QUIZ 1A HEMA LEC b.

Slightly alkaline
1. What is the name of the iron-containing protein c. Composed of mostly WBCs and platelets
that give red blood cells their color? d. Cooler than body temperature
a. Hemoglobin 11. Which of the ff. statements are true?
b. Myoglobin a. All statements are true
c. Hemocyanin b. Blood consists of plasma and formed elements
d. Pyrite c. Plasma is approximately 92%
2. A decrease in the oxygen-carrying capacity of the d. Plasma is straw colored clear liquid containing
blood for any reason, is a condition known as cellular elements and solutes
a. Leucocytosis 12. Function of blood includes
b. Polycthemia a. Maintains body temperature
c. Thrombocytopenia b. Defend against infection
d. Anemia c. All the options are correct
3. Which of the following WBC is capable of d. Transport oxygen
phagocytosis? 13. What is the normal pH of the blood?
a. Lymphocyte a. 7.30-7.40
b. Neutrophil b. 7.20-7.30
c. Basophil c. 7.45-7.55
d. Eosinophil d. 7.35-7.45
4. Which if the ff. statements about erythrocytes is 14. The major components of plasma is
correct? a. Gases
a. They lack a nucleus b. Proteins
b. They are produced in the spleen c. Nutrients
c. They clot blood d. Water
d. They fight infection 15. The most abundant formed elements in the blood
5. Which of the following is the function of WBC? a. Globulins
a. Transport oxygen b. Leukocytes
b. Defend against infection c. Erythrocytes
c. Maintain homeostasis d. Albumins
d. Produce haemoglobin 16. Erythrocytes
6. Which of the following leukocytes have the ability a. Contain large quantities of haemoglobin
to produce antibodies? b. Are biconvex disks
a. B lymphocytes c. Divide frequently
b. Erythrocytes d. Have several nuclei in each cell
c. Monocytes 17. The WBC called granulocytes are
d. Neutrophils a. Monocytes, leukocytes, and neutrophils
7. Which of the ff leukocytes respond when a person b. Lymphocytes and monocytes
is allergic to something? c. Neutrophils, basophils, and eosinophils
a. Basophils d. Erythrocytes, thrombocytes, and platelets
b. Neutrophils 18. What is haematocrit?
c. Eosinophils a. a medicine that helps the bleeding stop
d. Lymphocytes b. a sometime fata blood disease
8. What is the fluid portion of the unclotted blood c. the proportion of RBC compared with total blood
called? volume
a. Neither plasma nor serum d. a blood pressure measuring device
b. Both plasma and serum 19. Mean cell haemoglobin concentration is increased in
c. Serum which of the ff. conditions?
d. Plasma a. Megaloblastic anemia
9. What are the little cellular fragments called that b. Spherocytosis
are responsible for clotting blood? c. All the options are correct
a. Thrombocytes d. Macrocytic anemia
b. Erythrocytes
c. Leukocytes
d. Haemoglobin 20. An indicator of the average weight of haemoglobin
in individual cell
10. Blood is a. MCHC
a. Acidic b. MCH
c. RDW
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 d. MCV HEMA LEC QUIZ 2 LEC (25/25)
21. The parameters required in calculating the RBC 1. All of the ff. do not occur in the medullary phase,
indices include the ff. except EXCEPT
a. Haematocrit o Recognizable clusters of developing
b. Haemoglobin erythroblasts, granulocytes and monocytes begin
c. RBC count to form
d. WBC count o Yolk sac begins to disappear
22. Mean cell volume is decreased in which of the ff. o Myeloid:erythroid ratio reaches 3:1
conditions? o Production of gower 1 and 2 hemoglobin
a. Iron deficiency anemia 2. Which of the ff. statements might NOT be true?
b. Haemolytic anemia o In adults, the thymus is virtually atrophied and is
c. Liver disease therefore no longer functional
d. Megaloblastic anemia o Not all lymphocytes are leukocytes
23. The normal reference for WBC - Correct answer: All lymphocytes are
a. 4800-10,800 cells leukocytes but all leukocytes are not
b. 3000-7000 cells lymphocytes.
c. 150,000-400,000 cells o Pluripotent hematopoietic stem cells are
d. 4-6 million cells produced in the bone marrow
24. If there is an increased RBC Count leading to o Thymocytes are educated in the thymus
hyperviscosity of blood, the condition is known as: 3. Which organ do B lymphocytes mature?
not sures o Spleen
a. None of the options are correct o Bone marrow
b. Leukimia o Thymus
c. Anemia o Lymph nodes
d. Polycythemia 4. Which of the ff. cells surround the cavity of the yolk
25. Volume of blood in averaged size adult male is sac?
a. 6 to 7L o Erythroblasts
b. 5 to 6L o Angioblasts
c. 3 to 4L o Lymphoblasts
d. 4 to 5L o All of the cells are correct
5. Cellularity is described in adults as hypoplastic when
the
o The marrow has <30% hematopoietic cells
o The marrow has >70% hematopoietic cells
o The marrow has >30% hematopoietic cells
o The marrow has <70% hematopoietic cells
6. The paracortical area of the lymph node is occupied
by the _____ cells
o T
o All of the cells are correct
o B
o Natural killer
7. Which of the ff. is not true of medullary phase of
hematopoiesis?
o Begins in the bone marrow cavity
o Measurable levels of erythropoietin can be
detected
o Hematopoietic activity apparent
o Production of megakaryocytes begins in this
phase
8. Myeloid:erythroid ration in normal adult is
o 6:1
o 5:2
o 3:1
o 5:1
9. Which of the ff. cells is a product of the common
lymphoid progenitor?
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 o Granulocyte o All of the items are correct
o Megakaryocyte o Erythropoetin
o T lymphocyte o Hemoglobin F and A
o Erythrocyte 19. The lymph is the fluid portion of blood that escapes
10. Which organ is the site of sequestration of into the connective tissue and is characterized by
membrane damaged RBCs and removes them from o Both statements are correct
the circulation? o A low protein concentration
o Spleen o None of the options are correct
o Thymus o The absence of RBCs
o Bone marrow 20. During the second trimester of fetal development,
o Liver the primary site of blood cell production is the
11. In severe hemolytic anemias, the liver ____ the o Bone marrow
conjugation of bilirubin and ____ the storage of o Liver
iron o lymph node
o Increases, decreases o spleen
o Decreases, increases 21. culling and pitting are functions of
o Decreases, decreases o Lymph node
o Increases, increases o Bone marrow
12. Progenitor cell which can give rise to mast cells o Spleen
o Common lymphoid progenitor o Liver
o CFU-GEMM 22. Which of the following forms an extracellular matrix
o Common myeloid progenitor promoting cell adhesion and regulating
o Pluripotent stem cells hematopoietic stem cells?
13. Theory stating that all blood cells come from one o Stromal cells
origin stem cell o Mesenchymal cells
o Monophyletic o Reticular cells
o Instructive o Epithelial cells
o Stochastic 23. The site for primitive hematopoiesis?
o Polyphyletic o Bone marrow
14. Which organ is the site of sequestration of o Spleen
platelets? o Yolk sac
o Spleen o Liver
o Bone marrow 24. The process of formation and development of blood
o Liver cells is termed
o Thymus o Hemocytometry
15. As one ages, the size of the thymus o Hematopoiesis
o Stays the same o Hematorrhea
o Decreases o Hematemesis
o Depends on the rate of the development of 25. Active red marrow hematopoiesis occurs in the
infection following, EXCEPT
o Increases o Skull
16. Which of the following cells are found in the liver o Iliac crest
acting as macrophages? o Sternum
o Langerhan cells o Kidney
o All cells are correct
o microglial cells
o Kupffer cells
17. Which of the following are functions of the
hepatocytes in the liver?
o Coagulations factors synthesis
o All statements are correct
o Carbohydrate and lipid metabolism
o Protein synthesis and degradation

18. Which of the following are detected during the

hepatic phase of hematopoiesis?
o G-CSF
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QUIZ 3 HEMA LEC b. None
1. Most immature erythrocyte precursor? c. Parachromatin
a. Myeloblast d. Heterochromatin
b. Metamyelocyte 11. All are characteristic of the myeloblast except that
c. Pronormoblast it has
d. Promyelocyte a. Nucleoli
2. As the erythrocyte mature, the nucleolus b. Low N:C ratio
cytoplasmic ratio c. Large nucleus with fine chromatin
a. increases d. Dark blue, scantly cytoplasm without granules
b. cannot be determined 12. Which of the ff. morphologic changes occurs during
c. decreases normal blood cell maturation?
d. stays the same a. Condensation of nuclear chromatin
3. What is the youngest cell in the maturation series b. Appearance of nucleoli
of the neutrophils? c. Increase in cell cytoplasm
a. Promyelocyte d. Development of cytoplasm basophilia
b. Metamyelocytes 13. The metamycelocyte matures to this type of cells
c. Myeloblast a. Monocyte
d. Myelocyte b. Band neutrophil
4. Which of these is true regarding the maturation of c. Myelocyte
RBC precursors? d. Basophil
a. The proportion of nucleus increases; the 14. The cytoplasm of the blast cell tends to strain much
cytoplasmic proportion decreases darker blue because of
b. Cytoplasmic color change from pink to gray to a. Increased amount of DNA
blue b. Increased amount of specific granules
c. Nucleoli becomes more prominent c. Increased amount of RNA
d. The nuclear chromatin becomes coarser, d. Increased amount of azurophilic granules
clumped and condensed. 15. Which of the ff. involves in asynchronistic
5. The basis for determining cell’s maturity maturation of cells?
a. Neither the nucleus nor the chromatin a. Cytoplasm
b. Both the nucleus and chromatin b. All items are correct
c. Nucleus c. Nucleus
d. Chromatin d. Size
6. Euchromatin is described as 16. Classification test
a. Very delicate, fine, and linear Cell is large – immature cells
b. Increasingly coarse and clumped Nucleoli present – immature cells
c. Decreasingly coarse and clumped Low N:C ration – mature cells
d. Non-staining areas in the nucleus of old cells Chromatin coarse and clumped – mature cells
7. All of these are changes observed in the cytoplasm Cytoplasm rich in RNA – immature cells
of hematopoetic stem cells except
a. Increase in proportion (vs nucleus)
b. Increase in basophlia
c. Change in color
d. Possible appearance of granules
8. Which type of leukocyte can myeloid progenitor
cells not produce?
a. B cells
b. Eosinophils
c. Platelets
d. Monocytes
9. Characteristic of the myeloblast
a. Has dark blue, scanty cytoplasm
b. All the statements are correct
c. Has large nucleus with fine chromatin network
d. Has nucleoli
10. The non staining areas in the nucleus of old cells
are called
a. Euchromatin
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MT-HEMA 1.1 (Hematology) LEC  Blood volume is 5L to 6L (1.5 gal) in an
average sized adult male and 4L to 5L (1.2
Historical Developments and General Characteristics gal) in an average sized adult female
of Blood  Newborn’s blood volume in 200-250mL of the
total blood volume
Hematology  Normovolemia – normal blood volume
 Concerned with the diseases of blood and blood-  Hypervolemia – increased blood volume
forming tissues o Excessive fluid intake
o Blood transfusion
Blood o IV injection of fluids
 Vital, life-sustaining fluid circulating in a closed system  Hypovolemia – decreased blood volume
of blood vessel and the heart o Loss of blood (bleeding or hemorrhage)
 The systemic circulation provides the functional blood o Loss of erythrocytes (hemolytic anemias)
supply to all body tissue. It carries oxygen and o Loss of plasma (severe burns)
nutrients to the cells and picks up carbon dioxide and o Loss of body fluids (diarrhea, LBM,
waste products. excessive sweating)
 It carries oxygenated blood from the left ventricle  Functions
through the arteries to the capillaries in the tissues of - Transportation
the body  Respiration
 Characteristics  Nutrient carrier from GIT
- Color  Transportation of hormones from endocrine
 Arterial – bright red or bright scarlet red glands
color (high oxygen content)  Transports metabolic wastes
 Venous – very dark red (low oxygen content) - Regulation
- Viscosity  Regulates pH
 Whole blood – 4.5 to 5.5 times as viscous as  Adjusts and maintains body temperature
water, more resistant to flow than water  Maintains water content of cells
- Reaction - Protection
 Alkaline pH 7.35-7.45, average 7.4  WBC protects against disease by phagocytosis
 pH is controlled by the buffer system of the  Reservoir for substances like water,
blood electrolyte, etc.
- Temperature - 38°C (100.4°F)  Performs haemostasis
- Osmolality  Composition
 Measure of how much one substance has - Whole blood includes
dissolved in another substance. The greater erythrocytes, leukocytes,
the concentration of the substance dissolved, platelets, and plasma.. when a
the higher the osmolality specimen is centrifuges,
 Very salty water has higher osmolality than leukocytes and platelets make up
water with just a tint of salt. the buffy coat (small layer of
 A normal result is typically 275 to 295 cells lying between the packed
milliosmoles per kilogram. RBCs and the plasma)
 Increased or decreased osmolality calls for - Plasma is the liquid, cell-free portion of unclotted
evaluation of the patient’s fluid and blood that has been treated with anticoagulants.
electrolyte balance. Serum is the fluid that remains after coagulation,
 The normal range for serum osmolality is therefore devoid of clotting factors such as
280-300 mOsm/kg. fibrinogen.
 Above normal values may indicate conditions
such as dehydration, hyperglycemia, diabetes
insipidus, hypernatremia, uremia and renal
tubular necrosis
- Specific gravity – ratio of the density of a
substance to that of a standard substance (water)
 Whole blood – 1.052-1.061
 Plasma – 1.022-1.026
- Volume/amount
 RBCs and plasma, regulated by kidney
 20% of the extracellular fluid amounting to
8% of the total body mass

