US Army Medical Course - Hematology I - MD0853 PDF

U.S.
ARMY MEDICAL DEPARTMENT CENTER AND SCHOOL

FORT SAM HOUSTON, TEXAS 78234-6100
Hematology I
SUBCOURSE MD0853 EDITION 100

DEVELOPMENT
This subcourse is approved for resident and correspondence course instruction. It

reflects the current thought of the Academy of Health Sciences and conforms to printed
Department of the Army doctrine as closely as currently possible. Development and
progress render such doctrine continuously subject to change.
ADMINISTRATION
For comments or questions regarding enrollment, student records, or shipments,

contact the Nonresident Instruction Branch at DSN 471-5877, commercial (210) 221-
5877, toll-free 1-800-344-2380; fax: 210-221-4012 or DSN 471-4012, e-mail
accp@amedd.army.mil, or write to:
COMMANDER
AMEDDC&S
ATTN MCCS HSN
2105 11TH STREET SUITE 4192
FORT SAM HOUSTON TX 78234-5064
Approved students whose enrollments remain in good standing may apply to the
Nonresident Instruction Branch for subsequent courses by telephone, letter, or e-mail.
Be sure your social security number is on all correspondence sent to the Academy of
Health Sciences.
CLARIFICATION OF TRAINING LITERATURE TERMINOLOGY
When used in this publication, words such as "he," "him," "his," and "men" are intended
to include both the masculine and feminine genders, unless specifically stated otherwise
or when obvious in context.
.
TABLE OF CONTENTS
Lessons Paragraphs
INTRODUCTION
1 BLOOD
Section I. The Composition of Blood 1-1-1-4

Section II. Formation of Blood Cells 1-5-1-6
Section III. Normal Cell Maturation 1-7-1-9
Section IV. Abnormal Cell Maturation 1-10-1-12
Section V. Functions of Blood Cells 1-13-1-16
Exercises
2 MATERIAL EMPLOYED IN HEMOTOLOGY
Section I. Laboratory Reagents 2-1-2-3

Section II. Laboratory Glassware 2-4-2-7
Section III. Laboratory Equipment 2-8-2-9
Exercises
3 COLLECTION OF BLOOD AND PREPARATION OF

BLOOD SMEARS
Section I. Collection of Blood Specimens 3-1-3-4

Section II. Preparation and Staining of Blood
Smears 3-5-3-8
Exercises
4 MORPHOLOGY OF BLOOD CELLS
Section I. General Information 4-1-4-2

Section II. Erythrocytes 4-3-4-5
Section III. Leukocytes 4-6-4-11
Section IV. Thrombocytes 4-12-4-13
Section V. The Leukemias 4-14-4-18
Exercises
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TABLE OF CONTENTS (continued
Lessons Paragraphs
5 MANUAL CELL COUNTS
Section I. Manual Counts, Blood Specimens 5-1-5-4

Section II Manual Counts, Other Body Fluids 5-5-5-6
Exercises
6 HEMATOCRIT, ERYTHOCYTE SEDIMENTATION

RATE, AND HEMOGLOBIN
Section I. Hematocrit 6-1-6-2

Section II. Erythrocyte Sedimentation Rate 6-3-6-5
Section III. Hemoglobin 6-6-6-14
Exercises
APPENDIX
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CORRESPONDENCE COURSE OF THE
U.S. ARMY MEDICAL DEPARTMENT CENTER AND SCHOOL
SUBCOURSE MD0853
HEMATOLOGY I
INTRODUCTION
This subcourse is concerned with the blood tests performed in the hematology
section of the laboratory. The purpose of these tests is to aid the physician in
diagnosis. Thus, these tests are important and often essential to the health and life of
the patient. Thorough study of this subcourse should enable you to better fulfill your
role in health care.
Subcourse Components:
This subcourse consists of 6 lessons and an Appendix. The lessons are:
Lesson 1. Blood
Lesson 2. Material Employed in Hematology.
Lesson 3. Collection of Blood and Preparation of Blood Smears.
Lesson 4. Morphology of Blood Cells.
Lesson 5. Manual Cell Counts.
Lesson 6. Hematocrit, Erythrocyte Sedimentation Rate, and Hemoglobin.
Appendix. Glossary of Terms.
Credit Awarded:
To receive credit hours, you must be officially enrolled and complete an

examination furnished by the Nonresident Instruction Branch at Fort Sam Houston,
Texas. Upon successful completion of the examination for this subcourse, you will be
awarded 12 credit hours.
You can enroll by going to the web site http://atrrs.army.mil and enrolling under
"Self Development" (School Code 555).
A listing of correspondence courses and subcourses available through the

Nonresident Instruction Section is found in Chapter 4 of DA Pamphlet 350-59, Army
Correspondence Course Program Catalog. The DA PAM is available at the following
website: http://www.usapa.army.mil/pdffiles/p350-59.pdf.
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LESSON ASSIGNMENT
LESSON 1 Blood.
TEXT ASSIGNMENT Paragraphs 1-1 through 1-16.
LESSON OBJECTIVES After completing this lesson, you should be able to:
1-1. Select the statement that best describes the

composition of blood.

cellular and liquid constituent characteristics of
blood.
1-3. Select the characteristic features of normal and

abnormal blood cell formation and maturation.
1-4. Correctly select the functions of blood cells.
SUGGESTION After completing the assignment, complete the

exercises at the end of this lesson. These exercises
will help you to achieve the lesson objectives.
MD0853 1-1
LESSON 1
BLOOD
Section 1. THE COMPOSITION OF BLOOD
1-1. INTRODUCTION
Blood is a complex and unique fluid of variable composition circulating through

the heart, arteries, capillaries, and veins, known as the vascular system of the body. It
is a tissue in which cellular constituents are suspended in a liquid medium performing
specialized functions. The prime function of blood is to carry oxygen from the lungs to
the body tissues and carbon dioxide from the tissues to the lungs. Blood carries fluid to
and from the tissues, thus maintaining proper fluid balance throughout the body
(explained later in this paragraph). The average pH value of blood is 7.40. Blood also
carries nutrients or food supplies from the digestive system to the body cells or tissues
and transports waste products for the tissues to the kidneys and bowel for excretion to
prevent accumulation. In response to trauma or infection, blood cells and antibodies
are carried in the blood to a point protection against the causative agents of disease, or
to transport blood-clotting substances to a break in a blood vessel to promote the
clotting process when injury is caused by bleeding or hemorrhage. The blood also
carries hormones from the endrocrine glands to the target organs, and it assists in the
regulation of the body temperature by carrying excess heat from the interior of the body
to the surface layers of the skin, where the heat is dissipated to the surrounding air. To
perform these complex functions, blood is complex and is composed of two main parts.
One part is composed of blood cells, the particles suspended in the plasma, making up
approximately 45 percent of total blood volume and including erythrocytes (red blood
cells), leukocytes (white blood cells), and thrombocytes (platelets). The red and white
blood cells are known as corpuscles. The other part is plasma, the fluid portion of the
blood, which consists primarily of water in which are dissolved proteins and many
inorganic and organic substances carried by the blood to and from the tissues. Plasma
makes up 55 percent of the blood.
1-2. GENERAL CELLULAR STRUCTURE
a. A typical cell is composed of a single nucleus embedded in cytoplasm. The

living substance of the cell is a grayish, viscous liquid called protoplasm. Protoplasm is
enclosed in the cell interfaces by a cell membrane that selectively regulates the
interchange of materials between the cell and its environment. A typical cell tree is
diagramed in figure 1-1, however, all cells and forms or development of cells are not
depicted.
b. The nucleus is a spherical oval body surrounded by a thin membrane (nuclear

membrane). Contained in the nucleus is a sphere called the nucleolus. The nucleus is
thought to be an organizing center for the cell and can have the capacity for cell
production. The absence of nuclei signifies the end of cell development. Also found
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Figure 1-1. Typical cell structure.
within the nucleus is a network of nuclear fibrils made up of DNA (deoxyribonucleic
acid) and protein called chromatin. It is thought that the decreasing growth activity of a
cell during maturation is regulated by chromatin.
c. Surrounding the nucleus is a mass of protoplasm called cytoplasm.

Contained within the cytoplasm are numerous granules, filaments, and globules. These
structures are divided into two groups known as organoids (organelles) and inclusions.
The organoids are thought to perform most of the metabolic functions of the cell.
Mitochondria, Golgi apparatus, fibrils, centrioles, and the chromatin substance are
classified as orgnanelles. Cytoplasm inclusions are usually seen as granulation. The
granulation is an accumulation of proteins, lipids, carbohydrates, pigments, and
secretory granules.
1-3. CELLULAR CONSTITUENTS
a. Erythrocytes. An erythrocyte (red blood cell) is an elastic, non-nucleated,

biconcave disc having a diameter of approximately 7.2 microns. The mature red cell
contains about 34 percent hemoglobin (a complex iron-bearing pigment that transports
oxygen). Hemoglobin is contained in the interior of the cell, and the outer surface of the
cell is surrounded by a cell membrane. When unstained, the cell has a pale, greenish-
yellowish appearance. It is buff pink with an accented central zone of pallor when
stained with Wright’s stain. The production of erythrocytes or erythropoiesis, occur
primarily in the red marrow of the spongy bones. Erythrocytes make up the great
majority of cells found in the peripheral blood. Their vast surface area is important in
the transport of oxygen from the lungs to the tissues because of quick exchange of
oxygen in both sites that occurs across the red cell surface. Erythrocytes are subject to
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many alterations in shape, size, staining properties, and structure in different disease
processes. An adult female has approximately 4.8 million/cu mm red cells, and an adult
male has approximately 5.4 million/cu mm red cells. The erythrocytes have an average
life span of 80 to 120 days. See Table 1-1 for purposes of the blood cell tree as
described in this subcourse.
Table 1-1. Blood cell tree.
b. Leukocytes. Leukocytes are commonly known as white blood cells because

of their lack of color in unstained preparations. They are nucleated cells that have an
average diameter of 8 to 12 microns. There are 5,000-10,000 leukocytes per cubic
millimeter of blood. Leukocytes are divided into three groups: (1) granulocytes
(neutrophils, eosinophils, and basohils) that can phagocytose or ingest bacteria or other
particles; (2) lymphocytes which participate in humoral (B lymphocyte) and cell
mediated (T lymphocyte) immunity and (3) monocytes that phagocytose bacteria
cellular debris and interact with lymphocytes in the processing of antigens in the
immune reaction. In humans, there are 3 types of phagocytes: macrophages,
granulocytes, and monocytes. All three types are attracted to bacteria and certain other
particles by substances released by the particles (the process of chemotaxis). They
then pin the particle against a surface and engulf it. They are differentiated by the
specific nuclear and cytoplasmic staining properties.
c. Thrombocytes. Thrombocytes are found commonly known as platelets.

They are the morphologically recognizable precursors of platelets and are detached
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fragments of the cytoplasm-megakaryocytes that are found in the bone marrow (others
coming from red bone marrow include: promegakarycocytes and megakaryoblasts).
Unstained platelets appear as small hyaline structures with a diameter of approximately
two microns. When stained with Wright’s stain, they have a pale blue cytoplasm with a
dark granular center. Platelets are very fragile and live for a period of about 3 to 5 days.
1-4. LIQUID CONSTITUENTS
a. Plasma. Blood plasma is a clear, yellowish fluid that accounts for about 55
percent of the total volume of the blood. The chemical nature of plasma is very
complex. It consists of 92 percent water, 7 percent proteins (albumin, globulin, and
fibrinogen), carbohydrates (glucose), lipids (fats, lecithin, and cholesterol), dissolved
gases (oxygen, carbon dioxide, and nitrogen), non-protein nitrogenous substances, and
less than 1 percent of inorganic salts. Blood plasma is the fluid portion of the blood
before clotting occurs. Put another way, plasma from which fibrinogen has been
removed is called serum.
b. Serum. Serum is the fluid portion of blood after the clotting process is
complete. Fibrinogen, the precursor of fibrin, is removed from plasma to form the
framework of the blood clot.
Section II. FORMATION OF BLOOD CELLS
1-5. EMBRYONIC HEMATOPOIESIS
a. The primary source of blood cells is the mesenchyme connective tissue in the
embryo. Three phases of embryonic hematopoiesis merge, resulting in the formation of
blood (see figure 1-2).
b.
Fetal Development–in months
Figure 1-2. Embryonic hematophoiesis.
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b. During the first two months of embryonic development, the mesoblastic phase
occurs. The blood cells are formed in the blood islands of the yolk sac. Immature blood
cells develop, having large nuclei containing a vesicular chromatin meshwork. The
nuclei are surrounded by a thin rim of cytoplasm. The cells gradually develop
hemoglobin to become a nucleated red blood cell. Mitosis occurs and daughter cells
contain more hemoglobin. Finally the nucleus is lost and the nature erythrocyte is
produced.
c. At two months, the hepatic phase begins. Blood cell development shifts to
the body of the fetus as the organs of the reticuloendothelial system (liver, spleen,
thymus, etc.) are formed. During this phase, red cells begin their normal development.
Granulocytes and thrombocytes are formed in the liver while lymphocytes and
monocytes are formed in the spleen and thymus.
d. The myeloid phase begins during the fifth month of gestation. Blood cells are
formed in the bone marrow and lymphatic system that at the time of birth constitute the
total sources of hematopoiesis. The bone marrow is the principle source of production
of erythrocytes, granulocytes, and thrombocytes. The lymph nodes are the primary
sites of production of lymphocytes, monocytes, and plasmacytes.
1-6. POSTNATAL HEMATOPOIESIS
a. Blood formation at birth is confined primarily to the bone marrow (central

medullary structure of the bone). Blood cells multiply by mitosis and then mature to a
specific cell type. The mature cells lose the ability to reproduce and develop a definite
life span. Regeneration of blood cells after birth involves multiplication of precursor
cells, evolution of the definitive characteristics of each type, and release of mature cells.
b. Myelopoiesis is the production of blood cells and bone marrow by the bone
marrow (medullary site of production). The red bone marrow is the principle source of
production of red cells and white cells of the granulocytic series. At birth, the central
medullary structure of bones is red bone marrow and it is actively engaged in
hematopoiesis. At about 5 years of age non-hematopoietic type of marrow (fatty yellow
bone marrow) that is a reserve potential tends to replace most of the red bone marrow.
This partial replacement of red marrow is complete when the individual reaches maturity
(about 18 years of age) at which time, active hematopoietic centers in bone tissue are
limited almost exclusively to the sternum, pelvic area, vertebrae, skull, ribs, clavicle,
scapuli, and the epiphyses of the long bones.
c. Extramedullary hematopoiesis is blood production that occurs in sites other
than the bone marrow. Active sites are the spleen, thymus, lymph nodes, and other
lymphoid tissues. Cell production is largely limited to lymphopoiesis. The lymph nodes
are the primary source of lymphocytes and plasmacytes (see figure 1-3).
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Figure 1-3. Hematopoietic system in an adult 18-20 years of age.
MD0853 1-7
Section III. NORMAL CELL MATURATION
1-7. GENERAL FEATURES
In the course of blood cell maturation, certain specific features are developed
(see Figure 1-4). Each of the component parts of the cell undergoes a transformation
during maturation. The immature cell or blast cell contains a large nucleus, a small
amount of cytoplasm, and no granules. As the cell ages, the cytoplasm becomes less
basophilic and nuclear chromatin becomes heavier (darker stain). Reduction in size
and loss of nucleoli occurs as the cell becomes older. The three types of granulation
(neutrophilic, basophilic, and eosinophilic) become more specific and smaller as the cell
ages. Maturation, in general, involves: (1) cytoplasmic differentiation, (2) nuclear
maturation, and (3) reduction in cell size.
Figure 1-4. Development of blood cells.
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1-8. CYTOPLASM
The basophilia of a blast cell is proportional to the ribonucleic acid (RNA)

content. As the cell matures, the RNA content decreases and the cell becomes a paler
blue. In the myeloid cells, a specific type of granulation occurs. When granules appear
they are pinkish-red and few in number. The granules increase in number and
differentiate into three types upon maturation. As the cell matures, it develops an
affinity for the acid or basic portion of the stain (Wright’s stain). Basophilic granules
stain blue-black, eosinophilic granules stain red-orange, and neutrophilic granules stain
pinkish-purple. Lymphocytes are usually devoid of cytoplasmic granulation but they can
possess non-specific azurophilic (dark-purple) granules, usually characteristic of
monocytes and plasmocytes. Upon maturation, the erythrocyte develops a light orange
respiratory pigment called hemoglobin.
1-9. NUCLEUS
The nucleus of the young cell is large, round, and occupies most of the cell. As
the cell matures, the size of the nucleus decreases. Nuclei of early or primitive cells
usually have one or more nucleoli. The latter are small, round, homogeneous areas
that usually stain light blue with a darker boundary. In appearance nucleoli are
somewhat like craters in the nucleus. They are surrounded by strands of chromatin.
These nucleoli, plus a delicate reticular network of chromatin, are the principle
indicators of blood cell immaturity. As the cells mature, the nucleus gradually becomes
smaller, stains darker, and chromatin meshwork become “coarse” with the strands of
chromatin less fine and lacelike. In the course of cell development, the nucleus
changes its shape, particularly in the granulocytic series, where it becomes indented,
lobulated, segmented, or fragmented. As maturation or development progresses, the
nucleus, if still intact, becomes small, compact, usually dark and structureless, and can
completely disappear. The loss or shrinking of the nucleus is accompanied by a
decrease in cell size.
Section IV. ABNORMAL CELL MATURATION
1-10. GENERAL FEATURES
Abnormal cell maturation is asynchronous development as opposed to normal

cell maturation or synchronous development. Since the normal sequence of cell
maturation is upset, atypical cells will be present. Abnormal cells can be recognized by:
(1) abnormal cytoplasmic maturation, (2) abnormal nuclear maturation, and (3)
abnormal size.
1-11. CYTOPLASM
Asynchronism of the cytoplasm is most commonly seen in the granulocytes.

Granulation can be primitive or absent. In some instances, the granules fail to
differentiate. Erythrocytes show basophilia late in the series and retarded
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hemoglobination. Inclusions in the cytoplasm, such as Dohle bodies (infectious
diseases), Auer rods (leukemia), and toxic granulation (infection affecting the marrow)
are seen in the abnormal white cells.
1-12. NECLEUS
Abnormal cells often show two nuclei in severe disturbances, such as leukemia.
Nucleoli have a retarded reduction. The nucleus can have an irregular outline or
indentation (Rieder cells). Hypersegmented nuclei occur in the neutrophils in sepsis
and in pernicious anemia. Abnormal maturation of the nucleus often results in variation
in cell size.
Section V. FUNCTIONS OF BLOOD CELLS
1-13. INTRODUCTION
Each component of blood is uniquely capable of performing one or more

functions. Together, these components provide the maintenance of a relatively stable
environment of the body by a variety of mechanism. This maintenance of a relative
biological constancy or integrity is known as homeostasis. Once the blood cells reach
full maturity, they enter the bloodstream and begin fulfilling their functions.
1-14. ERYTHROCYTES
Hemaglobin is the main functioning component of the cell. It carries out the
transportation of oxygen to the tissues and the removal of carbon dioxide. Hemoglobin
also aids in the maintenance of the delicate acid-based buffer system of the body. The
erythrocyte must also supply energy to accomplish the active transport of glucose and
ions against a gradient across the red cell membrane.
1-15. LEUKOCYTES
Leukocytes remove invading antigens (for example, bacteria) and to some extent
transport and distribute antibodies. Monocytes and all of the granulocytes have been
shown to demonstrate directional movement. Their movement is subject to chemotaxis
or the response of living protoplasm to a chemical stimulus. As mentioned previously,
the attraction of the leukocytes to bacteria and certain other particles by substances
released by the particles is called chemotaxis. They either transport the particles or
engulf them. The chemotaxis process of engulfing and destroying bacteria, or
phagocytosis, is a prime function of leukocytes.
a. Monocytes. These cells will engulf bacteria and larger materials, including
even protozoa and red cells, and are transformed into and/or are called macrophages.
In this regard, monocytes are perhaps the most efficient phagocytes of all the cells.
Monocytes contain many of the lytic enzymes that are found in microphages
(granulocytes). In addition, monocytes contain lipases that enable them to dissolve the
lipoid capsules of certain bacteria.
MD0853 1-10
b. Neutrophils. Neutrophilic leukocytes are excellent microphages. That is,
they engulf bacteria and other microscopic particles. The particles are first surrounded
by cellular pseudopodia and then incorporated into a cell vacuole. There the foreign
bodies mix with substances released from the cytoplasm of leukocytes. In this way the
leukocyte is not injured by whatever "combat activity" is taking place in the vacuole.
Neutrophils are fully developed (mature) cells that are incapable of mitotic division.
They carry on active metabolism. Eventually the granulocytes disintegrate and in
inflammatory processes are succeeded by monocytes.
c. Eosinophils. The eosinophils are easily distinguishable by their large

cytoplasmic granules. They have some similarities with the neutrophil: they arise from
a common stem cell and share same morphologic features. Unlike the neutrophil,
however, eosinophils initially develop in the bone marrow, then are released into the
circulation, and undergo further maturation in the spleen. Their emergence from the
marrow seems to depend upon a stimulus provided by lymphocytes. Eosinophils are
found in tissue fluid as well as in peripheral blood, especially in areas where there is an
allergic reaction. Current thinking holds that eosinophils are involved in antigen-
antibody reactions, and have been shown to phagocytize antigen-antibody reactants.
Eosinophils are also thought to transport, or at least contain, lysins that act on fibrin. It
is suggested that eosinophils limit the action of substances such as histamine. How this
is accomplished is not yet clear. The mobilization of eosinophils from their reserve in
the bone marrow is at least in part under hormonal control. If the adrenal cortex is
functioning properly, an injection of adrenocorticotropic hormone (ACTH) results in a
marked decrease in the number of circulating eosinophils and in the number of
circulating lymphocytes. On the other hand, there is an increase in the number of
circulating neutrophils.
d. Basophils. The function of basophils in man has not been ascertained.

They quite possibly represent a vestige of evolution. Their granules have been found to
contain heparin and these cells frequently appear during the clot dissolution phase of an
injury. Hence, it has been suggested they may be involved in clot absorption.
e. Lymphocytes. The lymphocyte is now believed to be directly connected with

antibody production. Undoubtedly, the lymphocyte performs important immunologic
functions. According to very recent studies, many of the activities previously thought to
take place in the reticuloendothelial (RE) system actually take place in lymphocytic
tissue.
1-16. PLATELETS
Platelets possess metabolic system, expend energy, and respond to stimulus.

They contain many enzymes and undergo respiratory activity and glycolysis. They
possess coagulation factors usually designated as PF-1, PF-2, and on through PF-7.
The cells contain fibrinogen and vasoconstrictor substances, calcium, and many other
components that are either known or presumed to participate in the clotting mechanism.
Clot-promoting lipoproteins are also found in platelets. In addition, well-defined
MD0853 1-11
antigens have been found in platelets. The role of platelets in the blood coagulation
mechanism will be described in more detail in Subcourse MD0857 Lesson 2.
NOTE: See Table 1-2 for a summary of blood cell functions.
Table 1-2. Blood cell functions.
Continue with Exercises
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EXERCISES, LESSON 1
INSTRUCTIONS: Answer the following exercises by marking the lettered response that
best answers the exercise, by completing the incomplete statement, or by writing the
answer in the space provided at the end of the exercise. Some exercises may require
you to refer to the appendices located at the back of the subcourse.
After you have completed all the exercises, turn to "Solutions to Exercises" at the
end of the lesson and check your answers. For each exercise answered incorrectly,
reread the material referenced with the solution.
1. Plasma constitutes approximately _________________ percentage of the blood

and is composed mainly of ______________________.
a. 45 percent; water.
b. 50 percent; protoplasm.
c. 55 percent; water.
d. 70 percent; cytoplasm.
2. What does chromatin regulate in a cell during maturation?
a. Movement.
b. Growth.
c. Granulation.
d. Waste products.
3. Cytoplasmic granulation is usually due to cytoplasmic:
a. Fibrils.
b. Inclusions.
c. Organelles.
d. Parachromatin.
MD0853 1-13
4. When stained with Wright's stain, what is the color of the normal erythrocyte and
how is it accented?
a. Light orange I granular zone.
b. Pale green, central pale zone.
c. Pale, greenish-yellowish, central pale zone.
d. Buff pink, central zone of pallor.
INSTRUCTIONS: Exercise items 5 through 10 require you to select the name of

the particle, activity or definition of the typical cell structure as shown below.
Typical cell structure.
5. A grayish, viscous liquid living substance within the cell that regulates the
interchange of materials between the cell and the environment is called:
a. Cytoplasm.
b. Protoplasm.
c. Centriole.
d. Mitochondria.
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6. The nuclear fibrils that possibly regulate growth activity during maturation and are
made up of DNA (deoxyribonucleic acid) and protein are called:
a. Lysosome.
b. Golgi apparatus.
c. Chromatin.
d. Mitochondria.
7. Which chromatin substances are contained within cytoplasm and are classified as
organelles?
a. All granules, filaments, and globules inside the nucleus.
b. Mitochondria, Golgi apparatus, fibrils, and centrioles are some of them.
c. Some granules, filaments, and globules inside the nucleus.
8. Which organoid substance appears like a cork-screw cigar?
a. Golgi apparatus.
b. Fibrils.
c. Centrioles.
d. Mitochondria.
9. Mitochondria is defined in this subcourse as:
a. A meshwork of lipid containing fibrils within the cytoplasmic portion of a cell.
b. A spherical or oval body surrounded by a thin membrane (nuclear membrane)
c. Granular components of a cell cytoplasm active in oxidative processes
d. A minute cell organoid within the centrosome.
MD0853 1-15
10. Centriole is defined in this subcourse as:
a. A meshwork of lipid containing fibrils within the cytoplasmic portion of a cell.
b. A spherical or oval body surrounded by a thin membrane (nuclear membrane)
c. Granular components of a cell cytoplasm active in oxidative processes
d. A minute cell organoid within the centrosome.
11. Only nucleated red blood cells are formed in the islands of the yolk sac during the
___________________ of embryonic development.
a. First 2 months.
b. Third to fifth months.
c. Fifth to final months.
d. Entire duration.
12. The bone marrow is the principal organ of red blood cell production during the
myeloid phase as well as at birth and during the remainder of life.
a. First 2 months.
b. Third to fifth months.
c. Fifth to final months.
d. Entire duration.
13. As the child matures, myelopoiesis is increasingly confined to the:
a. Bones.
b. Medullary sites.
c. Ligaments.
d. Muscles.
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14. Which definition of myelopoeisis is correct?
a. It is the production of blood cells and bone marrow by the kidney.
b. It is the production of blood cells and bone marrow by the liver.
c. It is the production of blood cells and bone marrow by the bone marrow
(medullary site of production).
d. A decrease in the number of neutrophils in the blood.
15. During the formation of blood cells, which tissue is their primary source?
a. Striated tissue.
b. Mesenchyme connective tissue in the embryo.
c. Liver.
d. Tissue thromboplastin.
16. The three types of granulation occurring during normal cell maturation that
become more specific and smaller as the cell matures are:
a. Basophilic, hematopoietic, and eosinophilic.
b. Non-hematopietic, neutrophilic, and eosinophilic.
c. Netrophilic, basophilic, an eosinophilic.
d. Basophllic, neutrophilic, and idiopathic.
17. Immature blood cells that are so young that they can hardly be distinguished
morphologically from each other are called:
a. First cells.
b. Blast cells.
c. Band cells.
d. Monocytes.
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18. What occurs to the RNA content of the blast cell as it matures?
a. Increases.
b. Decreases.
c. All of the above.
19. Generally speaking, as normal cells maturate:
a. The nucleus decreases in size.
b. The cytoplasm differentiates.
c. The cell reduces in size.
d. All of the above.
20. What color does the blast cell develop into as it matures?
a. Green.
b. Dark purple.
c. Blue.
d. Pinkish-purple.
21. Lymphocytes during normal cell maturation are devoid of cytoplasmic granulation
but can possess nonspecific ________________________ granules.
a. Azurophilic.
b. Dark green.
c. Light pink.
d. Rich orange.
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22. What colors do basophilic and neutrophilic granules appear when Wright's stain is
applied?
a. Pink or brown-orange.
b. Red-orange; blue-black.
c. Blue-black; pinkish-purple.
d. Reddish-purple; blue-black.
23. With Wright’s stain, eosinophilic granules appear large and:
a. Pink or brown.
b. Red-orange.
c. Blue-black.
d. Reddish-purple.
24. With Wright’s stain, the nonspecific azurophilic granule is:
a. Pink or brown.
b. Red-orange.
c. Colorless.
d. Red/dark purple.
25. The presence of _________________________ shaped nucleoli is generally a

sign of a cell ___________________________.
a. Oblong; maturity.
b. Crater; immaturity.
c. Lacelike; cytoplasmic change.
d. Coarse; granulocytic change.
MD0853 1-19
26. Abnormal cytoplasmic maturation is seen most frequently in:
a. Monocytes.
b. Lymphocytes.
c. Granulocytes.
d. Erythrocytes.
27. What are Dohle bodies?
a. Greek food.
b. Atypical cells.
c. Abnormal cells.
d. Infectious diseases.
28. What another term for normal cell maturation?
a. Polychromasic.
b. Synchronous.
c. Asynchronous.
d. Rieder.
29. What term is used to describe abnormal cell maturation or uncoordinated cell
development?
a. Dohle bodies.
b. Asynchronous.
c. Binary fission.
d. Synchronous.
e. Azurophilic.
f. Sepsis.
MD0853 1-20
30. If asynchronism occurs in the cytoplasm, where would it most commonly be seen?
a. Granulocytes.
b. Nucleolus.
c. Lysosome.
d. Mitochondria.
31. When there is abnormal cell maturation, where does inclusion occur?
a. Cell membrane.
b. Nucleus.
c. Cytoplasm.
d. Fat droplet.
32 Which cytoplasmic cells show basophilia late in the series and hemoglobinization
retardation during abnormal cells maturation?
a. Monocytes.
b. Lymphocytes.
c. Granulocytes.
d. Erythrocytes.
e. Neutrophils.
f. Basophils.
MD0853 1-21
33. Leukemia is also known as:
a. Auer rods.
b. Rieder cells.
c. Juvenile cells.
d. Macrocytosis.
e. Aplastic anemia.
34. When a cell is maturing abnormally, how many nuclei might it have if it has a
severe disturbance such as leukemia?
a. 0.
b. 1.
c. 2.
d. 3.
35. The nuclei of abnormal cells often have:
a. Two nuclei, in severe disturbances, as in leukemia.
b. Reduced reduction of nucleoli.
c. Undifferentiating granules.
d. Rieder cell indentation.
e. Hypersegmented nuclei.
f. Cell variation.
g. All of the above.
h. None of the above.
MD0853 1-22
36. Hyposegmented nuclei occur in the neutrophils in _________________ and in
pernicious ______________________.
a. Sepsis; leukemia.
b. Sepsis; anemia.
c. Sepsis; hemotoma.
d. Sepsis; sickle cell anemia.
37. Homeostasis is defined as:
a. Biological constancy or integrity of blood or the checking of the flow of blood,

especially from a vessel.
b. The spitting of blood; coughing up blood.
c. Infection of the marrow; infectious disease.
d. Blood cells reaching full maturity and entering the mainstream to begin their
functions.
38. Which statement is correct concerning the function of the blood cell?
a. Individually, each component of blood provides the maintenance for a

relatively stable environment of the body by a variety of mechanisms.
b. Together, the blood components provide the maintenance for a relatively

stable environment of the body by a variety of mechanisms.
39. One function of the erythrocyte is to aid in the maintenance of the delicate acid-
base buffer system of the body. Which part of the cell performs this function?
a. Hemoglobin.
b. Membrane.
c. Lysosome.
d. Glycoproteins.
MD0853 1-23
40. Which are the oxygen-carrying cells of the body?
a. Monocytes.
b. Granulocytes.
c. Lymphocytes.
d. Erythrocytes.
41. _________________ mainly controls the level of oxygen in the tissues.
a. Hemoglobin.
b. Chemotaxis.
c. Phagocytosis.
d. Adrenocorticotropic hormone.
42. Erythrocytes, leukocytes, and platelets are considered to be the

_____________________ of (in) the blood.
a. Coagulation factors.
b. Immunologic functions.
c. Components.
43. Which cells possess the powers of locomotion and phagocytosis?
a. Erythrocytes.
b. Leukocytes.
c. Thrombocytes.
d. Platelets.
MD0853 1-24
44. Which cell is able to phagocytize the largest particles and because of this is called
macrophage?
a. Neutrophils.
b. Eosinophils.
c. Basophils.
d. Monocytes.
45. Which cell has the main function of transporting O2 to the tissues and removing
CO2?
a. Neutrophils.
b. Eosinophils.
c. Basophils.
d. Erythrocyte.
46. Which cell is surrounded by cellular pseudopodia and is not injured by “combat
activity” taking place in the vacuole?
a. Eosinophils.
b. Neutrophils.
c. Monocytes.
d. Basophils.
47. Which cell is incapable of mitotic division?
a. Eosinophils.
b. Neutrophils.
c. Monocytes.
d. Basophils.
MD0853 1-25
48. Which statement is correct?
a. Monocytes are cells containing foreign bodies with substances released from
the cytoplasm.
b. Lymphocytes are now believed to contain heparin.
c. Easinophils are involved with antigen-antibody reactions and eat antigen-

antibody reactants.
d. Basophils may be involved in attracting cells to substances and then either

transport or engulf them.
49. Cellular particles associated with the clotting of blood are:
a. Thrombocytes (platelets).
b. Hemoglobin particles.
c. White blood cells.
d. Red blood cells.
50. Which statement is correct?
a. Platelets possess metabolic systems, expend energy, respond to stimuli, and

then disintegrate.
b. Platelets possess metabolic systems, mobilize the bone marrow, expend

energy, and respond to stimuli.
c. Platelets possess carbon dioxide and metabolic systems, expend energy, and
respond to stimuli.
d. Platelets possess metabolic systems, expend energy, respond to stimuli, and

undergo glycolysis.
Check Your Answers on Next Page
MD0853 1-26
SOLUTIONS TO EXERCISES, LESSON 1
1. c (para 1-1)
2. b (para 1-2b)
3. b (para 1-2c)
4. d (para 1-3a)
5. b (para 1-2a)
6. c (para 1-2b)
7. b (para 1-2c)
8. d (figure 1-1)
9. c (appendix)
10. d (appendix)
11. a (para 1-5b)
12. c (para 1-5d)
13. a (para 1-6b)
14. c (para 1-6b)
15. b (para 1-5a)
16. c (para 1-7)
17. b (para 1-7)
18. b (para 1-8)
19. d (para 1-7)
20. c (para 1-8)
21. a (para 1-8)
22. c (para 1-8; figure 1-4; appendix)
MD0853 1-27
23. b (para 1-8; figure 1-4; appendix)
24. d (para 1-8; appendix)
25. b (para 1-9)
26. c (para 1-11)
27. d (para 1-11)
28. b (para 1-10)
29. b (para 1-10)
30. a (para 1-11)
31. c (para 1-11)
32. d (para 1-11)
33. a (para 1-11)
34. c (para 1-12)
35. g (para 1-11, 1-12)
36. b (para 1-12)
37. a (para 1-13)
38. b (para 1-13)
39. a (para 1-14)
40. d (para 1-14)
41. a (para 1-14)
42. c (paras 1-13 to 1-16)
43. b (para 1-15)
44. d (para 1-15a)
45. d (para 1-14)
MD0853 1-28
46. b (para 1-15b)
47. b (para 1-15b)
48. c (para 1-15c)
49. a (para 1-16)
50. d (para 1-16)
End of Lesson 1
MD0853 1-29
LESSON ASSIGNMENT
LESSON 2 Material Employed in Hematology.
2-1. Correctly list the procedures in preparing

laboratory reagents.
2-2. Select the safety precautions necessary to

ensure that reagents and equipment are labeled
and handled carefully and appropriately.
2-3. Select the statement that best describes

important characteristics and functions of
common hematological glassware.
2-4. Correctly identify the parts of a compound

microscope.
2-5. Correctly select the functions of a compound

microscope.
2-6. Select the statement that correctly describes the

important characteristics and functions of
equipment used in the laboratory.
2-7. Select the correct procedures for cleaning and

maintaining laboratory equipment.

