o Race or ethnic group

OUR LADY OF FATIMA UNIVERSITY

COLLEGE OF MEDICAL LABORATORY SCIENCE
HEMATOLOGY
SUMMARY o
o
Family history of disease
Neurologic symptoms
 Anemia o Previous medication
 Blood Collection o Previous episodes of
 Hematopoiesis jaundice
 Hemoglobinopathies/Thalassemia
 Leukemia  Iron deficiency can lead to an
interesting symptom called pica.
Patients with pica have cravings
ANEMIA for unusual substances such as
ice (pagophagia), cornstarch, or
clay.
 Functional definition of anemia is  Certain features should be
a decrease in the oxygen-carrying evaluated closely during the
capacity of the blood. It can arise physical examination to provide
if there is insufficient hemoglobin clues to hematologic disorders
or the hemoglobin has impaired such as:
function. o Skin (for petechiae)
 Anemia is defined operationally o Eye (for pallor, jaundice,
as a reduction in the hemoglobin and hemorrhage)
content of the blood that can be o Mouth (for mucosal
caused by a decrease in RBCs, bleeding)
hemoglobin, and hematocrit  The examination should also
below the reference interval for search for:
healthy individuals of similar age, o Sternal tenderness
sex, and race, under similar
o Lymphadenopathy
environmental conditions.
o Cardiac murmurs
PATIENT HISTORY AND o Splenomegaly
CLINICAL FINDINGS o Hepatomegaly
 To elucidate the reason for a  Jaundice is important for the
patient’s anemia, one starts by assessment of anemia, because it
obtaining a good history that may be due to increased RBC
requires carefully questioning the destruction, which suggests a
patient, particularly with a regard hemolytic component to the
to: anemia.
o Diet
Hemoglobin
o Drug
concentration
o Ingestion
Moderate 7 to 10 g/dL
o Exposure to chemicals
anemias
o Occupation
Severe 7 g/dL
o Hobbies
anemias
o Travel
o Bleeding history
Moderate anemias may cause:

1
 o Pallor of conjunctivae and nail
beds but may not produce clinical
Ineffective and Insufficient
symptoms if the onset of anemia
Erythropoiesis
is slow.
 Erythropoiesis is the term used
o However, depending on the
for marrow erythroid proliferative
patient’s age and cardiovascular
activity. Normal erythropoiesis
state symptoms such as:
occurs in the bone marrow and is
 Dyspnea
under the control of the hormone
 Vertigo
erythropoietin (produced by the
 Headache
kidney) and other growth factors
 Muscle weakness
and cytokines.
 Lethargy
 Ineffective erythropoiesis refers
Severe anemia usually produces: to the production of erythroid
o Tachycardia precursor cells that are defective.
o Hypotension  Several conditions that involve
o Other symptoms of volume loss ineffective erythropoiesis as a
mechanism of anemia:
Severity of anemia is gauged by the  Megaloblastic anemia
degree of reduction in hemoglobin, (deficient DNA synthesis
cardiopulmonary adaptation, and the due to vitamin B12 or folate
rapidity of progression of the anemia. deficiency)
 Thalassemia (deficient
PHYSIOLOGIC ADAPTATIONS globin chain synthesis)
 Sideroblastic anemia
Anemia Caused by Sudden Loss of (deficient protoporphyrin
Blood Volume synthesis
 Insufficient erythropoiesis
The following adaptations occur in refers to the decrease in the
minutes to hours: number of erythroid precursors in
 Increase in heart rate, respiratory the bone marrow, resulting in
rate, and cardiac output decreased RBC production and
 Redistribution of blood flow from anemia. Many factors can lead to
the skin and viscera to heart, the decreased RBC production,
brain, and muscle including:
 Deficiency of iron
Anemia Caused by Slow Loss of
(inadequate intake,
Blood
malabsorption, excessive
The following adaptations occur over
loss from chronic bleeding;
days to weeks:
 a deficiency of
 Decrease in hemoglobin-oxygen erythropoietin (renal
affinity by increasing the disease); or
production of 2,3-  loss of the erythroid
biphosphoglycerate precursors due to an
 Increase in erythropoietin autoimmune reaction
production by kidneys (aplastic anemia, acquired
pure red cell aplasia); or
 infection (parvovirus B19)

MECHANISMS OF ANEMIA LABORATORY DIAGNOSIS OF

 The life span of the RBC in the ANEMIA
circulation is about 120 days.  To detect the presence of anemia,

2
 the medical laboratory residual ribonucleic acid (RNA) to
professional performs a complete complete the production of
blood count (CBC) using an hemoglobin.
automated hematology analyzer
to determine the; Test Formula Adult
 RBC count Referenc
 Hemoglobin concentration e Interval
 Hematocrit Absolute = [reticulocytes 20-112 x
 RBC indices reticulocyt (%)/100] x 109/L
 White blood cell count e count RBC count (x
 Platelet count 1012/L)
Corrected = reticulocytes --------
 The RBC indices include the: reticulocyt (%) x patient’s
 mean cell volume (MCV), a e count HCT (%)/45
measure of the average
Reticulocyt = corrected In anemic
RBC volume in femtoliters
e reticulocyte patients,
(fL). The most important
production count/maturati RPI
Test Adult index (RPI) on time should be
Reference >3
Interval
Mean cell volume 80-100 fL
(MCV) (fL)
APPROACH TO EVALUATING
Mean cell 26-32 pg
ANEMIAS
hemoglobin (MCH) Morphologic Classification of
(pg) Anemia Based on Mean Cell Volume
Mean cell 32-26 g/dL  MCV is extremely important tool
hemoglobin and is the key in the
concentration morphological classification of
(MCHC) (g/dL) anemia.
RBC indices. - Microcytic anemia is
 mean cell hemoglobin characterized by an MCV of
(MCH) less than 80 fL with small
 mean cell hemoglobin RBCs.
concentration (MCHC) - Microcytosis is often
Red cell distribution width (RDW), an associated with hypochromia,
index of variation of cell volume in a red characterized by an increased
blood cell population. central pallor in the RBCs and
an MCHC of less than 32 g/dL.
Reticulocyte Count
- Microcytic anemias are
 Reticulocyte count serves as an
caused by conditions that
important tool to asses bone
result in reduced hemoglobin
marrow’s ability to increase RBC
synthesis.
production in response to an
 Most common microcytic
anemia.
anemia is Iron deficiency.
 The adult reference interval is
0.5% to 2.5% expressed as a
percentage of the total number of - Macrocytic anemia is
RBCs. characterized by an MCV
 The newborn reference interval is greater than 100 fL with large
1.5% to 6.0%. RBCs.
 Reticulocytes are young RBCs - Macrocytic anemia arises
that lack a nucleus but still contain from conditions that results in

3
 megaloblastic or autoimmune hemolytic anemia,
nonmegaloblastic red cell cold agglutinin disease,
development in the bone paroxysmal cold hemoglobinuria,
marrow. hemolytic transfusion reaction,
- Normocytic anemia is hemolytic disease of the fetus and
characterized by an MCV in newborn
the range oof 80 to 100 fL.  Nonimmune red blood cell injury:
- Some normocytic anemia microangiopathic anemia
develop due to the premature (thrombotic thrombocytopenia
destruction and shorten purpura, hemolytic uremic
survival of RBCs, and they are syndrome, HELLP syndrome,
characterized by an elevated disseminated intravascular
reticulocyte count. coagulation), macroangiopathic
hemolytic anemia (traumatic
cardiac hemolysis), infectious
PATHOPHYSIOLOGIC agents (malaria, babesiosis,
CLASSIFICATION OF ANEMIAS bartonellosis, clostridial species),
other injury (chemicals, drugs,
Anemia caused by Decreased venoms, extensive burns)
Production of Red Blood Cells  Blood loss: acute blood loss
Hematopoietic stem cell failure: anemia
acquired and inherited aplastic anemia
 Disturbance of DNA synthesis: Evaluating anemia in the laboratory
megaloblastic anemia Basic information
 Disturbance of hemoglobin  Size of Red Blood Cells:
synthesis: iron deficiency anemia, (small/normal/big)
thalassemia, sideroblastic  Abnormal cells on microscopin
anemia, anemia of chronic examination
inflammation  Status of leukocytes and platelets
 Disturbance of proliferation and  Reticulocytes and platelets
differentiation of erythroid  Reticulocyte count (ability of
precursors: anemia of renal marrow to respond to anemia)
failure, anemia associated with  Evidence of destruction (elevated
marrow infiltration LDH, indirect bilirubin)

Anemia caused by Increased Red Practical approach to Anemia

Blood Cell Destruction or Loss Size of RBCs - MCV (Mean Cell
Volume)
Intrinsic abnormality o Microcytic <80 fl
 Membrane defect: hereditary o Normocytic 80-100 fl
spherocytosis, hereditary o Macrocytic >100 fl
elliptocytosis, pyropoikilocytosis,
paroxysmal nocturnal
hemoglobinuria
 Enzyme deficiency: glucose-6-
phosphate dehydrogenase Differential diagnosis of Microcytic
deficiency, pyruvate kinase Anemia
deficiency  Iron deficiency
 Globin abnormality: sickle cell  Anemia of chronic disease
anemia, other  Thalassemias
hemoglobinopathies o Hemoglobinopathies
Extrinsic abnormality o Hereditary spherocytosis
 Immune causes: warm-type o Hereditary X-linked sireroblastic

4
 anemia
o Lead poisoning (usually mild
microcytosis)
Iron deficiency anemia
• Common nutritional deficiency
• Bleeding is a leading cause of
iron deficiency anemia
• Iron facts
– Body iron:
• 80% functional (Hgb,
myoglobin, cytochromes,
etc.)
• 20% storage
– Absorption: primarily in the
duodenum
– Transferrin: transports iron in
blood
– Ferritin: storage form of iron
– Hemosiderin: derived from
ferritin, long- term storage of
iron
Lab studies in iron deficiency
anemia
o Microcytic, hypochromic anemia
o Decreased MCV, MCH, & MCHC
o Iron studies
– Low serum iron
– High total iron binding capacity
(TIBC, transferrin concentration)
– Low % transferrin saturation
– Low ferritin
– Decreased bone marrow
storage iron (hemosiderin)
Thalassemia
• Hemoglobin is a tetramer; with
two alpha and two beta
• Due to abnormally low production
of alpha or beta-globin chains:
named for the chain which is
decreased or absent
• + Indicates diminished, but some
production of globin chain still
happens: e.g. β+
• 0 Indicates complete absence of
production production of globin
chain by gene: e.g. β0
Demographics: Thalassemia
• Found most frequently in the
Mediterranean, Africa, Western
and Southeast Asia, India and
Burma
• Distribution parallels that of
Plasmodium falciparum
Distinguishing features between iron
def (IDA) and thalassemia
– Mentzer index: MCV/RBC < 13
favors thalassemia
– England and Fraser Index: MCV –
(5x Hemoglobin)
– The RBC count in thalassemia is
more than 5.0 x 106/μL (5.0 x
1012/L) and in IDA is less than
5.0 x 106/μL (5.0 x 1012/L)
– MCV usually less than 70 in TT,
more than 70 in IDA
– The red cell distribution width
(RDW) in IDA is more than 17%
and in TT is less than 17%.
ANEMIA OF CHRONIC DISEASE
• Mild to moderate anemia due to
increased hepcidin, leading to iron
sequestration
– Body unable to use iron stores
• Mildly microcytic or normocytic
anemia
• Etiologies: chronic immune
activation
– Chronic infections
– Collagen vascular disease
– Malignancy
Macrocytic Anemia
• Non-megaloblastic
– Liver disease
– Myelodysplastic syndrome
– Increased reticulocyte count
– Hemorrhage
• Megaloblastic
– Vitamin B12 deficiency
– Folic acid deficiency
Anemia Due to Folate or Vitamin B12
(Cobalamin) Deficiency
• Folate and cobalamin required for
DNA synthesis
• Deficiency results in megaloblastic
anemia due to impaired DNA replication
– Impaired nuclear development but
abundant cytoplasm (nuclear-
cytoplasmic asynchrony)
– Large marrow progenitors
5
• Similar clinical features* in peripheral • Leukemia
blood and marrow morphology in folate • Myeloma
and cobalamin deficiency • Myelofibrosis
* Exception: Neurologic abnormalities in • Metastases
B12 deficiency – Myelodysplastic Syndrome D

Folate and Cobalamin Deficiency: Evaluation of Normocytic Anemia

Clinical and Laboratory Findings • PB smear, reticulocyte count
• Non-specific signs and symptoms • Screen for liver, endocrine, renal
of anemia disease
• Macrocytic anemia • Iron studies
• Relatively low reticulocyte count • Bone marrow biopsy
• Hypersegmentation of neutrophils
• Mild thrombocytopenia and/or Hemolytic Anemia
neutropenia • Inherited hemolytic anemia
• Megaloblastic changes in marrow • Acquired hemolytic anemia
• Neurological findings (B12
deficiency only): loss of position Inherited hemolytic anemia
sense, ataxia, psychomotor o Membrane defects (eg. hereditary
retardation, seizures spherocytosis)
o Globin defects (eg. Sickle cell
HYPERSEGMENTED anemia)
NEUTROPHILS AND BONE o Metabolic disorders
MARROW OF MEGALOBLASTIC – Glucose-6 phosphate
deficiency
ANEMIA – Pyrimidine 5’-nucleotidase
deficiency (basophilic
stippling).