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 - Part of the complete blood count (CBC) test. They
are used to help diagnose the cause of anemia, a
condition in which there are too few RBCs.
- MCV (Mean corpuscular volume)
 Reference range (SI/Conventional units) is
80-95 femtoliters (fL), newborn 96-180 fL
 An indicator of the average/mean volume of
RBCs
 Reflects RBC diameter on a Wright-stained
blood film
 Calculate using the hematocrit (Hct) and RBC
count
��� (%) 10
MCV (fL) =��� ������10 ( 2)
L
 Low MCV indicates microcytic (small average
RBC size) normal MCV indicates normocytic
(normal average RBC size), and high MCV
- Blood Plasma indicates macrocytic (large average RBC size)
 When the formed elements are removed from  Increased MCV can be seen in megaloblastic
the blood, straw-colored liquid called plasma anemia, hemolytic anemia with
is left. Plasma 91.5% water and 8.5% solutes, reticulocytosis, liver disease and normal
much of which (7%) are proteins. newborn.
 Some of the proteins in plasma are also found  Decreased MCV can be seen in iron
elsewhere in the body but those confined to deficiency anemia, thalassemia, sideroblastic
the blood are called plasma proteins. anemia and lead poisoning.
 These proteins play a role in maintaining - MCH (Mean Corpusular Hemoglobin)
proper blood osmotic pressure which is  Reference range (SI/conventional units) is
important in total body fluid balance. 26-34 picograms (pg)
 Most plasma proteins are synthesized by the  An indicator of the average weight of
liver, including the albumins (54% of plasma hemoglobin in individual RBCs
proteins), globulins (38%) and fibrinogen (hemoglobin amount per RBC)
(7%).  Expresses the mass of hemoglobin and
- Formed elements parallels the MCHC
 They are so named because they are enclosed  Calculate using the hemoglobin (Hgb) and
in a plasma membrane and have a definite RBC count
�� (�/��)� 10
structure and shape. MCH (pg) = �102
 All formed elements are cells, broadly ��� ����� ( L )

classified as RBCs, WBCs, except for platelets,  Increased MCH can be seen in macrocytic
which are tiny fragments of bone marrow anemia
cells.  Decreased MCH can be seen in microcytic,
hypochromic anemia
Red blood cells (erythrocytes) - MCHC (Mean Corpuscular Hemoglobin
 Anucleate, biconcave, discoid cells filled with a reddish Concentration)
protein, hemoglobin, which transports oxygen and  Reference range (SI/conventional units) is
carbon dioxide 32-37 g/dL (SI units) 320-370 g/dL
 Appear pink to red and measure 6-8µm in diameter  A measure of the average concentration of
 They have a zone of pallor that occupies one-third of Hgb in g/dL
their center  Reflects RBC staining intensity and amount of
 RBCs counted in measured volumes can detect anemia central pallor
or polycythemia  Calculate using Hgb and Hct values
��� (�/��) 100
 Anemia – loss of oxygen-carrying capacity and is often MCHC (g/dL) =
���
reflected in a reduced RBC count or decreased RBC  32-37 g/dL MCHC indicates normochromic
hemoglobin concentration. RBCs
 Polycythemia – increased RBC count reflecting  Lesser then 32 g/dL MCHC indicates
increased circulating RBC mass, a condition that leads hypochromic RBC, which is seen in iron
to hyperviscosity. deficiency anemia
 RBC indices  Greater than 37 g/dL MCHC indicates possible
- For the computation of RBC indices, three error in RBC or Hgb measurement, or the
numerical results—RBC count, Hgb and Hct presence of spherocytes
(packed cell volume) may be used.  White blood cells

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 Other RBC parameters  High levels could be indicative of the rare
- RDW (RBC distribution width) blood diseases, polycythemia. It causes the
 A measurement of the range in the volume body to make too many RBCs, causing the
and size of the RBCs blood to be thicker than usual.
 A normal range for RDW is 12.2-16.1& in  This can lead to clots, heart attacks, and
adult females and 11.8-14.5 in adult males. If strokes. It is a serious, lifelong condition that
you score outside this range, you could have a can be fatal if it’s not treated
nutrient deficiency, infection, or other  Parameters
disorder. However, even at normal RDW - All these parameters—RBC count, Hgb, Hct,
levels, you may still have a medical condition indices and RBC morphology—are employed to
 Determined from RBC histogram, a graphic detect, diagnose, assess the severity and monitor
representation of particle size distribution the treatment of anemia, polycythemia and the
 High RDW: it could be an indication of numerous systemic conditions that affect RBCs.
nutrient deficiency of iron, folate or vitamin - Automated hematology profiling instruments are
B12. These results could also indicate used in nearly all laboratories to generate these
macrocytic anemia when your body doesn’t data, although visual examination of the Wright-
produce enough red blood cells. stained blood film is still essential to verify
 A person may have high RDW and normal abnormal results
MCV. This suggests a deficiency of iron, B12  Reticulocytes
or folate. It may also indicate chronic liver - Polychromatic (polychromatophilic) erythrocytes,
disease. High RDW and low MCV. newly released from the RBC production site: the
 A low RDW means your RBCs are all about bone marrow
the same size - Indicate the ability of the bone marrow to
increase RBC production in anemia due to
blood loss or excessive RBC destruction

White Blood Cells (Leukocytes)

 Leukocytes protect their host from infection and
injury.
 They are transported in the blood from their source,
usually bone marrow or body cavity destination.
 WBCs are so named because they are nearly colorless
in an unstained cell suspension
 Leukopenia – decreased WBC count (fewer than
4500µL)
- Hematocrit  Leukocytosis – increased WBC count (more than
 Reference range for males (conventional 11,500µL)
units is 41-53 L/L) (SI units is 0.41-0.53 L/L),
for females (conventional units is 36-46%) (SI Platelets (Thrombocytes)
units 0.36-0.46 L/L). Reference range is age  Thrombocytes are true blood cells that maintain blood
and sex dependent. vessel integrity by initiating vessel wall repairs.
 The percentage of RBCs in a given volume  They are only 2-4µm in diameter, round or oval,
of whole blood. anucleate “cell fragments”
 The buffy coat layer of leukocytes and
 They appear insignificant (small size), but they are
platelets, not included in the measurement,
essential to life and are extensively studied for their
can be seen between plasma (upper) and RBC
complex physiology.
(lower) layers
 Thrombocytosis – elevated platelet count
 Calculate by many automated cell counters
using the MCV and RBC count  Thrombocytopenia – low platelet count
��� (��) ��� ����� (�
�102
)
 Thrombocythemia – life-threatening hematologic
Hct (%) = L disorder
10
- Hemoglobin  PLT (platelets)
 A protein in RBCs that carries oxygen. - Reference range (SI units – 150-450x109/L)
 Reference range for males (conventional (conventional units – 150,000-450,000/µL)
units is 13.5-17.5 g/dL) (SI unita 135-175  MPV (Mean Platelet Volume)
g/L); for females (conventional units – 12.0- - Reference range (SI/conventional units – 6.8-
16.0 g/dL) (SI units – 120-160 g/dL) 10.2 fL)
 Reference range for hemoglobin is age and
sex dependent.

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 Analogous to MCV for erythrocytes Relative and Absolute Blood Cell Counts
 Relative count – amount of a cell type in relation to
other blood components
- Relative polycythemia – an apparent rise of the
erythrocyte level in the blood, however the
underlying cause is reduced plasma volume
 Absolute count – actual number of each cell type
without respect to other blood components
- Absolute polycythemia – true increase in red cell
mass from any cause
 Complete Blood Count – CBC is performed on
automated hematology profiling instruments and
includes the RBC, WBC and platelet measurements
- RBC parameters
 RBC count
 Hgb
 Hct
 MCV
 MCH
 MCHC
 RDW
 Retic
- Platelet parameters
 PLT count
 MPV
- WBC parameters
 WBC count
 Neutrophil count: % and absolute
 Lymphocyte count; % and absolute
 Eosinophil and Basophil counts: % and
absolute

Basic Homeostasis Basic Hematology Terminology

 Homeostasis – body’s tendency to move toward  a- – without
physiological stability. In vitro testing of blood and  -blast – youngest/nucleated
other body fluids must replicate exact environmental  -chromic – colored
body conditions. These conditions should include the  -cyte – cell
following:  dys- – abnormal
- Osmotic concentration – the body/cellular water  -emia – in the blood
concentration, composed of 0.85% sodium  ferro- – iron
chloride. This normal osmotic concentration is  hyper- – increased
termed isotonic  hypo- – decreased
- Hypotonic solution – greater amount of water in  iso- – equal
relationship to lesser amount of solutes—water  macro- – large
enters the cell; the cell swells and may lyse
 mega- – very large, huge
- Hypertonic solution – lesser amount of water in
 micro- – small
relationship to greater amount of solutes – water
leaves the cell; cell may crenate  myelo- – marrow
 normo- – normal
 -oid – like
 -osis – increased
 pan- – all
 -penia – decreased
 -plasia – formation
 -poiesis – cell production
 poly- – many
 pro- – before
 thrombo- – clot

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Formed Elements and Sizes decreases granulocytic production and
involves itself solely in hematopoiesis
 Fetal hemoglobin (HgF) is the predominant
hemoglobin, but detectable levels of adult
hemoglobin (HgA) may be present
- Myeloid/medullary phase
 Prior to the 5th month of fetal development,
hematopoiesis begins in the bone marrow
cavity
 This transition is called medullary
hematopoiesis because it occurs in the
medulla or inner part of the bone
 Hematopoietic activity, especially myeloid
activity, is apparent during this phase and the
M:E ratio gradually approaches 3:1 (adult
Hematopoiesis levels)
 Measurable levels of erythropoietin (EPO),
Hematopoiesis granulocyte colony-stimulating factor (G-CSF),
 Hematopoiesis is a continuous regulated process of granulocyte-macrophage colony-stimulating
blood cell production that includes cell renewal, factor (GM-CSF), and hemoglobins F and A can
proliferation, differentiation and maturation be detected
 These processes result in the formation, development  Blood cell production, maturation and death occur in
and specialization of all the functional blood cells that the organs of the RES
are released from the bone marrow to the circulation - RES includes bone marrow, spleen, liver, thymus,
 In healthy adults, hematopoiesis is restricted primarily lymph nodes
to the bone marrow - RES functions in hematopoiesis, phagocytosis and
 During fetal development, the restricted sequential immune defense
distribution of cells initiates in the yolk sac and then  At birth, the bone marrow is very cellular with mainly
progresses in the aorta-gonad mesonephros (AGM) red marrow, indicating very active blood cell
region (mesoblastic phase), then to the fetal liver production
(hepatic phase) and finally resides in the bone - Red marrow is gradually replaced by inactive
marrow (medullary phase) yellow marrow composed of fat
- Mesoblastic (yolk sac) phase - Under physical stress, yellow marrow may revert
 These primitive yet transient yolk sac to active red marrow
erythroblast are important in early
embryogenesis to produce hemoglobin Pediatric and Adult Hematopoiesis
(Gower-1, Gower-2 and Portland) needed for  Bone marrow
delivery of oxygen to rapidly developing - Newborn – 80-90% of bone marrow is active red
embryonic tissues marrow
 Yolk sac hematopoiesis differs from - Young adult (age 20) – 60% of bone marrow is
hematopoiesis that occurs later in the fetus active. Hematopoiesis is confined to the proximal
and the adult. In that, it occurs ends of large flat bones, pelvis, and sternum
intravascularly, or within developing blood - Older adult (age 55): 40% of bone marrow is
vessels active; 60% is fat
- Hepatic phase - Cellularity – ratio of marrow cells to fat (red
 The hepatic phase begins at 5-7 gestational marrow/yellow marrow) and is described in
weeks and is characterized by recognizable adults as
clusters of developing erythroblasts,  Normocellular – marrow has 30-70%
granulocytes and monocytes hematopoietic cells
 The developing erythroblast in this phase  Hypercellular/hyperplastic – marrow has
signal the beginning of definitive >70% hematopoietic cells
erythropoiesis with a decline in the primitive  Hypocellular/hypoplastic – marrow has
hematopoiesis in the yolk sac <30% hematopoietic cells
 Lymphoid cells begin to appear in this phase,  Aplastic – marrow has few or no
hemopoiesis occurs extravascularly with the hematopoietic cells
liver remaining the major site of - M:E (myeloid:erythroid ratio) – ratio of
hematopoiesis during the second trimester of granulocytes and their precursors to nucleated
fetal life erythroid precursors. Normal ratio is between 3:1
 Production of megakaryocytes also begins and 4:1
during this phase. The spleen gradually

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  Granulocytes are more numerous because of  Hematopoietically inactive yellow marrow is scattered
their short survival (1-2 days) as compared to throughout the red marrow so that in adults, there is
erythrocytes with a 120 day life span approximately equal amounts of red and yellow
 Lymphocytes and monocytes are excluded marrow in these areas
from the M:E ratio  Yellow marrow is capable of reverting back to active
- Stem cell theory – hematopoiesis involves the marrow in cases of increased demand on the bone
production of pluripotent stem cells that develop marrow, such as excessive blood loss or hemolysis
into committed progenitor cells (lymphoid or  Adipocytes are large cells with a single fat vacuole;
myeloid) and finally mature blood cells role in regulating the volume of the marrow; also
 Progenitor cells secrete cytokines or growth factors that may stimulate
o Lymphoid – different into either B or T HSC numbers and homeostasis
lymphocytes in response to cytokines,  Others that are important in hematopoiesis
interleukins, growth factors, include: endothelial cells and reticular adventitial
lymphokines, CSFs cells, macrophages and lymphocytes, stromal cells
o Myeloid – give rise to the multipotential  Hematopoietic microenvironment or niche, plays
progenitor CFU-GEMM (colony-forming- an important role in nurturing and protecting HSCs
unit-granulocyte-erythrocyte- and regulating a balance among their quiescence, self-
macrophage-megakaryocyte), which will renewal and differentiation
differentiate into committed progenitor
 Stromal cells form an extracellular matrix in the niche
cells and finally mature blood cells in
to promote cell adhesion and regulate HSCs through
response to cytokines, interleukins,
complex signaling networks involving cytokines,
colony stimulating factors, growth factors
adhesion molecules and maintenance proteins
 Lymphoid tissue
- Primary lymphoid tissue Liver
 Bone marrow – site if pre-B cell  The liver serves as the major site of blood cell
differentiation production during the second trimester of fetal
 Thymus – site of pre-T cell differentiation development
 Ag-independent lymphopoiesis
 The hepatocytes in the liver have many functions:
- Secondary lymphoid tissue - Protein synthesis and degredation
 B and T lymphocytes enter the blood and
- Coagulation factor synthesis
populate secondary lymphoid tissue, where
- Carbohydrate and lipid metabolism
antigen contact occurs
- Drug and toxin clearance
 Includes lymph nodes, spleen, gut-associated
- Iron recycling and storage
tissue (Peyer’s patches)
- Hgb degredation in which bilirubin is conjugated
 Ag-dependent lymphopoiesis depends on
and transported to the small intestine for eventual
antigenic stimulation of T and B lymphocytes
excretion
 Kupffer cells are macrophages that remove senescent
Bone marrow
cells & foreign debris from the blood that circulates
 Bone marrow is one of the largest organs located
through the liver; also secrete mediators that regulate
within the cavities of the cortical bones
protein synthesis in the hepatocytes
 Two major components
 Liver pathophysiology
- Red marrow – active marrow consisting of
- In porphyrias, hereditary or acquired defects in
developing blood cells and their progenitor
the enzyme involved in heme biosynthesis result
- Yellow marrow – inactive marrow, composed
in the accumulation of the various intermediary
primarily of adipocytes (fat cells) with
poryphrins that damage hepatocytes, erythrocyte
undifferentiated mesenchymal cells and
precursors and other tissues
macrophages
- In severe hemolytic anemias, the liver increases
 During infancy and early childhood, all the bones in the conjugation of bilirubin and the storage of iron
the body contain primarily of red (active) marrow - The liver sequesters membrane-damaged RBCs
- Between 5 and 7 years of age, adipocytes become and removes them from the circulation
more abundant and begin to occupy the spaces in - It can maintain hematopoietic stem and
the long bones previously dominated by active progenitor cells to produce various blood cells
marrow (called extramedullary hematopoiesis) as a
 The process of replacing the active marrow by response to infectious agents or in pathologic
adipocytes (yellow marrow) during development is myelofibrosis
called regression - It is directly affected by storage diseases of the
- Eventually results in restriction of the active monocyte/macrophage (Kupffer) cells as a result
marrow in the adult to the sternum, vertebrae, of enzyme deficiencies that cause hepatomegaly
scapulae, pelvis, ribs, skull and proximal portion with ultimate dysfunction of the liver (Gaucher
of the long bones

