to 25 drops. Use the following table as guide.
Tube # 0.5% NaC1 (drops) Distilled H2O(drops)
25 25 0
24 24 1
23 23 2
22 22 3
21 21 4
20 20 5
19 19 6
18 18 7
17 17 8
16 16 9
15 15 10
14 14 11
5 Gently tap the side of each tube to mix contents.
6 Using a Sahli-Hellige pipette, deliver 0.02ml blood into each tube.
7 Cover all tubes with parafilm then mix all by inverting tubes once or twice.
Stand for two hours.
Examine each tube against a white background. Look for the first test tube that
8 shows initial hemolysis and for the first tube showing complete hemolysis.
Calculate for the salt concentration by multiplying the number of the tube that
9 shows initial and complete hemolysis by 0.02.
10 Record result with computation.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Reticulocyte count Properly Done

Steps: YES NO
1 Place equal amounts of anticoagulated blood and NMB stain in a tube. Gently tap the
tube’s side to mix.
2 Charge a capillary tube with blood and stain mixture.
3 Stand for exactly 10 minutes at room temperature.
4 Place a drop of the mixture on a slide and make a smear. Air dry.
5 Scan smear under oil immersion. Enumerate 1000 red cells while counting the number
of reticulocytes therein.
6 Calculate retic (reticulocytes) count using the following formula:
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Wedge Smear Preparation Properly Done

Steps: YES NO
1 On a clean, scratch-free glass slide, place a drop of blood 1-2 cm from one end.
2 Hold each end of the slide with the thumb and the middle finger of one end,
respectively.
3 On the other hand, hold another clean slide in similar way.
4 Place the lateral side of the clean slide against the slide with blood, such that both
slides form 30-45 angle and the blood drop is behind the clean slide.
5 Pull back the clean slide until the backside touches the blood, which will spread
laterally.
6 Keeping the angle constant, push the clean slide toward the other end of the slide
with blood. The blood is spread then with a thicker part at the starting point
becoming thinner towards the end of the motion and producing the so-called
“Feathery edge”.
7 Carefully lay slide on a flat surface to dry.
8 On the edge of the thick part of the smear, write the label with a lead pencil.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: PBS Staining Properly Done

Steps: YES NO
1 Fix blood smear in methanol for 30 sec. – 1 min.
2 Air dry smear and place it on a stain rack.
3 Cover entire smear with Wright’s stain using a dropper and count the number of
drops
of stain needed. Let stand for 1 minute.
4 Add equal number of drops of buffer solution.
5 Gently blow on the side of the slide to mix stain and buffer solution until a metallic
sheen appears on the surface of the mixture.
6 Let stand for 3-5 minutes (depending on the thickness of the smear and age of the
stain).
7 Using a spouted bottle of water, flush the stain gently until no more color comes
off with the water.
8 Gently blot-dry the smear on a piece of tissue paper.
9 Air-dry and examine under oil immersion objective.
10 Clean slide with xylene after examination to remove the oil immersion oil.
Comments: ______________________________________________________
Evaluator: __________________________________________


Laboratory Activity: WBC count Properly Done
Steps: YES NO
1 Perform standard finger stick on a patient. Wipe off the first drop of blood and
allow a good-sized drop of blood to form.
2 Aspirate blood up to mark 0.5 using the white cell pipette using the aspirator
provided.
3 Wipe-clean the tip of the pipette with a clean tissue paper but not reducing the
amount of blood in the stem.
5 Carefully suck diluting fluid up to mark 11.
6 Cautiously remove the rubber tube and place each of the pipette between the
thumb and middle finger, respectively.
7 Mix the solution by lateral figure-8 motion.
8 Discard the fluid that occupies the stem of the pipette and some few drops from
the bulb.
9 Charge the chamber and let stand for 30-60 seconds on a flat surface.
10 Place hemocytometer on a microscope and focus on the white cell counting area.
11 Count the cells. Calculate for the WBC count using the formula below.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Buffy coat preparation Properly Done

Steps: YES NO
1 Fill Wintrobe tube with well-mixed EDTA-anticoagulated blood.
2 Centrifuge for 6 minutes at 2000 rpm.
3 Remove plasma with a Pasteur pipette until a very small amount greater than the
buffy
coat layer remains.
4 Aspirate remaining plasma, buffy coat layer, and the very top of the mature red cell
layer and place them onto a watch glass or clean glass slide.
5 Rinse Pasteur pipette several times with the mixture to remove all cells possibly
sticking to the glass.
6 Mix sample well with a glass rod.
7 Transfer a drop of the mixture on a clean glass slide (or cover glass) and make a
smear.
Air-dry and stain.
8 Examine under oil immersion. Illustrate a portion of the smear (and label cells
observed) as seen under the microscope in the circle below.
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Platelet count Rees and Ecker Properly Done

Steps: YES NO
1 Fill red cell pipette with blood up to mark 0.5.
2 Wipe-off blood on the pipette tip without affecting the column of blood in the
pipette stem.
3 Aspirate the Rees-Ecker diluting fluid to mark 101.
4 Shake pipette thoroughly for 3 minutes.
5 Charge Hemocytometer poperly.
6 Place a water-moistened filter paper inside the petri dish.
7 Place Hemocytometer atop filter paper and cover petri dish.
8 Let stand for 5 minutes.
9 Focus the white blood cells counting area under low power.
10 Shift to high power. Count the platelets in 4 white cell squares.
11 Calculate platelet count using the formula below:
Comments: ______________________________________________________
Evaluator: __________________________________________

Laboratory Activity: Indirect Platelet count (Fonio's method) Properly Done

Steps: YES NO
1 Make a wedge smear of a finger-stick-obtained blood sample.
2 Air-dry and stain the smear.
3 Examine blood smear under oil immersion objective.
4 Observe 5 – 10 fields while counting the number of platelets in each oil immersion
field.
5 Calculate average number of platelets in the entire field examined.
6 Using the formula below, calculate platelet count.
Platelet count = average # of platelet x 20,000
Comments: ______________________________________________________
Evaluator: __________________________________________


Hematology 1 Module and Laboratory Mannual

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Normel C.

Adarve, RMT, MPH, IMT(ASCP)

COURSE TITLE: CLINICAL HEMATOLOGY 1

STUDENT LEARNING OUTCOMES:

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Alkali denaturation test ................................................................................................................................ 83

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Myelodysplastic syndrome ......................................................................................................................... 141

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LESSON INTENDED LEARNING OUTCOMES (LILO)

1. Discuss the Hematopoietic phases or periods. 3-hour lecture Learning Content:

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LESSON I: Hematopoiesis (Blood cell production)

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Types of Normal Globin chains Period present

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RBC nomenclature types

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Rubriblast Prorubricyte Rubricyte

Metarubricyte Reticulocyte Mature red cell

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Myeloblast Promyelocyte Myelocyte

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Monoblast Promonocyte Monocyte

Lymphoblast Prolymphocyte Small Lymphocyte

Large Granular Lymphocyte

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Megakaryoblast Promegakaryocyte Megakaryocyte Metamegakaryocyte

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LESSON INTENDED LEARNING OUTCOMES (LILO)

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LESSON II: Bone Marrow Examination and the Lymphoid Organs

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Normocellular Hypocellular Hypercellular

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LESSON INTENDED LEARNING OUTCOMES (LILO)

3. Differentiate the metabolic pathways of RBC.

5. Discuss the differences between normal Hgb from abnormal

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LESSON III: Red blood cell Physiology

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Enzymes involved in the catabolism of glucose