Normocytic Anemia
Differential Diagnosis of Normocytic
Anemia
o Increased reticulocytes
– Hemolytic anemia
– Post-hemorrhagic anemia
o Decreased reticulocytes
– Anemia of chronic disorders
– Endocrine disease
– Renal disease – Liver disease
– Hypoplastic anemia
– Marrow infiltration
Hereditary Spherocytosis
• Most common hereditary
hemolytic anemia
• Spherocytes--microcytic,
abnormal osmotic fragility
• May have broad RDW, but normal
to low MCV and usually increased
MCHC, depending on the degree
of reticulocytosis
• Autosomal dominant inheritance
(75%)
• Mutations in various structural
membrane proteins
• “Cured” by splenectomy
HEREDITARY SPHEROCYTOSIS
AND SICKLE CELL ANEMIA

6

Diagnosis of Hereditary
Spherocytosis
• Peripheral blood smear-
Spherocytes
• Osmotic fragility
– a laboratory test used in the
diagnosis of HS, is sensitive
but not specific. The test
measures the in vitro lysis of
RBCs suspended in solutions
of decreasing osmolarity.
Spherocytes are
characterized by membrane
loss and less redundancy to
withstand
Osmotic Fragility Test
Diagnosis Discontinued!!!
o Flow cytometry
– Greater than 95% sensitive and
specific for HS
– Labels patients RBCs with EMA
(eosin-5-maleimide)
– EMA binds specifically with band
3 protein
– EMA binding is affected by all
sorts of membrane protein
abnormalities, not just band 3
deficiency
Glucose-6-Phosphate Deficiency
(G6PD)
• Catalyzes the initial step in the
pentose phosphate pathway
• X-linked; more than 300 variants
identified • 130,000,000 probably
carry a mutant gene – Up to 20-
30% of Africans
– Up to 35% in Sardinia
– Also seen in Asians
Two types of hemolytic anemia
• Acute, acquired hemolytic anemia
– Associated with exposure to
primaquine, sulfa drugs
• Chronic mild hemolytic anemia
(common in Africans, Caucasians,
as compared to Mediterranean's)
G6DP HEINZ BODIES AND BITE
CELLS
Test for G6DP deficiency
• Fluorescent spot test
– Quick and cheap
– Detects generation of NADPH
from NADP+
– G6P and NADP added to a
drop of patient blood
– Blood spot fluoresces at 340
nm if NADPH is generated
– Only detects severe G6PD
deficiency (enzyme levels
below 30%)
• Enzyme activity assay
(spectrophotometric assay)
• *Caveats for non-PCR-based
tests:
– don’t perform right after a
hemolytic episode or blood
transfusion!
– May not detect heterozygous
females or mild deficiencies
• Mutation testing by PCR
– Useful in families with known
mutation
– Prenatal diagnosis
– Also useful in targeted
screening of populations with
high frequency of common
mutations, such as G6PD A- in
Africans & African-American
Hemolytic Anemia
• Acquired hemolytic anemia
– Immune mediated
7
 – Microangiopathic hemolytic – Reported in:
anemia • Prolonged marches
– Associated with infections • Competitive running
– Paroxysmal nocturnal • Conga drumming
hemoglobinuria • Karate
• Jack hammer operators
Immune-mediated Hemolytic Anemia
• Premature destruction of red Hemolytic Anemia Associated with
blood cells due to acquired Infection
antibodies directed against red o Clostridium sepsis: may be
cell antigens severe, overwhelming
• Direct Coombs test: detection of – Urgent identification and
immunoglobulin and/or treatment is necessary
complement molecules on the – Lecithinase
surface of red blood cells. o Malaria (Blackwater fever)
• Indirect Coombs test: incubates o Bartonella bacilliformis
normal red blood cells with patient o Babesia microcoti
serum, searching for unbound red o Trypanosomiasis
cell antibody in the patient serum. o Mycoplasma pneumonia (IgM
Microangiopathic Hemolytic Anemia against I antigen)
• Characterized by red blood cell o Infectious mononucleosis (IgM
fragments in the peripheral blood against i antigen)
(schistocytes)
Paroxysmal Nocturnal
• Differential Diagnosis:
Hemoglobinuria
– Thrombotic thrombocytopenic
 PNH characterized by pallor
purpura
(anemia), dark urine at night,
– HUS (renal, shiga toxin, E.
venous thrombi (especially large
coli O157)
vessels)
– Disseminated carcinoma
 Acquired clonal disorder, mutation
(Mucinous adenoca)
in the PIGA gene
– DIC – Malignant hypertension
 Defective synthesis of GPI-linked
– Giant hemangiomas – March
proteins • Detected by flow
hemoglobinuria
cytometry done on both RBC and
– Drugs (mitomycin-C)
WBC initially
 Treatment with eculizumab—
monoclonal Ab against C5
 Related to aplastic anemia and
MDS
Microangiopathic Hemolytic Anemia

BLOOD
COLLECTION
March hemoglobinuria
– Disorder of transient Blood
hemoglobinemia or The total blood
hemoglobinuria due to forceful volume in an
contact of body with hard adult is 5 to 6
surface liters, or 7 to 8%
of the body

8
weight. Approximately 45% of the
blood is composed of formed elements:
red blood cells, white blood cells,
and platelets. The red cells contain
hemoglobin, which binds oxygen; the
white blood cells defend the body
against foreign substances, such as
infections; and the platelets primarily
function in the stoppage of bleeding.
The remaining 55% of the blood is the
fluid portion, of which approximately
90% is water and 10% is composed
of proteins (albumin, globulin, and
fibrinogen), carbohydrates, vitamins,
hormones, enzymes, lipids, and salts.
 PH of Blood: 7.35-7.45 (ave.
7.40)
 Venous blood: 7.35 color: dark
purplish red
 Arterial blood: 7.45 color:
bright red
 Total Blood Volume (TBV) Adult
o Male : 5-6 L
o Adult Female : 4-5 L
o Newborn : 250-350 ml
visually using a microscope and
hemocytometer.
PLATELETS
Platelets, or thrombocytes, are true
blood cells that maintain blood vessel
integrity by initiating vessel wall repairs
Platelets rapidly adhere to the surfaces
of damaged blood vessels, form
aggregates with neighboring platelets to
plug the vessels, and secrete proteins
and small molecules that trigger
thrombosis, or clot formation. Platelets
are the major cells that control
hemostasis, a series of cellular and
plasma-based mechanisms that seal
wounds, repair vessel walls, and
maintain vascular patency (unimpeded
blood flow). Platelets are only 2 to 4
mm in diameter, round or oval,
anucleate and slightly granular.

RED BLOOD CELLS

RBCs are anucleate, biconcave,
discoid cells filled with a reddish
protein, hemoglobin (HGB), which
transports oxygen and carbon dioxide
RBCs appear pink to red and measure
6 to 8 mm in diameter with a zone of
pallor that occupies one third of their
center reflecting their biconcavity.

WHITE BLOOD CELLS

WBCs, or leukocytes, are a loosely
related category of cell types dedicated
to protecting their host from infection
and injury WBCs are transported in the
blood from their source, usually bone
marrow or lymphoid tissue, to their
tissue or body cavity destination. WBCs
are so named because they are nearly
colorless in an unstained cell
suspension. WBCs may be counted

9

Safety in the Hematology
Laboratory
Many conditions in the laboratory
have the potential for causing injury to
staff and damage to the building or to
the community. Patients’ specimens,
needles, chemicals, electrical
equipment, reagents, and glassware all
are potential causes of accidents or
injury. Managers and employees must
be knowledgeable about safe work
practices and incorporate these
practices into the operation of the
hematology laboratory. The key to
prevention of accidents and laboratory-
acquired infections is a well-defined
safety program.
STANDARD PRECAUTIONS
One of the greatest risks associated
with the hematology laboratory is the
exposure to blood and body fluids. In
December 1991, the Occupational
Safety and Health Administration
(OSHA) issued the final rule for the
Occupational Exposure to Bloodborne
Pathogens Standard. The rulethat
specifies standard precautions to
protect laboratory workers and other
health care professionals became
effective on March 6, 1992. Universal
precautions was the original term;
OSHA’s current terminology is standard
precautions. Throughout this text, the
term standard precautions is used to
remind the reader that all blood, body
fluids, and unfixed tissues are to be
handled as though they were potentially
infectious. Standard precautions must
be adopted by the laboratory. Standard
precautions apply to blood, semen,
vaginal secretions, cerebrospinal fluid,
synovial fluid, pleural fluid, any body
fluid with visible blood, any unidentified
body fluid, unfixed slides,
microhematocrit clay, and saliva from
dental procedures. Adopting standard
precautions lessens the risk of health
care worker exposures to blood and
body fluids, decreasing the risk of injury
and illness. Bloodborne pathogens are
pathogenic microorganisms that, when
present in human blood, can cause
disease. They include, but are not
limited to, hepatitis B virus (HBV),
hepatitis C virus (HCV), and human
immunodeficiency virus (HIV). This
chapter does not cover the complete
details of the standard; it discusses only
the sections that apply directly to the
hematology laboratory.
UNIVERSAL PRECAUTIONS may
be defined as a method for controlling
infection in which all blood and certain
body fluids are treated as if infected
with hepatitis B, human
immunodeficiency virus (HIV), or other
disease- producing blood-borne
pathogens. The reason for universal
precautions is that all patients infected
with blood-borne pathogens cannot be
readily identified. Therefore, certain
precaution techniques are used for all
patients.
Under universal precautions the
following policies are applicable to
laboratory personnel.
1. Skin and mucous membrane
exposure to blood and other body
fluids must be prevented.
2. Gloves must be worn when there
is any possibility of coming in
contact with blood or other body
fluids. When removing gloves
they must be disposed of in
biohazardous waste. Gloves must
never be reused.
3. Masks and protective eyewear
(goggles) or face shields are to be
worn if there is a possibility of
droplets or spattering of the blood
or body fluid. Use of plexi glass
shields in the work area is an
alternative.
4. Gowns or laboratory coats should
be worn when working with blood
or body fluids. These coverings
must be removed before leaving
the laboratory and may not be
taken home for washing.
5. Any skin surfaces that become
contaminated with blood or body
fluids should be washed
immediately. Hands must be
10
 washed upon removal of gloves.
6. It is of utmost importance that any
cuts or scratches be well
protected from contamination with
blood or body fluids.
7. During phlebotomy, used needles
should not be recapped, bent, or
broken by the technologist. The
unsheathed needle should be
placed directly into an
appropriately labeled puncture-
resistant bio- hazard container for
disposal.
8. All specimens for
centrifugation must be
centrifuged in a closed tube
(a top must be on every
tube).
9. Special care must be taken to
avoid leaking of specimen tubes
or containers.
10. All pipetting must be carried
out using mechanical pipet
devices.
11. All laboratory work benches
must be de- contaminated with
the appropriate germicide (10%
Clorox, for example) when work
has been completed (at least
once per shift).
12. Instrumentation must be
decontaminated prior to servicing.
If this is not possible, a warning
should be placed on the
equipment.
13. All materials coming in
contact with blood or body fluids
must be placed in biohazardous
waste containers when finished
with and disposed of according to
the institution's infective waste
disposal policy.
14. No precaution labels are
used on any patient specimens
because of the fact that all
specimens are considered
infectious and handled
accordingly.
HOUSEKEEPING
Blood and other potentially infectious
materials can contaminate work
surfaces easily. Contamination can be
caused by splashes, poor work
practices, and droplets of blood on the
work surface. To prevent
contamination, all work surfaces should
be cleaned when procedures are
completed and whenever the bench
area or floor becomes visibly
contaminated. An appropriate
disinfectant solution is household
bleach, used in a 1:10
volume/volume dilution (10%), which
can be made by adding 10 mL of
bleach to 90 mL of water or 1½ cups of
bleach to 1 gallon of water to achieve
the recommended concentration of
chlorine (5500 ppm).
LAUNDRY
If non-disposable laboratory coats are
used, they must be placed in
appropriate containers for transport to
the laundry at the facility or to a contract
service and not taken to the employee’s
home.
HEPATITIS B VIRUS
VACCINATION
Laboratory employees should receive
the HBV vaccination series at no cost
before or within 10 days after beginning
work in the laboratory.
TRAINING AND
DOCUMENTATION
Hematology staff should be properly
educated in epidemiology and
symptoms of bloodborne diseases,
modes of transmission of bloodborne
diseases, use of protective equipment,
work practices, ways to recognize tasks
and other activities that may result in an
exposure, and the location of the written
exposure plan for the laboratory.
Education should be documented and
should occur when new methods,
equipment, or procedures are
introduced; at the time of initial
assignment to the laboratory; and at
least annually thereafter.
11
Blood Collection Supplies and morphology of the red cell
Equipment
To make the phlebotomy procedure
easier for the technician, the following
suggestions should be implemented:
■ Prepare supplies and have
them readily available.
■ Review the minimally
acceptable volume of blood for an
individual assay or group of assays.
■ Determine the minimally
acceptable volume of blood for each
type of collection tube.
■ Develop a plan and an
alternative plan each time a phlebotomy
procedure is preformed.