MD0853 2-1
LESSON 2
MATERIAL EMPLOYED IN HEMATOLOGY
Section I. LABORATORY REAGENTS
2-1. PREPARATION
Various stains and solutions are utilized in routine hematological examinations.

These stains and solutions must be prepared with the utmost care and precisely
according to formulations. Detailed directions for the preparation of all reagents that are
required for performing procedures outlined throughout this subcourse are contained in
the respective procedure. Careful attention should be given to precise measurements
order in which reagents are added, control of temperature where indicated, filtration,
and aging. Particular attention must be given to storage of reagents particularly with
reference to requirements for refrigeration, incubation, and protection from intense light.
2-2. LABELING REAGENT CONTAINERS
Proper labeling of reagents is an extremely important detail. Labels should be

complete, securely attached, and neatly and legibly written or preferably typewritten.
Items recorded on the label should include all constituents and quantities utilized, date
of preparation, initials of the individual who prepared the reagent, and expiration date if
the solution deteriorates with age. Labels should be protected against damage from
water or other fluids by covering with a protective coating of cellophane tape over the
surface of the label.
2-3. SAFETY PRECAUTIONS
There are various precautions that must be taken in handling reagents in the
hematology laboratory. Among the most important are the following:
a. Once a portion of a reagent has been removed from the original container, it
should never be poured back because it can contaminate the remaining reagent.
b. Reagents are preferably stored in alphabetical order on shelving protected

from dust, moisture, and direct sunlight.
c. Never use a reagent that cannot be clearly identified from the label on the
container. Discard all reagents that cannot be accurately identified.
d. Always read the label before dispensing a reagent.
e. When working with newly prepared reagents, especially stains, ascertain

whether desired results are being obtained. Unsatisfactory solutions should be
discarded and replaced.
MD0853 2-2
f. All mixing containers, stirring rods, and containers used for storage of
reagents should be chemically cleaned prior to use.
g. During mixing and preparation, as well as in storage, it is good practice to

avoid contact of reagents with metals. Many reagents contain substances that will react
chemically with metals and produce changes that will render than unusable for
laboratory work.
h. Do not allow inexperienced personnel to prepare reagents without close

supervision.
i. Certain reagents are poisonous and adequate precautions should be taken to

prevent accidental poisoning. All highly toxic reagents should be conspicuously labeled
"POISON" and should be stored in a separate cabinet in the laboratory.
j. Commercial reagents should be checked with standards for purity. Record all
lot numbers in case a reagent is not pure.
k. Test all new reagents to assure that proper results are attainable.
Section II. LABORATORY GLASSWARE
2-4. DILUTING PIPETS
a. The blood cell diluting pipet (see figure 2-1) consists of a graduated capillary
tube having an arbitrary volume of one unit and marked in increments of that unit, each
designated as 0.1; note that this unit is not a standard measurement but merely an
arbitrarily selected unit. Above the capillary tube is a mixing bulb containing a color-
coded glass bead, and above the bulb another shorter capillary tube with an engraved
mark. The pipet for performing the white blood cell count has a white bead, the mixing
bulb is smaller than that of the red count pipet, and the marking above the bulb reads
"11." The pipet for performing the red blood cell count has a red bead in the mixing bulb
and the marking above the bulb is "101."
b. These pipets are used to take the specimen directly from a capillary puncture
or, after careful mixing, from a vial of fluid or of blood treated with an anticoagulant,
such as EDTA. The blood or fluid is drawn into the pipet to a predetermined point and
diluted to the correct mark with diluting fluid. After proper mixing, the diluted substance
is placed in the counting chamber and the cells are counted.
c. Usually, the technique for diluting the blood specimen with a pipet calls for
whole blood to be drawn exactly to the 0.5 mark and diluted only to the "11" or "101"
mark with appropriate diluting fluid dependent upon the type of cell count. Since the
volume of fluid in the stem does not enter into the dilution, the dilution is calculated on
the volume in the bulb, thus with the white blood cell count:
MD0853 2-3
Figure 2-1. Hematological pipets.
Whole blood = 0.5 = 1

Dilution = Volume in bulb 10 20
and in the RBC count
Whole blood = 0.5 = 1

Dilution = Volume in bulb 100 200
The dilution of the blood sample in the white blood count is 1 to 20 and the dilution
factor is 20. In the red blood count the blood sample is diluted 1 to 200 and the dilution
factor is 200. The permissible error of the red cell pipet is +5 percent and the white cell
pipet has a permissible error of +3.5 percent.
d. Blood cell diluting pipets are delicate pieces of equipment and should have
careful treatment. It is also important that clean, dry diluting pipets be used to prevent
dilution errors and hemolysis of cells.
e. If an aspirator is available, pipets can be easily washed by drawing cold tap

water (distilled water is preferred) through the pipet. If a deposit remains, it can be
dissolved by drawing dilute sodium hypochlorite (household bleach) through the pipet
followed by several washings with distilled water. If pipets become plugged through
neglect, the hole may be opened with a fine steel wire, and the above procedures may
then be carried out. When necessary, pipets may be dried rapidly by drawing a small
amount of alcohol through then and then ether or acetone followed by a stream of air
until the glass bead removes freely and no moisture remains in the pipet.
MD0853 2-4
f. All pipets should be inspected each time before use so as to prevent the use
of equipment that is dirty or that has chipped ends.
2-5. UNOPETTE SYSTEM
a. The Unopette System (Becton- Dickinson and Co.) consists of a disposable
uniform-bore glass capillary pipet with an attached plastic tab for handling. The pipet
(see figure 2-2) is attached to a plastic reservoir in which predetermined amounts of
diluting fluids, depending on their purpose, can be placed. The blood aspirated into the
diluting fluid can be mixed and dispensed with great ease and speed.
Figure 2-2. Unopette System.

b. The Unopette System can be used for a variety of hematological procedures.
In general, the Unopette System is used as follows:
(1) Puncture diaphragm. Using the protective shield on the capillary pipette,
puncture the diaphragm of the reservoir as follows:
(a) Place reservoir on a flat surface. Grasping reservoir in one hand,
take pipette assembly in other hand and push tip of pipette shield firmly through
diaphragm in neck of reservoir, then removed.
Figure 2-3. Unopetted filling procedure (continued)
MD0853 2-5
(b) Remove shield from pipette assembly with a twist.
(2) Add sample. Fill capillary with whole blood and transfer to reservoir as
follows:
(a) Holding pipette almost horizontally, touch tip of pipette to blood.

Pipette will fill by capillary action. Filling is complete and will stop automatically when
blood reaches end of capillary bore in neck of pipette.
(b) Wipe excess blood from outside of capillary pipette making certain
that no sample is removed from capillary bore.
(c) Squeeze reservoir slightly to force out some air. Do not expel any
liquid. Maintain pressure on reservoir.
MD0853 2-6
(d) Cover opening of overflow chamber of pipette with index finger and
seat pipette securely in reservoir neck.
(e) Release pressure on reservoir. Then remove finger from pipette

opening. Negative pressure will draw blood into diluent.
(f) Squeeze reservoir gently two or three times to raise capillary bore,
forcing diluent up into, but not out of, overflow chamber, releasing pressure each time to
return mixture of reservoir.
(g) Place index finger over upper opening and gently invert several
times to thoroughly mix blood with diluent.
Figure 2-3. Unopetted filling procedure (concluded).
MD0853 2-7
(h) Let stand for ten (10) minutes to allow red cells to hemolyze.
2-6. BLOOD CELL COUNTING CHAMBERS
a. The most common type of hemacytometer consists of two counting chambers
separated by grooves or canals. On the smooth glass surface of the counting
chambers are straight lines etched into glass in a gridwork pattern. The Neubauer
ruling, preferred for hematological work, consists of a gridwork with dimensions of 3 mm
by 3 mm. It is further divided into 9 smaller squares with dimensions of 1 mm by 1 mm;
4 of these squares are used for the white count. The 8 outer squares are further
subdivided into 16 squares 0.25 mm on a side. The central square is divided into 25
squares, 0.20 mm on a side, which are used for the red cell count. Thus the large
squares are 1 square mm, the 16 small squares in the outer large squares are 1/16
square mm and the 25 central squares are 1/25 square mm (see figure 2-4).
Figure 2-4. Rulings on a hemacytometer.
b. The cover glass must be free of visible defects and must be optically plane on
both sides within + 0.002 mm according to the United States (US) Bureau of Standards.
When the cover glass is placed on the platform, the space between it and the ruled
platform should be 0.1 mm.
2-7. CLEANING
Glassware can be cleaned in hot, soapy water and thoroughly rinsed in distilled
water. Blood dilution pipets can be washed by flushing water and acetone through
them. Ordinary household bleach can be used to remove blood clots in the bore of
pipets. Dilution pipets are dry when the bead moves freely in the bulb. Glassware to be
used for coagulation studies must be scrupulously clean. This glassware should be
MD0853 2-8
cleaned in nonorganic detergents and rinsed well with distilled water. The etched
surface of the hemacytometer should be rinsed in water and blotted dry with lens paper
to avoid marring or further etching of the lines on the surfaces. A method of cleaning
small bore tubes and pipets (such as Wintrobe sedimentation rate tubes) is to attach a
capillary pipet, by a rubber hose, to a water type suction pump. Attach the tube to the
flat end of the pipet and hold the tube under water. Blood is drawn from the tube as
water is drawn in. When the tube is clean, invert the tube, remove the residual water by
suction, and allow to dry. Occasionally, the tube should be cleaned with dilute sodium
hypochlorite (household bleach) to remove deposits of residual blood.
Section III. LABORATORY EQUIPMENT
2-8. MICROSCOPES
a. Introduction. A modern microscope for use in the hematology laboratory is

equipped with an illuminator system, a substage condenser system, an objective
system, a projector (eyepiece or ocular system), an iris diaphragm, nicol prisms, a
tubular barrel (monocular or binocular bodies), and a mechanical stage (see figure 2-5).
A compound microscope uses a combination of lenses, the objective lens (lens closer to
the object) and the ocular lens (lens closer to the eye) to project the image to the retina
of the eye. The objective lens acts much like a small projection lens which projects an
enlarged primary image near the top of the tubular barrel. This image, formed in air, is
known as an "aerial image”. This object is viewed through the projector or eyepiece that
acts like a magnifier except that it magnifies an aerial object instead of an actual object.
The final image projected on the retina of the eye is called a "virtual image" because the
light rays appear to come from the image. The rays are actually created by an increase
in magnification by the lens system.
b. Magnification.
(1) Magnification in a microscope is limited to the useful magnification that

can be achieved, that is, the ability to obtain fine detail of the object being examined.
This ability to render visible the fine detail is the resolving power of the microscope. The
resolving power of a microscope is dependent on the numerical aperture (N.A.) of the
objective lens and condenser lens. Therefore, proper adjustment of these lenses is
essential in order to obtain useful magnification.
(2) Microscopes in general use in medical laboratories are provided with

three objectives with focal lengths of 1.9 mm, 4 mm, and 16 mm, respectively.
Microscopes are usually provided with 5X and 10X (most common) oculars. Multiplying
the power of the ocular by the power of the objective gives the degree of magnification
of the object under observation. The degree of magnification is expressed in diameters
(refers to an increase in diameter). The ocular magnification, the millimeter length of
the objective, its magnification power, and the total apparent increase obtained using
oculars and objectives of the powers shown are given:
MD0853 2-9
Figure 2-5. Compound microscope.
Ocular Objective Magnification
10X 16 mm (10X) 100 diameters

10X 4 mm (43X) 430 diameters
10X 1.9 mm (97X) 970 diameters
Magnification is increased in practice by using a higher power objective. Most

microscopes are equipped with a revolving nosepiece, and selection of an objective
lens is done with ease.
c. Illumination.
(1) Correct illumination of the object under study is an extremely important
detail. Incorrect lighting of the object can lead to inaccurate results and conclusions.
Correct illumination can be obtained from a concave mirror or substage light.
(2) Illumination entering the microscope can be central or oblique. To
obtain central illumination the light from the source must be reflected from the mirror
directly into the microscope tube. Oblique lighting must be avoided because an object
in the center of the field will sway from side to side when the fine adjustment is rotated.
Oblique light is usually no problem with substage illuminators.
MD0853 2-10
(3) Regulation of the amount of light admitted is accomplished by the iris
diaphragm in the substage condenser. The size of the opening in the diaphragm is
controlled by a lever on the side of the condenser. The lever of the iris diaphragm
should never be forced to the full limit in either direction. Doing so may damage the
delicate leaves of the diaphragm. Generally, when observing liquid preparations under
low power, the diaphragm opening should be partially closed. Under the high dry
objective, the diaphragm is generally opened to a greater degree to allow more light to
pass through the material. When observing stained preparations under the oil
immersion objective, the iris diaphragm is usually opened wide.
(4) The substage condenser functions to direct a light beam of the desired
numerical aperture (N.A.) and field size onto the specimen. The size of the opening in
the condenser together with its position up or down controls the light entering the
system. When the condenser is close to the stage, concentration of light is greater; as
the condenser is moved downward, less light passes upward through the object under
observation.
(5) Improper illumination is indicated when: (1) dark points or shadows

appear in the field; (2) the outline of an object is bright on one side and dark on the
other; or (3) the object appears to be in a glare of light. This can usually be corrected
by changing the position of the mirror, by reducing the amount of light by adjusting the
size of the opening in the iris diaphragm, or by raising or lowering the condenser.
d. Focusing. Focusing can be defined as the adjustment of the relationship

between the optical system and the object so that a clear image is obtained. Several
important rules to be observed when focusing the microscope on the preparation are:
(1) After the object is mounted on the stage, the objective to be used is
turned into line with the eyepiece.
(2) Movement of the objective is accomplished by revolving the nosepiece.

The nosepiece is provided in order to enable rapid, convenient substitution of one
objective for another. This change is effected by grasping two of the objectives
between the thumb and forefinger of the right hand and rotating them until the desired
objective is brought into line with the axis of the body tube. It is very important that
exact alignment be obtained. The correct setting is indicated by a slight "click" as the
objective comes into position.
(3) Whenever the nosepiece is revolved, its movements should be observed

to make certain that the objectives do not come into contact with the object. Some
microscopes are not parfocal; that is, objects in focus under low power will not be in
focus when the nosepiece is rotated to a higher power of magnification. It may,
therefore, be necessary to refocus when changing to higher magnification. In
microscopes that are parfocal, it is possible to swing other objectives into place without
touching the coarse adjustment and with only a slight turn of the fine adjustment knob
required to restore perfect focusing.
MD0853 2-11
(4) To bring an object into focus, watch from the side and use the coarse
adjustment to lower the objective until it is below the point at which the object would
normally be expected to come into view.
NOTE: To avoid damage to slide or microscope, view from side for preliminary
focusing. Then, using the coarse adjustment and at the same time looking
through the ocular, raise the objective very slowly until the field comes into
view. Further adjust to the best image, using only the fine adjustment.
(5) In focusing upward with the fine adjustment, the object will first appear in
faint outline, then gradually more distinctly, and finally, sharply defined. If the
adjustment goes beyond the point of sharp definition, return to the point of greatest
clarity by using the fine adjustment.
(6) Never move an objective downward while looking through the eyepiece.
When the objective is moved downward, always observe the downward motion with the
eye held level with the microscope stage. Failure to observe these precautions can
result in damage to the lens of the objective or the object under study.
e. Care of the Microscope. The microscope is an instrument of precision with

many delicate parts, and it must be handled with the utmost care. Care should not be
confined to the optical elements alone. The microscope is a combination of optical and
mechanical excellence, one complementing the other. The following precautions should
always be observed in the care of the microscope:
(1) No unauthorized person should manipulate the microscope.
(2) Keep the microscope as free from dirt and dust as possible. Dusty
lenses produce foggy images, while dust in the focusing mechanisms causes excessive
wear of those parts.
(3) The microscope should be always covered when not in use.
(4) Care should be taken to prevent all parts of the microscope from coming
into contact with acid, alkali, chloroform, alcohol, or other substances that corrode metal
or dissolve the cementing substance by means of which the lenses are secured into the
objectives and oculars.
(5) Always carry the microscope with two hands by the arm and base.
(6) Avoid sudden jars I such as placing the microscope on the table with
undue force.
(7) No dust should be permitted to settle on the lenses nor should the finger
come in contact with any of the surfaces.
(8) The lens system should never be separated, as the lenses are liable to
become decentered and dust can enter.
MD0853 2-12
(9) Avoid all violent contact of the objective lens and the cover glass.
(10) Keep eyepieces in the microscope at all tines to keep free of dust.
(11) To remove dust, brush the lenses with a camel's hairbrush. Avoid hard
wiping, as dust is often hard and abrasive.
(12) Xylene is the only agent that should be used in cleaning lenses or
removing oil from objectives. Only small amounts of xylene are necessary and should
be used with care.
(13) When sewing machine oil is used to lubricate moving parts of the
microscope, all excess should be wiped off to prevent the collection of grit and dust.
(14) The microscope should be protected against direct sunlight and

moisture.
(15) In very warm, humid climates, microscopes should be stored in dry

cabinets when not in use. Such cabinets should be reasonably airtight, equipped with a
light bulb to supply heat, and several cloth bags containing a hygroscopic salt, such as
calcium chloride, to absorb moisture. In warm, humid climates, the lenses of
unprotected microscopes can be attacked by certain fungi that etch glass and ruin the
lenses.
(16) After use, always turn the nosepiece to a position, which brings the low
power objective into direct line with the opening in the substage condenser. If this
precaution is not taken, the longer, higher-powered objectives can accidentally come
into contact with the condenser lens.
(17) The entire microscope should be cleaned frequently to remove dust,

finger marks, oil, grease, and remnants of specimens. All parts of the microscope
should be kept scrupulously clean at all times.
(18) Never tamper with any of the parts of the microscope. If the instrument
does not seem to be functioning properly, immediately call the matter to the attention of
the laboratory supervisor.
(19) Maintenance of the microscope should be done in accordance with the
manufacturer's booklet of instruction.
(20) Immediately after use, the oil immersion objective must be wiped clean
of oil with a soft, absorbent lens paper.
f. Types.
(1) Binocular. This type of microscope is preferred since it gives a more

natural and restful condition of observation. A beam-dividing prism and three mirrors
MD0853 2-13
divide the light equally, sending half to the left eye and half to the right eye. The coating
on the mirrors is enhanced by aluminum to increase the reflectivity. The binocular body
is protected by cover glass seals to keep dust from entering. The binocular compound
microscope is the preferred microscope for routine hematology.
(2) Phase. Phase microscopy is becoming increasingly prevalent in platelet

counting. In bright-field illumination, a completely transparent specimen is difficult to
see in any detail. By using phase contrast, transparent living objects can be studied.
Phase microscopy operates on the principle that if a portion of light is treated differently
from the rest, and caused to interfere with the rest, it produces a visible image of an
otherwise invisible transparent specimen. Phase contrast accessories are available
from the standard optical companies.
2-9. CENTRIFUGES
These are laboratory devices or units that apply a relatively high centrifugal force
(up to 25,000 g) to a specimen, causing its separation into different fractions according
to their specific gravities. Centrifugation is the process of separating components of a
mixture (away from a center as in centrifugal force) on the basis of differences in
densities of the different components using a centrifuge.
a. Table Top Models. These units are mounted on rubber feet that absorb
vibration. The speed is controlled by means of a rheostat on the front panel. Top
speeds of centrifuges will vary and the top speed of a particular instrument should be
known in order to use the speed control device. Those centrifuges have adapters to
hold 6 tubes and adapters for 12 tubes.
b. Floor-Mounted Models. The heavier floor-mounted models accommodate a

large number of tubes at one time. The top speed of these instruments is higher than
that of table models. Because of their increased inertia, they are equipped with a brake
to facilitate stopping. In these units, the tubes are placed in balanced receptacles that
are mounted on spokes emanating from a central hub.
c. Microhematocrit Centrifuge. This centrifuge is a special type of high-speed
centrifuge employed to spin capillary tubes (see figure 2-6). The circular tube holder on
this centrifuge is flat, surrounded by a rubber ring. It has a capacity of 24 capillary
tubes. After a capillary tube is filled with blood, it is closed with a commercial plastic
sealing material. During centrifugation the sealed end is always placed in position
facing toward the outside of the holder plate. Most centrifuges of this type spin the tubes
at 10,000 rpm.
d. Precautions. In all instances where centrifugation is required, careful
attention must be given to balancing the units. This means that tubes must be placed
exactly opposite each other, they must be of identical weight, and they must contain the
same amount of fluid. If at all possible, centrifuges should be equipped with
tachometers so that speed nay be checked and controlled. Certain procedures, such as
hematocrits, require a critical relative centrifugal force (RCF or g). Relative centrifugal
MD0853 2-14
force is the weight of a particle in a centrifuge relative to its normal weight, the
centrifugal force per unit mass in gravities (g). To determine the RCF (or g) for these
procedures, consult the serology manual or a monograph. The inside of the centrifuges
should occasionally be cleaned to prevent dust particles from being blown into
specimens. The lid on the centrifuge should be closed and locked before and during
operation. Only open the lid when the centrifuge has stopped rotating.
Figure 2-6. Microhematocrit centrifuge and microhematocrit reader.
MD0853 2-15
EXERCISES, LESSON 2
answer in the space provided at the end of the exercise.
After you have completed all of the exercises, turn to "Solutions to Exercises" at
the end of the lesson and check your answers. For each exercise answered incorrectly,
1. When using laboratory reagents, what routine hematological care should be taken
in their preparation?
a. Follow detailed procedural directions.
b. Measure regents exactly.
c. Follow the order that reagents are to be added.
d. Control the temperature where indicated.
e. Use filtration of ingredients as stated.
f. Age the prepared solution as specified.
2. Particular attention must be given to storage of laboratory reagents, particularly

with reference to requirements for:
a. Refrigeration.
b. Incubation.
c. Protection from intense light.
MD0853 2-16
3. A reagent container is correctly labeled if the label contains the:
a. Constituents, initials of the individual who prepared the reagent in alphabetical

order, expiration date, quantity used, and date prepared.
b. Expiration date, initials of the individual who prepared the reagent,

constituents quantity used, temperature control, and date prepared.
c. Initials of the individual who prepared the reagent, stains, constituents,

expiration date, quantity used, and date prepared.
d. Constituents, expiration date, initials of the individual who prepared the

reagent, quantity used, and date prepared.
4. A reagent's container label is properly labeled and protected if the label is:
a. Complete and securely attached.
b. Neatly and legibly written or preferably typewritten.
c. Covered with a protective coating of cellophane tape over the surface of the
label.
5. Why must an unused portion of a reagent never be poured back into tile original
container?
a. Possible explosions.
b. Inefficiency.
c. Contamination.
d. Improper labeling.
MD0853 2-17
6. When storing reagents on shelving, protect them from:
a. Dust.
b. Moisture.
c. Direct sunlight.
7. What should you do if you cannot clearly read the reagent's label and/or identify
the contents of the container?
a. Use the contents.
b. Check with your colleague.
c. Discard it properly and use a reagent's container with a label that you can read
and the contents you can identify.
d. Save it for next time.
8. When preparing laboratory reagents for storage, which items should be chemically
cleaned prior to use?
a. Mixing containers.
b. Stirring rods.
c. Storage containers.
MD0853 2-18
9. At what point is it is a good, safe, practice to avoid contact of reagents with metals
since metals may become unusable for laboratory work?
a. Labeling and heating.
b. Refrigeration and labeling.
c. Preparation and mixing.
d. Preparation and heating.
10. Highly toxic reagents should be conspicuously labeled:
a. Reagent.
b. "POISON.”
c. Poison.
d. “REAGENT".
11. The white blood cell diluting pipet has a:
a. Smaller bulb, a red bead, and an "11.”
b. Smaller bulb, a white bead, and an “11.”
c. Larger bulb, a red bead, and a "101.”
d. Larger bulb, a white bead, and a “101.”
12. The red blood cell diluting pipet has a:
a. Smaller bulb, a red bead, and an "11."
b. Smaller bulb, a white bead, and an "11."
c. Larger bulb, a red read, and a "101."
d. Larger bulb, a white read, and a "101."
MD0853 2-19
13. The 0.5 mark on the RBC diluting pipet designates a volume equal to:
a. 0.5 ml.
b. 0.5 cu mm.
c. 1/20 of the dilution volume.
d. 1/200 of the dilution volume.
14. Which statement is correct concerning the use of the diluting pipets for cell
counting?
a. Blood or fluid is drawn into the pipet to a predetermined point and diluted to
the correct mark with diluting fluid. After proper mixing, the diluted substance
is placed in the counting chamber and the cells are counted.
b. Blood or fluid is drawn into the pipet to an arbitrary point and diluted to the
correct mark with diluting fluid. After proper mixing, the diluted substance is
placed in the counting chamber and the cells are counted.
c. Blood or fluid is drawn into the pipet to a predetermined point and diluted to a
guesstimated mark with diluting fluid. After gentle mixing, the diluted
substance is placed in the counting chamber and the cells are counted.
15. Usually, the technique for diluting the blood specimen with a pipet calls for whole
blood to be diluted only to the "11" or "101" mark with appropriate diluting fluid
dependent upon the type of cell count. How is the dilution calculated?
a. The volume of fluid is measured in the stem and then calculated.
b. Dilution is calculated on the volume in the bulb.
c. Both a and b.
d. None of the above
MD0853 2-20
16. In general, what is the proper procedure for puncturing the diaphragm using the
Unopette System?
a. Using the protective shield on the capillary pipette, puncture the diaphragm of
the reservoir.
b. Grasping the reservoir in one hand, take pipette assembly in other hand and
pull tip of pipette shield firmly through diaphragm in neck of reservoir, then
remove.
c. Grasping the reservoir in one hand, take pipette assembly in other hand and
push tip of pipette shield firmly through diaphragm in neck of reservoir, then
remove.
d. Remove shield from pipette assembly with a twist.
e. a, b, c, and d.
f. a, b, and d.
g. a, c, and d.
17. What phenomenon is used to fill the Unopette capillary with blood?
a. Gravity.
b. Capillary action.
c. Brownian movement.
d. Static electricity.
18. When using the Unopette system, at what point will the pipette automatically stop
filling and be complete? When:
a. The blood reaches the top reservoir.
b. Negative pressure is applied,
c. The blood reaches the end of the capillary bore.
MD0853 2-21
19. How many times should the reservoir be squeezed and with what pressure to raise
the capillary bore and force the diluent up into, but not out of, the overflow
chamber using the Unopette system?
a. 4 to 6; hard.
b. 3 to 5; even.
c. 2 to 3; gentle.
d. 1 to 6; steady.
20. Using the Unopette System, after the blood is thoroughly mixed with the diluent
and left to stand for 10 minutes, the red cells will:
a. Overflow.
b. Settle out.
c. Hemolyze.
21. What are the outer dimensions of the Neubauer ruling?
a. 0.20 by 0.20 mm.
b. 0.25 by 0.25 mm.
c. 1 by 1 mm.
d. 3 by 3 mm.
22. What configuration does the most common type of hemacytometer look like for
counting blood cells?
a. Two counting chambers separated by grooves or canals.
b. Three counting chambers separated by grooves or canals.
c. Two counting chambers separated by rough indentations.
d. Four counting chambers that are not separated.
MD0853 2-22
23. How many squares are used to count white cells, when the dimensions of the
Neubauer ruling are further divided into 9 smaller squares, with dimensions of
1 mm by 1 mm?
a. 2.
b. 3.
c. 4.
d. 5.
24. Using the Neubauer ruling for the red cell count, which portion and how many
mms are used?
a. Middle squares; 0 30 mm on a side.
b. Outer 8 squares; 0.15 mm on a side.
c. 10 outer squares; 0 05mm on a side.
d. Central square; 0.20 mm on a side.
25. When taking a blood cell count, the cover glass must be free of visible
____________________ and optically ____________________ on both sides.
a. Defects; plane.
b. Outer squares; plane.
c. Stains; rough.
d. Blood; clean.
MD0853 2-23
26. A method for cleaning Wintrobe sedimentation rate tubes, small bore tubes and
pipets, and remaining blood clots in the bore of a pipet is to:
a. Attach a capillary pipet by a rubber hose, to a water type suction pump.
b. Attach the tube to the flat end of the pipet and hold the tube under water.
c. Draw the blood from the tube as water is drawn in.
d. Ensure the tube is clean by inverting it, remove the residual water by suction,
and allow to dry.
e. All of the above.
f. None of the above.
27. What occasional method it used to clean small bore tubes and pipets?
a. Use dilute sodium hypochlorite (household bleach) to remove deposits of

residual blood.
b. Use distilled water.
c. Insert a small rubber hose.
d. Use a suction tube.
e. Absorbent lens paper and water.
28. Blood dilution pipets can be washed by:
a. Soaking in calcium chloride.
b. Soaking in ammonia.
c. Flushing water and acetone through them.
d. Rinsing in distilled water.
MD0853 2-24
29. Glassware, which is used for coagulation studies, must be __________________
cleaned in _______________________.
a. Gently; household bleach.
b. Freely; distilled water.
c. Scrupulously; non-organic detergent.
d. Somewhat organic soapy water.
e. Vigorously; in water with absorbent lens paper.
30. Select the correct items normally found on a modern microscope used in a
hematology laboratory.
a. A darkening system, a substage condenser system, an objective system, a

projector (eyepiece or ocular system), an iris diaphragm, nicol prisms, a
tubular barrel (monocular or binocular bodies), and a mechanical stage.
b. An illuminator system, a substage condenser system, an objective system, a

c. An illuminator system, a substage condenser system, an objective system, a

projector (eyepiece or ocular system), a round barrel (monocular or binocular
bodies), and a mechanical stage.
d. An illuminator system, a substage condenser system, an objective system, a