dane.
 disease, Nieman-pick disease, Tay-Sach’s - This occurs as a result of many conditions such as
disease) chronic leukemias, inherited membrane or
enzyme defects in RBCs, hemoglobinopathy,
Spleen thalassemia, malaria and the myeloproliferative
 Largest lymphoid organ and has three types of splenic disorders
tissue  Splenectomy may be beneficial in cases of excessive
- White pulp – scattered follicles with germinal destruction of RBCs such as autoimmune hemolytic
centers containing lymphocytes, macrophages and anemia when treatment with corticosteroids does not
dendritic cells effectively suppress hemolysis or in severe
- Marginal zone – surrounds the white pulp and hereditary spherocytosis
forms a reticular meshwork containing blood - It may also be indicated in severe refractory
vessels, macrophages, memory B cells and CD4-T immune thrombocytopenic purpura or in storage
cells disease/disorders with portal hypertension and
- Red pulp – composed primarily of vascular splenomegaly resulting in peripheral cytopenias
sinuses separated by cords of reticular cell - After splenectomy, platelet and leukocyte count
meshwork (cords of Billroth) containing loosely increase transiently
connected specialized macrophages  In sickle cell anemia, repeated splenic infarcts caused
 As RBCs pass through the cords of Billroth, there is a by sickled RBCs trapped in the small-vessel circulation
decrease in the flow of blood, which leads to of the spleen cause tissue damage and necrosis, which
stagnation and depletion of RBCs glucose supply. often results in autosplenectomy
These cells are subject to increased damage and stress  Hypersplenism is an enlargement of the spleen
that may lead to their removal from the spleen resulting in some degree of pancytopenia despite the
 The spleen also serves as a storage site for platelets. In presence of a hyperactive bone marrow
a healthy individual, approximately 30% of the total - The most common cause is congestive
platelet count is sequestered in the spleen splenomegaly secondary to cirrhosis of the liver
 Two methods of removing senescent or abnormal and portal hypertension
red blood cells from the circulation - Often causes thrombosis, vascular stenosis, other
- Culling – cells are phagocytized with subsequent vascular deformities such as aneurysm of the
degradation of cell organelles splenic artery and cysts
- Pitting – splenic macrophages remove inclusions
or damaged surface membrane from the Lymph nodes
circulating RBCs  Lymph nodes can be divided into an outer region
 Spleen Pathophysiology called the cortex and an inner medulla. An outer
- As blood enters the spleen, it may follow one or capsule forms trabeculae that radiate through the
two routes cortex and provide support for the macrophages and
 Slow-transit pathway – through the red lymphocytes located in the node
pulp, in which the RBCs have a more difficult  The lymph is the fluid portion of blood that escapes
time passing through the tiny openings into the connective tissue and is characterized by a
created by the interendothelial junctions of low protein concentration and the absence of RBCs
adjacent endothelial cells  After antigenic stimulation, the cortical region of some
o The combination of the slow passage and follicles develops foci of activated B cell proliferation
the continued RBC maturation creates an called germinal centers. Follicles with germinal
environment that is acidic, hypoglycemic centers are called secondary follicles, while those
and hypoxic without are called primary follicles
o Increased environmental stress on the  Located between the cortex and medulla is a region
RBCs circulating through the spleen leads called paracortex which contains predominantly T
to possible hemolysis cells and numerous macrophages
 Rapid-transit pathway – blood cells enter  The medullary cords lie toward the interior of the LN
the splenic artery and pass directly to the (consisting of plasma cells and B cells); three
sinuses in the red pulp and continue to the functions
venous system to exit the spleen - Site of lymphocyte proliferation from the germinal
 Aggregates of T-lymphocytes surround arteries that centers
pass through these germinal centers, forming a region - Involved in the initiation of the specific immune
called periarterial lymphatic sheath or PALS response to foreign antigens
 Interspersed along the periphery of the PALS are - Filter particulate matter, debris and bacteria
lymphoid nodules containing primarily B lymphocytes. entering the lymph node via the lymph
Activated B lymphocytes are found in the germinal  Lymph node pathophysiology
centers - Lymph nodes by their nature, are vulnerable to
 When splenomegaly occurs, the spleen becomes the same organisms that circulate through the
enlarged and is palpable tissue

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 - Sometimes, increased numbers of microorganisms condition are failure to thrive, uncontrollable
enter the nodes, overwhelming the macrophages infections, and death in infancy
and causing adenitis (infection of lymph nodes) - Adults with thymic disturbance are not affected
- More serious is the frequent entry into the lymph because they have developed and maintained a
nodes of malignant cells that have been broken pool of T lymphocytes for life
loose from malignant tumors  Medullary hematopoiesis – blood cell production
- These malignant cells may grow and metastasize within the bone marrow. Begins in the 5th month of
to other lymph nodes in the same group gestation and continues throughout life
 Extramedullary hematopoiesis – blood cell
Thymus production outside the bone marrow. Occurs when the
 The thymus tissue originates from endodermal and bone marrow can’t meet body requirements and
mesenchymal tissue and populated initially by mainly in the liver and spleen, with hepatomegaly and
primitive lymphoid cells from the yolk sac and the splenomegaly
liver
 In adults, T cell progenitors migrate to the thymus Theories on Blood Cell Formation
from the bone marrow for further maturation  Monophyletic theory – all blood cells come from one
 The thymus resembles other lymphoid tissue in that origin stem cell, the hemocytoblast rec
the lobules are subdivided into two areas: the  Polyphyletic theory – also known as the dualistic
cortex (a peripheral zone) and the medulla (central theory which suggest different groups of blood cells
zone) originate from different stem cells
- Both areas are populated with the same cellular - RBC, WBC, platelets – hemohistioblast
components—lymphoid cells, mesenchymal cells, - Monocytes, lumphocytes and plasma cells – tissue
reticular cells, epithelial cells, dendritic cells and hemohistioblast
many macrophages—although in different  Growth factors
proportions Committed Growth factor Mature cell
 The function of the cortex seems to be that of a progenitor
“waiting zone” densely populated with progenitor T CFU- Thrombopoietin, Thrombocytes
cells MEG GM-CSF
- When these progenitor T cells migrate from the CFU-GM CFU- GM-CSF, M-CSF, Monocytes
bone marrow and first enter the thymus, they M IL-3
have no identifiable CD4 and CD8 surface markers CFU-GM CFU- GM-CSF, G-CSF, Neutrophils
(double negative), and they locate to the G IL-3
corticomedullary junction CFU-GM CFU- Erythropoietin, Erythroid
- Under the influence of chemokines, cytokines and E GM CSF, IL-3
receptors, these cells move to the cortex and CFU-Eo GM-CSF, IL-3, IL- Eosinophils
express both CD4 and CD8 (double positive) 5
- Subsequently they give rise to mature T cells that CFU-Ba IL-3, IL-4 Basophils
expresss either CD4 or CD8 surface antigen as
they move toward the medulla Precursor cells
- Eventually, the mature T cells leave the thymus to
 Comprise the third marrow compartment
populate specific regions of other lymphoid tissue,
 Each type of unipotent stem cell matures into a blast
such as the T cell-depended areas of the spleen,
form
lymph nodes, and other lymphoid tissues
- Myeloblast
 The lymphoid cells that do not express the appropriate - Megakaryoblatst
antigens and receptors, or are self-reactive, dies in - Pronormoblast
the cortex or medulla as a result of apoptosis and are - Lymphoblast
phagocytized macrophages
 The medulla contains only 15% mature T cells and Principle of Normal Blood Cells Maturation
seems to be holding zone for mature T cells until they (synchronistic maturation)
are needed by the peripheral lymphoid tissues
 Blood cells mature synchronistically when its nucleus
 The thymus also contains other cell types including B and cytoplasm mature simultaneously. If ever one lags
cells, eosinophils, neutrophils, and other myeloid cells behind the other, asynchronistic maturation is taking
 Hardly recognizable in old age due to atrophy place
 The thymus retains the ability to produce new T cells,  Cytoplasmic changes
however, has been shown after irradiation treatment - Loss of basophilia
that may accompany bone marrow transplantation  The cytoplasm of an immature cell is usually
 Thymus pathophysiology blue or basophilic due to its ribonucleic acid
- Nondevelopment of thymus during gestation content
results in the lack of formation of T lymphocytes.  The more mature the cell, the less basophilic
Related manifestations seen in patients with this because of the lesser RNA content
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 - Elaboration of cytoplasmic granules - In erythrocytes, this is characterized by persistent
 In myeloid cells, the cytoplasm contain cytoplasmic basophilia and late
granules hemoglobinization
 These granules contain some enzymes which - Abnormal cytoplasmic inclusion bodies may be
distinguish the myeloid stem cells from other found in the cytoplasm of both erythrocytes and
cells leukocytes, especially in the granulocytes
 Those with affinity to red dye are called
acidophilic or eosinophilic granulocytes General Characteristics of “Blast” Cells
 Those with affinity to the blue dye are called  Size – large cells with high N:C ratio
basophilic granulocytes  Cytoplasm – very dark blue and small amount in
 Those with affinity to both acid and basic dyes comparison to the size of the nucleus. No granule is
are called neutrophilic granulocytes present
- Elaboration of hemoglobin  Nucleus – large in size as compared to the size of the
 This is a special feature of the maturation of cytoplasm
erythrocytes  Chromatin – reddish purple and indicates the
 At first, the immature cell contains no predominance of DNA, is very fine and very smooth,
hemoglobin resembling individual grains of sand or a very fine net
 Gradually the hemoglobin starts to appear as (euchromatin); no clumping of chromatin materials
the cell becomes mature until the most - Nucleoli which are present are pale light blue in
mature cell contains a standard and maximal staining
amount of it  Differences between immature and mature cells
 Mature red cells do not possess a nucleus Immature cells Mature cells
 Nuclear changes Cell is large Cell becomes smaller
- Structure and cytochemistry Nucleoli present Nucleoli absent
 The immature nucleus is round and the Fine and delicate Coarse and clumped
nuclear chromatin is very delicate, fine and chromatin chromatin
linear and is called eurochromatin
Round nucleus Round, lobulatd or
 As the cell matures, chromatin strands segmented nucleus
become increasingly coarse and clumped and
Dark blue cytoplasm (rich Light blue cytoplasm (less
are called heterochromatin
in RNA) RNA)
 There is reduction of nucleoli
High N:C ratio Low N:C ratio
 Non-staining areas in the nucleus of old cells
are called parachromatin
Cytoadhesion Molecules in Hematopoiesis
 The chromatin is considered as the best basis
of the maturity of the cell  Cytoadhesion molecules are required to modulate
many interactions between hematopoietic cells and
- Shape
 As the cell matures, the shape of the nucleus growth factors, stromal cells, endothelium and
changes too, and is especially true in extracellular matrix
granulocytes in which the nucleus divides into  These cell surface molecules influence induction,
segments or lobes on maturation differentiation and function of hematopoietic cells and
 The older the cell, the more segments or lobes often have different functions
the nucleus possess  Immunoglobulin supragene family, integrins, selectin
- Cell size changes (LEC-CAM)
 Reduction in cell size during maturation is a  Many extracellular matrix components interact with
feature of all cell lines except in receptors on hematopoietic cells. These incluse
megakaryocytic line fibronectin, thrombospondin, hyaluronic acid,
 Generally, all cell lines increase in number but hormonectin and heparan sulfate
the individual cell’s size decreases because
they undergo mitosis Development of Cells
 With the exception of the megakaryocytic Morphological features Usual development
cells, all mature blood cells are smaller than General cell size Decreases with maturity
the immature stages Nuclear cytoplasmic ratio Decrease with maturity
Nucleus Usual development
Principle of Abnormal Cell Maturation (asynchronistic Chromatin pattern Becomes more condensed
maturation) Presence of nucleoli Not visible in mature cells
 Pathologic hematopoiesis results in abnormal nuclear Color Progresses from darker
maturation, abnormal cytoplasmic differentiation and blue to lighter blue, blue-
abnormal size. The development may also be gray or pink
asynchronous Granulation Progresses from no
 Abnormal cytoplasmic differentiation granules to nonspecific to