ANTICOAGULANTS
1. EDTA/Versene/Sequestrene
• Most commonly used
• MOA: bind to non-ionized form
of Calcium then
chelates Calcium
3 forms:
 Dipotassium
EDTA
 Disodium EDTA
 Tripotassium EDTA

 Recommended amount: 1.2 mg/dl

of blood
 Used for: CBC, ESR, Platelet
count and PBS
 Can be used for modified
Westergren (ESR)
o 2 ml EDTA blood + 0.5 ml
of 3.8% of 0.129 M Na
Citrate
o 2 ml of EDTA blood + 0.5
ml NSS

DISADVANTAGES:
o Not recommended for
coagulation and platelet
satellitism
o Preparation of blood
smear from old EDTA
sample changes the

12
 transfusions
2. Sodium citrate o Causes agglutination or clumping
• Most common and of WBCs and platelets
preferred anticoagulant for o Not recommended for peripheral
coagulation studies blood smear preparation
• MOA: binds calcium
• Contained in light blue top
Evacuated tube
Anticoagulant buffer: 3.8% / 0.109 M
Na Citrate

3. Heparin
• Most commonly used for
Osmotic Fragility
Testing and
immunotyping
• MOA: binds
thrombin
2 forms:
 Lithium Heparin
 Sodium heparin
• Uses: RBC count, haemoglobin,
hematocrit, ESR and OFT
DISADVANTAGES:
o Not recommended for
coagulation studies because it
affects all stages of blood
coagulation
o Not for WBC
o Not recommended for blood
smear preparation
o Most expensive

4. Oxalate
• MOA: binds Calcium
• Recommended concentration:
1-2 mg/ml of blood
3 forms of oxalate:
 Double oxalate
 Lithium
Oxalate
 Sodium Oxalate
Double Oxalate
- salts of Ammonium and Potassium
- when used separately:
 Ammonium oxalate ( RBC Swells)
 Potassium oxalate ( RBC shrinks)
- Also known as “ Balance Oxalate”
-Uses: all RBC valuation tests, since
there is no effect on RBC
DISADVANTAGES:
o Not recommended for blood

13
 laboratory testing.
Patients with special  Requires both knowledge and skill
considerations to perform
 Blood sample collected: VENOUS
PEDIATRIC PATIENTS BLOOD
 Most widely used blood sample in
When working with children, it is
all laboratory tests
important to be gentle and treat them
with compassion, empathy, and 3 factors involved in a good
kindness. Attempt to interact with the venipuncture
pediatric patient, realizing that both the  Phlebotomist
patient and the parent (if present) may  Patient and his/her vein
have anxiety about the procedure and  The equipment needed
be unfamiliar with the new settings.
Acknowledge the parent and the child.
Be friendly, courteous, and responsive.
Allow enough time for the procedure.

ADOLESCENT PATIENTS
When obtaining a blood specimen from MOST COMMON SITES OF VEIN
an adolescent, it is important to be  Median cubital (Median vein)
relaxed and perceptive about any – the middle of the antecubital
anxiety that he or she may have. area.
General interaction techniques include – easiest to obtain blood; 1st
allowing enough time for the procedure, choice
establishing eye contact, and allowing – less painful and less prone to
the patient to maintain a sense of injury
control. – NOTE: it is located near a
tendon
GERIATRIC PATIENTS
It is extremely important to treat  Cephalic Vein
geriatric patients with dignity and – located on the outer-thumb
respect. Do not demean the patient. It is side of the antecubital area;
best to address the patient with a more 2nd choice
formal title such as Mrs., Ms., or Mr. – more prominent in obese
rather than by his or her fi rst name. patients.
Senior patients may enjoy a short – tends to be smallest but still
conversation. Keep a fl exible agenda accessible.
so that enough time is allowed for the
patient. Speak slowly because elderly  Basilic vein
patients are frequently hearing - located on the pinky side of
impaired. Allow enough time for the arm; 3rd choice
questions. The elderly have the right of - NOTE: It lies on top of, or
informed consent. Too many times this close to, an artery
fact is lost in dealing with any patient, -
but it seems more prevalent in dealing Selection of a Vein for Routine
with aging patients. Venipuncture
Two patterns of vein:
Venipuncture 1. “H” pattern
a. Median cubital vein –
 Manner of inserting a needle preferred vein
attached to a syringe to a b. Cephalic vein
palpable vein to collect blood for c. Basilic vein

14
 2. “M” pattern  Most widely used system for
a. Median vein – preferred collecting venous blood sample
vein  Consists of collection needle,
b. Accessory cephalic veins non-reusable needle holder and
c. Basilic vein a tube
 Method of blood collection using
a double sided needle whereby
the needle is attached to a
holder or adapter
 Comes in various (ml) size with
color-coded stoppers
 Intended for one-time use

METHOD OF COLLECTION 3. WINGED BLOOD

COLLECTION SET (Butterfly)
1. SYRINGE METHOD
 Often preferred over an
evacuated tube system
 Consists of a barrel, graduated
in milliliters, and a plunger
 Available with different types of
needle attachments and in
different sizes; may also be used
with winged infusion sets
 useful in drawing blood from
pediatric, geriatric, or other
patients with tiny, fragile, or
“rolling” veins
 NOTE: Attach the needle
securely to the syringe to
prevent air from entering

ORDER OF DRAW-SYRINGE
METHOD
1. Blood culture
2. Other sterile tubes
3. Light blue
4. Serum sterile tubes
5. Green
6. Lavender
7. Gray
8. Yellow
9. Red

2. ETS METHOD (EVACUATED

TUBE SYSTEM)

15
  Consists of a pressure cuff can aid in locating a
short needle vein (not higher than 40 mmHg
with plastic and no longer than 1 minute)
wings  Intravenous Lines - should be
connected to avoided if possible but if not ask
thin tubing an authorized caregiver to stop
 Used for infusion of IV fluids the infusion for 2 minutes before
 Useful in collecting specimens the specimen is drawn
from children or other patients  Dialysis Patients – blood should
from whom it is difficult to never be drawn from a vein in an
draw blood arm with a canula or fistula.
NOTE: Special precautions
REAGENTS AND EQUIPMENT needed to ensure that the patients
 Isopropyl alcohol, 70% (v/v), or do not bleed because they are
prepared 70% alcohol prep pads medicated with heparin
 Sterile gauze pads  Mastectomy Patients - CLSI
 Tourniquet requires physician consultation
 Gloves before blood is drawn from the
 Collection Tubes and Additives same side as a prior mastectomy
o Clot activators and bilateral mastectomies
o Anticoagulants - to prevent
blood clotting Sites should be avoided:
o Antiglycolytic Agent  Edema
o Separator Gel  Burned, Damaged, Scarred, and
 Sterile, disposable needle Occluded Veins
 Needle Holders
 Band-aid KEY NOTES TO REMEMBER ON
VENIPUNCTURE
SITES OF VENIPUNCTURE
 Angle of needle insertion
IN NEWBORN INFANTS UP TO 18 o Routine venipuncture – 45
MONTHS OLD degrees (15 when inserted)
 External jugular vein o WINGED BLOOD
 Temporal vein (scalp vein) COLLECTION – 15-30
 Superior longitudinal sinus degrees
o Two-way needle – 45
IN OLDER CHILDREN (18 MONTHS degrees (10-30 when
TO 3 years old) inserted)
 Femoral vein  Donor bleeding
 Long saphenous vein  Needle should be bevel up
 Popliteal vein  Length of needle - 1 to 1.5 inches
 Ankle vein Gauze vary from large (16-gauze)
3 YEARS OLD TO ADULT LIFE to much smaller (23-gauze)
 Wrist vein  Tourniquet – 1 min
 Dorsal vein of the hand  Blood pressure cuff – 40 - 60
 Dorsal vein of the ankle mmHg
 Number of attempts – maximum
of 2
 Position of the patient – DON’T
CHANGE THE POSISTION
SPECIAL SITE SELECTION
SITUATIONS
 Obesity - the use of a blood MNEMONICS!!! (order of draw)

16
 are appropriate sites for the
collection of small quantities of
blood
 earlobe may be used as a site of
last resort in adults
 Do not puncture the skin through
previous sites, which may be
infected
 The plantar surface (sole) of
the heel or big toe is an
appropriate site in infants or in
Microsample Technique special cases such as burn
victims.
Microsampling refers to blood collection
by skin puncture and is frequently used
on the following types of patients:
1. Infants less than 6 months of age
generally do not have a large
blood supply, and it is dangerous
to remove the volume of blood
involved in venipuncture.
2. In young children, if only a small
amount of blood is needed, a skin
puncture is performed on the
finger.
3. When an adult has poor veins,
when the veins cannot be used
because of intravenous (I.V.)
infusions, or in the case of a
severely burned patient, the finger
may be used as the phlebotomy
site.

REAGENTS AND EQUIPMENT

1. Isopropyl alcohol, 70% (v/v), or

prepared alcohol prep pads.
2. Sterile gauze pads.
3. Skin puncture device.
4. Appropriate capillary tubes,
Microtainers, Unopette and/or
pipets and diluting fluids.

SITES OF SKIN PUNCTURE

 fingertip (usually of the third or

fourth finger), heel, and big toe

17
 Note: The ideal site in infants is the must be inverted to mix the
medial or lateral plantar surface of additive and blood according to
the heel, with a puncture no manufacturer’s instructions.
deeper than 2.0 mm beneath the  Specimens should be transported
plantar heel-skin surface and no in an upright position to ensure
more than half this distance at the complete clot formation and
posterior curve of the heel. CLSI reduce agitation (which can also
recommendations are not to use result in hemolysis)
fingers of infants. The back of the  For certain tests, the specimens
heel should need to be chilled, not frozen, and
never be used should be placed in an ice-water
because of the bath to slow down cellular
danger of metabolism. (ei. ammonia, lactic
injuring the heel acid, parathyroid hormone, and
bone. The arch gastrin)
should never be  Other tests, such as the cold
punctured because tendons, agglutinin titer, require that
cartilage, and nerves may be injured specimens be kept warm.
in this area.  cells and serum must be
separated within 2 hours of
collection for tests such as those
measuring glucose, potassium,
and lactate dehydrogenase.

REASONS FOR SPECIMEN

REJECTION
 Test order requisition and the
tube identification do not match.
ORDER OR DRAW  Tube is unlabeled, or the labeling,
including patient identification
number, is incorrect.
 Specimen is hemolyzed.
 Specimen was collected at the
wrong time.
 Specimen was collected in the
wrong tube.
 Specimen was clotted, and the
test requires whole blood or
plasma.
 Specimen was contaminated with
intravenous fluid.
Specimen Handling  Specimen is lipemic.
 Blood collected into additive tubes

18
LEGAL ISSUES IN PHLEBOTOMY
There are many daily practices in health
care that, if performed without
reasonable care and skill, can result in
a lawsuit. Facilities have been and will
continue to be held legally accountable
for the actions of those who collect
blood for diagnostic testing. Two areas
of particular concern to phlebotomists:

1. breach of patient confidentiality

2. patient misidentification
To minimize the risk of legal action, the
phlebotomist should do the following:
1. Follow up on all incident reports.
2. Participate in continuing
education.
3. Become certified in the
profession.
4. Acknowledge the extent of liability
coverage.
5. 5. Follow established procedures.
6. Always exhibit professional,
courteous behavior.
7. Always obtain proper consent.
8. Respect and honor the Patients’
Bill of Rights.
9. Maintain proper documentation.

19
HEMATOPOIESIS
 Hematopoiesis is a termed
used to signify the production of
blood cells.
Three types of cellular elements
present in the blood:
 Red Blood cells (erythrocytes)
 White Blood cell (Leukocytes)
 Platelets (thrombocytes) – The
platelet is not considered a true
cell in that it does not contain a
nucleus.
In the fetus, hematopoiesis takes place
at various intervals in the liver, spleen,
thymus, bone marrow and lympnodes.
Within 2 weeks of embryonic life,
primitive red blood cells are produced in
the yolk sac. By the second month
granulocytes and megakaryocytes
begin to appear in the liver and spleen
become the primary sites of cell
development.
Lymphocyte production starts at
approximately the fourth month and
monocytes are produced by the fifth
month; at this time the bone marrow
has assumed its primary role, in
hematopoiesis.
At birth, and continuing into adulthood,
major blood cell production is confined
to the bone marrow and is known as
medullary hematopoiesis.
In the child, hematopoietic bone marrow
(red marrow) is located in the flat bones
of the skull, clavicle, sternum, ribs,
vertebrae, and pelvis and also in the
long bones of the arms and legs.
Eosinophil basophil neutrophil
 In certain disease states, the
bone marrow is unable to produce
sufficient numbers of
hematopoietic cells, and the liver
and spleen may then become the
sites of extramedullary
hematopoiesis.
 Hemolytic anemias – where there
is increased demand place on the
bone marrow.
In cases of, aplastic anemia and
leukemias blood cells are not produce
because of the fibrotic nature of the
bone marrow or infiltration with
malignant cells.
ORIGIN AND
INTERELATIONSHIP OF BLOOD
CELLS
- One of the more perplexing and
controversial problems in
hematology has concerned the
origin of the blood cells and their
relationship to one another.
 The hematopoietic or
multipotential stem cell though to
be the common precursor for red
cells, granulocytes, monocytes,
and megakaryocytes (platelets)
and gives rise to the committed or
unipotential stem cells through
stimulation by various
environmental factors within the
body.
CELL STRUCTURE
 The plasma or cellular membrane
surrounds the outer limits of the
cell and maintains the integrity of
the interior of the cell.
It is composed of three distinct
layer:
- A middle lipid layer located
between two layers of protein.
- These proteins—actin, myosin,
and tubulin—form microfilaments
and microtubules to maintain the
20
 shape (cytoskeleton) and
structure of the plasma
membrane.
- The “head” of the phospholipid
molecules ( adjacent to each
protein layer) is the water-soluble
portion and is positively charged.
- The inner ends of the lipids, or
“feet”, repel water (are water –
insoluble). Also present in the cell
membrane pores through which
substances may pass by osmosis,
diffusion, and actual transport.
The cytoplasm of the cell is containing
within the plasma membrane and is
composed of a variety of organelles.
 Mitochondria are rod-shaped
structures that supply a large
portion of the cell’s respiration
and energy requirements.
 Inner layer just out into the cavity
and forms projections called
cristae. The mitochondria do not
stain with wright’s stain.
 Golgi apparatus is located in the
cytoplasm of the cell near the
nucleus. It is an unstained area.
 Endoplasmic reticulum is
composed of Avast network of
tubules contained within a NUCLEAR MATURATION
membrane. One system of
tubules is termed rough surfaced
endoplasmic reticulum and
possess ribosomes adhering to
the membrane.
 Ribosomes contain ribonucleic
acid (RNA) and are active with
protein synthesis.
 Smooth surfaced endoplasmic
reticulum contains none of these
ribosome granules and is thought
to be the site of lipid synthesis.
There are small groups of RNA
molecules in the cytoplasm which
form polyribosomes.
 Nucleus of the cell consist
primarily deoxyribonucleic acid
(DNA) which controls cell function
and division. This chromosomal
material stains a dark purple color
with wright’s stain and called the
nuclear chromatin.
- Light or unstained areas within
the nucleus are termed the
pachromatin or euchromatin.
- The nucleolus stains a bluish
color with wright’s stain and is
composed of RNA.
NORMAL CELL MATURATION
- Progression from one stage to the
next is not abrupt, so frequently
the cell being studied maybe
between stages.
- As a cell transformed from the
primitive blast stage to the mature
form found in the blood, there are
changes in the cytoplasm,
nucleus, and cell size.
CYTOPLASMIC MATURITY
- The immature cytoplasm usually
stains a deep blue color
(basophilic) because of the high
content of RNA present.
- As the cell matures, therefore, a
lessening of the blue color.
- The nucleus of the immature cell
is round or oval and is large in
proportion to the rest of the cell.
- As the cell matures, the nucleus
decreases in relative size and
may or may not take on various
shapes (depending on the cell
type).
- The nuclear chromatin transforms
from a fine, delicate pattern to
become more coarse and
clumped in the mature form, and
the staining properties change
21
 from a reddish purple to a bluish Cytoplasm: Intensely basophilic.
purple. Nucleoli present in early Relatively amount of
stages of cell development cytoplasm increased over previous
usually dis-appear gradually as stage
the cell ages. (pronormoblast). Non-
granular.
CELL SIZE Nucleus: Relatively large. N/C ratio
- As a cell matures, it usually about 6:1. Round or slightly
becomes smaller in size. oval. Centrally
- The normal mature red blood cells located.
or small lymphocytes are Chromatin pattern is coarser than
generally of relatively constant in the previous stage.
size and may be used as a guide Nucleoli are usually not visible present.
for comparison
POLYCHROMATOPHILIC
RED BLOOD CELL NORMOBLAST (RUBRICYTE)
Produce in the bone marrow.