31. What combination of lenses does a compound microscope use?
a. Objective lens.
b. Monocular lens.
c. Aerial image magnifier.
d. a and b.
e. b and c.
MD0853 2-25
32. Select the best explanation of an "aerial image”.
a. An image formed in the air.
b. An image formed in the air. The object is viewed through the projector or
eyepiece that acts like a magnifier except that it magnifies an aerial object
instead of an actual object.
c. An image formed in the air. The object is viewed through the magnifying glass
except that it magnifies an aerial object instead of an actual object.
d. An image formed on a surface. The object is viewed through the projector or

eyepiece that acts like a magnifier except that it magnifies an aerial object
instead of an actual object.
33. The ability of a microscope to render fine detail is dependent upon the numerical
aperture and proper adjustment of which lens (es)?
a. Ocular and objective.
b. Ocular and condenser.
c. Objective and condenser.
d. Objective only.
34. When rotation of a microscope's fine adjustment causes an object in the center of
the field to sway from side to side, the lighting is:
a. Central.
b. Oblique.
c. Too dim.
d. Too intense.
MD0853 2-26
35. Name one item the resolving power of the microscope is dependent upon during
magnification?
a. Focal lengths.
b. Binocular bodies.
c. Arial image.
d. N.A. of the objective.
36. Preliminary focusing of a microscope should be observed from the:
a. Ocular.
b. Objective.
c. Top of the microscope.
d. Side of the microscope.
37. Since correct illumination of an object under study is an extremely important detail,
what can incorrect lighting cause?
a. Inaccurate results and conclusions.
b. Inaccurate steps and timings.
c. Faulty conclusions and recommendations.
d. Changing positions and recommendations.
38. What is the function of the substage condenser when illuminating slides under the
microscopic?
a. Indicates dark spots.
b. Reduces glare.
c. Directs a light beam.
d. Correct inconsistencies by changing the position left or right.
MD0853 2-27
39. Which statement about the substage condenser is true?
a. The substage condenser functions to direct a hair beam of the desired

numerical aperture (N.A.) and field size onto the specimen.
b. The size of the opening in the condenser together with its position up or down
controls the light entering the system.
c. When the condenser is open to the stage, concentration of light is greater.
d. As the condenser is moved upward, less light passes downward through the
object under observation.
40. Improper illumination is indicated when:
a. Light points appear on the outer edges of the slide.
b. The center of an object is bright on one side and dark on the other.
c. The object appears to be in dull light
d. Shadows appear in the field.
41. Which three parts of a microscope may be adjusted to control the illumination?
a. Mirror, iris diaphragm, and ocular.
b. Mirror, iris diaphragm, and objective.
c. Mirror, iris diaphragm, and condenser.
d. Ocular, objective, and condenser.
MD0853 2-28
42. When using a microscope the "virtual image" projected on the retina of the eye
is ________________________ the image.
a. Initial.
b. Intermediate.
c. Final.
d. Aperture.
43. Which of the following is used to clean microscope lenses?
a. Xylene.
b. Acetone.
c. Household bleach.
d. Saturated sodium hydroxide.
44. Which type of microscope is preferred for routine hematology?
a. Monocular.
b. Binocular.
c. Fluorescence.
d. Phase.
45. Because of dust, the lens system should:
a. Be separated.
b. Never be separated.
MD0853 2-29
46. After a capillary centrifuge tube is filled with blood, it is sealed with:
a. Plastic sealing material.
b. Paper.
c. Glass.
d. Wax.
47. Most microhematocrit centrifuges have a speed of about:
a. 500 rpm.
b. 1,000 rpm.
c. 5, 000 rpm.
d. 10,000 rpm.
48. Which centrifuge has the higher type of top speed for instruments?
a. Table top model.
b. Floor-mounted model.
49. Only which agent should be used to remove oil from a lens object?
a. An abrasive.
b. A rag.
c. Xylene.
d. Camel hairbrush.
MD0853 2-30
50. Whenever centrifugation is required, which precaution must always be followed?
a. Careful attention must be given to balancing the units. This means that tubes
must be placed exactly opposite each other, they must be of identical weight,
and they must contain the same amount of fluid.
b. Centrifuges should be equipped with a microhematocrit so that speed may be

checked and controlled.
c. Hematocrits require a regular force and can adapt for 6 to 19 tubes.
d. The heavier, floor-mounted models require rubber feet to absorb vibrations so

as to accommodate a large number of tubes, which are housed in mounted
receptacles on spokes from a distance hub.
MD0853 2-31
1. g (para 2-1)
2. d (para 2-1)
3. d (para 2-2)
4. d (para 2-2)
5. c (para 2-3a)
6. d (para 2-3b)
7. c (para 2-3c)
8. d (para 2-3f)
9. c (para 2-3g)
10. b (para 2-3i)
11. b (para 2-4a)
12. c (para 2-4a)
13. d (para 2-4c)
14. a (para 2-4b)
15. b (para 2-4c)
16. g (para 2-5b(1))
17. b (para 2-5b(2))
18. c (para 2-5b(2) (a))
19. c (para 2-5b(2)(f))
20. c (para 2-5b(2) (h))
21. d (para 2-6a)
22. a (para 2-6a)
MD0853 2-32
23. c (para 2-6a)
24. d (para 2-6a)
25. a (para 2-6b)
26. e (para 2-7)
27. a (para 2-7)
28. c (para 2-7)
29. c (para 2-7)
30. d (para 2-8a)
31. d (para 2-8a)
32. b (para 2-8a)
33. c (para 2-8b(1))
34. b (para 2-8c(2))
35. d (para 2-8b(1))
36. d (para 2-8d(4) NOTE)
37. a (para 2-8c(1))
38. c (para 2-8ac(4))
39. b (para 2-8c(4))
40. d (para 2-8c(5))
41. c (para 2-8c(5))
42. c (para 2-8a)
43. a (para 2-8e(12))
44. b (para 2-8f(1))
45. b (para 2-8e(8))
MD0853 2-33
46. a (para 2-9c)
47. d (para 2-9c)
48. b (para 2-9b)
49. c (para 2-8e(12))
50. a (para 2-9d)
End of Lesson 2
MD0853 2-34
LESSON ASSIGNMENT
LESSON 3 Collection of Blood and Preparation of Blood Smears.
3-1. Select the statement which best describes the

requirements for selection and care of blood
collection equipment.
3-2. Select the correct steps in collecting,

processing, and recording blood specimens.
3-3. Select the names and functions of commonly

used anticoagulants
3-4. Select the correct method and use the proper

procedures in staining blood films for the type of
blood cells found.
3-5. Select the correct function for each of Wright's

component stain and buffer solutions.
3-6. Select the correct steps to prepare a blood

smears.
3-7. Select the factors that affect the quality of

stained blood smears.

MD0853 3-1
LESSON 3
Section I. COLLECTION OF BLOOD SPECIMENS
3-1. INTRODUCTION
a. Hematological laboratory procedures are based upon the examination of

blood specimens. To obtain valid test results, specimens must be properly collected,
processed, and recorded. Blood specimens are usually obtained by either venous or
capillary puncture. The source of the specimen is determined chiefly by the quantity of
blood required to perform the laboratory procedures and the age and condition of the
patient.
b. There is generally little difference in blood counts performed on venous or

capillary blood if a free-flowing capillary blood specimen is obtained. Valid blood counts
cannot be made when capillary specimens are not taken from a free-flowing sample or
when they are obtained from cyanotic or calloused areas or areas of local stasis. White
blood cell counts made on blood obtained from such sources can vary as much as
1000-1500 cells per cu mm from their real value. For general purposes, however,
venous samples are preferable since they allow for multiple and repeated hematological
examinations and provide a sufficient quantity of blood for performing any other required
laboratory procedure. Further, with venous blood the chances of error are reduced
because operations are made under ideal conditions and repeat operations are
possible. In situations where there are limitations on the quantity of blood that can be
obtained, that is, in small infants or extensive burn cases, microquantitative methods
are satisfactory for performing an analysis on a specimen obtained by capillary
puncture.
3-2. VENIPUNCTURE
a. Site. To obtain blood by venipuncture, draw the specimen directly from a

patient's vein with a sterile hypodermic needle and syringe or a vacuum blood sample
device. In adults use the veins located in the proximal forearm or antecubital space as
illustrated in figure 3-1. In infants employ the jugular or femoral vein for the
venipuncture. The vein selected should be large, readily accessible, and sufficiently
close to the surface to be seen and palpated. If venipuncture poses a problem due to
the age of the patient, sclerotization due to repeated venipuncture, or any other unusual
circumstance, the technician should consult a physician concerning the procedure.
UNDER NO CIRCUMSTANCES SHOULD A TECHNICIAN WITHDRAW BLOOD
FROM A SAGITTAL SINUS, JUGULAR VEIN, OR FEMORAL VEIN. This should be left
to the discretion of the physician in charge. Occasionally, the best vein is found on the
hand, leg, or foot. These areas are more sensitive, and the veins are not as firmly
anchored as those of the arm. Veins can become distended and easier to enter by
allowing the arm to hang down for 2 or 3 minutes, by massaging the blood vessel
MD0853 3-2
Figure 3-1. Site of venipuncture.
toward the body, or by gently slapping the site of puncture. Young and vigorous
persons usually have elastic veins well filled with blood. Elderly or debilitated persons
can have sclerosed or fragile veins, which are hard to enter or which collapse easily.
b. Equipment. All syringes, needles, lancets, and other instruments used for
the collection of blood specimens must be sterile. Disposable syringes or blood
collection sets with vacuum tubes are available through normal supply channels. These
should be used whenever possible. Aseptic technique is necessary to prevent the
possible transmission of homologous serum hepatitis. The following equipment is
necessary to perform a venipuncture:
(1) Isopropyl alcohol, 70 percent, prep pads.
(2) Tourniquet.
(3) Sterile syringes or vacuum blood sample devices.
(4) Gauze pad, 2 x 2 inches.
(5) Needle, 1 to 1 ½ inches long, 19–23 gauge.
(6) Suitable blood collection tubes and labels.
(7) Gloves, latex.
MD0853 3-3
c. Preparation.
(1) Cleanse hands thoroughly with soap and water.
(2) Place an identifying label on the blood collecting tube.
(3) Assemble the sterile needle and syringe. If a vacuum system is used,
screw the needle into the plastic holder. Always leave the cap over the needle when
not in use.
(4) Check to be sure that the syringe works smoothly. The syringe must be
dry to avoid hemolysis of the red cells. The plunger must match the syringe and must
be pushed firmly to the bottom of the cylinder to prevent injection of air into the vein.
This can be fatal.
d. Syringe Procedure.
(1) Place a tourniquet around the patient’s arm above the elbow tightly
enough to check venous circulation, but not so tightly as to stop arterial flow. (If latex
tubing is used, place it approximately 2 inches above he proposed venipuncture site).
Form a loop with the longer end and draw the loop under the shorter end so that the
tails of the tubing are turned away from the proposed site (see figure 3-2a).
CAUTION: Do not allow the tourniquet to remain in place for more than 2 minutes.
Check the pulse at the wrist to make sure that arterial circulation is not
cut off.
Figure 3-2a. Venipuncture procedure: Locate the vein.
(2) Instruct the patient to make a tight fist.
(3) By inspection and palpation locate the desired vein, determine the
direction of its course, and estimate its size and depth (see figure 3-2a venipuncture
procedure, a through h).
MD0853 3-4
(4) Release tourniquet.
(5) Cleanse the skin over the selected vein with prep pads in 70 percent
isopropyl alcohol. Wipe off excess alcohol with a sterile dry gauze. Do not contaminate
the area after cleaning (see figure 3-2b).
Figure 3-2b. Venipuncture procedure: Clean the puncture site.
(6) Put on gloves.
(7) Replace tourniquet on arm and have the patient straighten out the arm
and make a fist.
(8) Grasp the syringe in the right hand and place forefinger on the hub of
the needle to guide it. Grasp the forearm with the left hand about 2 inches below the
area to be punctured and hold the skin taut with the thumb (see figure 3-2c).
3-2c. Venipuncture procedure: Guide needle toward the vein.
MD0853 3-5
(9) With the needle bevel up, parallel to, and alongside the vein, insert the
needle quickly under the skin and then into the vein. The insertion into the skin and
vein can be performed in one complete motion (see figure 3-2d). After entry into the
vein, blood will appear in the needle hub. Do not probe or move the needle horizontally,
as discomfort and possible nerve damage may result.
Figure 2-3d. Venipuncture procedure: Insert needle into the vein.
(10) Aspiration of the blood is accomplished by gently pulling upon the

syringe plunger (see figure 3-2e). The syringe barrel should be held steady during this
process. Withdraw the desired quantity.
Figure 3-2e. Venipuncture procedure: Aspirate the blood.
(11) Remove the tourniquet by pulling on the long, looped end of the tubing
only after blood is drawn into the syringe (see figure 3-2f).
Figure 3-2f. Venipuncture procedure: Remove the tourniquet.
MD0853 3-6
CAUTION: Do not remove the needle now. If the needle were remove prior to the
Tourniquet being removed, blood would be forced out of the
venipuncture site, resulting in blood loss and/or hematoma formation
(tumor-like cluster of blood forming under the skin).
(12) Place a sterile gauze pad over the point where the needle entered the
skin and deftly withdraw the needle simultaneously putting pressure on the site (see
figure 3-2g).
Figure 3-2g. Venipuncture procedure: Place a sterile pad over the site and
withdraw the needle.
(13) Have the patient extend the arm and maintain light pressure on the
gauze pad over venipuncture site (see figure 3-2h).
Figure 3-2h. Venipuncture procedure: Have the patient extend the arm and
maintain light pressure on the site.
MD0853 3-7
e. Vacutainer Procedure.
(1) Place the Vacutainer tube in the holder until the rubber stopper reaches
the guideline. The short needle should be embedded in the stopper, but the needle
must not break the vacuum (see figure 3-3).
Figure 3-3. Vacutainer system.
(2) Follow steps 1-6 in paragraph 3-2d.
(3) Enter the vein with the needle parallel to and alongside the vein.
Probing or horizontal movement of the needle while under the skin must be avoided.
(4) After entry into the vein push the tube all the way into the holder;
vacuum is broken, and blood flows freely into the tube. Release the tourniquet at this
time by pulling the long, looped end of the tube.
(5) If the multiple needle is used or more than one tube is required, release
the tourniquet after the first tube is filled; remove the filled tube and insert the next one.
CAUTION: Ensure the needle is not moved while tubes are being changed.
(6) Place a sterile gauze pad over the point where the needle enters the
skin and deftly withdraw the needle, placing pressure on the site.
(7) Have the patient extend the arm and maintain light pressure on the
gauze pad over the venipuncture site.
f. Discussion.
(1) Cleanliness is essential when performing a venipuncture.
(2) It is most important that correct technique be practiced in order to avoid

unnecessary pain to the patient, prevent tissue damage, secure a good representative
blood specimen, and prevent contamination of the specimen or infection of the patient.
(3) Syringes and needles must be thoroughly inspected for damage or

malfunction.
MD0853 3-8
(4) If difficulty is experienced in entering the vein or a hematoma begins to
form, release the tourniquet and promptly withdraw the needle and apply pressure to
the wound.
(5) Vigorous pulling on the plunger of the syringe can collapse the vein,
produce hemolysis of the blood specimen, or cause air to enter the syringe.
(6) When repeated venipunctures have to be performed on one patient, it is

advisable to select different sites for blood withdrawal.
(7) Remove the tourniquet as early as possible once a good flow of blood
has been established. Prolonged application of the tourniquet results in partial stasis of
blood and changes many quantitative values of blood components.
(8) Blood drawn by venipuncture is often stored for a period of time before it
is analyzed. For this reason, certain general precautions must be followed in order to
ensure a valid analysis. Before withdrawing blood from its container, make sure the
blood sample is thoroughly but gently mixed. Blood containers should Be tightly
stoppered at all times to prevent drying or contamination. Store the blood specimen in
the refrigerator. Blood counts should be done within 3 hours after collection. Under no
circumstances should blood taken for hematological examinations be stored overnight.
3-3. CAPILLARY PUNCTURE
a. Site. Several different sites are suitable for capillary puncture. Because it is
the most accessible, the palmer or lateral surface of the tip of the finger (preferably ring
finger) is the most common site in adults. However, certain problems can be
encountered such as heavy calloused areas or excessive tissue fluids (edema) that
tend to result in non-representative samples. The lobe of the ear can be used for
capillary puncture. However, differences in cell concentration do occur when blood is
obtained from this site, primarily because of higher lymphocyte concentrations in the ear
lobe. Because of the small amount of tissue on the fingers of infants, preferred site is
the heel or big toe. A modification of the normal technique that has proven quite
satisfactory when working with the heel of infants is to make two incisions in a
crisscross fashion or “T”.
NOTE: To be a valid report, work done on capillary blood must be from a FREE-
FLOWING puncture wound.
b. Equipment.
(1) Gauze pads 2 x 2 inches.
(2) Blood lancet.
(3) Glass slides, heparinized capillary tubes, and other devices to receive
the specimen.
MD0853 3-9
(4) Isopropyl alcohol, 70 percent, prep pads.
c. Procedure.
(1) The puncture site should be warm to assure good circulation of blood. If
it is cold, apply warm water (38o-40oC) for a few minutes. If blood is to be drawn from
the ear, the edge of the lobe, not the flat side, should be punctured.
(2) The site to be punctured is first rubbed with alcohol prep pads to remove
dirt and epithelial debris, increase circulation, and render the area reasonably
disinfected (see figure 3-4 capillary puncture procedure, a through d).
Figure 3-4a. Capillary puncture procedure: Clean the puncture site.
(3) Allow sufficient time for the circulation to equalize.
(4) While making a finger puncture, apply gentle pressure to the finger to
hold the skin taut. Hold the finger in one hand and the lancet in the other. The puncture
is made perpendicular to the lines of the fingerprints, which results in a more free-
flowing wound (see figure 3-4b).
Figure 3-4b. Capillary puncture procedure: Puncture the finger.
MD0853 3-10
(5) The first drop of blood that appears is wiped away before specimens are
taken (see figure 3-4c).
Figure 3-4c. Capillary puncture procedure: Wipe away first drop of blood.
(6) The blood must not be squeezed out since this dilutes it with fluid from
the tissues, thus altering the ratio of cellular elements to fluid, as well as the ratio of
cellular elements to each other.
(7) After the desired specimens have been collected, have the patient hold
a sterile dry gauze pad over the wound until bleeding stops (see figure 3-4d).
Figure 3-4d. Capillary puncture procedure: Apply pressure to the site.
d. Discussion.
(1) A disposable lancet is a very satisfactory instrument for puncture of the

skin. However, if this is not available a sharp pointed surgical blade is quite suitable.
(2) Do not use the finger on a hand, which has been hanging over the side
of the bed as it is likely to be congested. Edematous or cyanotic areas should not be
used.
MD0853 3-11
(3) The finger should be thoroughly dry prior to puncture; blood will not well
up on a finger that is moist. Furthermore, the alcohol or other antiseptic used can
coagulate the blood proteins causing cell clumping and erroneous values as well as
dilute cell volumes. This will result in incorrect counts and differentials.
(4) Finger punctures should be made along the lateral aspect of the
fingertip. More nerve endings are located on the fingerprint area of the fingers;
therefore, more pain results from punctures in this area. Scars can also form in these
sensitive areas, and difficulty may be encountered in puncturing a callous. All of these
difficulties are eliminated by drawing the blood from the lateral rather than the ventral
aspect of the finger.
3-4. ANTICOAGULANTS
a. Anticoagulants are used to prevent the clotting of the blood specimens and
the reagent employed should not bring about alteration of blood components.
Unfortunately, many anticoagulants can alter cell structures as well as coagulation. The
anticoagulants most often used are ethylene-diamine-tetra-acetate (EDTA), ammonium-
potassium oxalate (Heller and Paul double oxalate), and heparin.
b. The choice of anticoagulant will depend on the analysis to be made.

Ethylene-diamine-tetra-acetate (EDTA) is the anticoagulant of choice for most
hematological analyses. This anticoagulant causes a minimum of distortion to the cells
and platelets. It does not dissolve quickly in blood, however, so the tube must be
inverted four or five times after blood is added. The dipotassium salt is prepared as a 1
percent solution in distilled water, and a final concentration of 0.5 ml of anticoagulant for
each 5 ml blood is used. Another common anticoagulant is arnmonium-potassium
oxalate (Heller and Paul double oxalate). This combination of oxalates does not shrink
or enlarge the red blood cells appreciably. It is essential, however, to add an optimal
volume of blood to the oxalate, no less than 3.5 nor more than 6.0 mI.
c. Heparin does not alter the size of cellular components. It is, in fact, the
standard for comparison of anticoagulant distortion. Heparin is more expensive and
dissolves less readily than double oxalate salts. Approximately 0.5 to 1.0 mg is required
to anticoagulate 5 ml of blood for 72 hours. The quantity of anticoagulant noted above
in each case is sufficient to prevent clotting of the blood specimen. On the other hand,
an excess of anticoagulant should be avoided because too much will result in distortion
of cells and hemolysis. Ideally, differential blood smears should not be prepared from
blood that contains an anticoagulant.
d. If oxalate is added to vials and dried in an oven, take great care to avoid
temperatures above 80oC. Oxalates are converted to carbonates by prolonged
exposure to elevated temperatures. Under normal circumstances, it should not be
necessary to prepare your own oxalate solutions since prepared anticoagulant vacuum
tubes are available from Federal medical supply sources.
MD0853 3-12
e. Sodium citrate is the anticoagulant of choice for coagulation studies. It is
used in a concentration of 1 part 0.11 M sodium citrate to 9 parts whole blood. It
prevents coagulation by binding the calcium of the blood in a soluble complex.
f. Sodium oxalate is another anticoagulant widely used in coagulation studies.

It is used in a concentration of 1 part 0.1 M sodium oxalate to 9 parts whole blood. The
sodium oxalate combines with calcium in the blood to form insoluble calcium oxalate,
thereby, preventing coagulation.
NOTE: Under normal circumstances, it should not be necessary to prepare an

anticoagulant since prepared anticoagulant tubes are available through the
Federal supply system.
g. A correctly anticoagulated blood sample is essential to the proper

performance of a blood cell count. The cellular constituents must remain free in the
plasma and should be as similar as is possible to those remaining in the patient's
circulation.
Section II. PREPARATION AND STAINING OF BLOOD SMEARS
3-5. INTRODUCTION
a. The type of blood cells found in the peripheral blood smears may be of
diagnostic and prognostic importance. For this reason proper preparation and staining
of blood films is essential for the identification and study of different kinds of leukocytes.
The appearance of erythrocytes and thrombocytes will often give important clues that
help distinguish between different types of diseases or other physical changes.
b. There are two basic methods for the preparation of blood smears: the cover
slip and the slide methods. The cover slip method has certain advantages over the
slide method; distribution of cells is like that of the in vivo circulation. The principal
disadvantage of the latter method is that covers lips are very fragile and easily broken
during processing.
c. The slides and cover glasses must be chemically clean and dry. New slides
must first be washed with soapy water and rinsed thoroughly with distilled water. The
slides are then placed in a beaker of 95 percent ethyl alcohol. As the slides are
needed, dry them with a soft, lint free cloth. The slides may be reused by properly
cleaning them and making sure they are not chipped or scratched.
d. The foundation for the morphological study of blood was based on Ehrlich's
investigations of the aniline dyes, dating back to 1877, while he was still a student.
Originally, simple dyes were used in the clinical laboratory and tissues were stained
successively if more than one color was desired. The majority of the aniline dyes are in
the form of salts of acids and bases. During the process of staining, compounds are
probably formed between the basic dyes and the acid nuclear substances of cells and
MD0853 3-13
between the acid dyes (so called "neutral" dyes). In this way, the staining principles of
the original components were preserved; and, in addition, new staining properties
dependent upon the union of the component dyes were developed. These were,
therefore, termed polychromic dyes.
e. One modification of these polychromic stains is Wright's stain. This is the

stain most used in Army laboratories today. Wright's stain is a methyl alcohol solution
of an acid dye and a basic dye. The acid dye is known as eosin, which is red in color.
The basic dye, methylene blue, is blue in color. The white cells are mostly identified by
their preference for these dyes. In some cases the cells are even named for the dye
that they prefer. For example, cells that prefer a mixture of the acid and basic dye are
called neutrophils. In the staining process, a buffer solution is used to control the acid-
base balance of the stain. This is a most important function. If the buffer solution is too
acid it makes the acid dye too bright and the basic dye too faint. On the other hand, if
the buffer solution is too basic it makes the basic dye too bright and the acid dye too
faint. In either case, the result is a poorly stained slide. The acid-base balance of a
solution is measured by its pH value. A buffer solution should have a pH value between
6.4 and 6.8. This allows the best color contrast between acid and basic dyes.
f. When optimal staining conditions exist, Wright’s stain is very satisfactory and
easily differentiates cells. The eosin component stains cell cytoplasm, and the
methylene blue component stains nuclear material, granules, and inclusions. Both
stains oxidize rapidly because they are in alkaline solution. Giemsa, a purified
polychrome stain, is added to compensate for this defect by maintaining the azurophilic
staining property of the mixture.
3-6. SLIDE METHOD
a. Principle. A small drop of blood is placed near one end of a clean glass
slide. Using a second slide as the spreader, the blood is streaked into a thin film and
allowed to dry. It is then fixed and stained with modified Wright's stain.
b. Equipment.
(1) Venipuncture or finger puncture material.
(2) Clean glass slides.
c. Reagents.
(1) Wright's stain buffer.
(a) Solution A. Dissolve 9.47 grams disodium phosphate (Na2HPO4)

(dibasic), anhydrous, in a 1-liter volumetric flask containing about 750 ml of distilled
water. Dilute to the mark with distilled water.
MD0853 3-14
(b) Solution B: Dissolve 9.08 grams potassium acid phosphate
(KH2PO4) (monobasic), anhydrous, in a 1-liter volumetric flask containing about 750 ml
of distilled water. Dilute to the mark with distilled water.
(c) Mix 27 ml of the Na2HPO4 solution (Solution A) with 73 ml of the

KH2PO4 solution (Solution B). quarts to 1 liter. Final pH should be 6.4.
(2) Wright's stain solution.
(a) Add 9.0 grams of Wright's powder stain, 1.0 gram of Giemsa
powder stain, and 90 ml of glycerin to a mortar of suitable size. Triturate this mixture
thoroughly for 15-30 minutes.
(b) Transfer the mixture to a large open-mouth jar (a kitchen spoon is

ideal for this purpose). Cover the jar and incubate the glycerin stain mixture overnight
at 37oC.
(c) Transfer the glycerin/stain mixture to a large brown bottle and add
2,910 ml of acetone-free methanol.
(d) Age the Wright's stain solution approximately 2 weeks in the dark.
Mix daily to assure that the Wright's stain powder is completely dissolved.
NOTE: Commercially available Wright’s stain is available through the Federal supply
system.
3-7. SLIDE PREPARATION
a. Make a finger puncture or venipuncture in the usual manner.
b. Touch a drop of blood to a clean glass slide at a point midway between the
sides of the slide and a short distance from one end. If a venipuncture is made, use a
capillary tube to transfer a drop of blood from the tube to the slide. If a finger puncture
is made, dispense the drop of blood from the puncture site after discarding the first
drop.
NOTE: The drop of blood should be no larger than 1/8 to 3/16 inch in diameter (see
figure 3-5, side method for preparation of blood films, a through c.
c. Lay the specimen slide on a flat surface and hold it securely. Place a smooth,
clean edge of the spreader slide on the specimen slide at an angle of about 300 from the
horizontal (see figure 3-5a).
MD0853 3-15
Figure 3-5a. Side method for preparation of blood films: Place spreader slide
at an angle of about 300 from the horizontal.
d. Pull the spreader slide toward the drop of blood until contact is made within
the acute angle formed by the two slides as shown in figure 3-5b.
Figure 3-5b. Side method for preparation of blood films: Contact

blood with spreader.
e. Allow the blood to spread toward the sides of the slide.
f. Push the spreader slide smoothly and lightly toward the opposite end of the
specimen slide, drawing the blood behind it into a thin film (see figure 3-5c).
Figure 3-5c, Side method for preparation of blood films: Finished slide.
MD0853 3-16
g. Allow the blood film to air-dry completely. Do not blow on the slide in an effort
to enhance drying.
h. Using a lead pencil, write the name (or identification) of the patient in the thick
area of the smear. Do not use a wax pencil as it dissolves during the staining process.
3-8. SLIDE STAINING
a. Cover the slide completely with Wright's stain and allow it to remain on the
smear for about 2 minutes to fix the blood cells. The stain should cover the slide but
should not be allowed to overflow the edges; the stain must be replenished should it
begin to evaporate.
b. Add an equal volume of Wright's stain buffer directly to the stain and blow the
mixture gently to assure maximum mixing. Allow it to remain for about 4 minutes.
NOTE: The times recommended for staining and buffering are approximate and
should be adjusted with each fresh batch of stain to give the most satisfactory
results.
c. Using tap water, float off the mixture of stain and diluent from the slide to
avoid the deposition of metallic scum on the smear. The scum appears after the
addition of the buffer to the stain. Wash the slide thoroughly under cold, slowly-running
water.
d. Air dry the smear and wipe the excess stain from the under surface of the
slide.
e. Discussion.
(1) A properly prepared blood smear is margin-free; has no lines, ridges, or

holes; is placed centrally on the slide; has an adequate thin area; and has a uniform
distribution of leukocytes.
(2) It is preferable that blood smears not be made from blood containing
anticoagulants since the leukocytes change their staining characteristics, develop
vacuoles, engulf oxalate crystals, and show nuclear deformities. However, satisfactory
slides are made with blood anticoagulated with EDTA.
(3) Avoid the following errors:
(a) Thick films made from an excess amount of blood placed on the
slide.
(b) Delay in transferring the blood to the slide.
MD0853 3-17
(c) A spreader slide that has damaged or unpolished ends.
(d) The use of dirty, dusty, greasy, or scratched slides.
(4) If slides cannot be stained immediately, they should be dried and then
fixed in methyl alcohol for 30 minutes.
(5) In cases of marked leukopenia, smears can be prepared from the white
cell layer ("huffy coat") obtained by centrifuging the blood slowly in a Wintrobe
hematocrit tube at 500-800 rpm for 5 minutes.
(6) It is important that the blood film be completely dried before staining;
otherwise the wet areas will wash off the slide.
(7) Protect blood slides from insects such as flies, cockroaches, etc. They
can "clean" raw blood slides very rapidly.
(8) Protect slides from areas of high humidity. Excessive moisture tends to
hemolyze red blood cells.
(9) Slides should be stained as soon as possible after preparation. White

cells tend to become distorted and disintegrate very rapidly, thus causing considerable
difficulty in identification.
(10) Very little actual staining takes place during the fixation stage. Most of
the staining actually occurs during the buffering stage.
(11) During the buffering stage, it is important that only amounts of buffer
equal to the stain be added, otherwise there is a tendency to over-dilute, causing the
smear to stain weakly.
(12) After the staining is complete, do not blot the smear but air-dry it. To
speed up the drying process, the smear can be placed in the heat of the substage light.
It is important that the slide not be heated too intensely or too long since overheating
tends to darken the staining reaction.
(13) A good quality smear should macroscopically pinkish-gray in hue. It

should not be blue, green, or red. Microscopically, the red blood cells should be pink to
orange and the white blood cells bluish if they display their true staining color.
(14) If the RBCs are bluish or green, this indicates that the stain is too
alkaline. With an alkaline stain, the WBCs stain heavily and generally display fair
distinguishing characteristics. However, the heavy stain masks any abnormalities of the
RBCs. Heavy staining can be caused by:
(a) Blood smears which are too thick.
MD0853 3-18
(b) Over-staining (prolonged buffer action).
(c) Evaporation of the methanol in the stain.
(d) Stain or diluent which is alkaline.
(e) Alkaline fumes.
(15) If the red blood cells are bright red, the stain is too acid. In this condition
they stain well but the white blood cells (except eosinophilic granules) stain very poorly
if at all. Thus, the stain is of no value for differential studies. ''Tendency toward acid
staining is caused by:
(a) Incomplete drying before staining.
(b) Insufficient staining (insufficient buffer action).
(c) Overdilution of the stain with buffer.
(d) Prolonged washing of the slide after staining.
(e) Stain or buffer which is acid.
(f) Acid tunes.
(16) The staining reactions of blood are as follows:
Type of blood cell

or component Good stain Acid stain Alkaline stain
Erythrocytes Pink to orange Bright red Blue or green

All neclei Purple-blue Pale blue Dark blue
Eosinophilic granules Granules red Brilliant red, distinct Deep gray or blue
Neutrophilic Granules Violet-pink Pale Dark, prominent