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 specific granules normoblast erythroblast
Vacuoles Increase with age Orthochromic Metarubricyte Polychromatic
normoblast erythrocyte
Control of Hematopoiesis Polychromatic Polychromatic erythrocyte
 The entry of mature blood cells into the intravascular erythrocyte erythrocyte
space relies upon (reticulocyte)
- Multiplication of developing cells Erythrocyte Erythrocyte
- Gradual maturation  Progenitors
- Orderly release of cells from bone marrow - Bust-forming unit: erythroid (BFU-E)
 The vessels of the sinusoid system have no direct - Colony-forming unit: erythroid (CFU-E)
connections within the extravascular space, therefore, - Both committed to the erythroid cell line
some form of stimulus is required to either allow the  Estimates of time spent at each stage is about one
diapedesis of cells through the endothelium or week for the BFU-E to mature to the CFU-E
physical rupture of the wall or to engorge the  Another one week for the CFU-E to become a
extravascular space within mature cells pronormoblast, first morphologically identifiable RBC
precursor
Erythrocyte Production and Destruction  At the CFU-E stage, the cell completed approximately 3-
5 divisions before maturing further
Erythrocyte  It takes another 6-7 days for the precursor to become
 RBC or erythrocytes: classic example of the biologic mature enough to enter the circulation which
principles regarding their specialized functions and approximately is 18-21 days to produce a mature RBC
their structures which has specific functions from the BFU-E
 Function: carry oxygen from the lungs to the tissues  The mature erythrocytes have a life span of 120 days in
through attachment of oxygen to the hemoglobin (the the circulation
cytoplasmic component of RBCs).  In the erythrocytes cell line, there are typically three
- Other function: return carbon dioxide to the lungs and occasionally as many as five divisions with
and buffering the pH (secondary function only) subsequent nuclear and cytoplasmic maturation of the
 Protection: control the development, daughter cells, from a single pronormoblast there are
production, and normal destruction of RBC in 8-32 mature RBCs that usually result
order to avoid interruptions of oxygen
delivery even in the presence of harsh Important Events during Erythropoiesis
conditions such as loss of blood with Stage of Erythropoiesis Important Event
hemorrhage Proerythroblast Synthesis of hemoglobin
 Iron, RBC metabolism, membrane structure, and Early normoblast Nucleoli disappear
hemoglobin constitute to the foundation of the body’s Intermediate normoblast Hemoglobin sarts
response to diminished the oxygen capacity of the appearing
blood called anemia Reticulocyte Reticulum is formed; cell
enters the capillary from
Normoblastic Maturation site of production
 Erythroblasts: nucleated precursors in the bone Mature RBC Reticulum disappears; cell
marrow (Normoblast is the developing nucleated
attains biconcavity
cells with normal appearance)
 Morphologic identification of blood cell is well-stained
 Megaloblasts (erythroblasts): abnormal appearance
peripheral blood film or bone marrow smear with
of the developing nucleated cells in megaloblastic
modified Romanowsky stain: may be Wright or
anemia – large size
Wright-Giemsa stain which is the most commonly
 Three Nomenclature Used in Naming Erythroid used
Precursors
 Stage of maturation: careful examination of the nucleus
- Erythroblast: used primarily in Europe
and cytoplasm
- Normoblastic: used in the US and has an
- Qualities of Greatest Importance:
advantage of being descriptive of the appearance
 Nuclear chromatin pattern
of the cells
 Nuclear diameter
- Rubriblast: it parallels the nomenclature used for
 Nucleus:Cytoplasm ratio
granulocyte development
 Presence or absence of nucleoli
 Cytoplasmic color
Three Erythroid Precursors Nomenclature Systems
 As the RBCs mature, the appearance is affected
Normoblastic Rubriblast Erythroblastic
- The overall diameter of the cell decreases
Pronormoblast Rubriblast Proerythroblast
- The diameter of the nucleus decreases more
Basophilic Prorubricyte Basophilic rapidly than does the size of the cell; as a result,
normoblast eryhtroblast the N:C ratio also decreases
Polychromatic Rubricyte Polychromatic
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 - The nuclear chromatinpattern becomes coarser, Three General Important Characteristics of RBCs
clumped, and condensed o Oxygen transport, removal of metabolic wastes
 The nuclear chromatin of the RBCs is o Loss of nucleus is required for function
inherently coarser than that of the myeloid o Normal life span is 120 days
precursors
 It becomes even coarser and more clumped Erythrocyte Maturation
as the cell matures, developing a raspberry-  Pronormoblast (Rubriblast)
like appearance in which the dark-staining - Earlist TBC
chromatin is distinct from the almost white - N:C ratio of 8:1
appearance of the parachromatin - 1-3 nucleoli, nucleous has purple red chromatin
 Ultimately, the nucleus becomes quire - Chromatin: fine clumps
condensed with no parachromatic evident at - Deep or dark blue cytoplasm with no granules
all and the nucleus is said to be pyknotic  Basophilic normoblast (Prorubricyte)
- Nucleoli disappear, nucleoli represent areas - N:C ratio of 6:1
where the ribosomes are formed and are seen - Centrally located nucleus with 0-1 nucleoli
early in cell development as cells begin actively - Chromatin begins to condense, deep purple red
synthesizing proteins - Cytoplasm is deeper blue but intensely basophilic
 As RBCs mature, the nucleoli disappear which RNA
precedes the ultimate cessation of protein  Polychromatophilic Normoblast (Rubricyte)
synthesis - Size: up to 12um with an N:C ratio from 4:1 to 1:1
- The cytoplasm changes from blue/gray-blue to - Eccentric nucleus with no nucleoli
salmon pink - Chromatin shows significant clumping
 Blueness of basophilia – acidic components - Increase Hgb
attract the basic stain (methylene blue) - Cytoplasm: pink and blue – murky gray-blue
o The degree of cytoplasmic basophilia  OrthochromicNormoblast (Metarubricyte)
correlates with the amount of ribosomal - Size up to 10um with an N:C ratio of 0.5:1
RNA. These organelles decline over the - Eccentric nucleus with small, fully condensed
life of developing RBC and the blueness (pyknotic) nucleus, no nucleoli
fades - Pale blue to salmon cytoplasm
 Pinkness called eosinophilia or acidophilia - Hgb synthesis decreases
– due to accumulation of more basic  Reticulocyte (salmon-pink)
components attract the acid stain (eosin) - Size: up to 10um
o Eosinophilia of erythrocytes cytoplasm - Contains no nucleus with mitochondria and
correlates with the accumulation of Hgb ribosomes
as the cell matures
- Last stage to synthesize Hgb
o Thus, the cell starts out being active in - Last stage in bone marrow before release to the
protein production on the ribosomes that
blood
make the cytoplasm basophilic,
- Reference ranges: 0.5-1.5% (adults); 2.5-6.5%
transitions through a period in which the
(newborns)
red of Hgb begins to mmix with that blue
- Supravital stain – enumeration of reticulocytes
and ultimately ends with a thoroughly
- Reticulocyte count: one of the best indicators of
salmon-pink color when the ribosomes
bone marrow function
are gone only the Hgb remains
- Stress reticulocytes: young cells released from
bone marrow after older reticulocytes have been
released – a response to increased need
- Hgb continuous to be produced by the
reticulocytes for approximately 24 hours after
exiting the bone marrow
 Mature erythrocytes
- Size range: 6-8um
- Round, biconcave discocyte
- Salmon with central pallor (clearing in the center)
in Wright stained smear
 Normal cells have a central pallor that is one-
third the diameter of the cell
 Decreased central pallor is seen with
spherocytic disorders
 Central pallor greater than one-third the
diameter of the cell is seen in microcytic
anemias

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 RBC reference ranges in SI units - Is the entirety of erythroid cells in the body
- Females: 4.0-5.4 x 1012/L (conventional units 4.0- whereas the RBC mass refers only to cells in
5.4 x 106/uL) circulation
- Males: 4.5-6.0 x 1012/L (conventional units 4.6-6.0  Hypoxia
x 106/uL) - Too little tissue oxygen, stimulus to RBC
 Erythropoiesis is regulated by erythropoietin production
produced in the kidney. Additional regulation - Hypoxia of the peripheral blood us detected by the
includes: peritubular fibroblasts of the kidney, which
 Hypoxia due to high altitudes, heart or lung upregulates transcription of the EPO gene to
dysfunction, anemia increase the production of EPO
 Androgens (male hormones that appear to  Erythropoietin (EPO)
enhance the activity of erythropoietin) and - Primary hormone that stimulates the production
hemolytic anemias (increased erythrocyte of erythrocytes
destruction) - Able to rescue the CFU-E from apoptosis,
production of antiapoptotic molecules by the
Matured Erythrocytes erythroid progenitors
 Mature RBC has no nucleus, ribosomes, mitochondria - Shorten the time between the mitosis of the
 Small biconcave discs precursors
 Primary component: hemoglobin (1/3 of cell - Release reticulocytes from the marrow early
volume) - Reduce the number of mitoses of precursors
 Flexible because of stretchable fibers called spectrin - Produced in the kidney in response to hypoxia
through increased rated of transcription of the
Developmental Stages of RBCs gene
- Needs active bone marrow (no deficiency, no
primary bone marrow disease, and no
suppression by drugs or chronic diseases)
- Normal serum level is 20 IU/L
- Elevated in most anemias (up to thousands) but
lowered in anemia of chronic renal failure
 Apoptosis is the programmed cell death, the
mechanisms by which an appropriate normal level of
cell is controlled
- Fas, the death receptor, is expressed by young
normoblasts, and FasL, the ligand expressed by
the older normoblasts. As long as older cells
mature slowly in the marrow, induce death of
unneeded younger cells
Erythropoiesis
Microenvironment of the Bone Marrow
 Survival of RBC precursors in the bone marrow
depends on the adhesive molecules, such as
fibronectin and cytokines that are elaborated by
macrophages and other bone marrow stromal cells
 RBCs are found in erythroid islands, where
erythroblasts are various stages of maturation
surround a macrophage
 As RBC precursors mature, they lose adhesive
molecule receptors to stromal cells and can leave the
bone marrow timing of egress of RBCs
- Egress occurs between adventitial cells but
Erythrokinetics through pores in the endothelial cells
 Describing the dynamics of RBC production and
destruction Erythrocyte Destruction
 Erythron  Aged RBCs or senescent cells, cannot regenerate
- The collection of all stages of erythrocytes catabolized enzymes because they lack nucleus
throughout the body: the developing precursors in (single feature of normal RBCs responsible for limiting
the bone marrow and the circulating erythrocytes their life span)
in the peripheral blood and the vascular spaces  The semipermeable membrane becomes more
within specific organs such as the spleen permeable to water so the cell swells and becomes
spherocytic and rigid. Those cells that cannot squeeze

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 through the narrow spaces – trapped in the splenic
sieve
 Eryptosis is a nonnucleated version of apoptosis that
is precipitated by oxidative stress, depletion of energy
and other mechanisms that create membrane signals
that stimulate phagocytosis
 Extravascular or macrophage-mediated hemolysis
- Accounts for most normal RBC death
- The signals to macrophages that initiate RBC
ingestion may include
 Binding of autologous IgG to band 3
 Expression of phosphatidylserine on the outer
membrane
 Cation balance changes
 CD47-thrombospondin 1 binding
 Fragmentation or intravascular hemolysis
- Results when mechanical factors rupture the cell
membrane while the cell is in the peripheral
circulation
- This pathway accounts for a minor component of
normal destruction of RBCs

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HEMA LEC LONG QUIZ Question 5

Question 1 What is the youngest cell in the maturation series of the

neutrophils?
Volume of blood in an averaged size adult male is
O Metamyelocyte
O 5 to 6L
O Myelocyte
O 6 to 7 L
O Myeloblast
0 4 to 5 L
O Promyelocyte
O 3 to 4 L

Question 6
Question 2
A decrease in the oxygen-carrying capacity of the blood
What is the name of the iron-containing protein that
for any reason, is a condition known as
gives red blood cells their color?
O Leukocytosis
O Pyrite
O Thrombocytopenia
O Hemoglobin
O Anemia
O Hemocyanin
O Polycythemia
O Myoglobin

Question 7
Question 3
The normal reference range for red blood cells
The percentage of blood plasma in the blood is
O 4-6 million cells/μL
O 50%
O 150,000-400,000 cells/μL
O 45%
O 3000-7000 cells/uL
O 65%
O 4800-10,800 cells/uL
O 55%

Question 8
Question 4
Which of the following diseases has a deficiency of
If there is an increased RBC count leading to
enzymes causing hepatomegaly?
hyperviscosity of blood, the condition is known as
O Tay Sach's
O No option is correct
O All items are correct
O Polycythemia
O Gaucher's diseae
O Leukemia
O Niemann Pick
O Anemia
Question 9 O Plasma is approximately 92%

What are the little cellular fragments called that are O Blood consists of plasma and formed elements
responsible for clotting blood?
O Plasma is a straw colored clear liquid containing
O Hemoglobin cellular elements and solutes

O Leukocytes O All statements are true

O Thrombocytes

O Erythrocytes Question 14

At approximately___days, erythrocytes are removed

from the blood circulation by the reticuloendothelial
Question 10
system
Which of the following leukocytes respond when a
O 100
person is allergic to something?
O 120
O Basophils
O 150
O Lymphocytes
O 200
O Neutrophils

O Eosinophils
Question 15

If the MCV of an adult male is 100 fL, the cells are

Question 11
O Target cells
Rubricyte shows
O Microcytic
O 2 nucleoli
O Hyperchromia
O 1 nucleoli
O Macrocytic
O 0 nucleoli

O 3 nucleoli
Question 16

What is the shape of the erythrocytes when MCHC is

Question 12
increased?
In order to determine the MCH you have to divide the
O Microcytes
O Hgb by the RBC
O Target cells
O Hct by the RBC
O Sickle cells
O RBC by the Hgb
O Spherocytes
O Hct by the Hgb

Question 17
Question 13
Which of the following statements about erythrocytes is
Which of the following statements are true? correct?
O They lack a nucleus O Liver disease

O They fight infection O Megaloblastic anemia

O They are produced in the spleen

O They clot blood Question 22

The parameters required in calculating the RBC indices

include the following except
Question 18
O Hematocrit
Function/s of blood include
O Platelet count
O Defend against infection
O Hemoglobin
O Maintains body temperature
O RBC count
O All options are correct

O Transport 02
Question 23

Theory surrounding the source of stem cells which says

Question 19
that individual cell lineages have a specific stem cell
Cells that play a role in the body defense whether
O Monophylectic
cellular or humoral?
O Stoichastic
O Lymphocytes
O Polyphyletic
O Eosinophils
O Instructive
O Monocytes