PRONORMOBLAST (RUBRIBLAST)

Size: 14-20um in diameter

Cytoplasm: Deeply basophilic.
Relatively small amount appearing as a
band around the nucleus.
Nucleus: Relatively large. NI/C ratio is
about 8:1 Round or slightly
oval
Reddish purple in color.
Fine chromatin
pattern. Usually 1
to 2 nucleoli.
Nucleoli are larger than those
found in the myeloblast
and may stain with a
slightly bluish tint.

BASOPHILIC

NORMOBLAST (PRORUBRICYTE)
Size: 12 to 17 um in diameter.

22
 granular.
Nucleus: N/C ratio is about 1:2.
Small, pyknotic nucleus( a
homogeneous blue-black
mass with no structure). This is a
Size: primary difference between
the rubricyte and the
10- metarubricyte. Nucleus may be
15 um in diameter. bizzare in form and partially
Cytoplasm: Blue-gray to pink-gray extruded from
(Shows a large range in the cell.
color) due to the start of
hemoglobin production in the cell. Reticulocyte.
A slight increase in Size: 7 to 10 in diameter.
relative amount. Non (Approximately the same size or
- granular. slightly larger than the mature red blood
Nucleus: Round. Smaller than cell).
previous stage. N/C ratio is Cytoplasm: Pink to a slightly pinkish
approximately 4:1 gray. Contain a fine
More condensed. The chromatin basophilic reticulum of RNA, which
pattern is coarse and only stains with supravital
clumped. stain( see Reticulocyte
Stains a deeper blue-purple. Count in Chapter #).
Parachromatin Nucleus: Non present.
is distinct.
Mature Red Blood Cell.
Size: 6 to 8 in diameter.
Cytoplasm: pink in color.
The mature red blood cell is a non-
nucleated, round-bicon-cave cell.

Erythropoiesis
ORTHOCHROMIC NORMOBLAST - Normally, the rate of production of
(METARUBRICYTE) red blood cells determines the
hemoglobin level ( and red blood
cell count) in the peripheral blood
and shows little variations among
normal individuals.
- The back red blood cell volume
(hematocrit) is fairly consistent at
40 to 45% and is optimal for the
delivery of oxygen to the tissue.
- Red blood cell exist and develop
in the bone marrow as
Size: 7 to 9 um in diameter erythroblastic islands consisting of
Cytoplasm: Pinker than previous stage a macrophage surrounded y
because of increased concentric rings of maturing
hemoglobin production. normoblasts.
Increased in amount compared to - The macrophage serves to supply
previous stage. Non- the developing red cells with iron

23
 for hemoglobin synthesis.
- They also phagocytize extruded
nuclei and dying red cells. During
maturation of the erythroid cell,
three to four mitotic divisions
occur between the pronormoblast
and the polychromatophilic
normoblast stages. Thus,
polychromatophilic normoblast
stage.Thus, up to sixteen
erythrocytes may be produced
from each pronormoblast.
- The red blood cells than in the
bone marrow
- The red blood cells of the
circulating blood have a life span
of approximately 120 days + 20
days.
- Production of red blood cells
(erythropoiesis) is initiated by a
hormone, called erythropoietin,
which is produced mainly by the
kidney and found in the plasma.
- Erythropoietin, I conjunction with
interleukin-3 produced by T cell
lymphocytes, is responsible for
the formation of erythrocyte
colony forming units (CFU-E) from
burst forming units (BFU-E).
HEMOGLOBIN STRUCTURE
AND SYNTHESIS
- The hemoglobin molecule is
composed of four sub-units, each
containing heme and the protein,
globin.
- Every heme group is capable of
carrying 1 mole of oxygen, and
therefore each hemoglobin
molecule is able to transport 4
moles of oxygen.
- The process hen continues in the
cytoplasm of the cell, where two
molecules of delta-aminolevulinic
acid dehydrase to form
porphobilinogen.
- Four molecules of
porphobilinogen combine to form
urophyrinogen III in the presence
of uroporphyrinogen I synthetase .
- Coproporphyrinogen III is then
formed by the action of
uroporphyrinogen decarboxylase,
which removes for carboxyl
groups from the acetic acid side
chains.
- Heme is then produce in the
mitochondria, where
protoporphyrinogen IX is formed
by action of coproporhyrinogen
oxidase.
- Protoporphyrin IX is formed in the
presence of protoporphyrinogen
oxidase, and ferrous iron is
incorporated into the molecule in
the presence of the enzyme
ferrochelates (heme synthetase)
to form the heme molecule.
FUNCTION OF HEMOGLOBIN
- The red blood cell functions primarily
to supply oxygen to the tissues and
remove carbon dioxide.
- It is the hemoglobin molecule within
the red blood cell that is responsible for
supplying the tissues with oxygen.
ERYTHROCYTE MEMBRANE
- The normal mature red blood cell
may be described as a biconcave
disk.
- The distinctive allows the red cell
to have maximum membrane
surface area for its size, which
facilities the transfer of gases in
and out of the cell.
- The red blood cell membrane is
composed of protein (50%), lipid
(40%), and a small amount of
carbohydrate (10%) and can be
penetrated by most solutes.
METABOLISM OF THE RED
BLOOD CELL
- The mature red blood cell consist
of primarily of hemoglobin (about
90% of the dry weight). The
membrane is composed of lipids
24
 and proteins.
- In addition there are numerous
enzymes present which are
necessary for oxygen transport
and cell viability.
- The red blood cell derives its
energy from the breakdown of
glucose about 90% of the
glycolysis in the red blood cell
follows the anaerobic Embden
Meyerhof pathway.
- The methemoglobin reductase
pathway maintains the iron
present in the hestate (Fe⁺⁺) for
oxygen transport.
- The Rapoport-Leubering pathway
allows for the production of
(2,3DPG) which regulates the
affinity of the hemoglobin
molecule for oxygen.
- The 2,3 DPG combines reversibly
with the deoxygenated
hemoglobin, decreasing the
affinity of hemoglobin for oxygen.

BREAKDOWN OF THE RED

BLOOD CELL
- As a red blood cell ages, there is
a decrease in its enzymes, a
decrease in ATP, a decrease in
size, and an increase in density.
- As a result, the red cell
membrane loses deformability
and undergoes membrane
damage.
- Approximately 1% of the red
blood cells leave the circulation
each day and are broken down by
the mononuclear phagocytic
system (MPS).

25
 Glycolytic pathways in the red
ERYTHROPOIESIS
MEGALOBLASTIC
- A nuclear maturation defect
occurs in vitamin B₁₂ and folic
acid definciencies. As a result, the
red blood cell and its precursors
are much larger in sizer than
normal.
- The term megaloblast is used,
“megalo” meaning large.
- The maturation of the megaloblast
proceeds through the same
stages of development as the
normal red blood cell.

blood cell

BREAKDOWN OF
HEMOGLOBIN

26
 PROMEGALOBLAST

Size: 19 to 28 um in diameter
Cytoplasm: More abundant than in the
pronormoblast.
Deeply basophilic. Non-
granular.
Nucleus: Fine chromatin pattern.
Chromatin pattern is more open than in
the pronormoblast. 3-to 8 nucleoli.
N/C ratio 5:1.

27
 BASOPHILIC MEGALOBLAST

Size: 10 to 15 um in diameter.
Size: 17 to 24 um in diameter. Cytoplasm: Pink in color. More
Cytoplasm: Deeply basophilc. abundant than is found in
Non-granular. the orthochromic normoblast.
Nucleus: Chromatin may be clumped
Nucleus: No nucleoli visible. but is much less condensed
Chromatin than the normal
pattern is coarser than in orthochromic normoblast.
the basophilic normoblast. N/C N/C ratio is about 1:1.
ratio is 4:1

POLYCHROMATOPHILIC

MEGALOBLAST
Size: 15 to 20 um in diameter.
Cytoplasm: Blue gray to pinkish gray.
May contain
Howell-Jolly bodies
(nuclear fragments).
Nucleus: Chromatin pattern is coarser
and more open than in the
polychromatophilic
normoblast.
There may be a
breaking up of the
nucleus (Kayorrhesis).
N/C ratio is 2:1.

ORTHOCROMIC
MEGALOBLAST

28
 MACROCYTE

Size: 9 to 12 um in diameter.
The macrocyte may appear oval
on the stained blood smear.

RED BLOOD CELL

MORPHOLOGY
- In anemias and other disease
states, the mature red blood cells
of the peripheral blood may show
certain significant changes. The
terms applied to each of these
abnormalities are defined and
illustrated on the following pages.

NORMAL RED BLOOD CELLS

(discocytes)

- Are a

central pallor, and show only a

slight variation in size. (as the
relative amount of hemoglobin in
the red cell decreases [or
increases], the area of central
pallor will increase [or decrease]
accordingly).

29
MICROCYTIC RED BLOOD CELLS

- Shows a decrease in size and are - Denotes red blood cells with a large
found in thalassemia and a variety area of central pallor and is due to a
of anemias such as iron decreased concentration of hemoglobin
deficiency and hemolytic anemia. in the cell. Hypochromia is
characteristically present in iron
MACROCYTIC RED BLOOD CELLS deficiency anemia but it is also present
in other forms of anemia.

POLYCHROMATOPHILIA

- Are increased in size and may be

found in liver disease and the
megaloblastic anemias. When Indicates young red blood cells which
associated with vitamin B₁₂ or contain residual RNA. These cells are
folic acid deficiency, the generally largr than normal and stain
macrocytes may appear slightly pinkish gray to pinkish blue color (plate
oval in shape. V). When stained supravitally with
brilliant cresyl blue they show up as
ANISOCYTOSIS reticulocytes.

SPHEROCYTIC RED BLOOD CELLS

- Indicates a variation in the size of

the red blood cells.

HYPOCHROMIA They are not biconcave like a

normal red blood cell and do not
have the central area of pallor

30
which a normal red cell shows.
The spherocyte, therefore, has
less surface area for its size.
These cells are associated with
hemolytic anemia, ABO hemolytic
disease of the newborn, and
hereditary spherocytosis. In some
heredetary red cell enzyme
deficiencies, the spherocytes may
have many fine needle like
projection on the surface of the
cell.

31
 SPHEROIDOCYTES
- Are thicker than normal red blood
cells (more spherical than the
normal red cell, but less spherical
than the spherocyte), have a
higher than normal concentration
of hemoglobin and show a small
area of pallor than is usually of
center.
TARGET CELLS (LEPTOCYTES)
- Show a centrally stained area with
a thin outer rim of hemoglobin and
are associated with liver disease
and certain hemoglobinopathies
(abnormal hemoglobins ): sickle
cell anemia and hemoglobin CC,
E and SC diseases.
STOMATOCYTES
- Shows a slit like (rectangular)
area of central pallor. These red
blood cells have lost the
identation on one side and may
be found in liver disease,
alcoholism, electrolyte imbalance,
and hereditary stomatocytosis.
POIKILOCYTOSIS
- Indicates a variation in the shape
of the red blood cells.
OVALOCYTES
Are oval-shaped red blood cells.
Elliptocytes, more oval than ovalocytes,
are cigar-shaped. Both of these cells
are found in hereditary elliptocytosis in
large numbers. They are also present in
various anemias but at a much lower
concentration, no more than 6 to 10%
of the mature red blood cell population.
(these cells show normal shape in the
nucleated and reticulocyte stages).
TEARDROP SHAPED RED BLOOD
CELLS
32
Are found most notably in myelofibrosis, anemias caused by physical agents, as
pernicious anemia, yeloid metaplasia, in disseminated intravascular
thalassemia, and some hemolytic coagulation (DIC).
anemias.