Lymphocyte Blue Pale blue
Cytoplasm
(17) The technician should strive for a staining reaction that is neither too
alkaline nor too acid. Such a stain gives good distinguishing features for all the cells of
the blood system.
MD0853 3-19
(18) If the staining reaction is excessively alkaline, this can be corrected by
decreasing time of staining or neutralizing the stock stain solution with 1 percent acetic
acid or 1 percent hydrochloric acid. Add the acid a drop at a time. Check the results
after the addition of each drop of acid with trial slides.
(19) If the staining reaction is excessively acid, this may be corrected by

increasing the time of staining or neutralizing the stock stain solution with 1 percent
potassium bicarbonate or a weak solution of ammonia water. Add the ammonia water
or potassium bicarbonate one drop at a time. Check the results on trial slides after the
addition of each drop of the neutralizer.
(20) Staining reactions can also be varied by adjusting the proportions of

disodium phosphate and potassium acid phosphate used in preparing the buffer. For
example, increasing the proportion of disodium phosphate will make the buffer more
alkaline; reducing it will make the buffer more acid.
(21) A poorly stained smear can sometimes be saved by washing rapidly with
95 percent alcohol, washing quickly in water, then restraining.
MD0853 3-20
EXERCISES, LESSON 3
After you have completed all of these items, turn to "Solutions to Exercises" at the
end of the lesson and check your answers. For each exercise answered incorrectly,
reread the material referenced with the solution. Some questions have more than one
answer, so read them carefully.
1. To obtain valid blood test results, specimens must be properly:
a. Collected.
b. Processed
c. Recorded.
d. a and c.
e. a, b, and c.
2. Blood counts on venous and capillary blood are nearly the same if the capillary
puncture is:
a. Shallow.
b. Sterile.
c. Free-flowing.
d. Located on the finger.
MD0853 3-21
3. Valid blood counts cannot be made when:
a. Capillary specimens are not taken from a free-flowing sample.
b. When capillary specimens are obtained from cyanotic or calloused areas.
c. a and b.
d. When sources vary as much as 150 to 1550 cells per cu mm from the real
value.
4. Venous samples are preferred over capillary samples because they:
a. Allow for several and repeated hematological examinations.
b. Provide a sufficient amount of blood to perform the various laboratory tests

needed.
c. Provide for less chance of error because operations are made under better
conditions and repeated operations are possible.
d. a, b, and c.
5. Which blood count method would be performed if blood from extensive burn
victims was needed?
a. Venous.
b. Capillary (micro quantitative).
c. Neither method.
d. Both methods.
MD0853 3-22
6. When collecting blood for white blood cell counts, blood obtained from free-flowing
areas or areas of local stasis sources can vary as much as:
a. 400-1000 cells per cu mm from their real value.
b. 800-1200 cells per cu mm from their real value.
c. 1000-1300 cells per cu mm from their real value.
d. 1000-1500 cells per cu mm from their real value.
7. For adults, which veins should be used for venipuncture?
a. Veins located in the distal forearm or antecubital space.
b. Veins located in the proximal forearm or antecubital space.
c. The jugular vein.
d. The femoral vein or antecubital space.
8. For the elderly or debilitated persons, or those who may have sclerosed or fragile
veins, what should you do for the venipuncture?
a. Consult with a physician concerning the procedure.
b. Take blood from the veins located in the proximal forearm or antecubital
space.
c. Use the jugular vein.
d. Select the femoral vein.
MD0853 3-23
9. If blood is needed from infants, which veins should be used for the venipuncture?
a. Sagittal sinus area.
b. Veins located in the proximal forearm or antecubital space.
c. The collapsed vein.
d. The jugular or femoral vein. The vein selected should be large, readily
accessible, and sufficiently close to the surface to be seen and palpated.
10. If venipuncture poses a problem due to the age of the patient, sclerotization due to
repeated venipuncture, or any other unusual circumstance, which procedure is to
be followed?
a. Under some circumstances the technician should withdraw blood from a

sagittal sinus, jugular vein, or femoral vein.
b. Under no circumstances should a technician withdraw blood from a sagittal

sinus, jugular vein, or femoral vein.
c. The technician may withdraw blood from a sagittal sinus, jugular vein, or
femoral vein.
d. Withdraw blood from a sagittal sinus, jugular vein, or femoral vein.
e. If the need arises, the technician should withdraw blood from, a sagittal sinus
or jugular vein, but not from the femoral vein.
11. Which item of equipment is normally used for the collection of blood specimens?
a. Isopropyl alcohol, 40 percent.
b. Gauze pads, 6 x 6 inches.
c. Needle, 1 to 1 1/2 inches, 19-23 gauge.
d. Needle, large bevel.
MD0853 3-24
12. Veins are made easier to enter if:
a. The site of puncture is gently slapped.
b. The vein is massaged toward the heart.
c. a and b.
d. The arm hangs down for 4-6 minutes.
13. Generally speaking, veins from which group of people tend to collapse more
easily; and, therefore, greater care may be needed to select and puncture the
vein?
a. Children.
b. Middle-aged people.
c. Elderly.
14. Blood collection instruments should be:
a. Glass and disposable.
b. Plastic and calibrated.
c. Sterile and disposable.
d. Aseptic and anticoagulated.
15. The syringe and needle for venipuncture must be dry to avoid
_________________ of the red blood cells.
a. Hemolysis.
b. Coagulation.
c. Contamination.
d. Hemoglobin reduction.
MD0853 3-25
16. To prevent an injection of air into the vein, which can be fatal, what must the
technician do?
a. Use a longer plunger than the syringe.
b. Use a shorter plunger than the syringe.
c. It makes no difference.
d. The plunger must match the syringe.
17. If latex tubing is used as a tourniquet, how far above the venipuncture site should
it be secured?
a. 1 inch.
b. 2 inches.
c. 3 inches.
d. 4 inches.
18. Prolonged application of a tourniquet may change the concentration of many blood
components. The maximum period over which a tourniquet should be applied for
a venipuncture is:
a. 1 minute.
b. 2 minutes.
c. 4 minutes.
d. 6 minutes.
MD0853 3-26
19. Besides inspecting and palpating to locate the desired vein for venipuncture, on
what other two items should you focus?
a. Direction of vein course and estimate its size and depth.
b. Direction of vein course and estimate its length and color.
c. Direction of vein course and estimate its position and elasticity.
d. The vein’s thickness, length, and size.
20. When preparing for the venipuncture, what should be done with the needle?
a. Keep the cap on until ready to draw.
b. Place it on a sterile pad.
c. Throw it away.
21. What are the reasons for inspecting a possible puncture site?
a. Estimate the size and depth of the vein (some may be too small or shallow).
b. Determine the direction of the vein’s course (puncture with the grain, so to
speak).
c. Palpate the vein (for resiliency).
22. What may not be done once the puncture area is cleansed and excess alcohol
wiped off?
a. Grasp the forearm with the left hand.
b. Straighten the arm.
c. Contaminate the area.
d. Have the patient make a clenched fist.
MD0853 3-27
23. Puncture of the Vacutainer stopper is completed immediately:
a. Before the needle enters the vein.
b. After the needle enters the vein.
c. Before withdrawal of the needle.
d. After withdrawal of the needle.
24. Which way is the needle bevel supposed to be and how is it to be situated at time
of entry?
a. Bevel side down, parallel with, and alongside the vein.
b. Bevel side up, adjunct with, and alongside the vein.
c. Bevel side perpendicular to, and close to the vein.
d. Bevel side up, parallel with, and alongside the vein.
25. After the needle for a venipuncture is withdrawn, what must the patient do?
a. Take an iron compound.
b. Lie down for 10 minutes.
c. Keep his fist clenched for 5 minutes.
d. Maintain light pressure on the gauze pad over the site.
MD0853 3-28
26. What is an important vacutainer procedure?
a. The short needle should be embedded in the stopper, but the needle must not
break the vacuum.
b. Any needle should be embedded in the stopper, but the needle must not break
the vacuum.
c. The long needle should be embedded in the stopper, but the needle must not
break the vacuum.
d. The first needle should be embedded in the stopper, but the needle must not
break the vacuum.
27. If the multiple needle is used or more than one tube is required for venipuncture,
what is to be followed?
a. Tighten the tourniquet after the first tube is filled; remove the filled tube and
insert the next one.
b. Loosen the tourniquet after the first tube is filled; remove the filled tube.
c. Release the tourniquet after the first tube is filled; remove the filled tube and
insert the next one.
d. Tighten the tourniquet after the first tube is filled and insert the next one.
28. Why must you be careful not to remove the needle while tubes are being
changed?
a. The blood will continue to flow.
b. The skin may rip.
c. a and b may occur separately or at one tine.
MD0853 3-29
29. Why is it most important that correct venipuncture technique be practiced? To:
a. Avoid unnecessary pain to the patient.
b. Prevent tissue damage.
c. Secure a good representative blood specimen.
d. Prevent contamination of the specimen or infection of the patient.
f. a, b, and d.
30. What may occur to the donor if the tourniquet is not removed as early as possible
once the blood starts flowing well?
a. Coagulation of the blood.
b. Changes in the quantitative values of the blood components.
c. Hemolysis of the blood specimen.
31. The blood count should be performed within _________________ once the blood
is collected.
a. 30 minutes.
b. 3 hours.
c. 24 hours.
d. 48 hours.
32. The site of a capillary puncture should be:
a. Warm.
b. Cold.
MD0853 3-30
33. When is the tourniquet released and removed?
a. When a hematoma begins to form, the first drop of blood appears, or when it is
hard to enter the vein.
b. When it is hard to enter the vein, a hematoma begins to form, or the first drop
of blood appears, aspiration occurs.
c. When a hematoma begins to form, or it is hard to enter the vein.
34. What will occur if blood is squeezed from a capillary puncture?
a. Infections.
b. Unnecessary pain.
c. Free- flowing punctures.
d. Inaccurate test results.
35. Which aspect of the fingertip should be used as the site for a capillary puncture?
a. Dorsal.
b. Ventral.
c. Frontal.
d. Lateral.
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36. Sodium citrate is a good anticoagulant for coagulation studies because:
a. A concentration of one part 0.2 M sodium citrate is used to 9 parts of whole

blood.
b. It binds the calcium of the blood into a soluble complex to prevent coagulation.
c. It combines cellular constituents in the plasma.
d. A concentration of one part 0.1 M sodium citrate is used to 15 parts of whole

blood.
37. Heparin:
a. Does not alter the size of cellular components.
b. Dissolves more rapidly than double oxalate salts.
c. Is least expensive.
d. Can be used in excessive amounts.
38. EDTA ammonium-potassium oxalate, and heparin are commonly used:
a. Stains.
b. Buffers.
c. Fixatives.
d. Anticoagulants.
39. What are the two basic methods for the preparation of blood smears?
a. Cover slip and polychromic stains.
b. Slide and acid dye.
c. Slide and cover slip.
d. Methylene blue and slide.
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40. How many solutions are needed to perform a Wright's stain buffer?
a. 1.
b. 2.
c. 3.
d. 4.
41. What should be the final pH of the buffer used with Wright's stain?
a. 6.0.
b. 6.4.
c. 6.8.
d. 7.2.
42. Where on a dried blood smear is the name or identification of the patient written?
a. Side.
b. Middle.
c. Thin area.
d. Thick area.
43. The Wright's stain should remain on a blood smear _____________________

before the buffer solution is added and should not be allowed to ______________
the edges.
a. 2 minutes; overflow.
b. 4 minutes; extend beyond.
c. 6 minutes; remain on.
d. 8 minutes; reach out.
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44. If blood smears for the differential leukocyte count cannot be stained immediately,
they should be dried and then fixed ____________________in for _________
minutes.
a. EDTA; 10.
b. Aniline dye; 15.
c. Methyl alcohol; 30.
d. Buffer solution; 40.
45. When Smears for a differential leukocyte count contain a low concentration of
white blood cells, but marked leukopenia, they can be prepared from the
_______________________ layer by slowly centrifuging the blood specimen in a
_______________________ tube.
a. Top; volumetric.
b. Buffy coat; Wintrobe hematocrit.
c. Plasma layer; test.
d. Red blood cell layer; Vacutainer.
46. If areas of a blood smear are still wet when staining is to begin, they will:
a. Hemolyze.
b. Wash away.
c. Stain well.
d. Stain too heavily.
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47. Increasing the proportion of disodium phosphate in the buffer for Wright's stain will
make the buffer more:
a. Red.
b. Blue.
c. Acid.
d. Alkaline.
48. What is indicated if when staining the slide the RBCs are bluish or green?
a. The stain is too acidic.
b. The WOC stains very lightly.
c. An alkaline stain is observed.
d. The film is too thick.
49. Why should slides be stained quickly after preparation?
a. So buffers will appear unequal.
b. WBC distort and disintegrate quickly.
c. Lines and ridges will appear.
d. Acid fumes will develop.
50. Which errors should be avoided when staining slides?
a. Routinely transferring of blood to the slide.
b. Using an unpolished slide.
c. Using clean, dust free, and smooth slides.
d. Using thin films of blood and placing on slides.
MD0853 3-35
SOLUTIONS TO EXECISES, LESSON 3
1. e (para 3-1a)
2. c (para 3-1b)
3. c (para 3-1b)
4. d (para 3-1b)
5. b (para 3-1b)
6. d (para 3-1b)
7. b (para 3-2a)
8. a (para 3-2a)
9. d (para 3-2a)
10. b (para 3-2a)
11. c (para 3-2b)
12. c (para 3-2a)
13. c (para 3-2a)
14, c (para 3-2b)
15. a (para 3-2c(4))
16. d (para 3-2c(4))
17. b (para 3-2d(1))
18. b (para 3-2d(1), CAUTION}
19. a (para 3-2d (3))
20. a (para 3-2c(3))
21. d (para 3-2d(3))
22. c (para 3-2d(5)
MD0853 3-36
23. b (para 3-2e(4))
24. d (para 3-c(9))
25. d (para 3-2d(11), e(7))
26. a (para 3-2e(l))
27. c (para 3-2e(5))
28. a, b, c (para 3-2d (11)CAUTION, e(5)CAUTION)
29. e (para 3-2f(2))
30. b (para 3-2f(7))
31. b (para 3-2f(8))
32. a (para 3-3c(1))
33. c (para 3-2f(4))
34. d (para 3-3a, c(6), d(3)
35. d (para 3-3d(4))
36. b (para 3-4e)
37. a (para 3-4c)
38. d (para 3-4a)
39. c (para 3-5b)
40. b (para 3-6c(1)
41. b (para 3-6c(1)(c)
42. d (para 3-7h)
43. a (para 3-8a)
44. c (para 3-8e(4))
45. b (para 3-8e(5))
MD0853 3-37
46. b (para 3-8e(6))
47. d (para 3-8e(20))
48. c (para 3-8e(14))
49. b (para 3-8e(9))
50. b (para 3-8e(3))
End of Lesson 3
MD0853 3-38
LESSON ASSIGNMENT
LESSON 4 Morphology of Blood Cells.
4-1. Select the statement that best describes a

general rule of cell identification.
4-2. Select the stages of erythrocyte development,

the order of maturation and series, and
characteristics of each stage with variation.
4-3. Select the stages of leukocyte development, the

order of maturation and series, and
characteristics of each stage with variation.
4-4. Select the stages of thrombocyte development,

the order of maturation and series, and
characteristics of each stage.
4.5. Select the statement which best describes the

occurrence of leukemia and its classifications
and types. Select the appropriate signs and
symptoms for the stage or type of leukemia.
4-6. Select the appropriate signs and symptoms for

the stage or type of leukemia.

MD0853 4-1
LESSON 4
MORPHOLOGY OF BLOOD CELLS
Section I. GENERAL INFORMATION
4-1. BASIC CONCEPTS OF CELL MORPHOLOGY
a. In this lesson, all normal and most commonly seen abnormal blood cells are
morphologically described. Although general rules for identification are given along with
representative photographs and drawings, it is important to realize that no biological
entity fits the guidelines precisely.
b. The present classification of blood cells is man’s attempt at identifying stages

of maturation by assigning artificial steps to a continuing process. The process is a
smooth, continuing one, and therefore no one cell ever precisely fits the criteria for a
specific stage. These stages are artificial classifications that exist to simplify
identification.
4-2. GENERAL RULES OF CELL IDENTIFICATION
Certain general rules are applied to all cell maturation (hemopoiesis) either in the
erythrocyte, leukocyte, thrombocyte, or plasmocyte series. Although these rules are
broken by individual cells, they are an aid to classifying cells.
a. Immature cells are larger than mature cells and become smaller as they
mature.
b. The relative and absolute size of the nucleus decreases as the cell matures.
In some cell series the nucleus disappears.
c. The cytoplasm in an immature cell is quite blue in color and lightens as the
cell matures.
d. The young nucleus is reddish and becomes bluer as the cell ages.
e. Nuclear chromatin is fine and lacy (lacelike) in the immature cell It becomes
coarse and clumped in the more mature cells.
f. If there is doubt in the identity of a cell, classify to the more mature form.
Section II. ERYTHROCYTES
4-3. INTRODUCTION
a. In the normal development and maturation (erythropoiesis) of the erythrocytic

series, the red blood cell undergoes a graduation of morphological changes. This cell
MD0853 4-2
development is a gradual transition (as noted in ASCP terminology—they are listed from
the most immature to mature cells) and six different stages can be identified. The
nomenclature used to describe red blood cells is recommended by the American
Society of Clinical Pathologists and the American Medical Association. The terms with
some of their synonyms are as follows:
ASCP Terminology Synonyms
Rubriblast Pronormoblast
Prorubricyte Basophilic normoblast
Rubricyte Polychromatophilic normoblast
Metarubricyte Orthochromatic normoblast
Diffusely basophilic erythrocyte Polychromatic erythrocyte
Erythrocyte Normocyte
b. Erythropoiesis is regulated by the intake of substances to build the cells, the

storage of these substances, and their proper utilization. When normal erythropoiesis
occurs, both the cytoplasm and the nuclei of the cells grow at a synchronized rate.
Individual differences in physiology and physical structure of the erythrocyte account for
minor morphological changes so often encountered. In certain diseases, these
morphological changes may vary to a greater extent. These variations occur in size,
shape, staining, and inclusions in the erythrocyte.
4-4. ERYTHROCYTIC SERIES
a. Rubriblast. (See figure 4-1, Erythrocytes series, a through f).
Figure 4-1a. Erythrocytes series: Rubriblast.
(1) Size. Fourteen to 19 microns in diameter.
(2) Nucleus. This cell has a large round-to-oval purple nucleus that
occupies most of the cell. The nuclear chromatin is arranged in a close mesh network
forming a reticular appearance. There are 0-2 light blue nucleoli present within the
nucleus.
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(3) Cytoplasm. The cytoplasm is dark blue, granule-free, and limited to a
thin rim around the nucleus. There is no evidence of hemoglobin formation.
b. Prorubricyte.
Figure 4-1b. Erythrocytes series: Prorubricyte.
(1) Size. Ten to 15 microns in diameter.
(2) Nucleus. The nucleus is generally round, dark purple, am smaller than
the nucleus of the rubriblast. The chromatin is coarse am clumped giving the nucleus a
darker stain. Nucleoli are usually not present, but when they are, they appear more
prominent than in the rubriblast.
(3) Cytoplasm. The cytoplasm is royal blue and more radiant than in the
rubriblast. Cytoplasmic granules are not present.
c. Rubricyte.
Figure 4-1c. Erythrocytes series: Rubricyte.
(1) Size. Eight to 14 microns in diameter.
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(2) Nucleus. The nucleus is dark, round or oval, and smaller than the
prorubricyte nucleus. The chromatin material is found in dense, irregular clumps.
Nucleoli are not present.
(3) Cytoplasm. The cytoplasm is more abundant than in the precursor cells.
It is blue-pink (polychromatic), the pink resulting from the first visible appearance of
hemoglobin. Cytoplasmic granules are absent.
d. Metarubricyte.
Figure 4-1d. Erythrocytes series: Metrarubricyte.
(1) Size. Seven to 10 microns in diameter.
(2) Nucleus. This cell has a pyknotic nucleus (a homogeneous blue-black

mass with no structure) that is round. This is the main difference between the rubricyte
and the metarubricyte.
(3) Cytoplasm. The cytoplasm is abundant, bluish-buff to buff pink.
e. Reticulocyte (diffusely basophilic erythrocyte).
Figure 4-1e. Erythrocytes series: Reticulocyte.
(1) Size. Seven to 9 microns in diameter.
(2) Nucleus. The nucleus is absent.
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(3) Cytoplasm. The cytoplasm stains a bluish-buff with Wright’s stain and
there is no central light pallor as in the erythrocyte. With supravital staining, this cell will
show light blue reticulum strands in the cytoplasm.
f. Erythrocyte.
Figure 4-1f. Erythrocytes series: Erythrocyte.
(1) Size. Six to 8 microns in diameter.
(2) Nucleus. The nucleus is absent.
(3) Cytoplasm. The cytoplasm of the periphery is light orange with a central
zone of pallor. The appearance of the central zone of pallor is due to the biconcave
morphology of the cell, which allows more light through the center than through the
margin areas.
4-5. VARIATIONS IN ERYTHROCYTES
a. Size.
(1) Anisocytosis. Anisocytosis is a variation in the size of erythrocytes

beyond the normal limits. Cells of varying size are seen in the same fields.
(2) Macrocytes. Macrocytes are erythrocytes larger than nine microns in

diameter. These cells may be found in liver disease.
(3) Microcytes. These erythrocytes are smaller than 6 microns in diameter.

These cells are found in thalassemia and other anemias.
b. Shape.
(1) Poikilocytosis. This term describes a marked variation in the shape of

erythrocytes. Poikilocytes can be pear-shaped, comma-shaped, oval- shaped, or
various other bizarre forms. These cells are encountered in pernicious anemia and
many other types of anemia.
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Figure 4-2a. Variations in erythrocytes: Poikilocytosis: Sickle cells.
(2) Sickle cell (Drepanocytes). Sickle cells are abnormal erythrocytes that
assume a crescent or sickle-shaped appearance under conditions of reduced oxygen
tension. The presence of sickle cells is an inherited abnormality due to the presence of
hemoglobin S. Sickle cell anemia is encountered primarily in Blacks.
Figure 4-2b. Variations in erythrocytes:

a. Metarubricytes Anisocytosis
Polychromasia Spherocytosis
(3) Spherocytes. These are abnormal erythrocytes that are spherical in
shape, having a diameter smaller than normal, and a darker stain (without central pallor)
than normal erythrocytes. These cells are found in instances of hemolytic anemias and
are particularly characteristic of congenital hemolytic jaundice, a hereditary disorder.
Figure 4-2c. Variations in erythrocytes:

Marked Poikilocytosis.
Anisocytosis and Target Cells.
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(4) Ovalocvtes. These cells are abnormal erythrocytes that have an oval or
“sausage” shape. They can be found in hereditary elliptocytosis.
Figure 4-2d. Variations in erythrocytes:

Elliptocytes (Oval Erythrocytes).
(5) Target cells (leptocytes). Target cells are erythrocytes that have deeply
stained (pink) centers and borders, separated by a pale ring, giving them a target-like
appearance. They are associated with liver disease and certain hemoglobinopathies.
(6) Burr cells. Burr cells are triangular or crescent-shaped erythrocytes with
one or more spiny projections on the periphery. These cells are seen in uremia, acute
blood loss, cancer of the stomach and pyruvate kinase deficiency.
Figure 4-2e. Variations in erythrocytes:
Crenated RBC Burr Cells

Acanthocytes 2 Leukocytes
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(7) Acanthocytes. Acanthocytes are irregularity-shaped erythrocytes with
long spiny projections. They are seen in a congenital abnormality characterized by
serum concentration of low density (beta) lipoproteins.
Figure 4-2f. Variations in erythrocytes:

a. Metarubricyte
b. Target Cell
c. Crenated RBC.
(8) Crenated erythrocytes. This condition occurs when blood films dry too
slowly and the surrounding plasma becomes hypertonic. There is no pathological
significance when they are found in blood smears.
(9) Schistocytes. These are red blood cell fragments. Frequently these
cells have a hemispherical shape (helmet cells).
(10) Rouleaux formation. This phenomenon is adherence of erythrocytes to
one another presenting a stack-of-coins appearance. It occurs in conditions
characterized by increased amounts of fibrinogen and globulin.
c. Staining.
(1) Hypochramia. Hypochramia is a condition in which the normal central

pallor is increased due to decreased hemoglobin content. This condition is
characteristic of many anemias.
Figure 4-2g. Variations in erythrocytes:

Hypochromic macrocytic erythrocytes.
MD0853 4-9
(2) Polychromatophilia. This term describes non-nucleated erythrocytes
that show bluish coloration instead of light pink. Polychromatophilia is due to the fact
that the cytoplasm of these cells does not mature, resulting in the abnormal persistence
of the basophilic cytoplasm of the earlier nucleated stages.
d. Inclusions.
(1) HoweII-Jolly bodies. These are nuclear remnants found in the

erythrocytes of the blood in various anemias. They are round, dark violet granules
about one micron in diameter. Generally, only one Howell-Jolly body will be found in
any one red cell. However, two or more may sometimes be present. HoweII-Jolly
bodies generally indicate absent or non-functioning spleen. They occur in megalobastic
anemia and in other forms of nuclear maturation defects.
Figure 4-2h. Variations in erythrocytes:

a. Metarubricyte
b. Howell-Jolly Bodies
(2) Cabot's rings (ring bodies). These are bluish threadlike rings found in
the red cells in the blood of patients with severe anemias. They are interpreted as
remnants of the nuclear membrane and appear as ring or “figure-eight' structures.
Usually only one such structure will be found in any one red cell.
Figure 4-2i. Variations in erythrocytes:

a. Cabot Ring.
MD0853 4-10
(3) Basophilic stippling. Round, small, blue-purple granules of varying size
in the cytoplasm of the red cell represent a condensation of the immature basophilic
substance (see poly-chromatophilia) that normally disappears with maturity. This is
known as basophilic stippling. It can be demonstrated by standard staining techniques
in contrast to reticulocyte filaments that require a special stain. Stippling occurs in
anemias and heavy metal poisoning (lead, zinc, silver, mercury, bismuth) and denotes
immaturity of the cell.
Figure 4-2j. Variations in erythrocytes:

a. Basophilic Stippled Erythrocyte.
(4) Heinz-Ehrlich bodies. These are small inclusions found primarily in
those hemolytic anemias induced by toxins. They are round, refractile bodies inside the
erythrocyte and are visible only in unfixed smears. It is thought that they are proteins
that have been dematured and that they are an indication of erythrocyte injury.
(5) Siderocytes. These are erythrocytes containing iron deposits. These
deposits indicate an incomplete reduction of the iron from ferric to the ferrous state that
is normally found in hemoglobin. Prussian blue stain must be used to readily
demonstrate these cells.
Figure 4-2k. Variations in erythrocytes:

a. Rubricytes (Pernicious Anemia).
Figure 4-2l. Variations in erythrocytes:

a. Metarubricyte (Pernicious Anemia)
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e. Megaloblastic Erythrocytes. The development of megaloblastic cells is
caused by a deficiency of vitamin B12 or folic acid. Pernicious anemia is a disease
considered to be due to a deficiency in vitamin B12 and/or certain related growth factors.
With this deficiency, the erythrocytes do not mature normally and are generally larger
than normal. The most notable characteristic of this abnormal maturation is a difference
in the rates of maturation of the cytoplasm and the nucleus. The development of the
nucleus is slower than that of the cytoplasm, so that in the more mature of the nucleated
forms a spongy nucleus as well as an exceptionally large size may be observed.
Nuclear chromatin in the megaloblast is much finer and is without the clumps observed
in the rubriblast. Such development is termed asynchronism. The mature cell is large
(about 10 microns) and is termed a megalocyte. The younger cells of this series are
named by adding the suffix “pernicious anemia type," that is rubricyte, pernicious
anemia type, and so forth.
Section III. LEUKOCYTES
4-6. GRANULOCYTIC SERIES
The stages in the normal maturation of the granulocytes are: myeloblast,
promyelocyte, myelocyte (neutrophilic, eosinophilic, and basophilic), metamyelocyte
(neutrophilic, eosinophilic, and basophilic), band cell (neutrophilic, eosinophilic, and
basophilic), and segmented cell (neutrophilic, eosinophilic, and basophilic). As the
granulocytes mature, the granules increase in number. These granules later become
specific and differ in the affinity for various dyes. Neutrophilic granules do not stain
intensely with either dye. Basophilic granules have an affinity for the basic or blue dye.
Eosinophilic stain red with an affinity for the acid dye. The criteria for identification of
the various stages of the granulocytic series are: size of cell, nucleus-cytoplasm ratio,
nuclear shape, number of nucleoli, and the type and size of cytoplasmic granulation.
a. Myeloblast.
(1) Size. Fifteen to 20 microns in diameter.
(2) Nucleus. The nucleus is round or ovoid and stains predominantly
reddish-purple. The interlaced chromatin strands are delicate, well defined, and evenly
stained. Two or more pale blue nucleoli are demonstrable. The nucleus occupies most
of the cell with a nucleus-cytoplasm ratio of 6:1. It is separated from the cytoplasm by a
definite nuclear membrane.
(3) Cytoplasm. The cytoplasm is a narrow, deep blue, nongranular rim
around the nucleus.
Figure 4-3a. Granulocytic series: Myeloblast.
MD0853 4-12
b. Promyelocyte.
(1) Size. Fifteen to 21 microns in diameter.
(2) Nucleus. The nucleus is round or ovoid with coarse-clumping, purple

chromatin material. One to three oval, light-blue nucleoli are usually present. The
nucleoli are less distinct than in the myeloblast. This cell has a nucleus-cytoplasm ratio
of 4:1.
(3) Cytoplasm. The cytoplasm is light purple and contains varying numbers
and sizes of dark nonspecific granules that stain red to purplish-blue. The granules
usually overlie the nucleus.
Figure 4-3b. Granulocytic series:

a. Promyelocyte.
b. Promyelocyte with Auer Body.
c. Myelocyte. In the myelocytic stage the granules are definite and so

numerous that frequently they obscure nuclear detail. While promyelocytes are
sometimes distinguished as neutrophilic, eosinophilic, or basophilic, the differentiation is
generally considered as first occurring in the myelocytic stage.
Figure 4-3c. Granulocytic series: Myelocyte.
MD0853 4-13
d. Neutrophilic Myelocyte.
(1) Size. Twelve to 18 microns in diameter.
(2) Nucleus. The nucleus is round, oval, or flattened on one side. The
chromatin strands are light purple, unevenly stained, and thickened. Nucleoli are
usually absent. The nucleus is smaller than the earlier cells of this series with a
nucleus-cytoplasm ratio of 2:1.
(3) Cytoplasm. The cytoplasm is bluish-pink and contains a small relatively

light area of ill-defined, pink granules, which develop among the dark, nonspecific,
azurophilic granules of the promyelocyte. As the myelocyte ages, the dark granules
become less prominent and the light-pink-colored neutrophilic granules predominate.
e. Neutrophilic Metamyelocyte.
(2) Nucleus. The nucleus is indented or kidney-shaped. The nuclear

chromatin stains dark purple and is condensed into irregular strands. Nucleoli are
absent. The nucleus-cytoplasm ratio is approximately 1.5:1.
(3) Cytoplasm. The cytoplasm is pinkish-blue and covered with many small,
light pink granules.
Figure 4-3d. Granulocytic series:

a. Metamyelocyte.
b. Band Neutrophil.
f. Neutrophilic Band.
(2) Nucleus. The nucleus is shaped like a horseshoe with a dark pyknotic
mass at each poIe of the nucleus where the lobes develop. The nucleus-cytoplasm
ratio is approximately 1: 2.
MD0853 4-14
(3) Cytoplasm. The cytoplasm contains many small, evenly distributed light
pink granules.
g. Neutrophilic Segmented Cell.
(2) Nucleus. The nucleus has definite lobes separated by a very narrow
filament or strand. The nucleus-cytoplasm ratio is approximately 1: 3.
(3) Cytoplasm. The cytoplasm is light pink and the small, numerous, and
evenly distributed neutrophilic granules have a light pink color.
h. Development of the Eosinophilic Group. Cells of the eosinophilic group
are characterized by relatively large, spherical, cytoplasmic granules that have a
particular affinity for the eosin stain. The earliest eosinophil (myelocyte) has a few dark
spherical granules with reddish tints that develop among the dark, nonspecific granules.
As the eosinophilic cells pass through their various developmental stages, these
granules become less purplish-red and more reddish-orange. The dark blue,
nonspecific granules, characteristic of the promyelocyte and the early myelocyte stages,
disappear. Because the percentage of eosinophils is usually low in bone marrow
peripheral blood smears, no useful clinical purpose is served by routinely separating the
eosinophils into their various myelocyte, metamyelocyte, band, and segmented
categories. On the other hand, in situations such as eosinophilic leukemia in which the
eosinophils are greatly increased, an analysis of the incidence of the various stages
would be useful in diagnosis.
i. Mature Eosinophil.
(I) Size. Ten to 15 microns in diameter.
filament or strand. Seldom does an eosinophil have more than two lobes.
(3) Cytoplasm. The cytoplasm contains bright reddish-orange, distinct
granules. The granules are spherical, uniform in size, and evenly distributed throughout
the cytoplasm, but rarely overlie the nucleus.
Figure 4-3e. Granulocytic series: Eosinophil.
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j. Development of the Basophilic Group. These cells have round, indented,
band, or lobulated nuclei and are classified according to the shape of the nuclei, as
basophilic rnyelocytes, metamyelocytes, bands, and segmented forms. These cells are
so few in peripheral blood and bone marrow that there is little clinical value in
differentiation of the various maturation stages.
Figure 4-3f. Granulocytic series:

a. Basophil.
b. Neutrophil: Segmented.
k. Mature Basophils.
filament or strand. The nuclear details are obscured by the cytoplasmic granulation.
(3) Cytoplasm. The cytoplasm is covered by many blue to black granules.