O Basophils
Question 24

Single progenitor cell which can give rise to all blood

Question 20
cells
What is the normal pH of the blood?
O Pluripotent stem cell
O 7.35-7.45
O Common lymphoid progenitor
O7.20-7.30
O Common myeloid progenitor
O 7.30-7.40
O CFU-GEMM
O7.45-7.55

Question 25
Question 21
Which of the following is true regarding the mesoblastic
Mean cell volume is decreased in which of the following phase of hematopoiesis?
condition?
O Hematopoiesis occurs intravascularly
O Hemolytic anemia
O Start of definitive hematopoiesis
O Iron deficiency anemia
O Beginning synthesis of fetal hemoglobin (HbF)
O Peaks within 4 to 5 weeks age of gestation O Segmented neutrophil

Question 26 Question 30

All of the following do not occur in the medullary phase The best basis of determining cell's maturity
except
O Chromatin
O Yellow marrow replaces red marrow to long bones
O Neither the nucleus nor the chromatin
O Yolk sac begins to disappear
O Both the nucleus and chromatin
O Myeloid:erythroid ratio
O Nucleus
O Recognizable clusters of developing erythroblasts,
ganulocytes and monocytes begin to form
Question 31

The white blood cells called agranulocytes are

Question 27
O Monocytes, leukocytes, and neutrophils
All of these are changes observed in the cytoplasm of
hematopoietic stem cells except O Erythrocytes, thrombocytes, and platelets
O Decrease in basophilia O Lymphocytes and monocytes
O Decrease in proportion (vs nucleus) O Neutrophils, basophils, and eosinophils
O Change in color

O Possible appearance of granules Question 32

The non-staining areas in the nucleus of old cells are

called
Question 28
O None of the options are correct
Cellularity is described in adults as hypercellular when
the O Euchromatin
O The marrow has >30% hematopoietic cells O Parachromatin
O The marrow has <30% hematopoietic cells O Heterochromatin
O The marrow has >70% hematopoietic cells

O The marrow has <70% hematopoietic cells Question 33

The cytoplasm of the blast cell tends to strain much

darker blue because of
Question 29
O Increased amount of DNA
The metamyelocyte matures to this type of cells
O Increased amount of RNA
O Polymorphonuclear neutrophil
O Increased amount of azurophilic granules
O Band neutrophil
O Increased amount of specific granules
O Myelocyte
 Question 38

Question 34 Euchromatin is described as

What is hematocrit? O decreasingly coarse and clumped

O A sometimes fatal blood disease O non-staining areas in the nucleus of old cells

O A medicine that helps the bleeding stop O very delicate, fine and linear

O The proportion of red blood cells compared with O increasingly coarse and clumped
total blood volume

O A blood pressure measuring device

Question 39

During the fifth month of fetal development,

Question 35 hematopoiesis begins in the

The site for primitive hematopoiesis? O Spleen

O Bone marrow O Yolk sac

O Spleen O Liver

O Yolk sac O Bone marrow

O Liver

Question 40

Question 36 Which of the following are detected during the hepatic

phase of hematopoiesis?
Mean cell hemoglobin concentration is increased in
which of the following conditions? O All items are correct

O Macrocytic anemia O G-CSF

O Spherocytosis O Hemoglobin F and A

O All options are correct O Erythropoietin

O Megaloblastic anemia
Question 41

Which one of the following morphologic changes occurs

Question 37
during normal blood cell maturation?
A normal range for MCH is
O Increase in cell cytoplasm
O 34-43 pg
O Condensation of nuclear chromatin
O 26-34 pg
O Development of cytoplasm basophilia
O 50-57 pg
O Appearance of nucleoli
O 43-50 pg
Question 42 Question 46

An indicator of the average weight of hemoglobin in In severe hemol anemias, liver the conjugation of
individual cell bilirubin and the storage of iron

O MCV O Decreases, decreases

O MCH O Increases, decreases

O MCHC O Increases, increases

O RDW O Decreases, increases

Question 43 Question 47

Which of the following cells can release heparin in the After splenectomy, platelet and leukocyte count
blood? ________ transiently.

O Basophil O Have no effect

O Monocyte O Increase

O Eosinophil O Decrease

O Neutrophil O Slightly decreased

Question 44 Question 48

Which of the following reflects RBC staining intensity Which of the following forms an extracellular matrix
and amount of central pallor? promoting cell adhesion and regulating hematopoietic
stem cells?
O MCH
O Mesenchymal cells
O MCHC
O Reticular cells
O RDW
O Epithelial cells
O MCV
O Stromal cells

Question 45
Question 49
The lymph is the fluid portion of blood that escapes into
the connective tissue and is characterized by The degree of cytoplasmic basophilia correlates with
the amount of
O Both statements are correct
O ribosomal RNA
O None of the options are correct
O messenger RNA
O The absence of RBCs
O no option is correct
O A low protein concentration
O transfer RNA
Question 50 O Central pallor that is one-third the diameter of the
cell is seen in microcytic anemias
Orthochromic normoblast (metarubricyte) is
characterized by the following except

O N:C ratio of 0.5:1 Question 54

O Pale blue to salmon cytoplasm All of these are changes observed in the nucleus of
hematopoietic stem cells, except
O Hgb synthesis increases
O Decrease in size of the nucleus
O Eccentric nucleus with small, fully condensed
(pyknotic) nucleus, no nucleoli O Appearance of granules

O Chromatin condensation

Question 51 O Loss of nucleus

The signals to macrophages that initiate RBC ingestion

may include the following except
Question 55
O Binding of autologous IgG to band 3
Which of these is NOT a function of erythropoietin?
O Cation balance changes
O Increase of rate of surviving cells entering the
O Expression of phosphatidylserine on the outer circulation
membrane
O Delays the time for the reticulocytes to be released
O Binding of autologous IgM to band 3
O Increase in hemoglobin production

O Decreased in apoptosis
Question 52

Survival of RBC precursors in the bone marrow depends

Question 56
on adhesive molecules like
The "C" in CFU stands for
O Fibronectin
O Committed
O Both fibronectin and cytokines
O Corrected
O Cytokines
O Common
O Neither fibronectin nor cytokines
O Colony

Question 53
Question 57
Which of the following statements is correct?
Nucleoli are usually more prominent in these 2 RBC
O None of the statements are correct
stages
O Increased central pallor is seen w/ spherocytic
O Reticulocyte and rubriblast
disorders
O Prorubricyte and reticulocyte
O Normal cells have a central pallor that is one-third
the diameter of the cell O Pronormoblast and prorubricyte
O Polychromatic normoblast and metarubricyte

Question 58

Which of these is true regarding maturation of RBC

precursors?

O The proportion of nucleus increases, the cytoplasmic

proportion decreases

O Cytoplasmic color changes from pink to gray to blue

O Nucleoli becomes more prominent

O The nuclear chromatin becomes coarser, clumped

and condensed

Question 59

How many mature RBCs are produced from a single

pronormoblast? (di ko sure pota sabi 8-32)

0 18

02

O8

O4

Question 60

Mechanical hemolysis is

O Intravascular

O Extraembryonic

O Intrauterine

O Extravascular
HEMATOCRIT DETERMINATION 3. Centrifuge the blood at 10,000 rpm for 4-5
minutes using a microhematocrit centrifuge.
Hematocrit 4. Determine the level of packed RBCs using a
- the volume of packed red blood cells (PRBCs) after microhematocrit reader. Do not include buffy
coat in reading haematocrit because it will
centrifugation of a blood sample
- known as packed cell volume (PCV) or erythrocyte give false positive result.
volume fraction (EVF) Note: estimation of Hgb and RBC is possible on the basis of
- reported in percent (%), cell volume percent (CV%) or the Hct value under normal circumstances
volume percent (vol%)
- one of the simplest, most accurate, valuable tests in 1% haematocrit = 0.34 gm % hemoglobin
haematological investigation
- more useful than RBC count in detecting cases of = 107,000 RBC/mm^3
anemia
- RBC indices can be computed manually from RBC
count values, Hgb levels, and Hct level.

Materials needed

• EDTA blood
• Microhematocrit capillary tube (blue/red)
• Disposable sterile blood lancet
• Wintrobe tube
Other Methods
• Microhematrocrit centrifuge
• Sealing clay • Wintrobe Method
• Microhematocrit reader - This method utilizes a Wintrobe tube with
two calibrations, 0 to 10 (top to bottom)
Methods which is used for erythrocyte rate (ESR) and
• Adam’s Microhematocrit Method (most commonly 10 to 0 (bottom to top), which is used for
used) haematocrit
1. Fill around ¾ of the capillary tube with blood.
If the blood is from a skin puncture, use
heparinized (red) capillary tube. A non-
heparinized (blue) tube us used if blood
collected with anticoagulant

- Anticoagulant of choice is double oxalate

Procedure:

2. Seal one end of the capillary tube with 1. Fill Wintrobe tube with blood using Pasteur pipette.
sealing clay (about 3mm). press it slightly at Insert the pipette well and slowly raise it up to avoid
the clay. bubbles
2. Centrifuge tube at 3000 rpm for 30 minutes
 3. Read the volume of packed RBCs Procedure:
4. Compute for the Hct level using the formula:
1. Place 1 mL of anticoagulant into the tube
2. Add 5mL go benou blood and mix
3. Centrifuge mixture at high speed for 15 minutes

• Haden’s Modification
- The anticoagulant is 1.1% sodium oxalate in
distilled water.
- Uses a calibrated tube

Procedure: • Bray’s Method

- Anticoagulant is heparin and a Bray’s tube is
1. Place 1 mL of 1.1% sodium oxalate in the tube used.
2. Add 5 mL of blood. Mix well - This tube is calibrated on both sides similar
3. Centrifuge the mixture for 20 minutes at 3,000 rpm to the Winstrobe tube
4. Read the volume of packed RBCs - The calibration is from 10-50 mm. each
division is 1mm and the capacity is 5 mL

Procedure:

1. Fill the tube with heparinized blood

2. Let the tube stand at a vertical position for one hour
3. Read the volume of RBCs from the lower right side
calibration

• Van Allen Method

- Anticoagulant is 1.6% sodium oxalate in
distilled water.
- Uses a tube with bulb and calibration of 1 to
10 cm or 10 to 100 mm
Automated Method
Procedure:
• Coulter counter
1. Fill the tube with blood up to the 10th mark • Autoanalyzer
2. Dilute the blood with the diluting fluid up to the bulb
about half full
3. Seal the tube and centrifuge with shaft end down at
2500 rpm for 15 to 30 minutes
4. Read volume of packed RBCs

Note: each unit of division is equal to 1%

• Sanford-Magath
- Anticoagulant is 1.3% sodium oxalate, the
tube is calibrated at 1mm per division
- The tube is about 5 inches long with a
funnel-like mouth
ERYTHROCYTE SEDIMENTATION RATE

- Refers to the speed of the settling or RBCs in

anticoagulated blood
- It is the measure of the distance and speed of fall of
RBCs in the plasma in a tube of a standard bore and Westergren Method
length after standing perpendicularly
- Most important factor influencing ESR is the action of - Most sensitive for ESR determination
plasma proteins - Can be used for the serial study of chronic diseases
like tuberculosis, carcinoma, etc.
Stages of ESR
- Has a smaller bore
1. Initial period of aggregation of rouleaux formation – 10 Procedure:
minutes
2. Period of fast settling – 40 minutes 1. Fill the Westergen tube with blood using a rubber
3. Final period of packing – 10 minutes aspirator
2. Let the tube stand vertically on a Westergen rack
Total of 60 minutes / 1 hour 3. Record the ESR in millimetres after 1 hour
Importance of ESR

➢ It is used as an index of the presence of an active

infection
➢ It measures the suspension stability of RBCs
➢ It indicates abnormal concentration of fibrinogen,
globulin, and other plasma proteins

Materials Needed

• Unclotted blood
• Wintrobe tube
• Timer
• Test Tube Rack
• Pasteur pipette / syringe with cannula
• Westergen tube
• Westergen rack
• Rubber aspirator

Wintrobe-Landsberg Method

- Used in majority of cases and is quite accurate

- Advantages include outweighing of few drawbacks
- Uses the Wintrobe tube, calibrated on two sides (0-
10, 10-0
- Commercially available

Procedure:

1. Fill the Wintrobe tube with blood with a Pasteur

pipette or cannula attached to a syringe Let the blood go up by capillary action and let it stand for 1
2. Place the tube in a vertical position on a rack hour. Blood should be in the 0 mark, if it does and is
3. After letting the tube stand for one hour, record the irreversible, cancel the whole processes.
ESR in milimeters
 3. Allow the tube to stand in an upright position until the
RBCs settle at the 18mm mark
4. Note the time for this event to take place. Record in
minutes

B. Micro Methods
• Micro Laundau (a modification of
Linzenmeier-Raunert) – anticoagulant is 5%
sodium citrate, that uses a Micro-Landau
tube which is calibrated 0-50 mm and has
two graduation marks, one at 12.5 mm and
another at 62.5 mm, with a small bulb similar
to RBC and WBC pipettes
Other Methods of ESR Determination
Procedure:
A. Macro Methods 1. Draw 5% sodium citrate by turning the top-screw to
• Graphic or Cutter – anticoagulant of choice is 3% the left until the upper meniscus of the solution
sodium citrate, uses Cutler tube which has a 5 reaches the lower mark A
mL capacity; graduation of 0-50 mm 2. Draw up the blood until the height of the liquid
Procedure: reaches the upper meniscus of graduation B
3. Clean the tip of the tube with ether, then continue to
1. Add 0.8 mL of blood to 0.5 mL of 3% sodium citrate draw up the mixture into the bulb by turning the top-
2. Close the tube with paraffin-coated cork, then mix screw to the left until the lower meniscus of the blood
3. Allow the tube to stand for 1 hour, observing every column ends just a few millimetres below the lower
five minutes opening of the bulb. Do not draw the blood into the
4. Compare results with normal values in the bulb completely
sedimentation chart 4. Shake the tube carefully
5. Force the blood up and down twice very slowly by
• Linzenmeier method – anticoagulant of choice is turning the screw to the left then to the right
3% sodium citrate, uses Linzenmeier tube which 6. Set the tube vertically on a rack and read the ESR at
is 65 mm in length, 5 mm in diameter, and has a the end of one hour
capacity of 1 mL (with a mark of18 mm) • Smith Micro – used for infants and when
venipuncture children may not be practiced
Procedure:

1. Add 0.8 mL of blood of 0.02 mL of 3% sodium citrate

2. Mix and pour the mixture into the tube up to the 1 mL
mark.
 Procedure:

1. Fill the special pipette with 5% sodium citrate and

expel 0.04 mL
2. Draw capillary blood with the same pipette. Three
successive batches of 0.1 mL are collected and
expelled into the tube containing the citrate. Thorough
shaking is necessary to ensure adequate mixing and
prevent coagulation
3. The blood is transferred to the special sedimentation
tube using a capillary pipette and the test is
completed in the usual manner

• Crista / Hellige-Vollmer

C. Automated Methods
• Automated ESR system by Vega Biomedical

The automated ESR system is a fully automated

instrument for ESR determination. One mL of blood is
collected from an evacuated tube, containing liquid
sodium citrate. The tube is then placed in the Ves-
Matic analyser where it is automatically mixed,
allowed to settle, and read. Results are comparable to
the Westergren method, and it takes only 22 minutes
to finish.