ACANTHOCYTES
CRENATED RED BLOOD CELLS
(Echinocytes)

Are red blood cells with irregularly

- Have blunt spicules evently
distributed over the surface of the
red blood cell and are usually
artefactual due to faulty drying of
the blood smear, or, may be a
result of hyperosmorality.
BURR CELLS
spaced projections. These spicules vary
in width but usually contain a bulbous,
rounded end. These cells have a
decreased survival time and are found
in abetalipoproteinemia and certain liver
and lipid metabolism disorders.
SICKLE CELLS (drepanocytes)

- Are red blood cell with uniformly

spaced, pointed projections on
their outer edges. These cells
occur in uremia, acute blood loss,
cancer of the stomach, and
pyruvate kinase deficiency.

SCHISTOCYTES
- are red blood cell fragments and may
occur in microangiopathic hemolytic

33
 cell. They generally appear singly
in hemolytic anemia, following
splenectomy, and in cases of
splenic atrophy as in sickle cell
anemia. Multiple Howell- Jolly
bodies in ared blood cell occur in
megaloblastic anemia and in
other forms of nuclear maturation
- - Are red blood cells in the defects.
shape of a sickle or crescent due
to the formation of rod-like HEMOGLOBIN C CRYSTALS
polymers of hemoglobin S within
the cell. To be considered sickle
cells, they must come to a point at
one end. These cells are
associated with hemoglobin S and
are found in sickle cell anemia,
hemoglobin SC disease, and
hemoglobin SB thalassemia.

BASOPHILIC STIPPLING - And are tetragonal in shape and

Is present as many coarse or fine, may be found in patients with
purple-staining granules in the red homozygous hemoglobin C
blood cell. The granules result from disease and characteristically in
aggregation of ribosomes and are found patients with hemoglobin SC
in lead poisoning, anemias with disease.
impaired hemoglobin synthesis,
refractory anemias, alcoholism, and
megaloblastic anemias.

SIDEROCYTES

-
- Are
deposits of iron in the red blood
cell. They are generally seen near
the periphery of the cell and may
appear as a single granule or as
multiple granules. When present
on a Wright-stained smear, the
granules appear less vividly
stained than Howell Jolly bodies
and are termed Pappenheimer
bodies.

HOWELL-JOLLY BODIES
- Are round, purple staining nuclear
fragments (DNA) in the red blood

34
 CABOT RINGS blood once the drop has been
placed on the slide) or may be
due to the presence of high
- a r e p concentrations
u r of abnormal
p l
globulins or fibrinogen. This
formation of red cells is found in
multiple myeloma and
macroglobulinemia.

filaments in the shape of a ring in

the red blood cell. They are
thought to be microtubules from a
mitotic spindle and are seen
rarely in pernicious anemia and
lead poisoning. They probably
indicate abnormal erythropoiesis.

PLATELETS ON TOP OF RED

BLOOD CELLS

- Should not be confused with a red

blood cell inclusion body.
Compare the platelet with those in
the surrounding field. Also there is
generally a non-staining halo
surrounding the platelet when it is
positioned on top on the red blood
cell.

ROULEAUX FORMATION

- and represents erythrocytes

arranged in rolls or stacks. This
may be due to an artifact (as a
result of delay in smearing the

35
 AGGLUTINATION

of the red blood cell, is found in patients

who have a cold agglutinin (antibody),
or autoimmune hemolytic anemia. Note
the clumping of the red blood cells
rather than the stacking as found in
rouleaux formation. When agglutination
of the red blood cells is seen on a blood
smear, routine automated methods of
red blood cell counting and sizing
should not be utilized.

CRESCENT BODIES
- are faintly staining bodies in the
shape of a quarter moon. They
are probably ruptured red blood
cells. When examining a blood
smear, any of the preceding red
blood cell morphology or
inclusions should be noted.

WHITE BLOOD CELL AND

PLATELETS
Granulocytes
- There are three types of mature
granulocytes: the neutrophil,
eosinophil, eosinophil, and
basophil. These three cell lines
are distinguishable from each
other by the presence of specific
granules that appear in the
myelocyte stage.

36
 Size: 15-21 um in diameter. This
cell is normally slightly larger than
the myeloblast.
Cytoplasm: Pale blue to
basophilic.
Contains a few to many,
large blue to reddish
purple staining
nonspecific (primary)
granules.
Nucleus: Occupies half or more
of the N/C ratio of 3:1 to
2:1.
MYELOBLAST Oval or round.

MYELOCYTE This is the last

stage capable of cell devision.

Size: 15 to 20 um in diameter
Cytoplasm: Small amount in
relation to the rest
of the cell. Usually a
Size: 12 to 8 um in diameter.
moderate blue in color.
Cytoplasm: Moderate amount.
Texture is smooth
May
and usually
contain a few patches of
nongranular.
blue.
Nucleus: Round or slightly oval.
Few moderate
Occupies
number of
about four-fifths or the cell.
non-specific granules.
N/C ratio of 4:1.
Small, specific
Extremely fine chromatin
(secondary)
pattern.
granules begin to appear in
Reddish
this stage.
purple in color.
Contains two five nucleoli.

Neutrophil myelocyte: the pink

specific granules may be seen as
PROMYELOCYTE
pinkish or lighter staining areas in the
cytoplasm, usually appearing near the
nucleus first.

Eosinophil myelocyte: the specific

granules first appear as dirty orange to
blue. These granules are larger than
the specific and nonspecific granules of
the neutrophil.

37
Basophil myelocyte: the specific the metamyelocyte
granules are few, large, and stain a stage.
dark blue-purple. Chromatin pattern is
coarse and clumped.
METAMYELOCYTE SEGMENTED

NEUTROPHIL
Size: 10 to 15 um in diameter. Size: 9 to 15 um in diameter.
Cytoplasm: Moderate to abundant Cytoplasm: Full complement of
amount giving a pink to rose violet
decreased N/C ratio. specific granules.
A few non-specific Abundant amount.
granules. Full Few non-specific
complement of specific granules are
granules. present.
Neutrophil metamyelocyte: the Nucleus: Normally two to five
granules are pinker and more lobes connected
numerous. by thin nuclear
Eosinophil metamyelocyte: the filaments.
granules are a brighter orange-red and Coarse, clumped
more numerous. chromatin pattern.
Basophil metamyelocyte: the dark
purple to black granules are more
numerous.
Nucleus: Indented or kidney-shaped.
Chromatin pattern is coarse
and clumped and stains dark
purple.

BAND

EOSINOPHIL
Size: 9 to 15 um in diameter.
Cytoplasm: Contains the full
complement of large
reddish-orange granules
Nucleus: Usually has two lobes.
Coarse, clumped
chromatin pattern.
Size: 9 to 15 um in diameter.
Cytoplasm: Same as the
metamyelocyte.
Nucleus: Elongated or band-
shaped.
Deeply indented from

38
 BASOPHIL division and maturation occur and
will generally undergo a total of
three to five cell divisions over a
period of 6 to 7 days.
- During the promyelocyte stage
the cell produces primary
(azurophilic) granules. The
number of granules per cell will
decrease with each cell division.
At the myelocyte stage the cell
Size: 10 to 16 um in diameter.
produces secondary (specific)
Cytoplasm: Stains slightly pink to
granules. (Synthesis of primary
colorless.
granules stops when the cell
Contains specific dark
begins secondary granule
purple to blue-black
production.)
granules.
There are fewer
granules present
than found in the
eosinophil.
The granules are
water-soluble and
tend to wash out when
stained (probably because
of improper
fixation).
Nucleus: Does not appear as
coarse in the neutrophil
or eosinophil.
Generally
unsegmented or
bilobed, rarely has three or
four lobes.

NEUTROHIL GRANULOCYTES
PHYSIOLOGY AND FUNCTION
- Neutrophil production and
maturation occur in the bone
OF THE NEUTROPHIL
marrow. The mature neutrophil
moves into the lungs, spleen, and
liver from the pheripheral blood
and then into the tissues where it
carries out its major functions of
ingesting and killing invading
microorganisms.
NEUTROPHIL KINETICS
- As the myeloblast develops into
the mature segmented neutrophil,
the myeloblast, promyelocytes,
and myelocyte undergo cell
devision. These cells constitute
the mitotic pool (or proliferative
compartment) where both cell
- Neutrophils are metabolically
active. They are capable of both
aerobic and anaerobic glycolysis
for their source of energy. Their
major function is to stop or retard
the action of foreign material or
infectious agents by means of:
- (1) moving into the area of
inflammation or infection, (2)
phagocytosis of the foreign
material, and (3) killing and
digestion of the offending
material.
- The neutrophil is capable of both
random locomotion (chemotaxis)
and directed locomotion

39
 (chemotaxis). This locomotion is
possible on if the neutrophil is
attached to a surface. The
neutrophil is normally spherical in
shape but becomes bipolar or
ameboid when in contact with a
surface.
- The tail end, or uropod, is blunt
and adhesive; the front end, or
protopod, extends pseudopod,
which results in the creeping
movement of the neutrophil along
surface.
NEUTROPHILIA
(1) Extreme exercise and also the
administration of certain drugs
cause a decrease in the
proportion of neutrophils in the
marginal pool. These cells
become part of the circulating
pool and are reflected by an
increased white blood cell count
and an increased percentage of
neutrophils in the differential
count.
(2) In the presence of infection, an
increased number neutrophils are
present in the marginal pool.
These cells then enter the tissues
at faster rate. The influx of
neutrophils from the storage pool
increases until the rate of ouflow
to the tissues is exceeded by rate
of inflow from the storage pool in
the bone marrow.
(3) In Chronic infection, the high rate
of influx of neutrophils into the
peripheral blood and the
corresponding increased outflow
may remain unchanged and a
steady state of neutrophilia is
maintained.
NEUTROPENIA
- When the absolute neutrophil
count is less than 1000/ul, the
patient is prone to recurent
infections. However, when the
count drops to less than 500/ul
the patient is seriously open to
infectiously open to infection from
bacteria or fungi.
(1)Certain drugs cause an increased
number of neutrophils in the
circulating pool.
(2)In a severe infection, the outflow
of cells to the tissues may exceed
the output from the bone marrow
storage pool.
(3)Decreased production in the bone
marrow from congenital causes,
cytotoxic drugs, or aplastic
anemia gives rise to decreased
numbers of neutrophils available
to the blood.
(4)An increased loss of white blood
cells (as might occur with
splenomegaly), whereby the
spleen sequesters (removes from
the blood and holds) and destroys
the cells, may also lead to
neutropenia.
THE EOSINOPHIL
- The majority of eosinophilis are
produced in the bone marrow.
They most likely originate from
their own commited stem cell as
the neutrophils. The maturation
process of the eosinophils.
- The maturation process of the
eosinophil, however, closely
parallels that of the neutrophil,
however, closely parallel that of
the neutrophil.
- They are slightly larger than
neutrophils and the nuclei of the
cells average fewer lobes than
found in the mature neutrophil.
The eosinophil will average 2.1
lobes in the normal person.
BASOPHIL AND MAST CELLS
- Are appear to function similarly in
inflammatory processes. They
appear to participate in immediate
hypersensitivity immune reactions
and are also involved in some
delayed hypersensitivity
reactions.
MONOCYTE
The monocyte is produce in the
bone marrow.
40
 MONOBLAST Size: 14 to 20 um in diameter.
Cytoplasm: Abundant.
Blue-gray.
Outline may be
irregular because of
the presence of
pseudopods.
Many fine azurophilic granules,
giving a ground glass
Size: 1 to 20 um in diameter. appearance.
Cytoplasm: Moderately basophilic Vacuoles may sometimes be
to blue gray. present.
Nongranular.
Nucleus: Ovoid or round in Nucleus: Round, kidney
shape. shaped, or may show
Light blue-purple in slight lobulation. It may be
color. folded over on top of itself,
Fine, lacey chromatin. thus showing brainlike
One to tho nucleoli. convolutions.
N/C ratio is 4:1 to 3:1 No nucleoli are visible.
Chromatin is fine and
PROMONOCYTE lacey (not clumped),
arranged in skein-like
strands.

PHYSIOLOGY AND BIOLOGY

OF THE MONOCYTE
- The monocyte arises from the
same bipotential stem cell as the
(immature monocyte)
neutrophil, the CFU-G, M (colony
Size: 14 to 18 um in diameter.
forming unit granulocyte,
Cytoplasm: Blue-gray.
monocyte). From this stem cell
May contain fine
arises the CFU-M (monocyte), the
dustlike azurophilic
stem cell committed to forming
granules.
monocytes.
Ground glass
- The precursor to the monocyte is
appearance.
the monoblast and is
Moderate amount.
morphologically indistinguishable
Nucleus: Oval, may have a
from the myeloblast.
single fold or
Cytochemical stains may be used
fissure.
to differentiate the two cell lines.
One to five nucleoli.
Five chromatin
pattern. LIFE SPAN OF THE MONOCYTE
N/C ratio is 3:1 to 2:1. - The bone marrow contains the
developing monocyte precursors
MONOCYTE and supplies monocytes to the
peripheral blood.
- The proliferation of monocyte to
the bone marrow takes about 55
hours.
- They undergo no maturation in
the bone marrow and leave there
randomly, in no specific order.