These granules are unevenly distributed and vary in number, size, shape, and color.
4-7. AGRANULOCYTES
Agranulocytes are leukocytes devoid of specific granulation. These cells

generally originate in the lymphatic system, but can be found in normal bone marrow.
Agranulocytes include the lymphocytic series, monocytic series, and plasmocytic series.
4-8. LYMPHOCYTIC SERIES
The stages in the development of the lymphocytic series are: lymphoblast,

prolymphocyte, and lymphocyte. These cells are fragile and can show shape variants.
Lymphocytes usually have round contours, blue cytoplasm, and eccentrically located
round nuclei. Cells of this series are differentiated on the basis of the nuclear
chromatin.
MD0853 4-16
a. Lymphoblast.
(2) Nucleus. The nucleus has an oval or round shape and stains reddish-
purple. The nuclear chromatin is fine, well distributed, and coarser than in the
myeloblast. Chromatin is condensed at the edges of the nucleus to form a definite
nuclear membrane. One or more nucleoli are present. The nucleus is prominent with a
nucleus-cytoplasm ratio of 6:1.
(3) Cytoplasm. The cytoplasm is deep blue with a frequent perinuclear
clear zone.
Figure 4-4a. Lymphocytic series:

a. Lymphoblast.
b. Lymphocyte.
c. Smudge Cell.
b. Prolymphocyte.
(2) Nucleus. The nucleus is oval and slightly indented. The nuclear
chromatin is coarse, slightly clumped, am dark purple. One light blue nucleolus is
usually present. The nucleus- cytoplasm ratio is 5:1.
(3) Cytoplasm. The cytoplasm varies from light blue to dark blue and it can
show a few red-purple ( azurophilic) granules.
Figure 4-4b. Lymphocytic series: .Prolymphocyte.
MD0853 4-17
c. Lymphocyte.
(1) Size. The mature cell of this series varies greatly in size. Small
lymphocytes are 7 to 9 microns in diameter. The large lymphocytes are 10 to 18
microns in diameter.
(2) Nucleus. The nucleus is round or oval and can be slightly indented.
The nuclear chromatin is markedly condensed, dark purple-blue, and clumped. Nucleoli
are absent and a definite nuclear membrane ids present. The nucleus-cytoplasm ratio
is approximately 1.5:1.0.
(3) Cytoplasm. The cytoplasm is light blue to blue with a perinuclear clear
zone around the nucleus. A few azurophilic granules can be seen in the cytoplasm of
larger lymphocytes.
Figure 4-4c. Lymphocytic series:

Lymphomcyte.
Azurophilic Granulation.
4-9. MONOCYTIC SERIES
The stages in the development of the monocytic series are monoblast,
promonocyte, and monocyte. Cells of the series are slightly larger than granulocytes.
They are round with smooth margins and seldom show shape variants. The mature
monocyte is differentiated from the lymphocyte and metamyelocyte by the very fine,
light staining nucleus.
a. Monoblast.
(2) Nucleus. The nucleus is round or oval and red-purple in color. The
nuclear chromatin is fine and well distributed. One to two nucleoli are present. The
nucleus-cytoplasm ratio is 6:1.
(3) Cytoplasm. The cytoplasm is a clear, deep blue and follow a thin rim
around the nucleus.
MD0853 4-18
Figure 4-5a. Monocytic series:
a. Monoblast.
b. Stem (Ferrata Cell)
b. Promonocyte.
(2) Nucleus. The nucleus is oval or indented and light purple. The nuclear
chromatin is fine and spongy. One to five nucleoli may represent. The nucleus-
cytoplasm ratio is 5:1.
(3) Cytoplasm. The cytoplasm is gray-blue with fine dust like azurophilic
(red-purple) granules.
Figure 4-5b. Monocytic series: Promonocyte.
c. Monocyte
(2) Nucleus. The nucleus is round or kidney-shaped, but can be deeply

indented or have two or more lobes. One of the most distinctive features of the
monocyte is the presence of superimposed lobes, giving the nucleus the appearance of
brain-like convolutions. Heavy lines marking the edges of the folds and grooves are
MD0853 4-19
features that are not seen in other cells. Another feature of the nucleus, which is of
value in diagnosis, is the tendency for the nuclear chromatin to be loose with light
spaces in between the chromatin strands, giving a coarse linear pattern in contrast to
the Iymphocyte that has clumped chromatin. Nucleoli are absent. The nucleus-
cytoplasm ratio is approximately 3:1.
(3) Cytoplasm. The cytoplasm of the monocyte is dull gray-blue while the
cytoplasm of the neutrophils in the adjacent fields is definitely lighter and is pink rather
than gray-blue. The nonspecific granules of the monocyte are usually fine and evenly
distributed, giving to the cell a dull, opaque or ground-glass appearance. In addition to
the background of evenly distributed nonspecific granules, there may be a few unevenly
distributed larger azurophilic granules. Vacuoles are often demonstrable in the
cytoplasm.
Figure 4-5c. Monocytic series:

a. Neutrophil (Late Band)
b. Monocyte.
4-10. PLASMOCYTIC SERIES
Plasmocytes constitute approximately one percent of the white cells in the
normal bone marrow. These cells can represent in the peripheral blood in chronic
infections, granulomatous and allergic diseases, and multiple myeloma. The stages of
development are: plasmoblast, proplasmocyte, and plasmocyte.
a. Plasmoblast.
(1) Size. Eighteen to 25 microns in diameter.
(2) Nucleus. The nucleus is large, oval or round, and located off center in
the cell. Nuclear chromatin is fine, purplish, and reticulated. There are multiple nuclei
that may or may not be visible.
Figure 4-6a. Plasmocytic series: Plasmoblast.
MD0853 4-20
b. Proplasmocyte.
(2) Nucleus. The nucleus is ovoid and located eccentrically. The chromatin
is purple, coarser, and more clumped. One to two nucleoli are present. The nucleus-
(3) Cytoplasm. The cytoplasm is pale blue, nongranular. A lighter-staining
area in the middle of the cell (in the cytoplasm, next to the nucleus) may become visible.
This is termed a hof.
Figure 4-6b. Plasmocytic series: Proplasmocyte.

c. Plasmocyte.
(1) Size. Eight to 20 microns in diameter.
(2) Nucleus. The nucleus is small, ovoid, and eccentrically located. The
chromatin is coarse, lumpy, and purple. Nucleoli are not usually present. The nucleus-
(3) Cytoplasm. The cytoplasm adjacent to the nucleus is lightly stained in
contrast to the periphery of the cell that has a high saturation of red and blue dyes. In
same cells, the dark cytoplasm has a greenish or larkspur-blue color. The cytoplasm
contains multiple small and relatively unstained globules embedded in a bluish-red
filamentous matrix. It is the presence of these tapioca-like globules in the dark
surrounding medium that gives the plasmocyte its characteristic mottled and foamy
appearance and its brilliant translucency. In occasional cells, the globules can be quite
prominent and take a red or bluish-red stain. Such globules are called Russell or
fuchsin bodies, or eosinophilic globules. Vacuoles of various sizes are frequently
demonstrable.
Figure 4-6c. Plasmocytic series: Plasmocyte.
MD0853 4-21
4-11. VARIATIONS OF LEUKOCYTES
Variations of leukocytes occur as a result of abnormal maturation of the nucleus

and/or cytoplasm. These variations are induced by leukemic states, infectious
diseases, and toxicity. Described below are the most frequently occurring variations.
a. Dohle Bodies. Dohle bodies are light blue or blue-gray, small, round
inclusions found in the cytoplasm of neutrophilic leukocytes. The variation may occur in
toxic conditions such as severe infections, burns, poisoning, and following
chemotherapy.
b. Auer Rods. Auer rods are rods or spindle-shaped, cytoplasmic inclusions.

They stain red-purple and are 1 to 6 microns long and less than 1.5 microns thick. They
are frequently found in leukemias.
c. Toxic Granullation. Toxic granulation occurs in the neutrophilic

metamyelocyte, band, and segmented cells. These granules are distinguished from the
normal granulation because they are coarser and stain a dark purple. The variations
occur in toxic states, severe infections, and burns.
Figure 4-7a. Variations in leukocytes:

Band Neutrophil: Toxic Granulation.
d. Basket Cell. A basket cell is a ruptured leukocyte that has a network

appearance. These cells result from a partial breakdown of the immature and fragile
leukocytes. Basket cells are found predominantly in diseases with an acute shift toward
immature forms, for example, leukemias.
e. Vacuolated Cell. A vacuolated cell is a degenerated cell with holes or

vacuoles in the cytoplasm. Vacuolated cells can be seen in severe infections,
poisoning, and leukemias, and in cells that have been in Heller & Paul oxalate too long.
MD0853 4-22
f. Hypersegmentation. A normal neutrophilic segmented cell has a nucleus
with an average of three lobes or segments. In a hypersegmented cell the nucleus is
broken up into five to ten lobes or segments. This cell usually has a larger diameter
than a normal neutrophilic segmented cell. Hypersegmentation is often seen in
pernicious anemia and folic acid deficiency. They may also be found in chronic
infections.
Figure 4-7b. Variations in leukocytes:

Neutrophili: Hypersegmented.
g. Atypical Lymphocytes. These lymphocytes are characteristic of infectious

mononucleosis but they may also be seen in apparently healthy individuals and those
with certain other diseases. Atypical lymphocytes are larger than normal and vary in
appearance. Downey and McKinley described three types of atypical lymphocytes, but
this classification has no real clinical purpose.
(1) Size. Large, up to 20 microns in diameter.
(2) Nucleus. The nucleus is oval or kidney shaped with very coarse
chromatin strands not as lumpy as a normal lymphocyte.
(3) Cytoplasm. The cytoplasm is blue to dark blue. Often it is vacuolated

which gives rise to a foamy appearance.
Figure 4-7c. Variations in leukocytes:

Atypical Lymphocytes:
Infectious Mononucleosis
MD0853 4-23
h. L.E. Cells.
(1) Persons having lupus erythematosus, one of the “collagen” diseases,

have an abnormal; plasma protein that causes swelling and breakdown of certain blood
cell nuclei in vitro. This degenerated nuclear material attracts phagocytic cells,
particularly segmented neutrophils, which engulf this nuclear mass. The resulting
phagocyte and inclusion material is termed an "L.E.” cell.
(2) The nucleus of an L.E. cell is adjacent to the peripheral outline of the
inclusion material. The inclusion is smooth and silky or light purple and has no visible
chromatin network.
Figure 4-7d. Variations in leukocytes: L.E. Cells.
i. Rosettes. Rosette formation is the intermediate stage in the formation of an

L.E. cell. A rosette formation consists of neutrophilic leukocytes surrounding free
masses of lysed nuclear material.
j. Tart cells. A tart cell, which may be confused with the L.E. cell, contains
lysed nuclear material within its cytoplasm. It differs from an L.E. cell because the
inclusion retains characteristic nuclear structure. This inclusion is not smooth and has a
darker staining periphery. The significance of tart cells is not known but their presence
in an L.E. preparation does not signify a positive test for systemic lupus erythematosus.
Section IV. THROMBOCYTES
4-12. INTRODUCTION
a. The general pattern of thrombocyte maturation is slightly different from that of
leukocyte maturation. The cells of the megakaryocytic series tend to grow larger as
they mature until there is cytoplasmic fragmentation (or breaking off) to form the
cytoplasmic thrombocytes seen in the peripheral blood.
b. Azurophilic granulation begins to appear in the second stage of development
and continues until it almost obscures the nuclear lobes. The nucleus develops from a
fine single lobe to multiple ill-defined lobes. The stages in the normal maturation of the
megakaryocytic series are: megakaryoblast, promegakaryocyte, megakaryocyte, and
thrombocyte.
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4-13. MEGAKARYOCYTIC SERIES
a. Megakaryoblast.
(1) Twenty to 50 microns in diameter.
(2) Nucleus. One to two large oval or kidney-shaped nuclei are present.
There is a fine chromatin pattern. Multiple nucleoli may be present which stain blue.
The nucleus cytoplasm ratio is approximately 10:1.
(3) Cytoplasm. The cytoplasm is blue, nongranular, and may have small,
blunt pseudopods. It is usually seen as a narrow band around the nucleus.
Figure 4-8a. Megakaryocytic series:

Megakaryoblast: Bone marrow.
b. Promegakaryocyte.
(1) Size. Twenty to 50 microns in diameter.
(2) Nucleus. One-to-two indented round or oval nuclei are present. They
may show slight lobulation. The nuclear chromatin is purple, coarse, and granular.
Multiple nuclei are present but may be indistinct.
c. Megakaryocyte.
(1) Size. Forty to 100 microns in diameter.
(2) Nucleus. Two-to-sixteen nuclei may be visible or the nucleus may show
multilobulation. No nucleoli are visible. The nuclear chromatin is purple, coarse, and
granular.
(3) Cytoplasm. The cytoplasm is pinkish-blue in color and very granular.

Numerous blue-purple granules begin to aggregate into small bundles that bud off from
the cell to become platelets.
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Figure 4-8b. Megakaryocytic series: Megakaryocyte.
d. Thrombocyte (Platelet)
(1) Size. One to 4 microns in diameter.
(2) Nucleus. None.
(3) Cytoplasm. The cytoplasm is light blue to purple and very granular. It
consists of 2 parts:
(a) The chromomere, which is granular and located centrally.
(b) The hyalomere, which surrounds the chromomere and is

nongranular and clear to light blue.
Figure 4-8c. Megakaryocytic series: Thrombocytes.
MD0853 4-26
Section V. THE LEUKEMIAS
4-14. INTRODUCTION
a. Leukemia is an abnormal, uncontrolled proliferation of one or more of the

white-cell-producing cells. It is a disease of the blood-forming tissues that always
involves the bone marrow.
b. The condition was first discovered microscopically by Donne in 1839 but was
not recognized as a clinical entity until 1845. Leukemia was commonly called “white
blood” because in many cases, the number of leukocytes in the peripheral blood was so
great that upon separation of the blood elements, the buffy (or white cell layer) might be
ten times greater than normal.
4-15. 0CCURRENCE
Leukemia occurs at any age. Often during the first five years of life, and until the
age 21, the great majority of cases are acute. From this time until around 45 the tide
turns and chronic granulocytic leukemia becomes the predominating form. It is much
more difficult to ascertain the relative frequencies of the various forms of acute
leukemia. One school of thought is that acute lymphoblastic leukemia predominates in
childhood until about the age of puberty and approximately equals the sum of the cases
of myeloblastic and monocytic; that monocytic leukemia never occurs in the aged; or
that chronic leukemia is never found in children.
4-16. CLASSIFICATION
a. The leukemias are classified by the duration of the disease; by the number of
white cells present in the peripheral blood; and by the predominant cell type found in the
peripheral blood and bone marrow. On the basis of disease duration, leukemia may be
described as:
(1) Acute leukemia, a rapidly progressive disease that lasts several days to
6 months.
(2) Sabacute leukemia; 2 to 6 months.
(3) Chronic leukemia; the length of this disease is variable, depending on

the age of the patient and the type of cell involved. Most patients live a minimum of 1 to
2 years or more.
b. On the basis of the leukocyte count in peripheral blood, the leukemias are
classified as:
(1) Leukemic leukemia; white blood count is greater than 15,000.
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(2) Subleukemic leukemia; white blood count is less than 15,000 WBC per
cu mm with immature or abnormal forms of white cells present in the peripheral blood.
(3) Aleukemic leukemia; white blood count is less than 15,000 WBC cu mm
with no immature or abnormal white cells present in the peripheral blood. The
distinction between subleukemic and aleukemic leukemia will depend mostly on a
thorough search for abnormal cells.
c. Another classification is based on the type and the maturity of the cells found
to be predominant in the blood and bone marrow. In most cases, classification by cell
type makes it possible to distinguish between lymphocytic, neutrophilic, and other
leukemias. In acute blast cell leukemia a pure population of blast cells is seen, and the
cell type usually cannot be identified. Classification according to cellular maturation will
often parallel the classification according to duration; acute leukemias show a large
number of immature cells while the chronic cases have a significant number of well-
differentiated cells. Morphologically, a “subacute” leukemia will usually resemble the
acute stage more readily than the chronic condition.
4-17. TYPES OF LEUKEMIA
Several of the more common types of leukemia are discussed below:
a. Acute Lymphocytic Leukemia. At the time of diagnosis, the white count is

usually noticeably elevated, with 60 percent or more lymphoblasts and some
prolymphocytes present. Severe anemia and thrombocytopenia are characteristic. The
lymphoblasts have a round or oval nucleus composed of coarse granular or stippled
chromatin and usually contain one or two nucleoli.
b. Chronic Lymphocytic Leukemia. The white count is usually 20,000 to

200,000 per cu mm, with the peripheral blood smear showing 60 to 95 percent
lymphocytes. These cells are generally the small type of mature lymphocyte that often
show a small cleft or indentation in the shape of the nucleus. Lymphoblasts are
generally absent from the peripheral blood but a rare prolymphocyte may sometimes be
found. These lymphocytes are somewhat more fragile than normal, resulting in many of
the cells appearing to be entirely devoid of cytoplasm. A normocytic, normochromic
anemia generally develops as the disease progresses. The platelet count is usually
normal.
c. Acute Myelogenous Leukemia. The white blood count usually shows

moderate to marked elevation with 60 percent or more of the cells being myeloblasts.
Auer rods may or may not be present in the cytoplasm of these cells. Some cases of
this disease show micromyeloblasts, a much smaller myeloblast than normal. Severe
normocytic, normochromic anemia develops, along with thrombocytopenia. The
platelets that are present may be large and bizarre-looking.
MD0853 4-28
d. Acute Promyelocytic Leukemia. In this form of leukemia, the predominant
cell in the bone marrow and blood is the promyelocyte. Often, the nucleus of this cell is
more immature than usual and the cytoplasmic granules may be large and abnormal-
appearing.
e. Chronic Myelogenous Leukemia. The white count is usually 100,000 to

300,000 per cu nm at the time of diagnosis. Less than 10 percent myeloblasts are
present in the peripheral blood and there are numerous immature granulocytes.
Eosinophils and basophils are commonly increased, and the percentage of monocytes
may also show an increase. Mild normochromic anemia is generally present. The
platelet count is often increased and large forms of the platelets may be present.
f. Acute Monocytic Leukemia of Schilling. The white cell count is usually

moderately elevated with a predominant number of immature monocytes present. The
nucleus is even more lacy than in typical monocytes and it may appear to be folded or
segmented. One-to-five nucleoli may appear as fading or inconspicuous. The
cytoplasm is variable in amount, generally has few to no visible granules, and may have
a serrated-shaped border. Anemia and thrombocytopenia are usually present.
g. Acute Myelomonocytic Leukemia of Naegeli. At diagnosis, the white count

usually shows moderate to marked elevation. Anemia is commonly found and
thrombocytopenia may also be present. The abnormal cell found most commonly in this
disorder has characteristics of both the myeloblast and the monocyte. The nucleus is
monocytoid with a fine chromatin pattern and appears convoluted or folded. Granules
present in the cytoplasm show characteristics of granulocytes. All stages of these cells
are present in the bone marrow and the peripheral blood. Auer rods may be present in
the blast cell.
4-18. CLINICAL SIGNS
a. A patient with leukemia may appear to be in more or less perfect health, or, at
the extreme, when the disease is well advanced, the picture of long-drawn, progressive
ill health is noticeable; emaciation is usually apparent and pallor is well marked.
b. The major symptoms of leukemia are fever, weight loss, and increased
sweating. Enlargement of the liver, spleen, and lymph nodes may occur. The basal
metabolic rate is often elevated, and there may be hemorrhagic tendencies if marked
thrombocytopenia is present. When splenic enlargement exists, the contrast between
protuberant abdomen and the general evidences of weight loss produces a striking and
characteristic picture.
MD0853 4-29
c. In chronic myleocytic leukemia the spleen is usually markedly enlarged at the
time the patient presents himself to the physician. The size of the spleen may be
extreme and the organ may extend to the ilium and even to the right anterior superior
spine. The size does not appear to be related to the total number of leukocytes. The
entire course of development of the splenomegaly may be painless, but not infrequently
there is a history of distress in the left upper abdominal quadrant or in the posterior
portion of the body.
d. Pain, limitations of movement, and other symptoms that suggest arthritis,

gout, or osteomyelitis, (or those due to actual fracture of bone) may occur in acute or in
chronic leukemia. These symptoms are common in children. In chronic leukemia, there
may be no symptoms associated with the bones and joints. Bone tenderness can be
elicited if firm pressure is made over the sternum. The patient is frequently unaware of
any tenderness until pressure is applied at the proper point.
MD0853 4-30
EXERCISES, LESSON 4
After you have completed all of the exercises, turn to “Solutions to Exercises” at
1. The stages of blood cell maturation are:
a. Irrelevant categories.
b. Artificial classifications
c. Obsolete pigeonholes.
d. Strictly and universally applicable categories.
2. As a general role for cell identification, the cytoplasm in a mature cell is:
a. Green.
b. Dark green.
c. Blue.
d. Light orange.
3. Generally speaking, what are the texture and consistency of the nuclear chromatin
in an immature cell?
a. Fine and lacy.
b. Course and clumpy.
c. Rough and lacelike
d. Fine and crumbled together.
MD0853 4-31
4. Generally speaking, what is the size of the cell and the texture and consistency of
the nuclear chromatin in a mature cell?
a. Larger than an immature cell; fine and lacy.
b. Smaller than an immature cell; course and clumpy.
c. Smaller than an immature cell; rough and lace-like.
d. Larger than an immature cell; fine and crumbled together.
5. Which does NOT occur during the development of blood cells?
a. Nucleus disappears.
b. Nucleus reduces in size.
c. Cytoplasm lightens in color.
d. Nucleus becomes reddish in color.
6. The most immature cell in the erythrocytic series is the:
a. Rubricyte.
b. Rubriblast.
c. Prorubricyte.
d. Metarubricyte.
e. Erythrocyte.
f. Diffusely basophilic erythrocyte.
MD0853 4-32
7. The rubricyte cell has:
a. Cytoplasm staining a bluish-buff and a purple nucleus.
b. A large oval, homogeneous blue-black mass for a nucleus.
c. Dense, irregular clumpy chromatin and a small nucleus.
d. Light blue reticulum strands in the cytoplasm, but no nucleus.
8. Which cell has a nucleus and usually a few nucleoli?
a. Rubricyte.
b. Rubriblast.
c. Megakaryocyte.
d. Metarubricyte.
9. In which stage of erythrocyte development does hemoglobin first become visible?
a. Rubricyte.
b. Rubriblast.
c. Prorubricyte.
d. Metarubricyte.
10. A diffusely basophilic erythrocyte is a/an:
a. Erythrocyte.
b. Rubricyte.
c. Reticulocyte.
d. Metarubricyte.
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11. The metarubricyte has a/an ________________ nucleus.
a. Pyknotic.
b. Absent.
c. Round and small.
d. Purple.
12. The immediate precursor of the polychromatophilic normoblast erythrocyte is the:
a. Rubricyte.
b. Rubriblast.
c. Prorubricyte.
d. Metarubricyte.
13. Which cell does not have a nucleus?
a. Rubricyte.
b. Reticulocyte.
c. Prorubricyte.
d. Metarubricyte.
14. The term normocyte is synonymous with what blood cell?
a. Rubricyte.
b. Metarubricyte.
c. Prorubricyte.
d. Erythrocyte.
MD0853 4-34
15. The average diameter of a normal prorubricyte is:
a. 3.8 microns.
b. 5.3 microns.
c. 11.4 microns
d. 18.8 microns
16. Abnormal variation in the size of erythrocytes is called:
a. Hypochromia.
b. Ovalocytosis
c. Anisocytosis.
d. Poikilocytosis
17. Which cells are triangular in shape and are spiny looking?
a. Ovalocytes.
b. Sickle.
c. Acanthocytes.
d. Burr.
18. In microcytosis, the microcytes are erythrocyte variations that are:
a. Larger than normal.
b. Smaller than normal.
c. Abnormally varied in size.
d. Abnormally varied in shape.
MD0853 4-35
19. RBC fragments that are helmet shaped erythrocytes are called:
a. Crenated erythrocytes.
b. Schistocytes.
c. Drepancytes.
d. Poikilocytosis.
20. Which cell is particularly characteristic of congenital hemolytic anemia (called

hemolytic jaundice in the text)?
a. Target
b. Crenated erythrocyte.
c. Spherocyte.
d. Siderocyte.
21. Which cell does have an irregular outline?
a. Acanthrocytes.
b. Burr cells.
c. Target.
d. Crenated erythrocytes.
22. Which cell does NOT indicate a possible hereditary disorder?
a. Ovalocyte.
b. Spherocyte.
c. Sickle cell.
d. Crenated erythrocyte.
MD0853 4-36
23. An increase of globulin and fibrinogen presents a stack-of-coins appearance for
erythrocytes. This is called a/an:
a. Irregularly-shaped erythrocyte.
b. Pale ring.
c. “Sausage" shape.
d. Rouleaux formation.
24. In hypochromic erythrocytes, the normal central pallor is increased as a result of

_____________________________ like many __________________________.
a. Cellular immaturity; nucleated stages.
b. An increased hemoglobin content; sickle cell abnormalities.
c. A decreased hemoglobin content; anemias.
d. Basophilic cytoplasm; mature cells.
25. A megaloblastic cell is caused by what deficiency?
a. Vitamin B6.
b. Vitamin B12.
c. Vitamin B1.
26. An immediate precursor of the neutrophilic band cell is the:
a. Myeloblast.
b. Promyelocyte.
c. Neutrophilic myelocyte.
d. Neutrophilic metamyelocyte.
e. Neutrophilic segmented cell.
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27. In the granulocytic series, the immediate precursor of the promyelocyte is the:
a. Monoblast.
b. Myeloblast.
28. The neutrophilic segmented cell belongs to which series?
a. Monocytic series.
b. Plasmocytic series.
c. Erythrocytic series.
d. Granulocytic series.
29. Which cell is the least mature and stains unevenly?
a. Neutrophilic myelocyte.
b. Neutrophilic band cell.
c. Neutrophilic metamyelocyte.
d. Neutrophilic segmented cell.
30. Which cell has two or more blue nucleoli, no cytoplasmic granules, and the
nucleus occupying a ratio of 6:1 nucleus-cytoplasm?
a. Myeloblast.
b. Promyelocyte.
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31. Which cell has only dark nonspecific granules within the cytoplasm, with the
granules overlying the nucleus?
a. Myeloblast.
b. Promyelocyte.
32. A myeloblast cell has a:
a. Small, relatively light area of pink granules among dark azurophilic granules
and has a nucleus that is round, oval, or flattened on one side?
b. Nucleus that is indented and a nucleus-cytoplasm ratio of about 1:5:1.
c. Narrow, deep blue, nongranular rim around the nucleus.
d. Nucleus with narrow filament that separates the nucleus lobes.
33. Which cell has a kidney-shaped nucleus and many small, light pink granules within
the cytoplasm?
a. Myeloblast.
b. Promyelocyte.
34. The normal stages of granulocytes are:
a. Myeloblast, myelocyte, and neutrophilic.
b. Myeloblast, promyelocyte, and myelocyte.
c. Promyelocyte, myelocyte, and esoinophilic.
d. Neutrophilic, esoinophilic, and basophilic.
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35. Numerous blue to black granules obscure the nucleus of the:
a. Erythroblast.
b. Mature basophil.
c. Mature eosinophil.
36. The promonocyte is a part of what leukocyte series?
a. Monocytic.
b. Lymphocytic.
c. Plasmocytic.
d. Granulocytic.
37. The lymphocyte has:
a. No nucleus.
b. A segmented nucleus.
c. An indented, round or oval nucleus.
d. A spongy, sprawling nucleus.
38. What are the stages of the lymphocytic series?
a. Myeloblast, lymphoblast, and basophilic.
b. Lymphocyte, myeloblast, and lymphoblast.
c. Lymphoblast, lymphocyte, and monocyte.
d. Lymphoblast, prolymphocyte, and lymphocyte.
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39. Azurophilic (reddish-purple) granules may be found in the cytoplasm of:
a. Lymphocytes.
b. Erythrocytes.
c. Mature eosinophils.
.
d. Neutrophilic segmented cells.
40. Auer rods are frequently found in:
a. Anemia.
b. Leukemia.
c. Multiple myeloma.
d. Infectious mononucleosis.
41. Toxic granulation of neutrophilic cells occurs in:
a. All of the below.
b. Severe infections.
c. Chemical poisoning.
d. Burns.
42. Which leukocyte variation is often produced in blood that has been oxalated too
long?
a. Vacuoles.
b. Auer rods.
c. Hyposegmentation.
d. Toxic granulation.
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43. A hypersegmented neutrophilic cell has how many segments?
a. 1-3.
b. 3 - 4.
c. 1 - 5.
d. 5-10.
44. Vacuolated cytoplasm is common in the atypical __________________________

characteristic of infectious mononucleosis.
a. Monocyte.
b. Lymphocyte.
c. Plasmocyte.
45. A segmented neutrophil that has phagocytized a homogeneous mass of nuclear

material is called:
a. A rosette.
b. An L.E. cell.
c. A tart cell.
d. A Dohle body.
46. Platelets (thrombocytes) have a diameter of:
a. 1-4 microns.
b. 4-6 microns.
c. 6-8 microns.
d. 8-10 microns.
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47. Leukemias, classified by duration, are described by the most progressive to the
least rapid by:
a. Acute, chronic, and subacute.
b. Acute, subacute, and chronic.
c. Chronic, acute, and subacute.
48. Which type of leukemia has smaller than normal myeloblast, large and bizarre-
looking platelets?
a. Acute rnyelomonocytic leukemia of Naegeli.
b. Acute promyelocytic leukemia
c. Chronic myelogenous leukemia.
d. Acute myelogenous leukemia.
49. With acute monocytic leukemia of Schilling, the patient has:
a. A moderately high WBC, a convoluted fine chromatin pattern, 1 to 5 nucleoli

with immature granulocyte, and a serrated-shaped border.
b. Anemia, a moderately high WBC, a normal platelet count, convoluted fine

chromatin patter, 1 to 4 nucleoli with immature monocytes, and a serrated-
shaped border.
c. a moderately high WBC, a serrated-shaped border, mature lymphocytes, 1 to

5 nucleoli, thrombocytopenia, and immature granulocytes.
d. Cytoplasm with few visible granules; a moderately high WBC; 1 to 5 nucleoli

with immature, lacy monocytes; and a serrated-shaped border.
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50. In advanced leukemia the signs and symptoms include:
a. Enlarged lymph nodes.
b. Spleen extending to the ilium.
c. Fever.
d. Weight loss.
e. Increased sweating.
f. Elevated metabolic rate.
MD0853 4-44
SOLUTIONS TO EXERCISES, LESSON 4.
1. b (para 4-1b)
2. c. (para 4-2c)
3. a (para 4-2e)
4. b (para 4-2a, e)
5. d (para 4-2b-d)
6. b (para 4-3a; figure. 4-1)
7. c (para 4-4c)
8. b (para 4-4a(2))
9. a (para 4-4c(3))
10. c (para 4-4e)
11. a (para 4-4d(2))
12. c (para 4-3a)
13. b (para 4-4e(2))
14. d (para 4-3a)
15. c (para 4-4b(1)
16. c (para 4-5a(l))
17. d (para 4-5b(6))
18. b (para 4-5a(3))
19. b (para 4-5b(9))
20. c (para 4-5b(3))
21. c (para 4-5b(5))
MD0853 4-45
22. d (para 4-5b(8))
23. d (para 4-5b(10))
24. c (para 4-5c(1))
25. b (para 4-5e)
26. c (para 4-6)
27. b (para 4-6)
28. d (para 4-6)
29. a (para 4-6)
30. a (para 4-6a(2), (3))
31. b (para 4-6b(3))
32. c (para 4-6a)
33. d (para 4-6e(2), (3))
34. b (para 4-6)
35. b (para 4-6k(2), (3))
36. a (para 4-9b)
37. c (para 4-8c(2))
38. d (para 4-8)
39. a (para 4-8c)(3))
40. b (para 4-11b)
41. a (para 4-11c))
42. a (para 4-11e)
43. d (para 4-11f)
44. b (para 4-11g)
MD0853 4-46
45. b (para 4-11h(1))
46. a (para 4-13d(1))
47. b (para 4-16a)
48. d (para 4-17c)
49. d (para 4-17f)
50. g (para 4-18)
End of Lessson 4
MD0853 4-47
LESSON ASSIGMMENT SHEET
LESSON 5 Manual Cell Counts.
LESSON OBJECTIVES After completing this lesson, you should

be able to:

materials, procedures, and calculations needed
to perform manual counts for blood specimens
(WBC, total eosinophil, and reticulocyte cell
counts).