Mini-Ves = four samples at one time

Ves-Matic = 20 samples at one time, prints results

Ves-Matic 60 – 60 samples at one time, prints results,

identifies sample by a barcode reader

Clinical Significance of ESR

- The ESR is a non-specific test. It is raised in a wide

range of infectious, inflammatory, degenerative, and
malignant conditions associated with changes in
plasma proteins, particularly increases in fibrinogen,
immunoglobulins, and C-reactive protein
- The ESR is also affected by other factors like anemia,
pregnancy, hemoglobinopathies, hemoconcentration,
and treatment with anti-inflammatory drugs
ERYTHROCYTE METABOLISM AND MEMBRANE Energy Production and Anaerobic Glycolysis
STRUCTURE AND FUNCTION
- mature RBC relies on anaerobic glycolysis for its
- The RBC is the primary blood cell, circulating at 5 energy – lack of mitochondria
million RBCs per microliter of blood on average. - glucose enters RBC with no energy expenditure via
- It is anucleated and biconcave and has an average the transmembrane protein Glut-1
volume of 90 fL - erythrocyte metabolic processes requiring energy:
- The cytoplasm provides abundant hemoglobin, a • intracellular cationic gradient maintenance
complex of globin, protoporphyrin, and iron that • cytoskeletal protein deformability
transports elemental oxygen from the lung capillaries • prevention of the peroxidation of proteins
to the capillaries of organs and tissues. and lipids
- Hemoglobin, plasma proteins, and additional RBC • maintaining cytoplasmic cationic
proteins also transport molecular carbon dioxide and electrochemical gradients
bicarbonate from tissues to lungs
- Hemoglobin is composed of four globin molecules, Pathways of Hemoglobin
each supporting one heme molecule. Each heme
molecule contains a molecule of iron. • Embden-Meyerhof Pathway/EMP
- The biconcave RBC shape supports deformation or - anaerobic EMP metabolizes glucose to
flexibility, enabling the circulating cell to pass pyruvate, consuming two ATP molecules
smoothly through capillaries, where it readily - EMP subsequently generates four ATP
exchanges oxygen and carbon dioxide while molecules per glucose molecule, a net gain
contacting the vessel wall. of two.
- RBCs are produced through erythrocytic • Hexose-Monophosphate Pathway (HMP)
(normoblastic) maturation in bone marrow tissue. - Aerobically converts glucose to pentose and
- The nucleus while present in maturing marrow generates NADPH
normoblasts, become extruded or rejected as the cell - NADPH reduces glutathione, which reduces
passes from the bone marrow to peripheral blood. peroxides and protects proteins, lipids, and
- Cytoplasmic ribosomes and mitochondria disappear heme iron from oxidation.
24 to 48 hours after bone marrow release, eliminating • Methemoglobin Reductase Pathway
the cell’s ability to produce proteins or support - Converts ferric heme iron (valence 3+ iron,
oxidative metabolism methemoglobin) to reduced ferrous (valence
- ATP is produced within the cytoplasm through 2+ form) which bind oxygen
anaerobic glycolysis (Embden-Meyerhof • Rapaport-Luebering Pathway
pathway/EMP) for the lifetime of the cell - Generates 2,3 BPG and enhances oxygen
- ATP drives mechanisms that slow the destruction of delivery to tissues
protein and iron by environmental peroxides and
RBC Membrane Deformability
superoxide anions, maintaining hemoglobon’s
function and membrane integrity - Excess surface-to-volume ratio enables RBCs to
- Oxidation, however, eventually takes a toll, limiting stretch undamaged up to 2.5 times their resting
the RBC circulating life span to 120 days, whereupon diameter as they pass through narrow capillaries and
the cell becomes disassembled into its reusable through splenic pores 2 um in diameter (RBC
components globin, iron, and the phospholipids and deformability)
proteins of the cell membrane while the - Depends on RBC geometry and cytoplasmic (Hgb)
protoporyphrin ring is excreted as bilirubin. viscosity
- RBC energy production, the protective mechanisms
that preserve structure, function, deformability and RBC Membrane Lipids
maintenance of the cell membrane – form the basis
- RBC membrane is a lipid bilayer whose hydrophobic
for understanding RBC disorders (anemia)
components are sequestered from aqueous plasma
and cytoplasm
 - The phospholipid membrane provides a ➢ Serve as transport and adhesion sites
semipermeable barrier separating plasma from and signalling receptors
cytoplasm and maintaining an osmotic differential ➢ Channel ions, water, and glucose and
- Phospholipids are asymmetrically distributed anchor cell membrane receptors
o Phosphatidylcholine and sphingomyelin ➢ They also provide the vertical support
predominate in outer layer connecting the lipid bilayer to the
o Phosphatidylserine (PS) and underlying cytoskeleton to maintain
phosphatidylethanolamine in inner layer membrane integrity
- Distribution of these four phospholipids is energy - The shape and flexibility of the RBC are
dependent, relying on a number of membrane essential to its function depend on the
associated enzymes termed flippases, floppases, and cytoskeleton
scramblases, for their positions ➢ Cytoskeleton is derived from a group of
- When phospholipid distribution is disrupted as in peripheral proteins on the interior of the
sickle cell anemia and thalassemia or in RBCs that lipid membrane
have reached the 120 day life span, PS redistributes - The major structural proteins are a and B-
to the outer layer spectrin which are bound together and
- Membrane phospholipids and cholesterol may also connected to transmembrane proteins by
redistribute laterally so that the RBC membrane may ankyrin, actin, protein 4.1, adducing,
respond to stresses and deform as they pass through thropomodulin, dematin, and band 3
a narrow passage - Cytoskeletal proteins provide horizontal support
- In liver disease with low bile salt concentration, for the membrane
membrane cholesterol concentration becomes - RBC cytoplasm K+ concentration is higher than
reduced. As a result, the more elastic cell membrane plasma K+ whereas Na+ and Ca+
shows a “target cell” (codocyte) microscopically concentrations are lower
- Acanthocytosis (spur cells) and target cells (Hgb ➢ Disequilibria are maintained by
concentration in the center of the RBC and around the membrane enzymes K+ ATPase, Na+
periphery to resemble a bull’s eye) are associated ATPase, and Ca+ ATPase.
with abnormalities in the concentration and ➢ Pumpl failure leads to Na+ and water
distribution of membrane cholesterol and influx, cell swelling, and lysis
phospholipids • Spectrin – major cytoskeletal protein forming a lattice
- Glycolipids (sugar bearing lipids) may bear copies of at the cytoplasmic surface of the cell membrane,
carbohydrate-based blood group antigens of the ABH providing lateral support to the membrane and thus
and the Lewis blood group systems maintaining its shape
- Any disruption in transport protein function changes o Abnormalities account for hereditary
the osmotic tension of the cytoplasm, which leads to spherocytosis, ovalocytosis, and
rise in viscosity and loss of deformability pyropoikilocytosis
- RBC membrane cholesterol is replenished from the ▪ Hereditary spherocytosis arises
plasma from defects in proteins that provide
vertical support for the membrane
RBC Membrane Proteins ▪ Hereditary elliptocytosis is due
- any change affecting adhesion proteins permits RBCs defects in cytoskeletal proteins that
to adhere to one another and to the vessel walls provide horizontal support for the
promoting fragmentation (vesiculation) reducing membrane
membrane flexibility, shortening the RBC life span • Glycophorin A – transmembrane or integral
- signal transduction, a process whereby signalling membrane protein
receptors bind plasma ligands and trigger activation of o Abnormalities in the horizontal and vertical
intracellular signalling proteins which then initiate linkages of the transmembrane and
various dependent cellular activities cytoskeletal RBC membrane proteins may
• Transmembrane proteins be seen as shape changes
DIFFERENTIAL WHITE BLOOD CELL COUNT 3. Dip in solution 3 (methylene blue, basic dye) for 4
seconds
- the linear representation of the percentage of the 4. Dip in buffer solution / aged distilled water for 45
various types of leukocytes in the peripheral or seconds
venous blood, known as the hemogram 5. Air dry
- the determination of the percentage of each type of
WBCs in the peripheral blood Differential Counting
- consists of the enumeration of the relative proportion
of the various types of WBCs as seen as stained 1. Prepare a stained blood smear
blood smears 2. Place one drop of cedar oil on the feathery edge of
the stained blood smear
Steps in Making a Differential Count 3. Examine the smear using LPO of the microscope.
Focus on area where the RBCs are not too
1. Making the blood smear overlapping or too scanty
2. Staining the blood smear 4. Shift to OIO. Using the strip differential method, count
3. Counting the cells 100 WBCs while differentiating them.
4. Reporting the results

Fixation of Blood Smears

- To preserve the cell morphology, films must be fixed

ASAP after they have dried
- It is important to prevent contact with water before
fixation is complete
- Methyl alcohol (methanol) is the chose, although ethyl
alcohol (absolute alcohol) can be used
- Methylated spirit (95% ethanol) must not be used as it
contains water Counting the Cells
- To fix films, place them in a covered staining jar in
tray containing the alcohol for 2-3 minutes 1. Strip Differential: all the cells are counted in the
- In humid climates, it might be necessary to replace longitudinal strip that is, from the head to the tail of
the methanol 2-3 times per day, the old portions can the smear
be used for storing clean slides 2. Exaggerated battlement: the count starts at one edge
of the smear and counting all the cells, advancing
What are Needed: inward to 1/3 of the width of the smear, then on the
line parallel to the edge, then out of the edge, then
• Blood smear
along the edge
• Methanol 3. Two-field Meander method: the count is made by
• Eosin, methylene blue dividing the smear into two fields and proceeds as
• Compound light microscope exaggerated battlement method
• Buffer solution pH 7.2/aged distilled water 4. Four-field Meander method: the count is made by
• Differential counter dividing the smear into four fields and proceeds as
• Cedar wood oil exaggerated battlement method
• Xylol
• Xylol-alcohol

Staining Jar/DIP method

1. Dip in solution with fixative (methanol) for 30 seconds

2. Dip in solution 2 (eosin, acidic dye) for 6 seconds
Goal: 100 WBCs Monocytes

Neutrophilic Segmenter - Nucleus is spongy and sprawling with brain-like

convolutions
- Nucleus is broken into segments but still connected - Cytoplasm is gray. Vacuoles are sometimes present.
by a fine strand - NV of relative count: 2-11% (CU)
- Cytoplasm contains small pinkish granules - NV of absolute count: 450-1,300/cu. mm.
- NV of relative count: 50-70% (CU)
- NV of absolute count 2,300-8,100 / cu. Mm

Eosinophil

- Nucleus is usually bilobed

- Contains large, coarse, reddish, or orange granules
Neutrophilic Band (stab / staff) - NV of relative count: 1-3% (CU)
- Younger form of neutrophil with C, S, U, or horseshoe - NV of absolute count: 0-400/cu. mm.
shaped nucleus
- Nucleus is continuous, no cut or division
- NV of relative count: 0-5% (CU)
- NV of absolute count: 0-600/cu. mm.

Basophil

- Nucleus is usually indistinct and obscured by the

granules
- Cytoplasm contains large purplish-black or dark blue
Lymphocyte
granules
- Nucleus is compact or intact and usually round - NV of relative count: 0-2% (CU)
- Cytoplasm is light blue and scanty
- NV of relative count: 18-42% (CU)
- NV of absolute count: 800-4,800/cu. mm.
 - NV of absolute count: 0-100/cu. mm o Combinations have two essential ingredients
(i.e., methylene blue and eosin or azure)
o Most are prepared in methyl alcohol to
combine fixation and staining
o Includes Giemsa and Wright’s
▪ Giemsa stain is recommended and
most reliable procedure, excellent
for staining thin and thick blood
films (inclusion bodies and
intracellular parasites as well as for
staining WBCs)
➢ It is composed of eosin
and azure blue, methylene
blue in methanol and
glycerin. The eosin
component stains the
parasite nucleus red while
Staining of Blood Smears the methylene blue
component stains the
- The microscopic study of stained, peripheral blood cytoplasm blue.
smear constitutes the most important part of routine ▪ Wright’s stain is a histologic stain
haematological examination that facilitates the differentiation of
- Cytochemical stains are essential for the identification blood cell types.
of hematopoietic cells ➢ It is classically a mixture of
- The most commonly used stains are polychrome eosin azures and oxidized
stains, those belonging to the Romanowsky group methylene blue dyes.
- A polychrome stain is a stain of many colors and the ➢ It is used primarily to stain
original polychrome stain was discovered by PBS, urine samples, and
Romanowsky bone marrow aspirates
- Polychrome methylene blue and eosin stains are the which are examined under
outgrowth of the original time-consuming light microscope
Romanowsky method and are widely used o Panoptic stains – consists of Romanowsky
- They stain differently most normally and abnormal and another dye to improve cytoplasmic
structures in the blood granules
- Intravital stain is used to stain the tissue by a dye ▪ Examples: Wright’s-Giemsa,
which is introduced into a living organism and which, Jenner-Giemsa, May-Grunwald-
by virtue of selective attraction to certain tissues, will Giemsa
stain these tissues. • Methylene blue
- Supravital stain is used to stain and inspect living o Basic dye
cells which have been removed from the body. It o Has affinity for acidic component of the cell
enables the cells to remain alive and mobile, but it (nucleus)
does not stain the nucleus or cytoplasm. It does stain • Eosin/azure
significant structures in cytoplasm (ex. Reticulocyte o Acidic dye
count)