41
 The marginal pool of monocytes
in the peripheral blood is about
3.5 times the size of the
circulating pool. The mature
monocyte spends about 12 hours
in the peripheral blood before
going to the tissues.
DEVELOPMENT OF THE
MONOCYTE INTO THE
MACROPHAGES
- The monocytes moves via
diapedesis through the blood
vessel walls into the various
tissues and transforms into the
macrophage, at the same time
becoming actively phagocytic. It is
also thought that some
macrophages are produced by
cell division of existing
macrophages.
- As the macrophages develops
from the monocyte, the cell
increases in size as a result of
increased cytoplasmic content.
One or more nucleoli develop and
there is an increase in the
hydrolytic enzymes.
MACROPHAGE
- The macrophage is a large cell,
ranging size from 15 to 80 um in
diameter. It has an eccentric
nucleus that may be efgg-shaped,
indented, or elongated. The
chromatin appears spongy, and
there are generally one to two
nucleoli.
- The cell has abundant sky blue
cytoplasm which contains many
coarse azure granules.
PROPERTIES AND FUNCTIONS
OF THE MONOCYTE AND
MACROPHAGE
- As the monocyte transform into
an activated macrophage, the cell
surface becomes sticky and
develops numerous fan-like folds
for optimal phagocytosis.
- Both the monocyte and
macrophage show active
chemotaxis and necrotaxis
attraction to dead or dying cells);
however compared to neutrophils,
monocytes are much slower to
appear wever compared to
neutrophils, monocytes are much
slower to appear at inflammatory
sites.
- Pinocytosis and micropinocytosis
increase as the cell matures into
the macrophage. Phagocytosis of
antigens by the monocyte and
macrophage requires that certain
antigens be coated with an
antibody (opsonization).
- The primary functions of the
monocyte and macrophage are:
Defense mechanism, Removal of
damaged and old cells, plasma
proteins, and plasma lipids,
Participation in iron metabolism,
Processes antigen information for
lymphocytes, Production and
secretion of various subtances.
LYMPHOCYTE
- Lymphocytes are produced by the
lymph nodes, spleen, thymus, and
bone marrow.
LYMPHOBLAST (nonleukemic
lymphoblast)
Size: 10 to 18 um in diameter.
Cytoplasm: No granules present.
Appears
smooth.
Moderate to dark blue. May
stain deep blue at the
periphery and a lighter
blue near the
nucleus.
Nucleus: Chromatin pattern is
somewhat coarse.
Round or oval in shape.
Generally
contains one to two
distinct nucleoli.
42
 N/C ratio is 4:1 small lymphocyte.
Pale
PROLYMPHOCYTE to moderate blue.
Size: May be the same size as May or may not contain a
the lymphoblast or few nonspecific
smaller. azurophilic granules.
Cytoplasm: Moderate to dark blue. Nucleus: Round or oval in shape and
Usually may be slightly indented.
non-granular, but may Chromatin
contain occasional azurophilic pattern is clumped but not as
granules. dense lookin as in the small
More lymphocyte. No nucleoli are
abundant than in the visible.
lymphoblast.
Nucleus: Round oval, or slightly LARGE LYMPHOCYTE
indented. Chromatin Size: 12 to 16 um in diameter.
pattern is more clumped Cytoplasm: Abundant.
than in the lymphoblast. Clear, very pale blue.
May contain one to May or
two nucleoli. may not contain a few
nonspecific azurophilic granules.
LYMPHOCYTE Nucleus: Round or oval in shape and
- The lymphocytes found in the may be slightly indented.
peripheral blood occur in Chromatin
varying sizes. For purposes of pattern is coarse. No
description, they are divided nucleoli are visible.
into three categories: small, May be eccentrically
medium, and large, with a size located.
variation of 8 to 16 um in
diameter. In addition to
differing in size, the relative LIFE SPAN, CIRCULATION AND
amount of cytoplasm varies. RECIRCULATION OF
Generally, the larger the LYMPHOCYTES
lymphocyte, the more abundant - The majority of lymphocytes are
the cytoplasm. long-lived, with a life span of
SMALL LYMPHOCYTE about 4 years. Some lymphocytes
Size: 8 to 10 um in diameter. however, may live as long as 10
(approximately the size of years. The remaining
anormal red blood cell). lymphocytes, about 15%, are
Cytoplasm: Usually forms a thin rim short-lived, lasting 3 to 4 days.
round the nucleus. T AND B LYMPHOCYTE CELL
Moderate MARKERS
to dark blue.
- T and B lymphocytes may be
Nucleus: Chromatin pattern is dense
identified and differentiated from
and clumped Round or oval in
each other by the identification of
shape and may be
characteristics antigens and
slightly indented.
receptors on their membranes.
No nucleoli are visible.
- The cell membranes of the B
MEDIUM LYMPHOCYTE lymphocytes contain
Size: 10 to 12 um in diameter. immunoglobulin which may be
Cytoplasm: More abundant than in the indentified by immunofluorescent
techniques.

43
 Eccentrically
MORPHOLOGY OF LYMPHOCYTES placed.
IN DISEASE Chromatin is coarser and densely
Downey type I: The nucleus may be stained.
irregularly shaped. The
cytoplasm is relatively basophilic Occasional nucleoli may be seen.
and may at times be foamy.
Downey type II: There is an increased PLASMACYTE (PLASMA CELL)
amount of cytoplasm that Size: 8 to 20 um in diameter
also contains radial or peripheral Cell shape maybe
basophilia. The nuclear chromatin is oval.
generally coarser and more clumped. Cytoplasm: Moderately abundant, but
less than in the
Downey type III: This group of cells previous stage.
increases size and shows Deeply basophic.
basophilic cytoplasm. The Large, well-defined hot next
nucleus usually has visible to nucleus. Nongranular,
nucleoli. These cells usually.
resemble immature Nucleus: Chromatin is condensed
lymphocytes. and coarse. Exhibits
“cartwheel” like pattern.
PLASMA CELLS Round or oval in shape.
PLASMABLAST Eccentric.
Size: 18 to 25 um in diameter. No
Cytoplasm: Basophilic cytoplasm. nucleoli are visible.
Abundant. - In certain pathologic states and
when manufacturing
Nongranular. immunoglobulins, plasma cells
Nucleus: Nuclear chromatin is more may produce striking alterations in
clumped than in the their appearance. Some of the
reticular lymphocyte. changes possible are:
Eccentric. ( The nucleus is off 1. Red staining of the cytoplasm
center, located at one (flame cell).
side of the cell.) 2. Red staining, crystalline, rod
Perinuclear halo may be present. shaped bodies present in the
Round or oval in cytoplasm.
shape. 3. Large, red staining globules in
Multiple nucleoli that may or may the cytoplasm (Russell bodies).
not be visible. 4. Numerous globular bodies
present in the cytoplasm
PROPLASMACYTE (grape, berry, or morula cell).
Size: 15 to 25 um in diameter.
Cytoplasm: Intensely basophilic; usually MEGAKARYOBLAST
bluer than in the blast Size: 20 to 50 um in diameter.
stage. Cytoplasm: Varrying shades of blue.
Nongranular. Usually
More abundant than in darker than the myeloblast.
the blast stage. A lighter May have small, blunt
staining area in the cytoplasm, pseudopods. Small to
next to the nucleus, is often moderate amount. Usually a
visible. This is termed a hot narrow band around the nucleus.
or perinuclear halo. As the cell matures, the
Nucleus: Round or oval. amount of cytoplasm

44
 increases. little bundles, which bud
Usually non granular. off from the periphery to become
Nucleus: Round, oval, or may be platelets.
kidney shaped. Fine Nucleus: Multiple nuclei are present,
chromatin pattern. or the nucleus is
Multiple nucleoli that generally multilobulated. No
stained blue. nucleoli visible.
N/C ratio N/C ratio is less than 1:1.
is about 10:1
Platelet (thrombocyte)
PROMEGAKARYOCYTE Size: 1 to 4 um in diameter.
(STAGE II) Cytoplasm: Light blue to purple.
Size: 20 to 60 um in diameter Very granular.
Cytoplasm: More abundant than
previous stage. Less Consists of two parts: (1) the
basophilic than in the blast. cromomere,
Granules begin to form in the which is granular and
Golgi region. located centrally, and (2) The
hyalomere, which surrounds
Nucleus: Chromatin becomes more the chromomere and is
coarse. Multiple nucleoli nongranular and clear to light
are visible. Irregular in blue.
shape; may even show slight Nucleus: None present.
lobulation.
N/C ratio is 4:1 to 7:1
depending on the
ploidy.
WHITE BLOOD CELL AND
GRANULAR MEGAKARYOCYTE PLATELET MORPHOLOGY
(STAGE III)
Size: 30 to 90 um in diameter. TOXIC GRANULATION- (plate Iva)
Cytoplasm: Abundan. consist of dark blue black cytoplasmic
Pinkish blue in color. granules in the neutrophil. They are
Very fine though to be primary granules, shoe
and diffusely granular. increased alkaline phosphatase activity,
Usually has an irregular and are found in acute infections, drug
peripheral border. poisoning, and burn.
Nucleus: Small in comparison to cell
size. Multiple nuclei DOHLE BODIES- (Plate IVb) appear as
may be visible or the sigle or multiple light blue or grayish
nucleus may show multilobulation. staining areas in the cytoplasm of the
Chromatin is coarser neutrophil. They are rough endoplasmic
than in the previous stage. reticulum containing RNA and may
No represent localized failure of the
nucleoli are visible. cytoplasm to mature. They are found in
N/C ratio is 2:1 to 1:1. infections, poisoning, burns, and
following chemotherapy.
MATURE MEGAKARYOCYTE
(STAGE !V) HYPERSEGMENTED NEUTROPHILS-
Size: 40 to 120 um in diameter. (plate IVc) are neutrophils with a six or
more, lobed nucleus. This represents
Cytoplasm: Contain coarse clumps of an abnormality in the maturation of the
granules aggregating into neutrophil and may be acquired (as in

45
megaloblastic erythropoiesis) or
inherited (Undritz anomaly).
BARR (sex chromatin) body (Plate
IVd)- represents the second X
chromosome in females and may be
seen in 2 to 3% of the neutrophils in
females. It is a small, well-defined,
round projection of nuclear chromatin
that is connected to the nucleus of the
neutrophil by a sigle, fine strand of
chromatin.
DEGENERATED NEUTROPHIL WITH
PYKNOTIC NUCLEUS- (Plate IVe)
results from the condensing of nuclear
into a solid, structureless, mass with no
pattern. These cells are not counted in
a different cell count.
A VACUOLATED NEUTROPHIL-
(Plate IIr) results when the
degenerating cytoplasm begins to
acquire holes or as the result of active
phagocytosis (may reflect increased
lysosomal activity). This condition may
be found in septicemia and severe
infection.
GIANT NEUTROPHILS- May be seen
occasionally in a normal peripheral
blood smear. These cells are much
larger than normal neutrophils and are
generally hyperlobulated. They may be
found normally in a frequency of 1 in
every 20,000 neutrophils but may
increase somewhat in frequency in
disease states.
PELGER-HUER ANOMALY- (Plate V,
e, f, g) indicates a failure of the
neutrophil nucleus tc segment properly.
All of the neutrophils have no more than
a bilobed nucleus. The nuclear
chromatin is coarsely clumped. This
benign anomaly may be inherited or
acquired, as in certain leukemias. A
person heterozygous for this
characteristic shows numerous bilobed
(dumbbell shaped) nuclei, whereas the
homozygous person has round
neutrophil nuclei. The neutrophils in this
anomaly appear to function normaly.
CHEDIAK-HIGASHI SYNDROME-
(Plate V a, b) is a rare, fatal disorder
found in children. It is inherited as an
autosomal recessive characteristic. The
granulocytes usually contain several
very large, reddish-purple or greenish
gray staining granules in the cytoplasm.
In the monocytes in lymphocytes they
stain bluish purple and may be present
singly, or there may be several in one
cell. These granules represent
abnormal lysosomes. Anemia,
neutropenia, and thrombocytopenia
generally develop, and patients will
show increased susceptibility to
infection.
ALDER-REILLY ANOMALY- (Plate IVf)
shown heavy, coarse blue black
granulation of the neutrophils,
eosinophils, basophils, and sometimes,
the lymphocytes and monocytes . This
is an inherited condition and is
commonly associated with Hurler’s
syndrome and Hunter’s syndrome.
MAY HEGGLIN ANOMALY- is an
inherited anomaly affecting the
neutrophils and platelets. Larger than
usual Dohle-like inclusion bodies are
present in the neutrophils. Giant bizzare
platelets are present, and the platelets
may be decreased in number. Some
patients are asymptomatic, whereas
others may exhibit bleeding tendencies.
Platelet function may be abnormal.
AUER RODS- (Plate Vh) are rod-like
bodies representing aggregated
primarily granules that stain a reddish
purple. They are found in the cytoplasm
of myeloblasts, monoblasts, and
promyelocytes in acute monocytic or
acute myelogenous leukemia and and
erythroleukemia.
SMUDGE or BASKET CELL – (Plate
IV j, k) is the disintegrating nucleus of a
ruptured white blood cell.
ATYPICAL PLATELETS- (Plate III o,
p), abnormal in appearance, occur in
some diseased states. In such cases,
the platelet may have one or more of
the following characteristics.
46
 1. Large size (4 to 7 um) seen in
conditions associated with
thrombocytosis. Giant platelets
are 7 to 8 um or larger and are
seen in myeloproliferative
disorders.
2. Increased amount of hyalomere.
3. Granules decreased or absent
(platelet appears gray in color).
They stain very plae blue and are
difficult to see on Wright stained
smears.
4. Zoned appearance.
5. Bizarre or irregular shapes.
PLATELET SATELLITOSIS – 
(Platelets encircling the peripheral
boarders of neutrophils) is seen in are
patient whose blood is anticoagulated
with EDTA. This phenomenon is
thought to be due to a serum factor
which reacts in the presence of EDTA.
Hematopoiesis- is a continuous,
regulated process of blood cell
production that includes cell renewal,
proliferation, differentiation, and
maturation.
- During fetal development,
hematopoiesis progresses through the
aorta-gonad mesonephros (AGM)
region (mesoblastic phase), then to
the fetal liver (hepatic phase), and
finally resides in the bone marrow
(medullary phase).
3 PHASES
1.Mesoblastic Phase
- occurs intravascularly.
- begin around the nineteenth day of
embryonic development after
fertilization.
- cells from the mesoderm migrate to
the yolk sac then form erythroblasts in
the central cavity of the yolk sac remain
active for eight to twelve weeks.
- Gower 1, Gower 2 and portland are
produced by hemoglobin to deliver
oxygen.
- remaining cells become ”Angioblasts”
2. Hepatic Phase
 Occurs extravascularly; happens
in liver
 Begins at 5 - 7 gestational weeks
 Definitive Hematopoiesis (baby
began to take place) low activity
of yolk sac, active during 2nd
trimester of fetal life 
 Fetal liver peaks happen during 3
month of fetus.
 Thymus for T cells matures.
Bone Marrow for B cells matures.
 Spleen decreases the production
after 6months.
 Fetal Hemoglobin (Hbf):
Increases
HgA: fewer
3. Medullary or Myeloid Phase
 Occurs in the medulla or inner
part of the bone.
 Prior to 5 month of fetal
development begins in bone
marrow cavity.
 During this phase Mesenchymal
cells and Hematopoetic Stem
Cells (HSC) migrates into the core
of the bone.
 Myeloid Erythroid ration gradually
approach 3:1.
 Normal Ratio is 2:1 to 4:1
 Average Ratio is 3:1
 By the end of 24 weeks or 6
months of gestation, the bone
marrow
ADULT HEMATOPOIETIC TISSUE
Hematopoietic tissue is located in the
bone marrow, lymph nodes, spleen,
liver, and thymus.
Lymphoid development
Primary lymphoid tissue
- consists of the bone marrow and
thymus and is where T and B
lymphocytes are derived.
Secondary lymphoid tissue
- lymphoid cells respond to foreign
antigens, consists of the spleen,
lymph nodes, and mucosa-
associated lymphoid tissue.
Bone Marrow
- contains developing erythroid,
47
 myeloid, megakaryocytic, and functions
lymphoid cells - indiscriminate filter of the
Major Function: Production and circulating blood.
Proliferation of blood cells - serves as a storage site for
platelets
TWO MAJOR COMPONENTS:
1. Red Marrow (outer) 3 REGIONS OF SPLEEN
- Hematopoetically active 1.White pulp
consisting of developing blood cell - consists of scattered follicles with
and their progenitor germinal centers containing
lymphocytes, macrophages, and
2. Yellow Marrow ( inner) dendritic cells.
- Hematopoetically inactive ,composed 2. Red pulp
of adipocytes or fat cells - composed primarily of vascular
withmesenchymal cells and sinusoids and sinuses
macrophage separated.
- 5-7 years of age 3. Marginal zone
- Retrogression--replacing the active - surrounds the white pulp and forms a
marrow by adipocytes.(sternum,skull, reticular
pelvis, vertebrae, proximalportion of meshwork containing blood vessels,
long bones) macrophages, and
specialized B cells.
hematopoietic inductive
microenvironment
- a.k.a “niche”
- nurturing and protecting HSCs and
regulating a balance among their
quiescence, self-renewal, and
differentiation.