materials, procedures, and calculations needed
to perform manual counts for other body fluids
(cerebrospinal fluid cell count and spermatozoa).

materials and procedures to perform a semen
analysis.

exercises of this lesson. These exercises will help you
to achieve the lesson objectives.
MD0853 5-1
LESSON 5
MANUAL CELL COUNTS
Section I. MANUAL COUNTS, BLOOD SPECIMENS
5-1. INTRODUCTION
a. Blood cells are subject to quantitative variations as well as the qualitative

variations described in Lesson 4. Some diseases stimulate the production of blood cells
while others prevent or diminish the production of blood cells. For this reason a cell
count gives valuable information to the physician concerning his patient's condition.
Furthermore, in the case of the leukocyte count, the total count is necessary to calculate
absolute counts for each type of leukocyte. This is done by multiplying the total count
by the percentage of the particular cell type.
b. Cell counts can be performed by a variety of methods. Erythrocytes and

leukocytes are counted by manual methods or automated methods. Other cell counts
are performed only by manual methods. It is important when performing a cell count to
maintain good quality control. Great care should be taken when performing any cell
count.
c. The following paragraphs outline procedures for white blood cell (WBC)
count, total eosinophil count, and reticulocyte count. The WBC counts are routinely
done; they are performed either by the hemacytometer method (manually) or by
automated methods. Total eosinophil counts are performed by a hemacytometer
method (manually) using special diluting fluids to accentuate these cells. Reticulocytes
are demonstrated by using a supravital stain. Semen analysis and cerebrospinal fluid
(CSF) counts use a hemacytometer to perform the procedure as well. They are
included in the next section.
5-2. WHITE BLOOD CELL COUNT
a. Principle. A sample of whole blood is mixed with a weak acid solution that
lyses nonnucleated red blood cells. Following adequate mixing, the specimen is
introduced into a counting chamber where the white blood cells (leukocytes) in a diluted
volume are counted.
b. Reagent. White-count diluting fluid. Either of the following diluting fluids may
be used:
(1) Two percent acetic acid . Add 2 ml glacial acetic acid to a 100 ml volumetric
flask. Dilute to the mark with distilled water.
(2) One percent hydrochloric acid. Add 1 ml hydrochloric acid to a 100 ml

volumetric flask. Dilute to the mark with distilled water.
MD0853 5-2
c. Procedure.
(1) Draw well-mixed capillary or venous blood exactly to the 0.5 mark in a
white blood cell diluting pipet. This blood column must be free of air bubbles.
(2) Wipe the excess blood from the outside of the pipet to avoid transfer of
cells to the diluting fluid. Take care not to touch the tip of the pipet with the gauze.
(3) Immediately draw diluting fluid to the "11" mark while rotating the pipet
between the thumb and forefinger to mix the specimen and diluent. Hold the pipet
upright to prevent air bubbles in the bulb.
(4) Mix the contents of the pipet for 3-5 minutes to ensure even distribution
of cells. Expel unmixed and relatively cell-free fluid from the capillary portion of the
pipet (usually 4 drops).
(5) Place the forefinger over the top (short end) of the pipet, hold the pipet
0
at a 45 angle, and touch the pipet tip to the junction of the cover glass and the counting
chamber.
(6) Allow the mixture to flow under the cover glass until the chamber is
completely charged. Similarly, fill the opposite chamber of the hemacytometer.
NOTE: If the mixture overflows into the moat or air bubbles occur, clean and dry the
chambers, remix the contents of the pipet, and refill both chambers.
(7) Allow the cells to settle for about 3 minutes. Under low-power
magnification and reduced light, focus on the ruled area and observe for even
distribution of cells.
(8) Count the white cells in the four 1 sq mm corner areas corresponding to
those marked A, B, C, and D of Figure 5-1 in each of two chambers.
(9) Count all the white cells lying within the square and those touching the
upper and right-hand center lines. The white cells that touch the left-hand and bottom
lines are not to be counted. In each of the four areas, conduct the count as indicated by
the "snake-like" line in figure 5-1. A variation of more than 10 cells between any of the
four areas counted or a variation of more than 20 cells between sides of the
hemacytometer indicate uneven distribution and require that the procedure be repeated.
MD0853 5-3
Figure 5-1. Hemacytometer counting chamber (WBCs). Areas marked A, B, C,
and D are used to count white blood cells.
d. Calculation.
(1) Routinely, blood is drawn to the 0.5 mark and diluted to the 11 mark with
WBC diluting fluid. All the blood is washed into the bulb of the pipet (which has a
volume of 10). Therefore, 0.5 volumes of blood are contained in 10 volumes of diluting
fluid. The resulting dilution is 1:20. (These figures are arbitrary and refer strictly to
dilution and not to specific volumetric measurements.)
(2) The depth of the counting chamber is 0.1 mm and the area counted is 4
sq mm (4 squares are counted, each with an area of 1.0 sq mm therefore, 4 X 1.0 sq
mm = a total of 4 sq mm). The volume counted is: area X depth = volume. Four sq
mm X 0.1 mm = 0.4 cu mm.
MD0853 5-4
(3) The formula is as follows:
Average number of chambers (2) WBCs counted X dilution (20)

WBCs per cu mm =
Volume (0.4)
(4) For example:
First Chamber Second Chamber

Cells counted in Cells counted
each square in each square
35 45
40 37
44 36
39 44
158 WBCs counted 162 WBCs counted
Average of the 2 chamber counts. Total the amount and divide by 2:
158 320 - = 160 WBCs

+162 2
320
Then: 60 X 20 =8,000 WBCs

0.4
e. Sources of Error.
(1) Improper collection of blood specimens causes variable results.
(2) Wet or dirty pipets.
(3) Poor condition or inaccurate calibration of pipets. Pipets must be in

good condition and calibrated to have maximum error of + or -1percent.
(4) Poor pipetting technique causes high or low counts. Poor pipetting
technique includes:
(a) Undershooting desired line with blood or diluting fluid.
(b) Overshooting desired line with blood or diluting fluid.
(c) Air bubbles in the column on bulb.
(d) Failure to wipe tip free of blood.
MD0853 5-5
(e) Too slow manipulation following the withdrawal of the specimen
thus, allowing some of the blood specimen to coagulate.
(f) Failure to mix the blood and diluent properly.
(5) Failure to expel 2 or 3 drops in the pipet tips before charging the
hemacytometer.
(6) Overfilling the chamber of the hemacytometer, which causes

erroneously high counts.
(7) Wet or dirty cover glasses and hemacytometers.
(8) Uneven distribution of cells in the counting chamber causes erroneous

results.
(9) Inaccuracy or carelessness in marking counts.
(10) Diluent that which is cloudy or contains debris.
(11) Failure to mix anticoagulated blood thoroughly before use.
f. Discussion.
(1) The available error when four large squares are counted is +20 percent.
Counting eight large squares decreases the error to +15 percent.
(2) The importance of clean, dry diluting pipets cannot be stressed too much
as the greatest source of error in the counting of WBC is the use of wet and/or dirty
pipets.
(3) The counting chamber must be scrupulously clean and free of debris
that might be mistaken for cells.
(4) The minimum blood sample recommended for performing routine white
blood cell counts is that obtained using one pipet and counting two chambers as
previously outlined.
(5) In cases where the WBC count is exceptionally high, as in leukemia, the
dilution should be made in the red blood cell diluting pipet. The blood is drawn to the
“1.0” mark and the diluting fluid is drawn to the “101” mark. The resulting dilution is
1:100.
(6) In cases of leukopenia, the white pipet should be filled to the “1.0” mark
and diluted to the “11” mark with 2 percent acetic acid. The resulting dilution is 1:10.
MD0853 5-6
(7) If nucleated erythrocytes are present, the count is corrected by the
following formula:
observed count X 100

corrected count =
100 + percent nucleated erythrocytes
The percent nucleated erythrocyte is obtained from the differential count, which is
discussed in another subcourse.
g. Normal Values.
(1) Adults (both sexes): 4,500-11,500 WBCs per cu mm.
(2) Childhood: 6,000-14,000 WBCs per cu mm.
(3) Birth: 9,000-30,000 WBCs per cu mm.
h. Unopette Procedure for White Blood Cell Count.
(1) Follow procedure described in para 2-5 for blood dilution.
(2) Prepare diluted specimen for count.
(a) Mix diluted blood by inverting reservoir to resuspend cells.
(b) Convert to dropper assembly by withdrawing pipet from reservoir

and reseating securely in reverse position.
(c) To clean capillary more, invert reservoir, gently squeeze sides, and
discard first three to four drops.
(d) Carefully load hemacytometer with diluted blood by gently

squeezing sides of reservoir to expel contents until chamber is properly filled.
(e) Place hemacytometer in moisture chamber, let stand for 3 to 5

minutes (10 minutes for platelets) to allow cells to settle.
(3) Counting and calculation of leukocytes.
(a) Under 100X (low power) magnification, count leukocytes in all nine
large squares of the counting chamber.
(b) Add 10 percent of count to total number of cells counted. This step
simplifies the calculation that actually entails dividing the number of cells by the number
of squares counted and multiplying by 10 to correct for the depth of the chamber.
MD0853 5-7
(c) Multiply this figure by 1000 to get total leukocyte count.
5-3. TOTAL EOSINOPHIL COUNT
a. Principle. A sample of blood is diluted with a solution that selectively stains

the eosinophils and eliminates all other leukocytes and erythrocytes from view.
Following mixing, the specimen is introduced into the counting chamber and the number
of eosinophils in a known volume of blood is counted.
b. Reagent. Pilot's Solution: Add 50 ml propylene glycol, 40 ml distilled water,

and 1.0 ml sodium carbonate (10 percent aqueous) to a 150-ml beaker. Mix well and
filter. Add 10 ml of phloxine (1 percent aqueous) just prior to use. The addition of 10
units of heparin to this solution prevents clumping of cells.
c. Procedure.
(1) Draw capillary (or venous) blood to the “1.0” mark in each of two white
cell diluting pipets.
(2) Draw Pilot's solution to the “11” mark of each pipet.
(3) Mix by gently shaking the pipets for 30 seconds. Prolonged and harsh
shaking will tend to cause rupturing of the eosinophils.
(4) Expel the cell-free liquid from the capillary portion of the pipets.
(5) Using one pipet, charge both chambers of a hemacytometer and with
the other pipet charge both chambers of the second hemacytometer.
(6) Allow both hemacytometers to stand for 15 minutes to permit staining of

the eosinophils. To prevent evaporation, the hemacytometers are placed on a damp
towel and covered with Petri dish covers.
(7) Under low-power magnification, count the red-stained eosinophils in the

entire ruled area (9 sq mm) each of the four chambers (a total area of 36 sq mm). The
chamber has a depth of 0.1 mm so the total vo1ume is 3.6 cu mm.
d. Calculations.
Number of eosinophils counted X dilution (10)

Number of eosinophils =
Per cu mm volume (3.6)
e. Source of Error. See paragraph 5-2e.
MD0853 5-8
f. Discussion.
(1) Fuchs-Rosenthal or levy hemacytometer is preferable to a standard

counting chamber since its greater volume (4.0 X 4.0 X 0.2 mm) allows for counting of
more cells, thereby reducing the statistical error. Counting two of these chambers is
equivalent in accuracy to seven standard chamber counts.
(2) In eosinopenia, it is necessary to set up more chambers to provide an

optional number of cells to be counted.
(3) The eosinacetone diluting fluids are unsatisfactory and should not be
used.
(4) Estimation of eosinophils on a stained blood smear is too inaccurate for

use because of poor cellular distribution.
(5) The propylene glycol in Pilot's solution renders the erythrocytes invisible,
and the sodium carbonate causes lysis of all the leukocytes except the eosinophils.
The phloxine stains the eosinophils.
(6) In the Thorn test an eosinophil count must be made prior to the initiation
of the test proper. This establishes the patient's total eosinophil count, to which the
response of the adrenal cortex to adrenocorticotropic hormone (ACTH) can be judged.
The ACTH is then injected and, at an interval of 4 hours, another eosinophil count is
made. The interpretation of this test is as follows:
Normal--approximately a 50 percent drop in eosinophils.
Cushing's disease (hyperadrenalism)--0-30 eosinophils per cu mm.
Addison's disease (hypoadrenalism) no change in eosinophil count.
(7) Nasal smears are also submitted for eosinophil evaluation. These
smears are stained with Wright's stain and examined for the presence of eosinophils.
g. Normal Value. Normal value: 150-300 eosinophils per cu mm.
5-4. RETICULOCYTE COUNT
a. Principle. Nonnucleated immature erythrocytes contain nuclear remnants of

RNA and the cell is known as a reticulocyte. To detect the presence of this RNA, the
red cells must be stained while they are still living. This process is called supravital
staining. With supravital staining, the RNA appears as a reticulum within the red cell.
MD0853 5-9
b. Reagent.
(1) New Methylene Blue Solution. Dissolve 0.5 grams of new methylene
blue, 1.4 grams of potassium oxalate, and 0.8 grams of sodium chloride in distilled
water. Dilute to 100 ml. Filter before use.
(2) Brilliant Cresyl Blue Solution. Dissolve 1.0 grams of brilliant cresyl blue
in 99 ml of .85 per cent sodium chloride. Filter before use.
c. Procedure.
(1) Place 3 or 4 drops of new methylene blue and 3 or 4 drops of blood

(venous or capillary) in a small test tube.
(2) Mix the tube contents and allow to stand for a minimum of 15 minutes.
This allows the reticulocytes adequate time to take up the stain.
(3) At the end of 15 minutes, mix the contents of the tube well.
(4) Place a small drop of the mixture on a clean glass slide and prepare a
thin smear.
(5) Counterstain with Wright's stain, if desired.
(6) Allow smear to air-dry.
(7) Place the slide on the microscope stage and, using the low power
objective, locate the thin portion of the smear in which the red cells are evenly
distributed and are not touching each other.
(8) Switch to oil immersion magnification and count the number of

reticulocytes in 5 fields of 200 RBCs.
d. Calculation.
Number of reticulocytes counted

= % reticulocytes
10
(1) Equal volumes of blood and stain give optimum staining conditions. An
excess of blood causes the reticulum to understain. An excess of stain usually
obscures the reticulum.
MD0853 5-10
(2) Crenated erythrocytes and rouleaux formation make an accurate count
difficult to perform.
(3) Stain precipitated on erythrocytes causes them to appear as

reticulocytes
(4) Dirty slides cause uneven spreading.
(5) The dye solution should have adequate time to penetrate the cell and
stain the reticulum.
f. Discussion.
(1) Reticulocytes are nonnucleated erythrocytes that exhibit blue reticulum

strands within their cytoplasm when stained supravitally. When stained only with
Wright's stain, they are buff-pink in color and larger and darker than erythrocytes.
(2) Reticulocytes serve as an index of the activity of the bone marrow in

blood regeneration. As such, these counts are of value in following anti-anemia
therapy. Satisfactory response to therapy is evidenced by an increase of reticulocytes
in the peripheral blood. Increased reticulocyte counts also occur whenever there is
rapid bone marrow activity as in leukemia or blood regeneration associated with
hemorrhage or hemolysis. Decreased reticulocyte counts occur in conditions in which
the bone marrow is not producing adequate red blood cells, such as aplastic anemia.
(3) Several methods for staining and counting reticulocytes are in common
use. Compared to the use of alcoholic solutions of dye, methods employing saline
solutions of new methylene blue can give slightly higher values for reticulocytes. For
comparative studies, the same method should be used throughout the work.
(4) Precipitated stain is often confused with reticulum but can be recognized
by its presence throughout the smear and apart from the red cells. Precipitation can be
eliminated as a source of error by frequently filtering the stain.
(5) An alternate method of counting reticulocytes utilizes the Miller disk that
is placed inside the microscope eyepiece. This disc consists of 2 squares as shown
below in figure 5-2. The area of the smaller square (B) is a tenth that of square A.
Therefore, if there are 40 red cells in square A, there should be four red cells present in
square B. When employing this method to count reticulocytes, the red cells in square B
are counted in successive fields on the slide, until a total of 500 red cells have been
counted. At the same time, the reticulocytes in square A are enumerated. At the
completion of the count, theoretically, the reticulocytes obtained in this way are divided
by 50, in order to obtain the percent reticulocytes present in the blood.
MD0853 5-11
Figure 5-2. Miller disc.
g. Normal Values.
(1) Birth. Two and one-half to 6.0 percent, but falls to adult range by the
end of second week of life.
(2) Adults (both sexes). Five-tenths to 1.5 percent.
Section II. MANUAL COUNTS, OTHER BODY FLUIDS
5-5. CEREBROSPINAL FLUID CELL COUNTS
a. Principle. Cerebrospinal fluid is delivered to a counting chamber and

examined microscopically for blood cells. Normally, spinal fluid is clear. If it is cloudy,
dilute before charging the counting chamber.
b. Reagent. Cerebrospinal Fluid Diluting Fluid: Add 10 ml of glacial acetic acid

and 0.2 grams of crystal violet to a 100-ml volumetric flask. Dilute to the mark with
distilled water.
c. Procedure.
NOTE: Set up cell counts on spinal fluids within 30 minutes after withdrawal of the
specimen.
(1) Clear spinal fluid is set up as follows:
(a) With a capillary pipet introduce a drop of well-mixed spinal fluid into
one counting chamber of a hemacytometer.
CAUTION: Avoid contamination by careful handling of spinal fluid.
MD0853 5-12
(b) Examine the entire ruled area for the presence of cellular elements.
If both leukocytes and erythrocytes are observed, note the condition of the red cells
(fresh or crenated).
(c) Count all cells in the entire ruled area (0.9 cu mm).
(2) Turbid spinal fluid is set up as follows:
(a) Draw cerebrospinal fluid diluting fluid to “1.0” mark of the white
blood cell diluting pipet.
(b) Carefully draw a well-mixed specimen of spinal fluid to the “11”

mark.
(c) Shake the pipet for two minutes to mix the specimen.
(d) Discard the fluid in the capillary portion of the pipet.
(e) Charge the counting chamber and allow the cells to settle for five
minutes.
(f) Under low-power magnification count all cells in the entire ruled
area (0.9 cu mm).
(g) Switch to high-power and perform a rough differential count diluting

pipet.
(3) For very cloudy spinal fluid a white blood cell dilution is made as follows:
(a) Draw spinal fluid to the “0.5” mark in the white blood cell diluting
pipet.
(b) Draw cerebrospinal fluid diluting fluid to the 11 mark.
(c) Perform steps (c) through (g) in paragraph 5-6c(2).
d. Calculations.
(1) Clear spinal fluid:
Number of cells counted

= cells per cu mm
volume (0.9)
MD0853 5-13
(2) Turbid spinal fluid:
Number of cells counted X dilution (10)

= cells per cu mm
volume (0.9 cu mm)
(3) Very clouded spinal fluid:
Number of cells counted X dilution (20)

= cells per cu mm
volume (0.9 cu mm)
e. Source of Error. See paragraph 5-2e.
f. Discussion.
(1) If more than 100 leukocytes per cu mm are present, centrifuge the
undiluted specimen, make a smear, and stain with modified Wright's stain. Perform a
routine differential count and also estimate the ratio of erythrocytes to leukocytes.
NOTE: It may be necessary to use egg albumin or cell-free serum to make the
sediment adhere to the slide.
(2) Normally the spinal fluid is water clear. It can be turbid if cell count is
500 or more cells per cu mm. If there is fresh blood with spontaneous clotting, the
indications are those of a bloody tap. Xanthochromia develops after subarachnoid
hemorrhage has been present for a few hours and is due to disintegration of blood
pigments. Xanthochromia may also develop from tumors, abscesses, and
inflammation.
(3) Cell counts above ten are considered to be evidence of intracranial

disease. The predominant cell in most viral infections, syphilis, and tuberculous
meningitis is the lymphocyte. Bacterial infections due to meningococcus,
pneumococcus, and so forth, usually result in a predominance of the neutrophil.
Cerebral and extradural abscesses as well as subdural hemorrhages produce a
neutrophilic response although bacteria are not demonstrated.
(4) Biochemical, bacteriological, virological, serological, and hematological

ands are all necessary to reflect the true condition of the cerebrospinal fluid. The
current laboratory standing operating procedures should give guidance for the most
efficient method to accomplish all the necessary ands.
g. Normal Value: Zero to five cells per cu mm (chiefly lymphocytes).
MD0853 5-14
5-6. SEMEN ANALYSIS
a. Principle. Semen analysis involves gross examination (volume, color,

turbidity, viscosity, and pH) and microscopic examination (motility and spermatozoa
count).
b. Reagent. Spermatozoa Fixative Solution: Add 5 grams sodium bicarbonate

(NaHCO3) and 1 ml of formalin to a 100 ml volumetric flask. Dilute to the mark with
distilled water.
c. Collection Instructions. A physician will usually give the instructions;

however, the patient should be reminded of several critical points.
(1) The patient may be required to abstain from intercourse for a period
directed by the physician.
(2) The specimen is collected in a clean container that has been pre-
warmed to body temperature.
(3) The specimen should be delivered to the laboratory within 30 minutes.
(4) The specimen must be kept at body temperature (37oC) and not
subjected to extremes of heat or cold.
d. Gross Examination.
(1) Record. the time of collection and receipt of the specimen.
(2) Measure and record the volume.
(3) Observe and record the color (white, gray, yellow, and so forth), turbidity
(clear, opalescent, opaque, and so forth), and viscosity (viscid, gelatin, liquid).
(4) Determine the pH with a pH reagent strip and record this.
e. Motility Examination.
(1) When the specimen becomes fluid (within 15 to 30 minutes after

collection, the semen liquifies by the action of fibrinolysin), place 1 drop on a slide (pre-
warmed to 37oC) and place a cover slip on it.
(2) Under high dry power, count motile and nonmotile spermatozoa in two or
more areas until a total of at least 200 spermatozoa have been observed. It is
necessary to focus through the entire depth of a given field so as to include nonmotile
spermatozoa that may have settled to the bottom of the slide. Only those that move
forward actively are considered motile. Record the percent of motile spermatozoa seen.
MD0853 5-15
(3) Repeat this procedure in three hours and six hours, using a new drop
from the original specimen each time.
f. Spermatozoa Count.
(1) Draw the liquefied semen to the “0.5” mark on white blood cell diluting
pipet.
(2) Draw fixative solution to the “11” mark on the WBC pipet .
(3) Let the mixture stand until the mucus dissolves.
(4) Shake the pipet thoroughly and charge a hemacytometer.
(5) Count the spermatozoa in the same manner as you would count white
blood cells.
(6) After counting the sperm, examine the morphology and report the
percent of abnormal forms. Morphologically normal sperm are quite uniform in
appearance. Any sperm with rounded, enlarged, small, or bilobed heads are abnormal.
Abnormal tails are enlarged, small, irregular in length, absent, or multiple. See
Figure 5-3 for morphology of spermatozoa.
Figure 5-3. Morphology of spermatozoa.
MD0853 5-16
g. Calculations.
(1) Number of sperm

counted X dilution (20)
= sperm per cu mm
volume (0.4)
(2) Sperm per cu mm X 1000 = sperm per ml.
h. Sources of Error.
(1) Delay in analysis results in a lower percentage of motile forms and a

lower count.
I (2) Temperature extremes cause spermatozoa to die.
(3) See paragraph 5-2e for sources of error when counting.
i. Discussion.
(1) Semen analyses are usually performed as part of infertility studies or

following a vasectomy.
(2) Semen analysis can be performed for medico-legal cases involving rape
or to support or disprove a denial of paternity on the grounds of sterility.
(3) Semen is derived from the following: testes, seminal vesicles, prostate,
epididymides, vasa deferentia, bulourethral glands, and urethral glands.
j. Normal Values.
(1) Volume: 1.5-5.0 ml.
(2) pH: 7.2-7.6.
(3) Motility: 60-90 percent.
(4) Spermatozoa Count: 60-150 million per ml.
MD0853 5-17
EXERCISES, LESSON 5
INSTRUCTIONS: Answer the following exercises by marking the lettered responses

that best answer the exercise, by completing the incomplete statement, or by writing the
1. For a WBC count, after drawing blood into the diluting pipet, it is necessary to wipe
any excess blood from the outside of the pipet in order to avoid:
a. Wasting cells.
b. Dirtying the pipet.
c. Dirtying the hemacytometer cover glass.
d. Transferring cells to the diluting fluid.
2. When doing a WBC count, to what mark should the diluting fluid be drawn?
a. 3.
b. 7.
c. 11.
d. 13.
3. When performing a WBC count, which reagents may be used as dilutants?
a. Acetic acid or sulfuric acid.
b. Acetic acid or citric acid.
c. Acetic acid or hydrochloric acid.
d. Acetic acid or trisphosphoric acid.
MD0853 5-18
4. For the WBC count, immediately after the contents of the pipet have been mixed
for about three minutes, it is necessary to:
a. Use a mechanical mixer.
b. Expel three to four drops.
c. Observe for even distribution of cells.
d. Refill both chambers of the hemacytometer.
5. After the WBCs have settled for about three minutes during a manual WBC count,
which powered magnification and lighting arrangements are used to focus on the
ruled area to observe for even distribution of WBC?
a. Low-power (10X); reduced light.
b. High-power (43X); bright light.
c. Oil immersion (97X); bright light.
d. High-power (43X); reduced light.
6. When counting WBCs, a variation of more than ___________ cells between any of
the four areas counted or a variation of more than _____________cells between
sides of the hemacytometer indicate uneven distribution and require that the
procedure be repeated.
a. 6; 12.
b. 7; 9.
c. 10; 20
d. 10; 18.
MD0853 5-19
7. Why is the pipet held upright immediately after drawing the diluting fluid to the 11"
mark and mixing it with the specimen?
a. To indicate a "snake-like" line.
b. To wash the blood.
c. To dilute the blood better.
d. To prevent air bubbles in the bulb.
8. Which WBCs are counted?
a. Those touching the inner left-hand bottom lines.
b. All WBCs outside the squares.
c. All WBCs within the square and those touching the upper and right hand
center lines.
d. All WBCs within the square and those touching the upper and left- hand center
lines.
9. How many 1-sq-mm comer areas and chambers are used to count WBCs?
a. 3; 4.
b. 4; 2.
c. 2; 6.
10. Which chemical is mixed with whole blood when obtaining a WBC count?
a. Sodium chloride.
b. Weak acid.
c. Weak base.
d. Calcium carbonate.
MD0853 5-20
11. Blood is drawn to the __________ mark and diluted to the __________ mark for a
WBC count.
a. 0.5, 10.
b. 0.1,20.
c. 0.5, 11.
d. 0.10, 20 .
12. The usual blood dilution for the manual WBC count is:
a. 1:10.
b. 1:20.
c. 1:100.
d. 1:200.
13. The volume is the:
a. Area X width.
b. Width X length.
c. Width X depth.
d. Area X depth.
MD0853 5-21
14. Using the hemacytometer counting chamber, the formula for calculating the WBC
count is:
a. Average number of WBCs counted X Dilution.

= WBCs per cu mm
Volume
b. Average number of WBCs counted X Dilution.

= WBCs per sq in
Volume
c. Average number of WBCs counted X Volume

= WBCs per cu mm
Dilution
d. Average number of WBCs per cu mm X WBCs counted

= Dilution
Volume
15. If blood is drawn to the “0.5” mark on a WBC diluting pipet, and diluting fluid to the
“11” mark, what is the WBC count of the patient if the average of two chamber
counts is 163?
a. 3,260 WBCs per cu mm.
b. 8,530 WBCs per cu mm.
c. 8,150 WBCs per cu mm.
d. 10,320 WBCs per cu mm.
counts is 214?
MD0853 5-22
counts is 198?
18. What is the maximum allowable error rate when using the four large
hemacytometer squares in the WBC count?
a. 10 percent.
b. 15 percent.
c. 20 percent.
d. 25 percent.
19. What is the maximum allowable error rate for the manual WBC count when 8
square areas are employed?
a. 10 percent.
b. 15 percent.
c. 20 percent.
d. 25 percent.
MD0853 5-23
20. When performing a WBC count, what is done when the white cell count is
exceptionally reduced as in the case of leukopenia?
a. The dilution should be made in the red blood cell diluting pipet.
b. The blood is drawn to the “1.0” mark and the diluting fluid is drawn to the “11”
mark. The resulting dilution is 1:100.
c. The white pipet should be filled to the “1.0” mark and diluted to the “11” mark
with two percent acetic acid. The resulting dilution is 1:10.
d. The count is corrected calculating the observed count x 100 divided by 100 +
the percent of nucleated erythrocytes.
21. When performing a WBC count, what is done when the white cell count is
exceptionally high as in the case of leukemia?
a. The dilution should be made in the red blood cell diluting pipet. The blood is
drawn to the “1.0” mark and the diluting fluid is drawn to the “101” mark. The
resulting dilution is 1:100.
b. The white pipet should be filled to the “1.0” mark and diluted to the “11” mark
with 2 percent acetic acid. The resulting dilution is 1:10.
c. The count is corrected calculating the observed count x 100 divided by 100 +
the percent of nucleated erythrocytes.
d. Add 10 percent of the count to the total number of cells counted + the percent
of nucleated erythrocytes.
22. If blood for a WBC count is drawn to the “1.0” mark on a RBC diluting pipet, and
diluting fluid to the “101” mark, what is the WPC count of the patient if the average
of two chamber counts is 356?
MD0853 5-24
23. If blood for a WBC count is drawn to the “1.0” mark on an RBC diluting pipet, and
diluting fluid is drawn to the “101” mark, what is the WBC count if the average of
two chamber counts is 290?
a. 72,500 WBCS per cu mm.
counts is 153?
d. 76,500 WBCS per cu mm.
25. If the WBC count is 10,210 and the differential indicates there are 19 nucleated
RBCs per 100 WBCs, what is the corrected WBC count?
a. 8,650.
b. 8,580.
c. 9,580.
d. 1,021,000.
MD0853 5-25
26. If the WBC count is 9,640 and the differential indicates there are 14 nucleated
RBCs per 100 WBCs, what is the corrected WBC count?
a. 7,390.
b. 8,256.
c. 8,456.
d. 946,000.
27. What is the normal range of the WBC count in adults?
a. 4,500 -11,500 WBCs per cu mm.
b. 6,000 -14,000 WBCs per cu mm.
c. 9,000 -30,000 WBCs per cu mm.
d. 4.2 -5.4 million WBCs per cu mm.
28. If 88 eosinophils are counted in a 36-sq mm area of a hemacytometer using a 1:10

dilution, what is the eosinophil count?
a. 20 eosinophils per cu mm.
b. 150 eosinophils per cu mm.
c. 244 eosinophils per cu mm.
d. 300 eosinophils per cu mm.
29. An unchanged eosinophil count 4 hours after the injection of ACTH is indicative of:
a. Hypoadrenalism.
b. Hyperadrenalism.
c. Cushing's disease.
d. Normal adrenocortical function.
MD0853 5-26
30. Which stain is used to evaluate eosinophil nasal smears?
a. Pink.
b. Orange.
c. Supravital.
d. Wright's.
31. The reticulocyte is an immature:
a. Rubriblast.
b. Erythroyte.
c. Prorubricyte.
d. Metarubricyte.
32. If 15 reticulocytes are counted in a total of 1,000 erythrocytes, what percentage of

reticulocytes should be reported?
a. 0.015 percent.
b. 1.15 percent.
c. 2.5 percent.
d. 1.5 percent.
33. If 86 reticulocytes are counted in a total of 1,000 erythrocytes, what percentage of

reticulocytes should be reported?
a. 8.6 percent.
b. 4.6 percent.
c. 66 percent.
d. 86 percent.
MD0853 5-27
34. In tuberculous meningitis, the predominant WBC type usually found in the spinal
fluid is the:
a. Monocyte.
b. Neutrophil.
c. Eosinophil.
d. Lymphocyte.
e. Bone marrow activity.
f. Hemolysis.
h. Hemacytometer placement.
35. If 198 cells are counted in an undiluted spinal fluid, what is the cell count?
a. 2.2 per cu mm.
b. 22 per cu mm.
c. 220 per cu mm.
d. 2,200 per cu mm.
36. If 47 cells are counted in a spinal fluid diluted 1:10, what is the cell count?
a. 4.7 per cu mm.
b. 52.2 per cu mm.
c. 470 per cu mm.
d. 522 per cu mm.
MD0853 5-28
37. White blood cell counts on spinal fluid that are above ___________________ are
usually considered indicative of some type of intracranial disease.
a. 10 per cu mm.
b. 15 per cu mm.
c. 20 per cu mm.
d. 25 per cu Im1.
38. In most viral infections, the predominant cell usually found in the spinal fluid is the:
a. Neutrophil.
b. Basophil.
c. Eosinophil.
d. Lymphocyte.
39. In subdural hemorrhages, the predominant cell type found in spinal fluid is usually
the:
a. Lymphocyte.
b. Neutrophil.
c. Monocyte.
d. Segmented lymphocyte.
40. The neutrophil cell is predominant in which disease or infection?
a. Tuberculous meningitis.
b. Syphilis.
c. Bacterial infections.
d. Hodgkin’s disease.
MD0853 5-29
41. Except for the diluting fluid used, the spermatozoa count is almost identical in
procedure to the:
a. RBC count.
b. WBC count.
c. Reticulocyte count.
d. Total eosinophil count.
42. What is the patient required to abstain from prior to having a Semen analysis
collected?
a. Water intake.
b. Using a pre-warmed container.
c. Intercourse.
d. Using the Miller disk.
43. What three factors should be observed and recorded during gross and of the
semen specimen?
a. Color, viscosity, and temperature.
b. Color, amount of blood, and viscosity.
c. Mucus dissolved, temperature, and color.
d. Viscosity, color, and turbidity.
44. During a motility and of spermatozoa, which cells are considered to be motile?
a. The entire mixture.
b. The entire depth of the field.
c. All active ones moving forward.
d. Only those that are floating.
MD0853 5-30
45. When should the motility procedure be repeated when examining spermatoza
specimens?
a. Every 15 minutes.
b. In 3 hours and 6 hours.
c. Within 30 minutes of collection.
46. Fibrinolysin causes what type of change to the semen?
a. It solidifies.
b. It is modified with Wright's stain.
c. It causes it to liquify.
d. Nothing.
47. Semen analysis can be performed for ____________________ cases involving

rape or in support or denial of paternity on the grounds of ___________________
a. Medico-legal; sterility.
b. Medico-vasa deferentia; sterility.
c. Abnormal; epididymides.
48. From which of the following male body parts is semen derived?
a. Testes, seminal vesicles, prostate, epididymides, vasa deferentia,

reticulocytes, and urethral glands.
b. Testes, seminal vesicles, prostate, erythrocytes, vasa deferentia, bulbourethral

glands, and urethral glands.
c. Testes, motility spermatozoa, seminal vesicles, prostate, epididymides, vasa

deferentia, bulbourethral glands, and urethral glands
d. Testes, seminal vesicles, prostate, epididymides, deferentia, bulbourethral

glands, and urethral glands.
MD0853 5-31
49. Which normal value of spermatoza is correct?
a. Volume: 0.5-5.0 ml.
b. pH: 7.4-7.6.
c. Motility: 60-90 percent.
d. Spermatozoa Count: 25-150 million per ml.
50. Which normal value of spermatoza is correct?
a. Volume: 1.5-5.2 ml.
b. pH: 7.2-7.6.
c. Motility: 60-94 percent.
d. Spermatozoa Count: 60-170 million per ml.
MD0853 5-32
1. d (para 5-2c(2))
2. c (para 5-2c(3))
3. c (para 5-2b)
4. b (para 5-2c(4))
5. a (para 5-2c(7))
6. c (para 5-2c(9))
7. d (para 5-2c(3))
8. c (para 5-2c(9))
9. b (para 5-2c(8))
10. b (para 5-2a)
11. c (para 5-2c(1), (3); 5-2d(1))
12. b (para 5-2d(1))
13. d (para 5-2d(2))
14. a (para 5-2d(2), (3))
15. c (para 5-2d(2), (3))
16. d (para 5-2d(2) , (3))
17. b (para 5-2b(2), (3))
18. c (para 5-2f(1))
19. b (para 5-2f(1))
20. c (para 5-2f(6))
21. a (para 5-2f(5))
22. c (para 5-2f(5))
MD0853 5-33
23. a (para 5-2d(3), (4))
24. a (para 5-2d(3), (4))
25. b (para 5-2f(7))
26. c (para 5-2f(7))
27. a (para 5-2g(1))
28. c (para 5-3d)
29. a (para 5-3f(6))
30. d (para 5-3f(7))
31. b (para 5-4a)
32. d (para 5-4d)
33. a (para 5-4d)
34. d (para 5-5f(3))
35. c (para 5-5d(1))
36. d (para 5-5d(2))
37. a (para 5-5f(3))
38. d (para 5-5f(3))
39. b (para 5-5f(3))
40. c (para 5-5f(3))
41. b (para 5-6f(5))
42. c (para 5-6c(1))
43. d (para 5-6d(3))
44. c (para 5-6e(2))
45. b (para 5-6e(3))
MD0853 5-34
46. c (para 5-6e(1))
47. a (para 5-6i(2))
48. d (para 5-6i(3))
49. c (para 5-6j(3))
50. b (para 5-6j(2))
End of Lesson 5
MD0853 5-35
LESSON ASSIGMMENT
LESSON 6 Hematocrit, Erythrocyte Sedimentation Rate, and