Various Stains for Peripheral Blood Film:

• Romanowsky Stain
o Employed for staining blood films
 o Has affinity for basic component of the cell - A pump tube set is installed to transport
(cytoplasm) each solution
- Tubing 1 = stain solution; Tubing 2 =
buffer solution; Tubing 3 = rinse solution
c. Hema-Tek 2000 Slide Stainer
- Stainer employs the same principle as
Hema-Tek 1000.
- The innovation is the improved staining
system through the use of new pumps
and volume controls.
- The operator can electronically adjust the
stain, buffer, and rinse solution

Stations in Automated Method

Station 1 – methanol (500mL)

Station 2 – Wright’s or Wright’s-Giemsa stain (500mL)

Station 3 – stain buffer mixture, Wright’s or Wright’s-Giemsa

(80 mL), phosphate buffer (420 mL)

Station 4 – deionized water (1000 mL)

Station 5 – phosphate buffer (500 mL)

Station 6 – warm air

Other Methods of Staining

1. Staining dish method involves placing the blood

smear in a rack positioned on a dish.
Staining of Two-coverglass
2. Automated method
a. Hemastainer automatic slide stainer
- Freshly prepared staining solutions are
used daily or every 4-8 hours during
operations
b. Hema-Tek 1000 Slide stainer
- Bottles of the Stain-Pak (stain, buffer,
and rinse solutions) are opened by
making a small hole and a cannula is
inserted into each solution.
Methods of Classification of Cells in Differential Count Shifting Processes

1. Schilling hemogram • Shift to the left – if there is an increase in younger

- In this method, all leukocytes are grouped forms of WBCs particularly classes I and II; seen in
according to maturity of cells into pyogenic infections
• Granulocytes – neutrophils, • Shift to right – if there is an increase in older forms of
eosinophils, basophils leukocytes particularly classes IV and V; seen in
• Non-granulocytes – lymphocyte and megaloblastic anemia and in convalescence
monocytes
- The PMNs are further classified according to
myelocytes, metamyelocytes, bands or 3. Haden’s classification
stabs, segmenters - Classifies the neutrophil according to the
presence of filaments.
- These neutrophils whose lobes are
connected by thin filaments are classified as
filamented, while those that are not
connected by filaments are grouped under
non filamented cells
• Filamented cells = 60%
• Non-filamented cells = 7%
• Eosinophils = 3%
2. Arneth’s classification
• Basophils = 1%
- The PMNs are classified according to the
• Lymphocytes = 21%
number of lobes which their nuclei possess.
• Monocytes = 8%
The more lobes, the older the cells.
• Class I – with lobe or indented Automated Differential Count
nucleus (5%)
• Class II – 2 lobes (35%) Two general principles:
• Class III – 3 lobes (41%)
1. Digital image processing – a uniformly made and
• Class IV – 4 lobes (17%) stained blood film is placed on a microscope slide,
• Class V – oldest with 5 lobes (2%) which is driven a motor. A computer controls the
Under the traditional unit, the results in differential leukocyte movement, scanning the slide and stopping it when
count are reported in percentage. leukocytes are in the field. The optical images are
then recorded by television camera, analyzed by
Under the SI unit, the proportion of each type of cell is reported computer and converted to digital form
as a decimal fraction and is called leukocyte type number 2. Flow through system – this system analyze the cells
fraction. suspended in a liquid. In photo-optical system,
measurement of light scattering and f light absorption
• Regenerative shift to the left – if predominating cells are made while the cells are being counted
are younger forms with the presence of myelocytes
and metamyelocytes and increase in band cells and it Overstained Smears
is accompanied by a high leukocyte count.
Causes:
• Degenerative shift to the left – if predominating cells ➢ Too thick smears
are younger forms, with an increase in band cells but ➢ Insufficient washing
without myelocytes and metamyelocytes and it is ➢ Too prolonged staining time
accompanied by low WBC count. ➢ Excessive alkalinity of the stain, buffer or water
Appearance of Cells:

➢ Erythrocyte stains blue or green

➢ Cytoplasm of the lymphocytes become gray or
lavender
➢ Granules of neutrophils are intensely overstained
➢ Eosinophilic granules become deep gray or blue

Understained Smears

Causes:

➢ Too thin smears

➢ Excessive washing of the smears
➢ Excess acidity of the stain, buffer, or water

Appearance of Cells:

➢ Nuclear chromatin is stained pale blue rather than

vivid blue
➢ Erythrocyte stains bright red or orange rather than
pink
➢ Eosinophilic granules stain brilliant red

Precipitated Stain Between Cells

Causes:

➢ Unclean slide or coverglass

➢ Faulty washing because of failure to hold the slide
horizontally and to float off the scum
➢ Permitting dust to settle on the film

Poor Staining

➢ Alkaline slides and alkaline distilled water

➢ Acid slides and acid distilled water
➢ Unclean slides
➢ Evaporation of the stain
➢ Incorrect buffer pH
➢ Imperfect polychroming of the stain
➢ Incomplete reaction of the staining fluid
➢ Error of the operator
Hemoglobin Metabolism (HEMA LEC 6TH WEEK) deoxygenated (not carrying an oxygen
molecule)
Heme Structure • The complete hemoglobin molecule is spherical,
has four heme groups attached to four
• Heme consists of a ring of carbon, hydrogen and
polypeptide chains, and may carry up to four
nitrogen atoms called protoporphyrin IX, with a
molecules of oxygen
central atom of divalent ferrous iron
• Each of the four heme groups is positioned in a Hemoglobin synthesis
pocket of the polypeptide chain near the
surface of the hemoglobin molecule • 65% hemoglobin synthesis occurs in immature
• The ferrous iron in each heme molecule nRBCs
reversibly combines with one oxygen molecule • 35% hemoglobin synthesis occurs in
• When the ferrous irons are oxidized to the ferric reticulocytes. Heme synthesis occurs in the
state, they no longer can bind oxygen. mitochondria of normoblasts and is dependent
on glycine, succinyl, coenzyme A, aminolevulinic
• Oxidized hemoglobin is also called
acid synthase (aminolevulinate synthase), and
methemoglobin
vitamin B6 (pyridoxine)
• Four identical heme groups, each consisting of a
protoporphyrin ring and ferrous iron
• Four globin (polypeptide) chains
o Alpha chains have 141 amino chains
o Beta chains and delta chains have 146
amino acids
• The amino acid sequence of the globin chain
determines the type of hemoglobin, normal
adult hemoglobin consists of two alpha and two
nonalpha chains in pairs