Liver
- serves as the major site of blood cell
production during the second trimester
of fetal development
- Lumen of the sinusoids contains
Kupffer cells that maintain contact with
the endothelial cell lining.

Kupffer cells- macrophages that

remove senescent cells and foreign
debris from the blood that circulates
through the liver
- secrete mediators that regulate
protein synthesis in the
hepatocytes

Spleen
-  spleen is the largest lymphoid LINEAGE-SPECIFIC
organ in the body. HEMATOPOIESIS
- It is located directly beneath the LINEAGE-SPECIFIC
diaphragm behind the fundus of
the stomach in the upper left
HEMATOPOIESIS
Erythropoiesis- process of producing
quadrant of the abdomen
red blood cells
- It is vital but not essential for life

48
Leukopoeisis- process of producing Size: 8-12 micrometers
white blood cells Nucleus: round, smaller than
Myelopoeisis- process of producing prorubricyte; eccentric;
granulocytes thick membrane
Lymphopoeisis- process of producing Chromatin: coarse and clumped;
lymphocytes Irregularly
Thrombopoeisis- process of producing condensed; distinct
thrombocystes or blood platelets parachromatin
Nucleoli: no longer visible
NORMOBLASTIC MATURATION Cytoplasm: relatively more abundant;
RUBRIBLAST varying mixture of red
Synonyms: Proerythroblast or and blue to diffusely lilac
Pronormoblast NC: 4:1
Size: 14 - 19 micrometers
Nucleus: round or oval; thin
membrane; central or
slightly eccentric
Chromatin: fine to coarse; scanty
and Indistinct METARUBRICYTE
Nucleoli: 1-2; usually pink Synonyms: Orthochromatic erythroblast
Cytoplasm: small, moderately Orthochromatic normoblast
basophilic, Late erythroblast
homogenous and opaque Late normoblast
N/C ratio: 8:1 Size: 7-10 micrometers
Nucleus small, shrunken, solid
PRORUBRICYTE black; degenerated
Synonyms: Early erythroblast Chromatin: non-linear clumped and
Basophilic normoblast condensed
Basophilic erythroblast Nucleoli: none
Size: 10 - 15 micrometers Cytoplasm: red with minimal amounts of
Nucleus: smaller than pronormoblast, residual blue
round and slightly eccentric
with thin membrane RETICULOCYTE
Chromatin: Irregular and coarse; Synonyms: Diffusely basophilic
sparse but distinct erythrocyte
Nucleoli: 0-1 Polychromatophilic
Cytoplasm: apparently abundant; erythrocyte
royal blue and opaque, Vital granulated erythrocyte
with varying amounts of Vital staining erythrocyte
hemoglobin; with Reddish Pro-erythrocyte
tinge Size: 8-10 micrometers
N/C ratio: 6:1 Nucleus: none, instead, after
supravital stain center of
RUBRICYTE cell shows network of fibrillae
Synonyms: Polychromatophilic and dots or Reticulum of
erythroblast bluish tint (residue of RNA)
Polychromatophilic Chromatin: None
normoblast Nucleoll: None
Intermediate erythroblast Cytoplasm: pink to reddish brown
Intermediate normoblast

49
ERYTHROCYTE
Synonyms: Normocyte, akaryocyte, red METAMYELOCYTE
blood cell, erythroplastid Synonym: Juvenile
Size: 6 - 8 micrometers Size: 10 - 28 micrometers
Nucleus: None Nucleus: Indented and kidney -
Chromatin: None shaped
Nucleoli: None Chromatin: Deep purple; coarse and
Cytoplasm: pink, darker in periphery more clumped; scanty
than in the center parachromatin
N/C ratio: 1:5:1
MATURATION OF Nucleoll: Not visible
GRANULOCYTE Cytoplasm: Fairly abundant; pink
MYELOBLAST with eosinophilic or
Size: 10 - 20 micrometers basophilic or neutrophilic granules
Nucleus: Round or oval, thin nuclear
membrane; stippled
finely reticulated; light purple BAND
Chromatin: abundant chromatin, pale Synonyms: Stab, Staff cell
blue or pink Size: 10 - 15 micrometers
parachromatin Nucleus: Sausage shaped; band
Nucleoli: 2-5 round or oval; pale blue shaped or shaped like
Cytoplasm: sparse, deeply basophilic, letters C,S,Z; curved rod
no granules Chromatin: coarse chromatin;
NC ratio: 71 deep purplish blue
Nucleoll: Not visible
PROMYELOCYTE Cytoplasm: Abundant; pale blue
Synonyms: Progranulocyte or pink with
Size: 14 - 20 micrometers neutrophilic, eosinophilic or
Nucleus large, round or oval, thin basophilic granules
membrane N/C ratio: 1:2
Chromatin: slightly clumping
Nucleolit 1-3; pale blue; SEGMENTED GRANULOCYTE
round or oval Synonym: Polymorphonuclear
Cytoplasm: sparse; basophillic, contains neutrophil, basophil or
slight purplish eosinophil
granules Size: 10 - 15 micrometers
NC ratio: 5:1 Chromatin: Coarse, dense, deep
purplish blue
MYELOCYTE Nucleus: Two or more lobes or
Size: 10 - 19 micrometers segments connected
round or oral by filaments
Nucleu: round or oval Nucleoli: Not visible
Chromatin: Coarse condensed Cytoplasm: Abundant; blue or pink
chromatin with blue or with characteristic
pink parachromatin eosinophilic (red,
cytoplasm: eosinophilic or basophilic or neutroph|lic (lilac) or
neutrophilic in Basophilic (dark blue or
the late or more mature blackish) granules
stage. N/C ratio: 1:3
N/C ratio: 2:1 Eosinophils rarely

50
have more than 2 Large lymphocyte - 8 - 18
segments; usually nucleus lies micrometers
over the granules are Nucleus: Round or oval, slightly or
coarse and red. Basophils deeply indented,
usually have indented eccentric, heavy membrane,
nucleus, rarely more than closely knit
2 segments, usually the Chromatin: Large coarse clumps
granules are found lying over the of chromatin blending
nucleus; granules are into sparse pale blue to pink
coarse and dark blue. parachromatin
Neutrophils have nuclel with Nucleoli: Generally none,
more than 2 segments as occasionally present
much as 5. Granules are Cytoplasm: Clear, homogenous
lilac and finer. Robin's egg blue or sky
blue with occasional azurophilic
MATURATION OF LYMPHOBLAST granules
N/C ratio: 5:2
LYMPHOBLAST
Size: 10 - 18 micrometers
Nucleus: Centrally located; definite MATURATION OF MONOBLAST
membrane, round or
oval MONOBLAST
Chromatin: Thin strands or light Size: 12 - 18 micrometers
red purple chromatin Nucleus: pale staining and indented;
Light blue sharply fine strands of
demarcated chromatin, abundant parachromatin
parachromatin Nucleoll: Usually none; occasionally
Nucleoll: 1-2 small pale blue one
Cytoplasm: Homogenous and Cytoplasm: Gray blue; opaque
moderately with numerous fine
basophilic; sparse with no granule dustlike lilac granules
N/C ratio: 7:5 N/C ratio: 6:1

PROLYMPHOCYTE PROMONOCYTE
Size: 10 - 18 micrometers Size: 14 - 18 micrometers
Nucleus: Round or oval; slightly Nucleus: Moderately Indented; thin
Indented membrane
Chromatin: Reddish - purple Chromatin: Fine, thread - like
densed chromatin; chromatin; abundant
parachromatin not well defined parachromatin
Nucleoll: One, round, blue and well Nucleoli: 0-1
outlined Cytoplasm: Opaque, gray blue
Cytoplasm: Moderately abundant; with very fine lilac
pale blue to medium granules
dark blue N/C ratio: 5:1
N/C ratio: 5:1
MONOCYTE
LYMPHOCYTE Size: 12 - 18 micrometers
Size: Small lymphocyte - 6 - 8 Nucleus: Indented or folder over;
micrometers delicate pale staining;

51
kidney shaped; with brain - like Cytoplasm: Abundant, pale with
Convolutions; sprawling with fine blue granules and
blunt pseudopods showing pseudopod – like
Chromatin: Fine strands; projections
abundant and distinct N/C ratio: 1:2
parachromatin - The granular megakaryocyte is
Nucleoli: Occasionally blue; usually characterized by spreading of the red
none pink granules diffusely through most of
Cytoplasm: Abundant; slightly the cytoplasm and further increase and
gray ground glass spreading of nuclear lobes. In the
appearance mature megakaryocyte, the nucleus is
N/C ratio: 4:1 more compact, basophilia has
disappeared, and the granules are
MATURATION OF clustered into small aggregates.
MEGAKARYOBLAST -At an ultrastructural level, this is
produced by proliferation in invaginated
MEGAKARYOBLAST surface membrane (demarcating
Size: 25 - 35 micrometers membrane) that separates the
Nucleus: Large oval or round cytoplasm into individual platelets.
Chromatin: Delicate purple Platelets are ultimately shed as
chromatin; sparse cytoplasmic fragments by fusion of
parachromatin demarcation membranes. In the
Nucleoli: 2-6; small and indistinct marrow, megakaryocytes are adjacent
Cytoplasm: Scanty; irregular; blue to sinus walls, and platelets are
non-granular with blunt released into the lumen.
pseudopods with various
shades of Blue; may contain THROMBOCYTE
azurophilic granules Synonym: Blood platelet
N/C ratio: 10:1 Size: 2-5 micrometers
Nucleus: None
PROMEGAKARYOCYTE Nucleoli: None
Size: 25-60 micrometers Cytoplasm: Light blue with
Nucleus: Irregularly large; single but granules at the center
may appear lobulated (granulomere, chromomere),
or multilobulated marginal
Nucleoll: 2-6 small indistinct zone - Hyalomere
Cytoplasm: Basophilic; prominent
blue to reddish purple MATURATION OF PLASMABLAST
granules and marginal bubbly
cytoplasm PLASMABLAST
N/C ratio: 6:1 Size: 15-25 micrometers
Nucleus: Round or oval; eccentric
MEGAKARYOCYTE Chromatin: Coarse and
Size: 40 - 150 micrometers reticulated; moderate
Nucleus: multiform resembling amount and distinct parachromatir
staghorn calculi; irregular Nucleoll: 2-4
bizarre Cytoplasm: Fairly abundant,
Chromatin: Coarse; irregularly moderately or deeply
clumped basophilic with no granules
Nucleoli: Usually not visible N/C ratio: 1:2