Hemoglobin.
TEXT ASSIGMMENT Paragraphs 6-1 through 6-14.
TASK OBJECTIVES After completing this lesson you should be able to:

procedures used to perform the microhematocrit
test.

procedures used to perform the erythrocyte
sedimentation using the Wintrobe-Landsberg
and modified Westergren techniques.
6-3. Select the statement that best describes

hemoglobin, the compounds of hemoglobin, and
the variations of hemoglobin.
6-4. State the four basic ways to measure

hemoglobin concentrations and correctly
describe the cyanmethemoglobin method.
6-5. Select the procedures required to perform the

detection of hemoglobin S and to demonstrate
the sickle cell phenomenon.

purpose of hemoglobin electrophoresis.

procedures to perform a fetal hemoglobin test.

exercises of this lesson. These exercises will help you
to achieve the lesson objectives.
MD0853 6-1
LESSON 6
HEMATOCRIT, ERYTHROCYTE SEDIMENTATION RATE, AND HEMOGLOBIN
Section I. HEMATOCRIT
6-1. INTRODUCTION
a. The hematocrit or the packed-cell volume is the percentage of the total

volume of red blood cells in relation to the total volume of whole blood. The term
"hematocrit" is derived from two Greek words: Hemato, meaning blood; and Krites,
meaning to judge (then reduced to crit, meaning to separate). The procedure is
performed by filling a capillary tube with blood and centrifuging at a constant speed for a
constant period of time. The packet cell volume is then measured. The hematocrit can
also be determined by automated sequential analyzers but is usually a calculated value.
b. The hematocrit is the most useful single index for determining the degree of
anemia or polycythemia. It can be the most accurate (2-4 percent error) of all
hematological determinations. In contrast, the direct red blood cell chamber count has
a percent error of 8-10 percent. The hematocrit is, therefore, preferable to the red blood
cell count as a screening test for anemia. The values for the hematocrit closely parallel
the values for the hemoglobin and red blood cell count.
6-2. MICROHEMATOCRIT
a. Principle. A capillary tube is filled with whole blood by capillary action to

within 1 to 2 cm of the end. The unfilled end is sealed and the tube is centrifuged. After
centrifugation, the capillary tube is placed in a reading device and the hematocrit value
determined.
b. Procedure.
(1) If anticoagulated venous blood is the specimen, fill a plain capillary tube
with blood. If blood without anticoagulant is used, fill a heparinized capillary tube with
the blood specimen. A heparinized capillary tube is identified by red line on the tube
specimen. A heparinized capillary tube is identified by a red line on the tube.
(2) Allow blood to enter two capillary tubes until they are approximately 2/3
filled with blood. (Air bubbles denote poor technique, but do not affect the results of the
test.
(3) Seal the unfilled ends of the tubes with clay.
(4) Place the two hematocrit tubes in the radial grooves of the centrifuge
head exactly opposite each other, with the sealed end away from the center of the
centrifuge.
MD0853 6-2
(5) Screw the flat centrifuge head cover in place.
(6) Centrifuge at 10,000 rpm for 5 minutes.
(7) Remove the hematocrit tubes as soon as the centrifuge has stopped
spinning. Determine the hematocrit values with the aid of a microhematocrit reader.
Results should agree within +2 percent. If they do not, repeat the procedure.
NOTE: Since there are a variety of readers available, it is necessary that the
technician carefully follow the directions of the manufacturer for the particular
device utilized.
c. Sources of Error.
(1) Inadequate mixing of the blood prior to sampling.
(2) Improper sealing of the capillary tube causes the blood to blow out of the
capillary tube during centrifugation.
(3) Capillary tubes must be properly identified. Numbered holders for

capillary tubes are available. Place the tubes in slots on the holder and record the
numbers on the laboratory request slip.
(4) Improper centrifugation leads to varied results. For good quality control,
maintain prescribed centrifuge speed and time.
(5) Misreading the red cell level by including the buffy coat causes elevated
values.
d. Discussion.
(1) The microhematocrit technique is advantageous because of speed, and

because only a small quantity of blood is necessary for the determination. An additional
advantage is the ease with which this procedure is adapted to infants and small
children. The microhematocrit technique requires only a simple capillary puncture
whereas in the Wintrobe method venous blood oust be used. Another advantage is the
use of disposable capillary tubes.
(2) If the microhematocrits cannot be read promptly, the capillary tubes

must be properly identified and placed in a vertical position. Slanting of the cell layer
will occur if tubes are left in a horizontal position for more than 30 minutes.
(3) Hematocrit results may also re obtained or computed through use of an

electronic cell counter (Coulter Models). Results of computed hematocrits are generally
1.3 to 3.0 percent higher due to their method of derivation. An accurate erythrocyte
count (three simultaneous electronic counts averaged) is multiplied by an accurate MCV
MD0853 6-3
measurement resulting in the area that the red cells would occupy if packed. The
computation makes no allowance for the trapped plasma that always occurs to sate
degree in manual packing procedures.
e. Normal Values.
(1) Birth: 50–62 percent.
(2) Age 1: 31--39 percent.
(3) Adult males: 42–52 percent.
(4) Adult females: 36–46 percent.
Section II. ERYGHROCYTE SEDIMENTATION RATE
6-3. ERYTHROCYTE SEDIMENTATION RATE
a. Introduction. When anticoagulated whole blood is allowed to stand for a

period of time, the red blood cells settle out frail the plasma. The rate at which the red
cells fall is known as the erythrocyte sedimentation rate (ESR). The ESR is affected
mainly by three factors: erythrocytes, plasma, and mechanical and technical factors.
b. Erythrocytes. Size and shape of erythrocytes cause the ESR to fluctuate.

Microcytes tend to settle slower than normal cells, while macrocytes fall more rapidly.
An increase in spherocytes and/or bizarrely-shaped red cells retards the sedimentation
rate. With a decreased hematocrit there is less retardation of sedimentation by the
erythrocytes themselves and they tend to settle faster. Corrections for anemic blood
are available; however, most experts consider this correction useless and invalid. An
increased hematocrit (above 55 percent) retards the sedimentation rate.
c. Plasma Composition. In certain diseases plasma proteins, namely

fibrinogen and globulin, may be altered, causing rouleaux formation. The speed of the
sedimentation corresponds to the length of the rouleaux formation. Increases in
fibrinogen or globulin will produce long rouleaux that are difficult to disperse. This leads
to a larger mass and an increased sedimentation velocity.
d. Mechanical Technical Factors. It is important that the ESR tube be exactly

perpendicular. A tilt of 30 can cause errors up to 30 percent. Vibration or movement of
the ESR tube or holding rack can affect the ESR as can large changes in temperature.
e. ERYTHROCYTE SEDIMENTATION RATE. Determination of the ESR is

performed by many methods of the more common methods; the Wintrobe-Landsberg
and modified Westergen methods are stated in this lesson.
MD0853 6-4
6-4. DETERMINATION OF SEDIMENTATION RATE (WINTROBE-LANDSBERG)
a. Principle. Anticoagulated blood is placed in a narrow tube. The blood cells

settle out of the suspension, leaving clear plasma above them.
The distance that the erythrocytes fall within a given interval of time is measured.
b. Procedure.
(1) Draw 5 ml of blood by venipuncture and place in a test tube containing

ethylenediaminetetraacetic acid (EDTA) (lavender top vacuum tube).
(2) Thoroughly mix the blood and anticoagulant by gently inverting the tube
several times, being careful not to cause bubbles.
(3) Draw the blood into the capillary pipet and fill the Wintrobe tube to the
“0” mark. This is done by inserting a capillary pipet to the bottom of the Wintrobe tube,
while holding it at an angle of 45°. As the tube is filled, slowly withdraw the pipet so that
the tip is always just below the level of the blood. The blood count must be free of
bubbles.
(4) Place the filled Wintrobe tube in a rack in an exactly vertical position and
note the time and roan temperature.
(5) At the end of exactly 1 hour, read the level to which the red cells have
settled on the descending scale etched on the tube. Each mark equals 1.0 mm while
each numbered mark equals 10 mm (1 cm). The figure obtained is reported in mm per
hour as the "uncorrected" erythrocyte sedimentation rate.
(6) If a "corrected" sedimentation is requested, perform a hematocrit.

Calculators to correct for sedimentation rate are available in the Federal Supply
Catalog.
c. Sources of Error.
(1) The blood specimen must be properly mixed with the proper
anticoagulant to obtain an undiluted representative sample.
(2) The test should be set up within two hours after the blood sample is
collected to avoid a false low sedimentation rate.
(3) Increase in temperature accelerates the rate. Desirable temperature

range is 22ºC to 27º C.
(4) The tube must be vertical. A 3º variation from the vertical rate
accelerates the rate by 30 percent.
MD0853 6-5
(5) Dirty Wintrobe tubes or capillary pipets can decrease the rate.
(6) Tubes should be placed free from vibration or disturbance.
d. Normal View.
(1) Males: Zero to nine mm per hour.
(2) Females: Zero to 20 mm per hour.
(3) Children: Zero to 13 mm per hour.
6-5. DETERMINATION OF SEDIMENTATION RATE
a. Principle. Well-mixed, whole blood is diluted with 0.85 percent sodium

chloride placed in a Westergren pipet, and allowed to stand for exactly one hour. The
number of millimeters the red cells fall during this tie period constitutes the ESR result.
b. Procedure.
(1) Mix the whole blood and the anticoagulant by gently inverting the tube
several times or by placing on a rotator for two minutes.
(2) Place 0.5 ml of 0.85 percent sodium chloride in a plain 13X 100 mm test
tube.
(3) Add 2.0 ml of well-mixed whole blood to the test tube.
(4) Mix the tube for two minutes.
(5) Make certain that the Westergren ESR rack is exactly level.
(6) Fill the Westergren pipet to exactly the “0” mark, making certain there
are no air bubbles in the blood.
(7) Place the pipet in the rack. Be certain the pipet fits snugly and evenly
into the grooves provided for it.
(8) Allow the pipet to stand for exactly 60 minutes.
(9) At the end of 60 minutes, record the number of millimeters that the red
cells have fallen. This result is the erythrocyte sedimentation rate in millimeters per
hour.
c. Sources of Error. See para 6-4c.
MD0853 6-6
d. Normal Values.
(1) Males: Zero to 15 mm per hour.
(2) Females: Zero to20 mm per hour.
(3) Children: Zero to10 mm per hour.
e. Discussion.
(1) The erythrocyte sedimentation rate is a nonspecific test that suggests

the possibility of a disease process and tissue damage in the body. It is not diagnostic
but is extremely useful in following the course of some diseases.
(2) The rate is usually increased in inflammatory infections, toxemia, cell or

tissue destruction, severe anemia, active tuberculosis, syphilis, acute coronary
thrombosis, rheumatoid arthritis, and malignant processes.
(3) Sickle cell anemia, polycythemia, hypofibrinogenemia, and certain drugs

usually decrease the rate.
Section III. HEMOGLOBIN
6-6. GENERAL INFORMATION
Hemoglobin is a conjugated protein composed of the basic protein globin linked

to 4 heme molecules. Ninety-eight percent of all the iron found in the blood is contained
in hemoglobin. Hemoglobin transports oxygen and carbon dioxide. This important
substance reacts with oxygen to form oxyhemoglobin. In the tissues, oxygen is
released and reduced hemoglobin is formed. Hemoglobin can react with acids, bases,
and oxidizing and reducing agents. It also can exist in a variety of forms. These
hemoglobin compounds and variants are discussed briefly in the following paragraphs.
For more detailed information, refer to the standard hematological texts.
6-7. COMPOUNDS OF HEMOGLOBIN
a. Oxyhemoglobin. Oxygen combines loosely with iron (ferrous state) in

hemoglobin. The loosely attached oxygen diffuses into the tissues for oxidative
processes. The hemoglobin then binds carbon dioxide and exists as reduced
hemoglobin.
b. Carboxyhemoglobin. Hemoglobin combines with carbon monoxide to form

carboxyhemoglobin. Carbon monoxide has an affinity 200 times greater for hemoglobin
than oxygen does. Hemoglobin in this combination is incapable of oxygen transport.
MD0853 6-7
c. Methamoglobin. This compound is formed when the ferrous state of the
heme is oxidized to the ferric state. This compound is incapable of oxygen transport.
d. Sulfhemoglobin. This compound results from the combination of inorganic

sulfides and hemoglobin. This compound is incapable of oxygen transport. This is an
irreversible reaction.
e. Cyanmethemoglobin. This compound results when methemoglobin

combines with the cyanide radical. This compound is used in hemoglobinometry.
6-8. VARIATIONS OF HEMOGLOBIN
The variations of hemoglobin occur due to structural differences in the globin

protein. These differences are genetically controlled. The normal hemoglobin
components are hemoglobin A (HbA), hemoglobin A2 (HbA2), and fetal hemoglobin
(HbF). HbA constitutes most of the hemoglobin of a normal adult while HbA2
constitutes a much smaller amount. HbF is present during the first 4 to 6 months of life
and not normally present in adults. Hemoglobin S and hemoglobin C are the most
commonly occurring abnormal hemoglobins. Others (D, E, H, etc.) are found in rare
occurrences associated with several types of anemia. The various types of hemoglobin
are separated by electrophoresis.
6-9. HEMOBLOGINOMETRY
The hemoglobin concentration is directly proportional to the oxygen-combining

capacity of blood. Therefore, the measurement of the hemoglobin concentration in the
blood is important as a screening test for diseases associated with anemia and for
following the response of these diseases to treatment. There are four basic ways to
measure the hemoglobin concentration: (1) measurement of the oxygen-combining
capacity of blood (gasometric), (2) measurement of the iron content (chemical method),
(3) colorimetric measurement of specific gravity (gravimetric method), and (4) method is
the most widely used. The cyanmethemoglobin method is the method of choice and is
recommended by the Technical Subcommittee on Hemoglobinometry of the
International Committee for Standardization in Hematology.
6-10. CHYANMETHEMOGLOBIN METHOD
a. Principle. Blood is diluted with a dilute solution of potassium ferricyanide and

potassium cyanide at a slightly alkaline pH. The ferricyanide converts the hemoglobin
to methemoglobin. The cyanide then reacts with the methemoglobin to form the stable
cyanmethemoglobin. The color intensity is measured in a spectrophotometer at a
wavelength of 540 mm. The optical density is proportional to the concentration of
hemoglobin.
MD0853 6-8
b. Discussion.
(1) Cyanmethemoglobin is the most stable of the various hemoglobin

pigments showing no evidence of deterioration after 6 years of storage in a refrigerator.
The availability of prepared standards is a distinct advantage of this technique. All
hemoglobin derivatives are converted to cyanmethemoglobin with the exception of
sulfhemoglobin.
(2) This method is highly accurate and is the most direct analysis available
for total hemin or hemoglobin iron. Its disadvantage is the use of cyanide compounds,
which, if handled carefully, should present little hazard.
(3) For accuracy in hemoglobin determinations, it is absolutely necessary

that the spectrophotometer and Sahli pipets be accurately calibrated.
(4) Venous samples give more constant values than capillary samples.
(5) If the procedure is performed properly, the degree of accuracy is +2 to 3

percent.
c. Normal Values.
(1) Infants at birth: 17–23 g hemoglobin per deciliter.
(2) Childhood: 12–14 g hemoglobin per deciliter.
(3) Adult males: 14–17 g hemoglobin per deciliter.
(4) Adult females: 13–15 g hemoglobin per deciliter.
6-11. DETECTION OF HEMOGLOBIN S AND NON S SICKLING HEMOGLOBINS
a. Principle. Erythrocytes are introduced into a phosphate buffer solution

containing a reducing agent and hytic agent. The red cells are lysed and the
hemoglobin is reduced. Reduced sickling types of hemoglobin are insoluble in
phosphate buffer and turbidity results. On addition of urea, hemoglobin S dissolves.
b. Reagents.
(1) Stock phosphate buffer solution. Dissolve 160.48 grams anhydrous

potassium dihydrogen phosphate (KH2PO4), AR, and 28.88 grams anhydrous
potassium monohydrogen phosphate (K2HPO4) and AR in a 1-liter volumetric flask
containing 500 ml of distilled water. Dilute to 1-liter with distilled water.
(2) Dithionite reagent. Add 20.0 grams dithionite (Ha2S404 2H20) and 0.25
grams saponin to a 100-ml volumetric flask. Add 80 ml of stock phosphate buffer
MD0853 6-9
solution. Mix well. Dilute to the mark with distilled water. This reagent remains stable
under refrigeration at 4°C for approximately 1 month. See figure 6-1.
Figure 6-1. Dithionite tube test interpretation.
WARNING: Sodium dithionite reagent may ignite if allowed to become damp.

Use only clean dry utensils in handling.
(3) Urea (USP).
c. Procedure.
(1) Pipet 2 ml of dithionite reagent in 12 x 75 mm test tube.
(2) Add 0.02 ml of well-mixed anticoagulated blood (collected in EDTA).
(3) Mix the contents and al1a.v to stand at roan temperature for 5 minutes
(see figure 6-1).
(4) After 5 minutes examine the tube for turbidity against a lined reader.
Hemoglobin 5, if present, produces turbidity in the tube .
d. Sources of Error.
(1) The use of 10 x 75 mm test tubes could cause a false negative result.
(2) The dithionite reagent has a limited stability. The freshness of this
reagent must be checked with positive and negative controls. The test should show the
blue-pink color of reduced hemoglobin and adequate lysis of erythrocytes.
MD0853 6-10
(3) Unstoppered tubes containing dithionite reagent decompose when left
out at room temperature.
(4) False negative results could occur if the blood sample for testing is
drawn within 4 months of transfusion.
e. Discussion.
(1) Hemoglobin S is an inherited type of hemoglobin found primarily in

blacks and people from Mediterranean areas.
(2) The degree of erythrocyte sickling is dependent on the concentration of

hemoglobin. SS, SC, and SD cells sickle more rapidly than AS cells. Newborns with
sickle cell anemia have erythrocytes more resistant to sickling due to the presence of
hemoglobin F.
(3) The dithionite test also detects other sickling types of hemoglobin. Urea
causes hemoglobin S (and structural variants of hemoglobin S) to dissolve. Other
hemoglobins remain turbid in the presence of urea.
(4) This test is a rapid screening test for hemoglobin S. All positive
dithionite tests should be electrophoresed for confirmation.
f. Interpretation. Hemoglobin S causes turbidity in the tube. Hemoglobin A is

soluble in the phosphate buffer.
6-12. DEMONST4RATION OF THE SICKLE CELL PHENOMENON
a. Principle. Erythrocytes of persons with sickle cell anemia or trait will assume
a sickle shape when the oxygen tension is lowered. This may be demonstrated by
mixing a drop of blood with a reducing agent such as sodium metabisulfite.
b. Reagent. Sodium metabisulfite, 2 percent. Add 2 g of sodium metabisulfite

to a 100-ml volumetric flask. Dilute to the mark with distilled water. This solution, if
stored at 3º or 4ºC remains effective for about 1 week.
c. Procedure.
(1) Place 1 drop of capillary (or venous without anticoagulants) blood on a

clean glass slide.
(2) Add 1 or 2 drops of 2 percent aqueous sodium metabisulfite and mix.
(3) Place a cover glass on the preparation and express the excess blood by
gently pressing the cover glass. The gentle pressure will produce a film thin enough to
permit examination of individual red cells. It is not necessary to seal the preparation.
MD0853 6-11
(4) Observe immediately and at intervals of 15 to 30 minutes after
preparation for signs of sickle cell information.
d. Discussion.
(1) The sickling phenomenon is a consequence of an inherited, abnormal

type of hemoglobin (hemoglobin S). The severity of the anemia as compared to the trait
will vary with the proportion of defective to normal hemoglobin. This abnormal
hemoglobin can be identified electrophoretically.
(2) When a fresh solution of sodium rnetabisulfite is used, positive cases

should show 10 to 75 percent sickling within 15 minutes. There is a positive correlation
between the degree of sickling and the severity of the disease.
(3) Normal blood treated in this way shows no sickling.
6-13. HEMOGLOBIN ELECTROHORESIS (CELLULOSE ACETATE)
a. Principle. Hemoglobin fractions are separated by the rate of their protein

migration in an electrical medium. The fractions are stained with ponceau S and
quantitated on a densitometer. The order of mobility from the cathode toward the anode
is A3> AI> F> S-D>C-A2.
b. Discussion.
(1) A2 hemoglobin migrates identically to hemoglobin C. They are

distinguished by the quantity present. If this band is 40 percent or more of the total
hemoglobin, it is C. A2 hemoglobin should always be less than 20 percent.
(2) Two slow-moving, nonhemoglobin components are see using this

technique. These fractions are carbonic anhydrases I and II (CAI and CAII)
(3) Hemoglobin A2 is elevated in thalassemia minor.
(4) Genotype SS is found in patients with sickle cell anemia.
(5) Genotype AS is found in patients with sickle cell trait.
(6) This method separates hemoglobin A2 in the presence of hemoglobin S

in patients manifesting sickle-thalassemia disease.
(7) Hemoglobin F is quantitated by the alkali denaturation test because it

migrates close to the hemoglobin A1 fraction on the electrophoretic pattern.
(8) Include known A, S, and C controls in each analysis.
MD0853 6-12
c. Normal Values
(1) A3 Hemoglobin: One and three tenths to 3 percent.
(2) Genotype: AA.
(3) F Hemoglobin: Zero to 2 percent (except in infants).
6-14. FETAL HEMOGLOBIN (ALKALI DENATURATION TEST)
a. Principle. Fetal hemoglobin (HbF) is more resistant to denaturation in

alkaline solution than adult hemoglobin (HbA). Alkali converts HbA to alkaline hematin.
Alkaline hematin is insoluble and precipitates. HbF is quantitated by measuring the
hemoglobin concentration before and after denaturation.
b. Reagents.
(1) Drabkin’s reagent. Available in pellets or powder in the Federal Supply

Catalog.
(2) Sodium hydroxide (NaOH) 0.10 N. Dilute 5.55 saturated NaOH to 1-liter
with distilled water. Mix well and store in a polyethylene bottle at room temperature.
(3) Ammonium sulfate, saturated. Add 400 g ammonium sulfate, AR, to 500
ml of distilled water. Stir mechanically for 10 minutes. Heat until all the salt has
dissolved and filter rapidly through a Whatman number one filter paper. Store in
polyethylene bottle.
c. Preparation of Hemolysate. The procedure is as follows:
(1) Dilute 0.2 ml of hemolysate with 1.8 of Drabkin's reagent and mix gently.
(2) Place 2.5 ml of 0.1 N NaOH in a 13 X 100 mm test tube. At zero time,
add. 0.5 ml of diluted hemolysate and mix rapidly.
(3) Exactly 2 minutes after addition of the hemolysate, add 2.0 ml of

saturated ammonium sulfate. Mix by inverting six times. DO NOT SHAKE!
(4) Filter through a 7 or 9 cm Whatman number 42 filter paper. If the filtrate

is not crystal clear, filter again. This is the fetal hemoglobin solution.
(5) Prepare a total hemoglobin solution by adding 0.05 ml of diluted

hemolysate to 5.0 ml distilled water. Mix well by inversion. DO NOT SHAKE!
(6) Prepare a fetal hemoglobin blank by adding 0.5 ml of Drabkin’s reagent

to 4.5 ml of distilled water. Mix as above.
MD0853 6-13
(7) Prepare a total hemoglobin control by adding 0 05 ml of Drabkin's
reagent to 5.0 ml of distilled water. Mix as above.
(8) Measure the absorbances of the fetal and total hemoglobin solutions
against the appropriate blanks.
d. Calculations.
Absorbance fetal hemoglobin solution