Complete Hemoglobin Molecule

• The hemoglobin molecule can be described by

its primary, secondary, tertiary, and quaternary
protein structures
• Primary structure refers to the amino acid
sequence of the polypeptide chains
• Secondary structure refers to chain
arrangements in helices and non-helices • Globin synthesis occurs in the ribosomes and it
• Tertiary structure refers to the arrangement of is controlled on chromosome 16 for alpha
the helices into a pretzel like configuration chains and chromosome 11 for all other chains
• Quaternary structure is also known as tetramer • Each globin chain binds to a heme molecule in
which describes the complex hemoglobin the cytoplasm of the immature RBC
molecule. The globin chains dissociate into
alphaBeta dimmers Hemoglobin/Erythrocyte Breakdown
• Globin chain amino acids in the cleft are
Intravascular hemolysis (10%)
hydrophobic, whereas amino acids on the
outside are hydrophilic, which renders the - It occurs when hemoglobin breaks down in the
molecule water soluble. blood and free hemoglobin is released into the
• This arrangement also helps iron remain in its plasma
divalent ferrous form regardless of whether it is - Free hemoglobin binds to haptoglobin (major
oxygenated (carrying an oxygen molecule) or free hemoglobin transport protein), hemopexin
 and albumin. And all of them are phagocytized o Functions in a reduced oxygen
by the liver macrophages environment Predominates at birth
- Laboratory: Increased plasma hemoglobin, (80%)
serum bilirubin, serum LD and urine o Gamma chain production switches over
urobilinogen; hemoglobinuria and to chain production and is complete by
hemosiderinuria present, decreased serum 6 months of age
haptoglobin. o We can detect the presence of the fetal
hemoglobin in the laboratory through:
Extravascular hemolysis (90%)
▪ Alkali denaturation test
- Occurs when senescent or old RBC are Kleihauer-Berke acid elution
phagocytized by macrophages in the liber or (Hgb F is resistant to
spleen denaturation or elution)
- Protoporphyrin ring metabolized to bilirubin ▪ Column chromatography
and urobilinogen excreted in urine and feces ▪ Radial immunodiffusion
- Globin chains are recycled in the amino acid ▪ Column chromatography and
pool for protein synthesis Radial immuno diffusion are
- Iron binds to transferrin and is transported to usually done in Clinical section
bone marrow for the production of new RBC in Chemistry and serology
o A compensatory hemoglobin and can be
Hemoglobin and Iron increased in homozygous and
hemoglobinopathies and beta-thalassemia
• Most iron in the body is in hemoglobin and major
must be in the ferrous state (Fe²+) to be used.
• Adult
Fe2+ binds to oxygen for transport to lungs and o Hgb A
body tissues ▪ 2 alpha and 2 beta-globin chains
• We can determine or detect extravascular or ▪ Subdivided into glycosylated
the presence of the transferrin. Transferrin fully fractions
saturated or we can also measure storage iron ▪ A1c fraction reflects glucose levels
in tissues and bone using the following in the blood
laboratory assays ▪ It monitors individuals with
• Ferric iron (Fe³+) is not able to bind to diabetes mellitus Blood is examined
hemoglobin, but does bind to transferring after 3 months in order for us to
• Iron is an essential mineral and is not produced reflect the glucose levels in the
by the body blood and to indicate if the patient
o Serum iron measures the amount of has diabetes mellitus
Fe3+ bound to transferring o Hgb A2
o Total iron-binding capacity (TIBC) ▪ 2 alpha and 2 delta-globin chains
measures the total amount of iron that o Reference range for adults is 97% Hb A, 2%
transferrin can bind when fully Hb A2 and 1% Hb F
saturated
o Serum ferritin is an indirect Different Forms of Hemoglobin
measurement of storage iron in tissue
• Oxyhemoglobin - with Fe²+ O2 seen in arterial
and bone marrow
circulation
Types of Hemoglobin • Deoxyhemoglobin with Fe2+ but no 0₂ seen in
venous circulation
• Hgb F (Fetal Hemoglobin) • Carboxyhemoglobin
o Two alpha and two gamma-globin o With Fe2+ and carbon monoxide (CO)
chains o Has 200x affinity for CO than O2, so CO
is carried instead of O2
 o Result in death but is reversible if given This conformation of the hemoglobin molecule
pure O2 (gives red color to the blood) in the oxygenated form is termed the relaxed
• Sulfhemoglobin (R) state
o With S • In this condition, the faces of hemoglobin
o Cannot transport oxygen dimers have moved apart to bind oxygen.
o Seldom reaches fatal levels However, in the veins and tissues, the oxygen
o Caused by drugs and chemicals tension is lower. The hemoglobin molecule
o Irreversible Not measured by the picks up and binds oxygen while in the capillary
cyanmethemoglobin method system of the lungs
• Methemoglobin • As the hemoglobin travels through the tissue
o With Fe³+ capillaries, in which the oxygen concentration is
o Cannot transport O2 decreased, it releases this oxygen to the tissues.
o Increased levels cause cyanosis anemia The deoxygenated form of hemoglobin is called
o We can see an increase levels in the tensed (T) state (lowest affinity for oxygen)
cyanotic and anemic patients
Oxygen Dissociation Curve
Hemoglobin
• The oxygen affinity is the ability of hemoglobin
• Cyanmethemoglobin to bind or release oxygen. Expressed in terms of
o Reference method for hemoglobin assay oxygen tension at which hemoglobin is 50%
o Principle saturated with oxygen
▪ Lysing agent (cyanmethemoglobin • The relationship between oxygen tension and
reagent) frees hemoglobin from hemoglobin saturation with oxygen is described
the RBCs. Free hemoglobin by the oxygen dissociation curve
combines with the potassium • If we're going to plot the oxygen dissociation
ferricyanide (cyanmethemoglobin curve, it may have sigmoidal shape (S) and the
reagent), converts hemoglobin steep is at 50%
iron from ferric state to form o Right shift decreases oxygen affinity,
methemoglobin, then combines more oxygen release to the tissues
with pigment o High 2,3 biphosphoglycerate level or
cyanmethemoglobin. Measured increased body temperature; decreased
potassium cyanide to form the body pH
stable using spectrophotometer) • Left shift increases oxygen affinity, less oxygen
▪ The cyanmethemoglobin color release to the tissues
intensity is proportional to • Low 2,3 biphosphoglycerate level or decreased
hemoglobin concentration, is body temperature; increased body pH
measured at 540 nm
spectrophotometrically and Introduction to Iron
compared with a standard
• Iron
▪ Other instruments used sodium
o Critical for transport and use of oxygen
lauryl sulfate (SLS) to convert
that the body conserves and recycles it
hemoglobin to
o Does not have a mechanism for its
SLSmethemoglobin. This method
active excretion
does not generate toxic wastes
o Most essential trace element
Function of Hemoglobin o Free radical production by iron ions
severely damages cells and thus
• When the oxygen tension in arterial blood is demands regulation. The body adjusts
high (about 95mmHg), the hemoglobin its iron levels by intestinal absorption,
molecule is about 95% saturated with oxygen.
 depending on need. Iron is distributed
into three compartments
o The largest percentage of body iron,
nearly 65% of it, is held within
hemoglobin in red blood cells of various
stages while about 25% of body iron is
in storage, mostly within macrophages
and hepatocytes.
o The remaining 10% is divided among
muscles, the plasma, the cytochromes
of cells, and various iron-containing
enzymes within cells.
o Three compartments
▪ Functional compartment - the
largest percentage of body iron,
nearly 65% of it, is held within • Iron chemistry
hemoglobin in RBCs of various o The metabolic functions of Iron depend
stages, iron in myoglobin on its ability to change its valence state
(muscles), and cytochromes (in from reduced ferrous (Fe+2) Iron to the
all cells) oxidized ferric (Fe+3) state
▪ Storage Compartment (25%) - o Thus, it is involved in oxidation and
the iron that is not currently reduction reactions such as the electron
functioning but is available transport within mitochondrial
when needed. The major cytochromes
sources of this stored iron o In cells, ferrous iron can react with
(ferritin and hemosiderin) are peroxide via the Fenton reaction,
the macrophages and forming highly reactive oxygen
hepatocytes, but every cell, molecules
except mature red blood cells, o The resulting hydroxyl radical (OH), also
stores some iron known as a free radical, is especially
▪ Transport Compartment (10%) reactive as a short lived but potent
- the iron that is in transit from oxidizing agent, able to damage
one body site to another in the proteins, lipids, and nucleic acids
plasma and transferrin carries • Iron Kinetics
iron in its ferric form o Systemic body iron regulation
▪ The total amount of iron
available to all body cells,
systemic body iron, is regulated
by absorption into the body
because there is no mechanism
for excretion
▪ In the lumen of the small
intestine, ferrous iron is carried
across the luminal side of the
enterocyte by divalent metal
transporter 1 (DMT 1)
o Ferroportin
▪ Once iron has been absorbed
into enterocytes, it requires
 another transporter,
ferroportin, to carry it across
the basolaminal enterocyte
membrane into the
bloodstream, thus truly
absorbing it into the body
▪ Is the only known protein that
exports iron across cell
membranes
▪ When the body has adequate
stores of iron, the hepatocytes
sense that and will increase
production of hepcidin, a
protein able to bind to
ferroportin (transports iron out
of macrophages, hepatocytes,
and enterocytes), leading to its
inactivation
▪ As a result, iron absorption into
the body decreases
▪ When the body iron begins to
drop, the liver senses that
change and decreases hepcidin
production
▪ As a result, ferroportin is once
again active and able to
transport iron into the blood.
Thus, homeostasis of iron is
maintained by modest
fluctuations in liver hepcidin
production in response to body
iron status and regulates
transfer of iron from the
enterocyte into the plasma
▪ The mechanism by which the
hepatocytes are able to sense
iron levels and produce
hepcidin is highly complex, with
multiple stimulatory pathways
likely involved
• Functions and locations of proteins involved in • Iron transport
body iron sensing and hepcidin production o Ferrous iron is exported from the
enterocyte into the blood is ferrous and
Protein Function
must be converted to the ferric form for
Hemochromatosis A protein that is bound transport in the blood
(HFE) to transferrin receptor o Hephaestin, a protein on the
1 (TfR1) until released basolaminal enterocyte membrane, is
by the binding of able to oxidize iron as it exits the
transferrin to TfR1 enterocyte
o Once oxidized, the iron is ready for
plasma transport, carried by a specific
Transferrin A hepatocyte
receptor 2 transferrin receptor protein, apotransferrin (ApoTf)
that is able to bind o Once iron binds, the molecule is known
freed HFE to initiate an as transferrin (Tf). Apotransferrin binds
internal cell signal for two molecules of ferric iron
hepcidin production • Cellular iron absorption and disposition
o Individual cells adjust the number of
transferrin receptors on their surface to
Bone morphogenic The ligand secreted by regulate the amount of iron they
protein (BMP) macrophages that absorb; receptor numbers rise when
initiates signal the cell needs additional iron but
transduction when it
decrease when iron in cell is adequate
binds to its receptor in
o Truncated soluble transferrin receptors
a cell membrane
are also shed into the plasma in
Bone morphogenic A common membrane
protein receptor receptor initiating proportion to their number of cells
(BMPR) signal transduction o Cells store iron as ferritin when they
within a cell when its have an excess. Iron can be released
ligand (BMP) binds from ferritin when needed by
degradation of protein by lysosomes
o Partially degraded ferritin can be
Hemojuvelin (HJV) A coreceptor acting detected in cells as stainable
with BMPR for signal hemosiderin
transduction o Ferritin is secreted in to the plasma by
macrophages in proportion to the
amount of iron that is in storage
Sons of mothers A second messenger of o Ferritin is elevated in plasma by the
against signal transduction
acute phase response, unrelated to the
decapentaplegic activated by BMPR HJV
amounts of stored iron
(SMAD) complex, able to
migrate to the nucleus • Iron recycling
and upregulate o When cells die, their iron is recycled.
hepcidine gene Multiple mechanisms salvage iron from
expression dying cells. The largest percentage of
- When BMPR binds to HJV, there is initial recycled iron comes from red blood
transduction. Together with second messenger cells
they will now be able to migrate to the nucleus o Senescent (aging) red blood cells are
then they will be upregulate the hepcidine gene ingested by macrophages in the spleen
expression (remember culling and splitting). The
hemoglobin is degraded, with the iron
 being held by the macrophages as membrane carrier, divalent metal transporter
ferritin (DMT1)
o Like enterocytes, macrophages possess • However, most dietary iron is ferric, especially
ferroportin in their membranes. This from plant sources. As a result, it is not readily
allows macrophages to be iron absorbed. Furthermore, other dietary
exporters so that the salvaged iron can compounds can bind iron and inhibit its
be used by other cells absorption. These include oxalates, phytates,
o The exported iron is bound to plasma phosphates, and calcium
apotransferrin, just as if it were newly • Release from these binders and reduction to
absorbed from the intestine the ferrous form are enhanced by gastric acid,
o Haptoglobin and hemopexin are plasma acidic foods (e.g., citrus), and an enterocyte
proteins able to salvage free luminal membrane protein, duodenal
hemoglobin or heme, respectively, cytochrome B (DcytB)
preventing them from urinary loss at • Thus, although the U.S. diet contains on the
the glomerulus and returning the iron order of 10-20mg of iron/day, only 1-2mg is
to the liver. (Actually, iron is not absorbed
secreted in the urine but in some • This amount is adequate for most men, but
disorders like protic syndrome - loss of menstruating women, pregnant and lactating
transferrin results to increase loss iron women, and growing children usually need
in the urine) additional iron supplementation to meet their
o There are iron disorders of iron increased need for iron
metabolism like iron deficiency • Heme with its bound iron is more readily
(reduction in the rate of hemoglobin absorbed. than ionic iron. Thus meat, with
synthesis and erythropoiesis leading heme in both myoglobin of muscle and
also to a disease called iron deficiency hemoglobin of blood, is the most bioavailable
anemia) and iron overload (disorders source of dietary iron
associated with this are acquired or
hereditary diseases, hemosiderosis, and Laboratory Assessment of Body Iron Status
hemochromatosis significant tissue
• Disease occurs when body iron levels are either
destruction occurs)
too low or too high
o Like macrophages, hepatocytes are
• The tests used to assess body iron status are
important to iron salvage. They also
able to detect both conditions
possess ferroportin so that the salvaged
iron can be exported to transferrin and • They include the traditional or classic iron
ultimately to other body cells studies: serum iron (SI), total iron-binding
capacity (TIBC), percent transferrin saturation,
Dietary Iron, Bioavailability, and Demand Prussian blue staining of tissues, ferritin assays,
soluble transferrin receptor (STfR), and
• Under normal circumstances, the only source of hemoglobin content of reticulocytes The results
iron for the body is from the diet of these measured parameters can be
• Foods containing high levels of iron include red combined to calculate an sTfR/log ferritin ratio
meats, legumes, and dark green leafy or graph a Thomas plot
vegetables, cereals Although some foods may • Finally, zinc protoporphyrin is another assay
be high in iron, that iron may not be readily with special application in sideroblastic anemia
absorbed and thus is not bioavailable • Diagnostically, the tests can be organized to
• Iron can be absorbed as either ionic iron or assess each of the iron compartments as
nonionic iron in the form of heme. Ionic iron indicated here:
must be in the ferrous (Fe+2) form for
absorption into the enterocyte via the luminal
Laboratory Typical Adult Diagnostic Use day and between-day variability; it also
Assay Male Compartment increases after recent ingestion of iron-
Reference Assessed containing foods and supplements
Interval o To avoid the apparent diurnal variation,
Serum Iron 50-160 µg/dL Indicator of the standard practice has been to
Level available collect the specimen fasting and early in
transport iron the morning when levels are expected
to be highest
o A diurnal variation in hepcidin has been
Serum 250-300 µg/dL Indirect
Transferrin indicator of detected that may explain some of the
Level iron stores serum iron variability and may still
support the early morning phlebotomy
Transferrin 20%-55% Indirect practice
Saturation indicator of • Total iron-binding capacity (TIBC)
iron stores with o The amount of iron in plasma or serum
transport iron will be limited by the amount of
Serum Ferritin 40-400 ng/mL Indicator of transferrin that is available to carry it
Level iron stores o To assess this, transferrin is maximally
Bone marrow Normal iron Visual saturated by addition of excess ferric
or liver biopsy stores qualitative iron to the specimen
with Prussian visualized assessment of o Any unbound iron is removed by
blue staining tissue iron
precipitation with magnesium
stores
carbonate powder
Soluble 1.15-2.75 mg/L Indicator of
transferrin functional iron o Then the basic iron method as
receptor (sTfR) available in described above is performed on the
level cells absorbed serum, beginning with the
sTfR/log ferritin 0.63- 1.8 Indicator of release of the iron from transferrin
index functional iron o The amount of iron detected represents
available in all the binding sites available on
cells transferrin-that is, the total iron-binding
RBC zinc <80 µg/dL of Indicator of capacity (TIBC)
protoporphyrin RBCs functional iron o It is expressed as an iron value,
level available in although it is actually an indirect
RBCs measure of transferrin.
Hemoglobin 27-34 pg/cell Indicator of • Percent transferrin saturation
content of functional iron
o Since the TIBC represents the total
reticulocytes available in
number of sites for iron binding and the
developing
RBCs SI represents the number bound with
• Serum iron (SI) iron, the degree to which the available
o Serum iron can be measured sites are occupied by iron can be
colorimetrically using any of several calculated
reagents such as ferrozine The iron is o The percent of transferrin saturated
first released from transferrin by acid, with iron is calculated as:
and then the reagent is allowed to react SI/TIBC X 100% = % transferrin
with the freed iron, forming a colored saturation
complex that can be detected It is important that both the SI and TIBC
spectrophotometrically be expressed in the same units
o The serum iron level has limited utility o A convenient rule of thumb evident
on its own because of its high within- from the table is that about one third
 (1/3) of transferrin is typically saturated o Under normal conditions, the number
with iron of circulating reticulocytes represents
• Prussian blue staining the status of erythropoiesis in the prior
o Prussian blue is actually a chemical 24-hour period; the amount of
compound with the formula Fe7(CN)18. hemoglobin in reticulocytes provides a
The compound forms during the staining near real-time assessment of iron
process, which uses acidic potassium available for hemoglobin production
ferrocyanide as the reagent/stain • Soluble transferrin receptor/log ferritin
o The ferric iron in the tissue reacts with o Although ferritin and sTfR values alone
the reagent, forming the Prussian blue can point to iron deficiency, the ratio of
compound that is readily seen sTfR to ferritin or STfR to log ferritin
microscopically as dark blue dots improves the identification of iron
o Tissues can be graded or scored deficiency when values are equivocal
semiquantitatively by the amount of o Because the sTfR rises in iron deficiency
stain that is observed and the ferritin (and its log) drops,
o Prussian blue stain is considered the gold these ratios are especially useful when
standard for assessment of body iron one of the parameters has changed but
o Staining is conducted routinely when is not outside the reference interval
bone marrow or liver biopsies are taken • Thomas plot
for other purposes o Demonstrated that when the sTfR/log
o Although ferric iron reacts with the ferritin is plotted against the
reagent, ferritin is not detected, likely hemoglobin content of reticulocytes, a
due to the intact protein cage four-quadrant plot results that can
o However, hemosiderin stains readily improve the identification of iron
• Ferritin deficiency
o The level of serum ferritin has been o In instances where there is true iron
shown to correlate highly with stored deficiency, the sTfR will rise and the
iron as indicated by Prussian blue stains ferritin will drop so that the sTfR/log
of bone marrow ferritin will be high and the hemoglobin
o Increase in ferritin can be induced content of reticulocytes will be low
without an increase in the amount of o Patient results will plot to the lower
systemic body iron. These rises may not right quadrant
be outside the reference interval but still o In instances where the ferritin may be
high enough to elevate a patient's falsely elevated by inflammation, the
ferritin above what it would otherwise sTfR/log ferritin will be normal despite
be reduced availability of iron for
• Soluble transferrin receptor (stfr) hemoglobin production -- thus low a
o Increase in the sTfR reflect either hemoglobin content in reticulocytes
increases in the amounts of TfR on o In this instance, patient values will plot
individual cells, as in iron deficiency, or to the lower left quadrant called
an increase in the number of cells each functional iron deficiency because the
with a normal number of TfRs systemic body stores are adequate but
o The latter occurs during instances of not available for transport and use by
rapid erythropoiesis, such as a response cells
to hemolytic anemia o As iron deficiency develops, other cells
• Hemoglobin content of reticulocytes are starved before erythrocytes;
o It is analogous to the mean cell production of hemoglobin in
hemoglobin (MCH), but just for reticulocytes remains at a normal level
reticulocytes for as long as possible
o However, the body's other iron-starved • Zinc protoporphyrin
cells will increase sTfR production and o Zinc protoporphyrin (ZPP) accumulates
systemic iron stores of ferritin will be in red blood cells when iron is not
depleted, thus elevating the sTfR/log incorporated into heme and zinc binds
ferritin value instead to protoporphyrin IX. It is easily
o These early iron-deficient patients’ detected by fluorescence
results will plot to the upper right o Although ZPP will rise during iron-
quadrant called latent iron deficiency deficient erythropoiesis, the value of
o Plotting the ratio of soluble transferrin this test is greatest when the activity of
receptor to log ferritin (sTfR/log ferritin) the ferrochelatase is impaired, as in
against the hemoglobin content of lead poisoning
reticulocytes produces a graph with
four quadrants
o Patients with values within the
reference intervals for each assay will
cluster in the upper left quadrant
o Those with functional iron deficiency,
like the anemia of chronic
inflammation, will cluster at the lower
left. (low hemoglobin content of
reticulocytes, normal sTfR/log ferritin)
o Latent iron deficiency, before anemia
develops, will cluster to the upper right
with frank iron deficiency in the lower
right quadrant
o Latent iron deficiency - normal
hemoglobin content of reticulocytes,
increased sTfR/log ferritin)
o Iron deficiency - low hemoglobin
content of reticulocytes, increased
sTfR/log ferritin