X 10 = Percent fetal hemoglobin
Absorbance total hemoglobin solution
Report percent to nearest tenth (0.1).
(1) If the hemoglobin concentration of the hemolysate is less than 5 g per

the sensitivity of the procedure is decreased.
(2) Poor pipetting technique causes a significant error due to the small
volumes involved.
(3) If the absorbance of the fetal hemoglobin solution is greater than 0.700,
dilute 1.0 ml of the fetal hemoglobin solution with 9.0 ml distilled water. Read the
absorbance against the total hemoglobin blank. Multiply the calculated result by 10.
f. Discussion.
(1) The alkali denaturation test can be performed on bloody rectal

discharges from infants. This test is used to determine whether the discharge is due to
ingested maternal blood or a gastrointestinal lesion. The procedure is as follows:
(a) Add a small amount of bloody discharge to 10 ml of distilled water.
(b) Add 0.2 ml 10 percent sodium hydroxide solution.
(c) If the solution turns pink, it is fetal blood. A muddy brown solution
indicates blood of maternal origin.
(2) Fetal hemoglobin constitutes approximately 80 percent of a newborn

infant's hemoglobin. This decreases to approximately 5 percent at 6 months.
MD0853 6-14
(3) Fetal hemoglobin is increased in adults with thalassemia major and
minor, hereditary persistence of HbF, erythroleukemia, aplastic anemia, and
spherocytosis.
g. Normal Values.
(1) Infants (less than 6 months): Up to 80 percent.
(2) Adults: Zero to two percent.
MD0853 6-15
EXERCISES, LESSON 6
INSTRUCTIONS: Answer the following exercises by marking the lettered responses

that best answer the exercise, by completing the incomplete statement, or by writing the
1. When blood is centrifuged, the volume percentage occupied by the packed red
cells is known as the:
a. Erythrocyte sedimentation rate (ESR).
b. Hematocrit.
c. Hemoglobin concentration.
d. Mean corpuscular volume.
2. The procedure for determining hematocrit is performed by:
a. Filling a capillary tube capillary with blood.
b. Centrifuging at constant speed for a constant period of time.
c. Measuring the packed-cell volume.
d. Automated sequential analyzers (the results are usually a calculated value).
f. a, c, and d.
MD0853 6-16
3. The hematocrit is the most useful single index in determining the degree of:
a. Anemia.
b. Hypochromia or anemia.
c. Leukopenia.
d. Thrombocytopenia or thrombocvtosis.
4. The hematocrit error rate for determining the degree of polycythemia is:
a. 1-3 percent.
b. 2-5 percent.
c. 2-4 percent.
d. 3-6 percent.
5. In contrast to hematological determinations, what is the percent error rate for the
direct red blood cell chamber count?
a. 2-4 percent.
b. 5-9 percent.
c. 6-8 percent.
d. 8-10 percent.
6. The hematocrit values closely parallel the values for the:
a. Packed WBCs and hemoglobin.
b. Packed-cell blood count and reagent.
c. WBC count and hemoglobin.
d. Hemoglobin and RBC count.
MD0853 6-17
7. Which microhematocrit principle is correct?
a. A capillary tube is filled with plasma by capillary action to within 1 to 2 cm of

the end. The unfilled end is sealed and the tube is centrifuged. After
centrifugation, the capillary tube is placed in a reading device and the
hematocrit value determined.
b. A capillary tube is filled with whole blood by capillary action to within 1 to 2 cm

of the end. The unfilled end is sealed and the tube is centrifuged. After
c. A capillary tube is filled with whole blood by capillary action to within 2 to 4 cm

of the end. The unfilled end is sealed and the tube is centrifuged. After
d. A capillary tube is filled with whole blood by capillary action to within 1 to 2 cm

of the end. The filled end is sealed and the tube is centrifuged. After
centrifugation, the capillary tube is read and recorded.
8. Centrifugation for the microhematocrit lasts:
a. 30 seconds.
b. 30 minutes.
c. 1 minute.
d. 5 minutes.
9. During the microhematocrit test, blood without anticoagulant is identified by a

heparinized capillary tube with a ___________________ line.
a. Green.
b. Red.
c. Yellow.
d. Pink.
MD0853 6-18
10. When performing the microhematocrit test, if blood contains anticoagulant, how far
up should the capillary tube re filled with blood?
a. One forth.
b. Halfway.
c. Three fourths.
d. Completely.
11. Measuring the microhematocrit test, when blood is allowed to enter the two
capillary tubes to approximately 2/3 full and air bubbles appear, what does this
signify?
a. A poor technique was used but it does not affect the results of the test.
b. The heparinized capillary tube line was passed.
c. The tubes were dirty.
d. The seal was broken.
12. At what rpm and for how long are the 2 hematocrit tubes centrifuged for the
microhematocrit test?
a. 4,000 rpm; 2 minutes.
b. 5,000 rpm; 4 minutes.
c. 10,000 rpm; 5 minutes.
d. 15,000 rpm; 7 minutes.
MD0853 6-19
13. When using the microhematocrit reader, the results should agree within
_______________________________. If they do not, then what should occur?
a. 1 percent; nothing.
b. 2 percent; nothing.
c. 2 percent; repeat the procedure.
d. 3 percent; repeat the procedure.
14. Hematocrit results computed with an electronic cell counter are generally
______________________ than a manual hematocrit.
a. 1 per cent lower.
b. 4-6 percent higher.
c. 1-4 percent lower.
d. 1-3 percent higher.
15. The rate at which red blood cells fall when anticoagulated whole blood is allowed
to stand is known as:
a. Plasma composition.
b. Erythrocyte sedimentation.
c. Coulter models.
d. Spherocytosis.
16. Erythrocyte sedimentation is retarded when the hematocrit exceeds:
a. 35 percent.
b. 40 percent.
c. 45 percent.
d. 55 percent.
MD0853 6-20
17. The normal hematocrit readings for adult males and adult females are
respectively:
a. 38-47 percent and 34-41 percent.
b. 44-64 percent and 34-41 percent.
c. 44-64 percent and 40-54 percent.
d. 42-52 percent and 36-46 percent.
18. Size and shape of the erythrocytes cause the erythrocyte sedimentation rate
(ESR) to:
a. Increase.
b. Remain the same.
c. Decrease.
d. Fluctuate.
19. During erythrocyte sedimentation and with certain diseases, what kind of formation
may form in the plasma protein if the plasma protein fibrinogen and globulin are
altered?
a. Round.
b. Long.
c. Rouleaux.
d. Short.
20. What happens to the mass of plasma and to the sedimentation rate when the
plasma protein of fibrinogen and globulin are altered?
a. Mass enlarges; rate increases.
b. Mass decreases; rate decreases.
c. Mass shrivels; rate increases.
d. Mass enlarges; rate decreases.
MD0853 6-21
21. Keeping in mind the mechanical and technical factors, why is it important that the
ESR tube be exactly perpendicular?
a. A tilt of 50 can cause errors up to 55 percent.
b. A tilt of 30 can cause errors up to 30 percent.
c. A tilt of 10 can cause errors up to o. 05 percent.
d. There are no other factors that effect the ESR tube.
22. What other mechanical and technical factors are important and why when working
with the ESR tube or holding rack?
a. Spilling the ESR tube or tilting holding rack can affect the ESR, as can large
changes in temperature.
b. Static electricity or movement of the ESR tube or holding rack can affect the
ESR, as can large changes in temperatube.
c. Vibration or movement of the ESR tube or holding rack can affect the ESR as
can large changes in temperature.
d. There are no other factors.
23. When determining the ESR, using the Wintrobe-Landsberg method, what happens
to the anticoagulated blood and how is this procedure measured?
a. The anticoagulated blood is placed in a narrow tube/
b. The blood cells settle out of the suspension, leaving clear plasma above them.
c. The distance that the erythrocytes fall within a given interval of time is
measured.
d. a, b, and c all happen.
e. a and d happen.
f. None of the above.
MD0853 6-22
24. With the Wintrobe-Landsberg method, which tube is used to draw 5 ml of blood by
venipuncture?
a. Green top vacuum tube.
b. Lavender top vacuum tube.
c. Red lined tube.
d. Blue lined tube.
25. After the Wintrobe tube is placed in a rack in an exactly vertical position and the
time and room temperature are noted, when is a reading taken and what is
observed?
a. At the end of exactly 1 hour, read the level to which the red cells have settled
on the descending scale etched on the tube.
b. At the end of exactly 2 hour, read the level to which the red cells have settled
on the descending scale etched on the tube.
c. Within 15 minute, read the level to which the red cells have settled on the
ascending scale etched on the tube.
d. In 5 minutes, read the level to which the red cells have settled on the
descending scale etched on the tube.
26. If measurement of the ESR is delayed more than 2 hours after blood collection,
the reading may be inaccurate because of a:
a. False varied sedimentation rate.
b. False low sedimentation on rate.
c. False high sedimentation rate.
d. Varied sedimentation rate.
MD0853 6-23
27. For the Wintrobe-Landsberg method, to determine the ESR fill the Wintrobe tube
to the ___________ mark while holding it at _________ angle.
a. 0, 30 degrees.
b. 0, 45 degrees.
c. 5, 10 degrees.
d. 10,50 degrees.
28. If the tube is at a 30 variation from vertical this is a source of error and will
accelerate the ESR by _________________ percent.
a. 30 percent.
b. 30 percent.
c. 40 percent.
d. 50 percent.
29. When using the modified Westergren method, whole blood is diluted with
_________ percent sodium chloride and mixed for _________ minutes.
a. 0.85 percent, 2.
b. 0.90 percent, 3.
c. 0.95 percent, 4.
d. 0.80 percent, 2.
30. Using the modified Westergren method, what is the normal value ESR for
children?
a. 0-15 mm per hour.
b. 0-20 mm per hour.
c. 0-10 mm per hour.
d. 0-25 mm per hour.
MD0853 6-24
31. Once hemoglobin gives up its oxygen to the tissues, it is known as:
a. Methemoglobin.
b. Carboxyhemoglobin.
c. Cyanmethemoglobin.
d. Reduced hemoglobin.
32. Hemoglobin reacts with oxygen to form:
a. Oxyhemoglobin.
b. Methemoglobin.
d. Carboxyhemoglobin.
33. Which compound results when methemoglobin combines with the cyanide radical?
a. Oxyhemoglobin.
b. Sulfhemogobin.
d. Carboxyhemoglobin.
34. As ferrous iron in hemoglobin is oxidized to the ferric state, which of the following
is produced?
a. Methemoglobin.
b. Carboxyhemoglobin.
c. Carbaminohemglobin.
d. Reduced hemoglobin.
MD0853 6-25
35. Which constitutes most of the hemoglobin of a normal adult?
a. Hemoglobin F.
b. Hemoglobin A2.
c. Hemoglobin A.
d. Hemoglobin S.
36. Which is normally present in infants of less than 6 months but not normally present
in adults?
a. Hemoglobin A.
b. Hemoglobin A2.
c. Hemoglobin F.
d. Hemoglobin S.
37. When hemoglobin combines with oxygen, its iron must be in what state?
a. Ferrous.
b. Globulin.
c. = Anemic.
d. Active.
38. How many basic ways are there to measure the hemoglobin concentration?
a. 2.
b. 3.
c. 4.
d. 5.
MD0853 6-26
39. Which method is the most widely used to measure the hemoglobin concentration
of blood?
a. Gasometric.
b. Cyanmethemoglobin.
c. Chemical.
d. Specific gravity.
40. What does the spectrophotometer's 540 mm wavelength measure during the
hemoglobin reaction using the cyanmethemoglobin method?
a. Specific gravity.
b. Proportionalism.
c. Color intensity.
d. Concentration.
41. AI though the cyanmethemoglobin method is accurate, what is a disadvantage of

using it?
a. It is not the most direct method.
b. If the cyanide compounds are handled incorrectly, they can be hazardous.
c. Venous samples give erratic values.
d. Its hemoglobin pigments are not stable.
42. The normal concentration of hemoglobin in blood of the adult male is:
a. 10-15 g/dl.
b. 12-16 g/dl.
c. 14-17 g/dl.
d. 18-27 g/dl.
MD0853 6-27
43. Reduced hemoglobin S is insoluble in:
a. Urea.
b. Anticoagulated blood.
c. Cyanmethemoglobin standards.
d. Phosphate buffer solution.
44. What may happen to tile sodium dithionite reagent if it becomes damp?
a. Dissolve.
b. Enlarge.
c. Ignite.
d. Remain the same.
45. Which cells sickle more rapidly than AS cells?
a. SS cells.
b. SC cells.
c. SD cells.
d. a, b, and c.
e. a and c.
46. Erythrocytes of persons with sickle cell anemia or trait will assume a sickle shape
when:
a. The oxygen tension is lowered.
b. The oxygen tension is raised.
c. An electrophoretic pattern is run.
d. Highly oxygenated blood is observed.
MD0853 6-28
47. Sickle cell anemia is caused by:
a. Endocrine disorders.
b. Massive hemorrhage.
c. Chronic hemorrhage.
d. An inherited protein abnormality of hemoglobin.
48. Which reagent is added and mixed with the blood to determine if sickle cells are
present?
a. Sodium rnetabisulfite, 2 percent.
b. Diathionite.
c. Sodium metabisulfite, 9 percent.
d. Acetic acid. 4 percent.
49. In hemoglobin electrophoresis, how is hemoglobin A2 differentiated from

hemoglobin C?
a. Quantity present.
b. Color after staining.
c. Degree of mobility.
d. Symptoms of patient.
50. Dithionite is:
a. Totally stable.
b. Very stable.
c. Not stable.
d. Limited in stability.
MD0853 6-29
SOLUTIONS TO EXCERCISES, LESSON 6
1. b (para 6-1a)
2. e (para 6-1a)
3. a (para 6-lb)
4. c (para 6-lb)
5. d (para 6-lb)
6. d (para 6-lb)
7. b (para 6-2a)
8. d (para 6-2b(6))
9. b (para 6-2b(1))
10. d (para 6-2b(1))
11. a (para 6-2b(2))
12. c (para 6-2b(6))
13. c (para 6-2b(7))
14. d (para 6-2d(3))
15. b (para 6-3a)
16. d (para 6-3b)
17. d (para 6-2e)
18. d (para 6-3b)
19. c (para 6-3c)
20. a (para 6-3c)
21. b (para 6-3d)
22. c (para 6-3d)
MD0853 6-30
23. d (para 6-4a)
24. b (para 6-4b(1))
25. a (para 6-4b(5))
26. b (para 6-4c(2))
27. b (para 6-4b(3))
28. a (para 6-4c(4))
29. a (para 6-5a,b(4))
30. c (para 6-Sd(3))
31. d (~as 6-6,6-7a)
32. a (para 6-7a)
33. c (para 6-7e)
34. a (para 6-7c)
35. c (para 6-8)
36. c (para 6-8)
37. a (para 6-7a)
38. c (para 6-9)
39. b (para 6-9)
40. c (para 6-10a)
41. b (para 6-10b(2))
42. c (para 6-10c(3))
43. d (para 6-11a)
44. c (para 6 -11b (2) WARNING)
45. d (para 6-11e(2))
MD0853 6-31
46. a (para 6-12a)
47. d (paras 6-8,12d(1))
48. a (para 6-12b)
49. a (para 6-13b(1))
50. d (para 6-1lb(2))
End of Lesson 6
MD0853 6-32
APPENDIX
GLOSSARY OF TERMS
Agranulocyte: A leukocyte without definite cytoplasmic granules.
Agranulocytosis: Complete or nearly complete absence of the granular leukocytes

from the blood and bone marrow.
Aleukemic Leukemia: A fatal condition of the blood-forming tissues, characterized by

marked proliferation of immature cells in the bone marrow, without their presence, in
any great numbers, in the blood steam.
Anemia: A condition in which the blood is deficient in quantity or quality of erythrocytes.
Anisocytosis: Variation in size of the erythrocytes.
Anomaly: Abnormality.
Anoxemia: Lack of normal proportion of oxygen in the blood.
Antecubital Space: The area on the forearm frontal to the elbow.
Anticoagulant: A substance that prevents the coagulation of blood. Commonly used

ones are potassium oxalate, sodium oxalate, sodium citrate, EDTA and heparin.
Aplasia: Incomplete or defective blood development; cessation of blood cell formation.
Aplastic Anemia: Anemia characterized b incomplete or effective blood development.
Asynchronous: Uncoordinated development as in abnormal cell development.
Azurophilic Granule: Rounded, discrete, reddish-purple granule, smaller than the

granules of neutrophils; 1-10 are common in lymphocytes, and they are very numerous,
and smaller, in the cytoplasm of monocytes
Band Form: In the Schilling classification, a neutrophil with the nucleus unsegmented
and ribbonlike; also stab, staff, nonfilamented.
Basket Cell: A degenerated primitive cell which has ruptured and in which the cell
nucleus appears as a pale staining smear without prescribed form or shape.
Basopenia: An abnormal decrease in the number of basophils.
Basophil: A granular leukocyte, the granules of which have affinities for the basic dye
of Wright stain (methylene blue). The granules are large, irregular and blue-black in
color.
MD0853 A-1
Basophilia: An abnormal increase in the number of basophils.
Basophilic: Staining readily with basic dyes, for example, blue with Ramanovsky type
stains.
Binary Fission: Simple cell division.
Bleeding Time: The time required for a small standardized wound, made in the
capillary bed of the finger or ear lobe, to stop bleeding.
Blood Dyscrasia: A disease of the blood or blood-forming organs.
Buffy Coat: The layer of leukocytes that collects immediately above the erythrocytes in
sedimented or centrifuged whole blood.
Cabot's Rings: Lines in the form of loops or figures-of-eight seen in erythrocytes in

severe anemias.
Centriole: A minute cell organoid within the centrosome.
Centrosome: An area of condensed cytoplasm active in mitosis.
Chemotaxis: the phenomenon of movement of leukocytes caused by a chemical

influence.
Chromatin: The more stainable portion of the cell nucleus contains genetic materials.
Clot Retraction: The rate and degree of contraction of the blood clot.
Coagulation Time: The time required for venous blood, in the absence of all tissue
factors, to clot in glass tubes under controlled conditions.
Cocatalyst: A substance that works in tandem with another group of chemicals to

accelerate a reaction velocity without being used up in the reaction.
Color Index: The ratio between the amount of hemoglobin and the number of red
blood cells.
Complete Blood Count: A hematology study which consists of a red cell count, white
cell count, hematocrit, hemoglobin, and blood smear study including differential white
cell count.
Congenital: Born with a person; existing at or before birth.
MD0853 A-2
Cooley’s Anemia (Mediterranean Disease or Thalassemia): A chronic progressive
anemia commencing early in life and characterized by many normoblasts in the blood,
unusual facies, splenomegaly and familial and racial incidence. Target type red blood
cells are often present in the peripheral blood.
Crenation: The scalloped or notched appearance of the periphery of erythrocytes

found when the cells are suspended in a hypertonic solution. Also found in smears,
caused by dirty glassware, slow drying, and poor smearing technique.
Cytoplasm: Protoplasm of a cell excluding the nucleus.
DNA: Deoxyribonucletic acid.
Differential Count: An en1.meration of the types of white blood cells seen on a stained
blood smear.
Discrete: Separate.
Dyscrasia: Abnormality.
Ecchymosis: Subcutaneous extravastion of blood covering a large area.
Endothelial Leukocyte: Monocyte.
Eosinopenia: An abnormal decrease in eosinophils.
Eosinophil: A granular leukocyte, the granules of which have an affinity for the acid
dye of Wright’s stain (eosin). The granules are large, round, uniform in size, red-orange
in color and are shiny and refractile.
Eosinophilia: A relative or absolute leukocytosis in which the main increase is in

eosinophils.
Eosinophilic: Readily stained with eaosin, red-orange stain.
Epigastric: Pertaining to the upper middle portion of the abdomen.
Erythremia: A disease marked by persistent polycythemia and increased blood volume

also polychythemia vera.
Erythrocyte: Red blood cell.
Erythrocytosis: An increase in the total number of erythrocytes.
Erythrogenic: Producing erythrocytes.
MD0853 A-3
Erythroleukemia: An abnormal condition characterized by proliferation of
erythroblastic and myeloblastic cells.
Erythropenia: A decrease in the number of red cells in the blood.
Erythropoiesis: The production of erythrocytes.
Etiology: The theory of the causation of a disease.
Extravascular: Occurring outside the blood vessels.
Extrinsic: Originating outside of the particular area.
Fibril: A microscopic filament often composed of fibrin.
Fibrin: The end product of the clotting mechanism that forms a network of fibers that
enmesh the formed elements of blood.
Fibrinogen: The precursor of fibrin that is present normally in the plasma and
produced by the liver.
Fragility Test (Osmotic): A test devised to measure the resistance of the erythrocytes
to break down (hemolyze) when subjected to varying concentrations of hypotonic salt
solutions.
Fulminating: Sudden and severe.
Golgi Apparatus: A meshwork of lipid containing fibrils within the cytoplasmic portion
of a cell.
Granulocyte: A white blood cell that contains specific cytoplasmic granules

(neutrophils, eosinophils, and basophils); these granules are peroxidase positive.
Granulocytosis: The presence of increased numbers of granulocytes in the blood.
Granulocytopenia (Granulopenia): A decrease in the number of granulocytes in the

blood.
Granulopoiesis: The production of granulocytes.
Hemacytometer: A calibrated chamber in which blood cells are counted.
Hematin: A brown or blue-black amorphous iron substance that unites with globin and
forms hemoglobin.
Hematocrit: The packed cell volume (PVC) of red blood cells obtained by globin and
forms hemoglobin.
MD0853 A-4
Hematology: The branch of medicine that deals with the study of blood cells, blood
producing organs and the manner in which these cells and organs are affected in
disease.
Hematoma: Subcutaneous effusion of blood with resulting swelling, pain, and

discoloration, forming a tumorlike mass.
Hematopoietic (Hemopoietic): Blood forming.
Hemoglobin: The coloring matter of the red blood cells. A complex iron-bearing
pigment that carries oxygen and carbon dioxide.
Hemoglobinuria: The presence of free hemoglobin in the urine.
Hemogram: The blood picture.
Hemolysis: The dissolution or dissolving of the erythrocytes.
Hemolytic Anemia: That type of anemia characterized by excessive intra-vascular

destruction of red cells.
Hemophilia: A hereditary disease characterized by a prolonged coagulation time and

repeated hemorrhages, occurring only in males and transmitted only by females and
affected males. The cause is a deficiency in a plasma factor (antihemophilic globulin or
thromboplastinogen) resulting in a defect in thromboplastic activity.
Hemoptysis: The spitting of blood; coughing up blood.
Hemostasis: The checking of the flow of blood, especially from a vessel.
Hepatic: Originating from the liver.
Heterozygous: Derived from germ cells unlike in respect to one or more factors.
Hemeostasis: Stability in normal body states.
Homozygous: Derived from germ cells that are alike.
Howell-Jolly Bodies: Small basophilic particles sometimes found in erythrocytes,

remnants of nuclear material.
Hygroscopic: Readily taking up and retaining water.
Hyperplasia: An increase in cell formation.
Hypertonic: Greater than isotonic concentration.
MD0853 A-5
Hypertrophy: Enlargement of an organ or part due to increase in the size of the
constituent cells.
Hypochromia: A decrease in color of the erythrocytes, hence a decrease in their

hemoglobin content.
Hypoplasia: A decrease in cell formation.
Hypotonic: Less than isotonic concentration.
Idiopathic: Disease of unknown cause.
Inclusion: Usually lifeless, an accumulation of fats, proteins, crystals pigments or

secretory granules within a cell cytoplasm.
Inhibitor: A substance, directed against a coagulation factor or factors, which interferes

with the coagulation process.
Intravascular: Occurring within the blood vessels.
Intrinsic: Situated within the particular part.
In Vitro: Within a test tube (glass, etc.).
In Vivo: Within the living organism, as in life.
Isotonic: Solutions with the same osmotic pressure.
Jaundice: Yellow mass of the skin and eyes due to the presence of blood pigments in
the blood; follows excessive destruction of the blood, obstruction of the bile passage,
diffuse liver disease, certain infections, toxic chemical agents and drugs.
Juvenile Cell: In the Schilling classification, the cell between the myelocyte and band
forms; also metamyelocyte.
Karyolysis: Apparent destruction of the nucleus of a cell.
Karyorrhexis: Fragmentation of the nucleus; a degenerative process usually followed

by karyolysis.
L.E. Cell: A large segmented neutrophil or eosinophil that contains ingested autolyzed
nuclear fragments in its cytoplasm.
Leukemia: An ultimately fatal disease of the blood-forming organs characterized by

increased numbers of leukocytes and associated anemia.
MD0853 A-6
Leukemoid Crisis or Reaction: A temporary appearance of immature leukocytes in
the blood stream, with a marked increase in the total white count. In the laboratory
sometimes temporarily indistinguishable from leukemia.
Leukocyte: White blood cell.
Leukocytosis: An increase in leukocytes in the blood.
Leukopenia: A reduction in the number of leukocytes in the blood.
Leukopoiesis: Leukocyte formation.
Lymphoblast: The parent cell of the lymphocytic series.
Lymphocyte: A white blood cell having a round or oval nucleus and sky blue
cytoplasm. The nuclear chromatin is densely clumped but separated by many clear
areas giving a "hill and valley" effect. A few red-purple (azurophilic) granules may be
present in the cytoplasm.
Lymphocytosis: A relative or absolute increase in the number of circulating

lymphocytes.
Lymphopenia: An abnormal decrease in the number of lymphocytes.
Lysis: Destruction by a specific agent.
Macrocyte: An erythrocyte larger than normal.
Macrocytosis: An increase in the number of macrocytes.
Mast Cell: A basophil or a true tissue cell.
Maturation Factor: A substance that will cause cells to ripen and care to maturity.
Mean Corpuscular Hemoglobin (MCH): The average amount of hemoglobin in the

red blood cell.
Mean Corpuscular Hemoglobin Concentration (MHC): The average percent

hemoglobin saturation in the red blood cell.
Mean Corpuscular Volume (MCV): The volume of the average red blood cell.
Megakaryoblast: The parent cell of the megakaryocytic series.
MD0853 A-7
Megakaryocyte: An extremely large cell with an irregular lobed, ring or doughnut-
shaped nucleus that stains blue-purple. The cytoplasm is abundant, light blue and is
packed with fine azurophilic granules. This cell gives rise to thrombocytes.
Megaloblast: The type of red cell precursor found in pernicious anemia. This differs
from the normal erythrocyte precursor (normoblast) in that the megaloblast is larger and
the nuclear chromatin has a fine meshwork or scroll design.
M.E. Ratio: The ratio of myeloid to erythroid cells in the bone marrow.
Mesentery: The fold of peritoneum that attaches the intestine to the posterior
abdominal wall.
Metamyelocyte: Juvenile cell of Schilling.
Metarubricyte: An erythrocyte with a pyknotic, contracted nucleus. Also called

orthochromatophilic normoblast.
Methemoglobin: A spectroscopically detected compound of hemoglobin found in

nitrobenzol, and other poisonings. The blood is a chocolate brC1.oln color to the eye.
Microcyte: An erythrocyte smaller than normal.
Microcytosis: An increase in the number of microcytes
Micron: One-thousandth of a millimeter, the common unit of microscopic measure.
Mitochondria: Granular components of a cell cytoplasm active in oxidative processes.
Mitosis: A series of changes through which the nucleus passes in indirect cell division.
A tissue showing many cells in mitosis indicates rapid gro.&1th of that tissue.
Monoblast: The parent cell of the monocytic series.
Monocyte: A large white blood cell with a pale blue-gray cytoplasm containing fine
azurophilic granules. The nucleus is spongy and lobulated.
Monocytosis: A relative or absolute increase in the number of circulating monocytes.
Mucosa: Mucous membrane.
Myeloblast: The parent cell of the granulocytic or myelocytic series.
Myelocyte: The stage in development of the granulocytic series that is characterized

by the first appearance of specific granules (eosinophilic, neutrophilic or basophilic) and
a round nucleus.
MD0853 A-8
Myeloid Cells: The granular leukocytes and their stem cells.
Myelopoiesis: Formation of bone marrow and the blood cells that originate in the bone
marrow.
Myeloproliferative: Rapid production of bone marrow constituents.
Necrosis: The death of a circumscribed portion of tissue. Simple necrosis is

degeneration of the cytoplasm and nucleus without change in the gross appearance of
the tissue.
Neutropenia: A decrease in the number of neutrophils in the blood.
Neutrophil (Polymorphonuclear Neutrophil or Segmented Neutrophil): A

granulocyte having fine neutrophilic (pink-violet) granules in the cytoplasm. The nucleus
is divided into two or more lobes; each lobe is usually connected by a filament.
Neutrophilia: An increase in neutrophils.
Normoblast. The nucleated precursor of the normal red blood cell. Also called a
rubriblast.
Normocyte (Erythrocyte): A red blood cell of normal size.
NRBC: Nucleated red cell, usually a metarubricyte when seen in the peripheral blood
smear.
Nucleolus: An intranuclear pale blue body, surrounded: by a dense condensation of

chromatin.
Occult Blood: The presence of blood that cannot be detected except by special
chemical tests.
Oligochromemia: A decrease in hemoglobin.
Oligocythemia: A decrease in the number of erythrocytes.
Organoid: Structures present in cells resembling organs.
Ovalocyte: An elliptical erythrocyte.
Oxyhemoglobin: The bright red hemoglobin that is loosely combined with oxygen and
found in arterial blood.
Pancytopenia: A reduction in all three formed elements of the blood, namely, the
erythrocytes, leukocytes and thrombocytes.
MD0853 A-9
Pathologic Increase (Or Decrease): Due to abnormal function or disease, as
contrasted to physiological (due to normal body function).
Pernicious Anemia: A chronic, macrocytic anemia caused by a defect in production of

“intrinsic factor” by the stomach. There is accompanying megaloblastic erythropoiesis,
poikilocytosis, granulocytic hypersegmentation, achlorhydia, and neurological
disturbances.
Petichiae: Small spots on the skin formed by subcutaneous effusion of blood (also
purpura and ecchymoses).
Phagocytosis: The destruction of organisms and extraneous matter by a process of

envelopment and absorption.
Plasma: The fluid portion of the blood composed of serum and fibrinogen, obtained
when an anticoagulant is used.
Plasma Cell: A lymphocyte-like cell with an eccentrically placed deep-staining nucleus.

The nuclear chromatin is distributed in a "wheel-spoke" fashion. The cytoplasm is deep
blue with a lighter halo about the nucleus.
Platelet: Thrombocyte.
Poikilocyte: A red blood cell having abnormal shape (pear-shape, sickle-shape, etc.).
Poikilocytosis: Increased number of abnormally shaped erythrocytes.
Polychromasia: Diffuse basophilia of the erythrocytes.
Polychromatophilia: The presence in the stained blood smear of immature,

nonnucleated, bluish-staining red blood cells.
Polycythemia: An increase in the total number of erythrocytes. (See erythremia.).
Precursor: A substance from which another substance is formed.
Promyelocyte: The precursor of the myelocyte having nonspecific azurophilic (red-

purple) cytoplasmic granules.
Prorubricyte: The second stage of development of the red cell.
Prothrombin: The inactive precursor of thrombin that is formed in the liver and present
normally in the plasma. Its formation depends upon adequate vitamin K.
Punctate Basophilia: Small basophilic aggregates in the erythrocytes that stain blue
with the basic dye of Wright's stain; also basophilic stippling.
MD0853 A-10
Purpura: Small spots on the skin formed by subcutaneous effusion of blood.
Pyknosis: A condensation and reduction in size of the cell and its nucleus.
Reduced Hemoglobin: A combination of hemoglobin and carbon dioxide that is found

in venous blood.
Reticulocyte: A red blood cell showing a reticulum or network when stained with vital
dyes (for example, brilliant cresyl blue). The stage between the nucleated red cell and
the mature erythrocyte.
Reticulocytosis: An increase above normal values of reticulocytes in peripheral blood.
RNA: Ribonucleic acid.
Rouleaux Formation: The arrangement of red cells with their flat surfaces facing, in
which they appear as figures resembling stacks of coins.
Rubricyte: Polychromatophilic normoblast.
Sedimentation Rate, Erythrocyte (ESR): The rate at which red cells will settle out in
their own plasma in a given time under controlled conditions.
Serum: The fluid portion of the blood, after clot formation.
Shift to the Left: A term used to designate that condition in which the immature forms
of the neutrophils are increased above their normal number.
Shift to the Right: Increase in mature, pyknotic, and hypersegmented neutrophils.
Sickle Cell: A sickle- or crescent-shaped erythrocyte.
Sickle Cell Anemia: This is a hereditary and familial form of chronic, hemolytic anemia
essentially peculiar to Negroes. It is characterized clinically by symptoms of anemia,
joint pains, leg ulcers, acute attacks of abdominal pain and is distinguished
hematologically by the presence of distinct hemoglobin, peculiar sickle-shaped and oat-
shaped red corpuscles, and signs of excessive blood destruction and active blood
formation.
Smudge Cell: A ruptured white cell; also basket cell, or degenerated cell.
Spherocyte. A red blood cell that is more spherical, smaller, darker, and more fragile
than normal.
Stasis: A stoppage of blood flow.
MD0853 A-11
Supravital Stain: A stain of low toxicity that will not cause death to living cells or
tissues.
Synchronous: Occurring at the sane time and in a regular pattern.
Target Cell (Leptocyte): An abnormal, thin erythrocyte characteristic of Cooley's or

Mediterranean anemia.
Triturate: To grind together.
Thrombin: This is an enzyme formed from prothrombin that converts fibrinogen to

fibrin. This is not present in circulating blood.
Thrombocyte: A blood platelet.
Thrombocytopenia: A decrease in blood platelets; also thrombopenia.
Thrombocytosis: An increase in blood platelets.
Thromboplastin: The substance that initiates the process of blood clotting. It is

released from injured tissue and/or formed by the disintegration of platelets in
combination with several plasma factors.
Thrombopoiesis: The production of thrombocytes.
Thrombosis: Formation of a thrombus, or blood clot.
Vacuole: A space or cavity formed in the protoplasm of a cell.
Venipuncture: The act of puncturing a vein in order to remove a sample of blood.
Viscous Metamorphosis: Friction between molecules resulting in a structural change.
Vitamin K: A vitamin constituent of the normal diet requiring bile salts for absorption.
The liver in the production of prothrombin utilizes this vitamin.
Xanthochromia: A yellowish discoloration, usually associated with spinal fluid.
End of Appendix
MD0853 A-12

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U.S.

ARMY MEDICAL DEPARTMENT CENTER AND SCHOOL

SUBCOURSE MD0853 EDITION 100

This subcourse is approved for resident and correspondence course instruction. It

For comments or questions regarding enrollment, student records, or shipments,

CLARIFICATION OF TRAINING LITERATURE TERMINOLOGY

Section I. The Composition of Blood 1-1-1-4

2 MATERIAL EMPLOYED IN HEMOTOLOGY

Section I. Laboratory Reagents 2-1-2-3

3 COLLECTION OF BLOOD AND PREPARATION OF

Section I. Collection of Blood Specimens 3-1-3-4

4 MORPHOLOGY OF BLOOD CELLS

Section I. General Information 4-1-4-2

5 MANUAL CELL COUNTS

Section I. Manual Counts, Blood Specimens 5-1-5-4

6 HEMATOCRIT, ERYTHOCYTE SEDIMENTATION

Section I. Hematocrit 6-1-6-2

This subcourse consists of 6 lessons and an Appendix. The lessons are:

Lesson 2. Material Employed in Hematology.

Lesson 3. Collection of Blood and Preparation of Blood Smears.

Lesson 4. Morphology of Blood Cells.

Lesson 5. Manual Cell Counts.

Lesson 6. Hematocrit, Erythrocyte Sedimentation Rate, and Hemoglobin.

Appendix. Glossary of Terms.

To receive credit hours, you must be officially enrolled and complete an

A listing of correspondence courses and subcourses available through the

TEXT ASSIGNMENT Paragraphs 1-1 through 1-16.

1-1. Select the statement that best describes the

1-2. Select the statement that best describes the

1-3. Select the characteristic features of normal and

1-4. Correctly select the functions of blood cells.

SUGGESTION After completing the assignment, complete the

Section 1. THE COMPOSITION OF BLOOD

Blood is a complex and unique fluid of variable composition circulating through

1-2. GENERAL CELLULAR STRUCTURE

a. A typical cell is composed of a single nucleus embedded in cytoplasm. The

b. The nucleus is a spherical oval body surrounded by a thin membrane (nuclear

c. Surrounding the nucleus is a mass of protoplasm called cytoplasm.

1-3. CELLULAR CONSTITUENTS

a. Erythrocytes. An erythrocyte (red blood cell) is an elastic, non-nucleated,

Table 1-1. Blood cell tree.

b. Leukocytes. Leukocytes are commonly known as white blood cells because

c. Thrombocytes. Thrombocytes are found commonly known as platelets.

1-4. LIQUID CONSTITUENTS

Section II. FORMATION OF BLOOD CELLS

1-5. EMBRYONIC HEMATOPOIESIS

Figure 1-2. Embryonic hematophoiesis.

1-6. POSTNATAL HEMATOPOIESIS

a. Blood formation at birth is confined primarily to the bone marrow (central

Figure 1-4. Development of blood cells.

The basophilia of a blast cell is proportional to the ribonucleic acid (RNA)

Section IV. ABNORMAL CELL MATURATION

1-10. GENERAL FEATURES

Abnormal cell maturation is asynchronous development as opposed to normal

Asynchronism of the cytoplasm is most commonly seen in the granulocytes.

Section V. FUNCTIONS OF BLOOD CELLS

Each component of blood is uniquely capable of performing one or more

c. Eosinophils. The eosinophils are easily distinguishable by their large

d. Basophils. The function of basophils in man has not been ascertained.