52
PROPLASMACYTE Regions of Lymph Nodes
Size: 15 - 25 micrometers Medulla
Nucleus: Oval or rough; eccentric; - inner region
- consists primarily of T lymphocytes
moderately coarse
and plasma cells
Chromatin: Intermediate
Nucleoli: 1-2; very large when Paracortex
abnormal - region between cortex and medulla
Cytoplasm: Brilliant blue; opaque - contains predominantly T cells and
N/C ratio: 1:2 numerous macrophage

PLASMACYTE Thymus
Synonym: Plasma cell - originates from endodermal and
Size: 10 - 20 micrometers mesenchymal tissue
- the thymus is populated initially by
Nucleus: Round or oval; eccentric,
primitive lymphoid cells from the yolk
dense and concentrated in
sac and the live
the periphery creating the - At birth, the thymus is an efficient
so called "cart-wheel" or "spokes of consists of two lobes, each measuring
a wheel" arrangement 0.5 to 2 cm in diameter
Nucleoli: None - Is the organ responsible in the
Cytoplasm: Dark blue; ovoid and conditioning of T lymphocytes
somewhat fibrillary with Stem Cell Theory
pale clear perinuclear Till and McCulloch
zone; non-granular but may - In 1961, conducted a series of
contain secretory experiments in which they irradiated
globules called "Russell spleens and bone marrow of mice,
bodies" which may be colorless, creating a state of aplasia.
red, pink, blue or green in - proposed that hematopoiesis is a
color. If globules fill the random process whereby the HSC
cyloplasm, cell is called "grape or randomly commits to self renewal or
berry or morula" cell. The differentiation
intracytoplasmic
deposition or Colony Forming Unit- Spleen
amorphous material gives rise to - aplastic mice were given an
Mont cell or Flame cell intravenous injection of marrow cells 2.
N/C ratio: 1:2 -Colonies of HSCs were seen 7 to 8
days later in the spleens of the
Lymph Nodes irradiated (recipient)
- organs of the lymphatic system
located along the lymphatic capillaries Stem Cell
- bean-shaped structures (1 to 5 mm in 1. Cells that have extensive proliferative
diameter) capacity
Functions - Ability to give rise to new stem cell
- Play a role in the formation of new - Ability to differentiate into any blood
lymphocytes from germinal centers cells lines
- Involved in the processing of specific 2. HSC are BMC that are capable of
Ig producing all types of blood cells
-Involved in the filtration of particulate 3. They differentiate into one or another
matter, debris, and bacteria entering the type of committed stem cells
lymph node via the lymph ( progenitor cells)

53
 and functional activation
TWO STEM CELL THEORY Source : monocytes and fibroblast
1. Polyphyletic theory
- suggest that each blood cell lineages M-CSF
- stimulates monocytes and
came from its own unique stem cell
macrophages production activity
sources is monocytes, fibroblast,
2. Monophyletic theory Endothelial cells.
- suggest that all blood cell came from
single progenitor stem cell
- pluripotential stem cell as the Meg-CSF
progenitor cell - mono, fibroblast , megakaryocytes

Hematopoietic Stem Cells
- are capable of self renewal
- HSCs in the bone marrow, 6 billion
blood cells per kilogram of body weight
are produced each day for the entire life
span of an individual.
Three possible fates:
- Self renewal
- Differentiation
-Apoptosis
- refers to programmed cell death
- a normal physiologic process that
eliminates unwanted, abnormal, or
harmful cells
Erythropoietin (EPO)
- Stimulates proliferation , growth and
differentiation of erythroid precursors
and may have minor effects on
megakaryocytes.
- Target cells are pronormoblast and
CFU-Erythroid cells
- Source: Kidney
Thrombopoietin
- regulates production platelets.
Control of Hematopoiesis
- The entry of mature blood cells into
the intravascualr space relies upon :
1. Multiplication of developing cells
2. Gradual maturation
3. orderly release of cell from bone
marrow

Cytokines
- is a group of specific glycoproteins
called growth factors that regulates the
proliferation differentiation, and
maturation of hematopoietic precursor
cells
– it includes:
1. Interluekins (Ils)
2. Lymphokines
Hematopoietic Growth Factors 3. Monokines
1. CSF : Colony Stimulating Factors 4. Interferons
Specific for various cell lines: 5 .Colony Stimulating Factors (CSFs)
6.Chemokines
GM-CSF Positive Influence
- a pan myeloid growth factor that 1. IL-1
stimulates Granu, - mono, 2. IL-3
megakaryocyte and eosinophil 3. IL-6
progenitors Source : fibroblast, T cells 4. IL-9
and endothelial cells 5. IL11
G-CSF 6. GM-CSF
- stimulates granulocytes production 7. Kit Ligand

54
8. FLT3 ligand
9. GM-CSF
Negative Influence
1. transforming growth factor-b
2. tumor necrosis factor-a
3. interferons

55
HEMOGLOBINOPATHIES
AND
THALASSEMIA
HEMOGLOBINOPATHY refers to a
disease state (opathy) involving the
hemoglobin (Hb) molecule.
Hemoglobinopathies are the most
common genetic diseases, affecting
approximately 7% of the world’s
population.
-All hemoglobinopathies result from a
genetic mutation in one or more genes
that affect hemoglobin
synthesis. The genes that are mutated
can code for either the proteins that
make up the hemoglobin molecule
(globin or polypeptide chains) or the
proteins involved in synthesizing or
regulating synthesis of the globin
chains.
SICKLE CELL DISEASE
 Sickle cell anemia (Hb SS), the
most common form of
hemoglobinopathy, is an
expression of the inheritance of a
sickle gene from both parents.
 It must be inherited from both
parents.
 Hb S is different from Hb A
because of a single nucleotide
change (GAT to GTT) that results
in the substitution of valine for
glutamic acid at the sixth position
on the b chain of the hemoglobin
molecule. This results in
abnormalities in polymerization
(or gelation), with deoxygenation
that leads to sickling.
Epidemiology
 SCD is found most commonly in
persons of African ancestry, but it
also affects persons of
Mediterranean, Caribbean, South
and Central American, Arab, and
East Indian ancestry.
 The sickle cell carrier state
confers a selective advantage to
Plasmodium falciparum malaria,
because of preferential sickling of
only the parasitized cells.
 One of the most common genetic
diseases in the United States.
 Hb SC disease affects an
estimated 1 in 835
AfricanAmerican births, and SC b-
thalassemia (S b-thalassemia)
affects 1 in 1,667 African
American live births.
Pathophysiology
 In Hb S, a substitution of T for A
in the sixth codon of the b-globin
gene leads to the replacement of
a glutamic acid residue by a
valine residue. On deoxygenation,
Hb S polymers form, causing cell
sickling and damage to the
membrane. Some sickle cells
adhere to endothelial cells,
leading to vasoocclusion.
Clinical Signs and symptoms
 In children, it is not present unless
the patient is older than 6 months.
Vasoocclusive disease develops
between the ages of 12 months
and 6 years. splenic dysfunction
is a potentially lifethreatening
complication that develops during
infancy. Chronic manifestations in
children include a progressive lag
in growth and development after
the fi rst decade of life and
chronic destruction of bone and
joints, with ischemia and infarction
of the spongiosa.
 In pregnancy, there is no increase
in disease manifestation but there
is an increase in maternal
mortality of 20% and fetal
mortality of 20%.
 In adults, Red cells with relatively
low Hb F levels are likely to
become sickled cells and,
therefore, have short life spans.
Acute chest syndrome is a
significant cause of death in
patients of all ages who have
sickle cell anemia.
56
 Experimental treatment approaches
General signs and symptoms include
 Pain or called vasoocclusive 1. Gene therapy. Gene therapy
crises involves inserting a normal gene
 Pulmonary complications into the bone marrow of patients
 Stroke with sickle cell anemia to produce
normal hemoglobin. Another
Laboratory testing approach is to attempt to turn off
 Decreased hemoglobin (5 to 9.5 the defective gene while
g/dL) reactivating another gene
 Hematocrit Count responsible for the production of
 Red Blood Cell Count Hb F.
 Persistent increase in the white 2. Butyric acid. Butyric acid is
blood cell (WBC) count of 12,000 normally used as a food additive
to 15,000 × 109/L but it may increase the amount of
 Red cell morphology on Hb F in the blood.
peripheral blood smear can 3. Clotrimazole. This over-the-
include moderate to significant counter antifungal medication
anisocytosis, poikilocytosis, and helps prevent a loss of water from
hypochromia red blood cells, which may reduce
 Other diagnostic laboratory tests the number of sickle cells that
include thin-layer isoelectric form.
focusing (IEF) or high 4. Nitric oxide. Abnormal function of
performance liquid the cells lining blood vessels may
chromatography (HPLC), contribute to the complications of
Hemoglobin electrophoresis, DNA SCD. Disruption in the synthesis
Analysis, Pre Natal Diagnosis, of nitric oxide, an important
and Screening of newborns for regulator of blood vessel
sickle cell anemia and carriers. relaxation, contributes to these
abnormalities. Treatment with
nitric oxide may prevent sickle
cells from clumping together.
5. Nicosan. This is an herbal
treatment in early trials in the
United States. Nicosan has been
used to prevent sickle crises in
Nigeria, West Africa.
Treatment
 Bone marrow transplant offers the SICKLE B-THALASSEMIA
only potential cure for sickle cell  It is variable in its clinical
anemia. manifestations but tends to be
 General supportive care includes milder in blacks than in
daily oral folate supplementation, Mediterranean persons.
antibiotic prophylaxis in childhood,  Patients have moderately severe
Pneumovax, Haemophilus hemolytic anemia.
influenzae vaccine,  Those with S b+ thalassemia can
meningococcal vaccine, a yearly make a small amount of Hb A and
flu shot, a yearly eye examination, have less extensive hemolysis
prompt treatment of infections, and vasoocclusive phenomena.
and avoidance of dehydration.  It can be diagnosed by examining
the blood fi lm and through
hemoglobin electrophoresis.

57
SICKLE-C DISEASE
 The patients have only Hb S and
C, with an absence of Hb A and
normal or increased levels of Hb
F.
 The complications associated with
this disorder are less severe than
SCD with three exceptions:
proliferative retinopathy, aseptic
necrosis of femoral heads, and
acute chest syndrome secondary
to fat emboli in the final months of
pregnancy.
 In SC red cells, the intracellular
hemoglobin concentration is
significantly elevated because of
the presence of Hb C.
 SC red cells have at least a 10%
higher level of Hb S than SA red
cells.
 Complications commonly include
retinopathy, hematuria from
medullary infarcts, and aseptic
necrosis of the femoral head
Sickle cell trait
 Approximately 8% of black
Americans are heterozygous for
Hb S. Their red cells contain Hb S
and A.
 It provides a survival advantage
over individuals with normal
hemoglobin in regions where
malaria, P. falciparum, is
endemic.
 Clinical signs and symptoms
associated with sickle cell trait
include hematuria, splenic
infarction at high altitude (over
10,000), hyposthenuria,
bacteriuria, and pyelonephritis in
pregnancy.
There are no manifestations of anemia,
red blood cell abnormalities, increased
risk of infection, or increased mortality
associated with sickle cell trait
Thalassemias are a diverse group of
inherited disorders caused by genetic
mutations that reduce or prevent the
synthesis of one or more of the globin
chains of the hemoglobin (Hb) tetramer.
-Seven years later, Whipple and
Bradford published a paper outlining the
detailed autopsy studies of children who
died of this disorder
-Because of the high incidence of
patients of Mediterranean descent with
this disorder, Whipple called the
disease Thalassic (Greek for “great
sea”) anemia, which was subsequently
changed to thalassemia.
-Thalassemia results from a reduced or
absent synthesis of one or more of the
globin chains of hemoglobin.
-Thalassemias are named according to
the chain with reduced or absent
synthesis.
-Mutations affecting the a- or b-globin
gene are most clinically significant
because Hb A (a2b2) is the major adult
hemoglobin.
Demographics
 Thalassemia is a growing global
public health problem, with an
estimated 900,000 births of
clinically significant thalassemia
disorders expected to occur in the
next 20 years.
Etiology
 Thalassemias are caused by an
abnormality in the rate ofsynthesis
of the globin chains. This is in
contrast to the true
hemoglobinopathies (e.g., Hb S
58
 and Hb C) that result from an
inherited structural defect in one
of the globin chains that produces
hemoglobin with abnormal
physical or functional
characteristics.
Pathophysiology
 Thalassemias are characterized
by the absence or decrease in the
synthesis of one of the two
constituent globin subunits of a
normal hemoglobin molecule.
 In a-thalassemia, decreased
synthesis of a-globulin results in
accelerated red cell destruction
because of the formation of
insoluble Hb H inclusion in the
mature erythrocyte.
 The more severe b-thalassemia
refl ects the extreme insolubility of
a-globin, which is present in
excess in the red cell because of
decreased b-globin synthesis.
Studies of RNA metabolism in
erythroid cells have suggested
that many patients with b-
thalassemia have a defect in RNA
processing. This defect affects effi
cient RNA splicing during protein
globin synthesis.
TWO MAJOR TYPES OF
THALASSEMIA
Alpha
 A condition in which the body
does not produce enough alpha
globin (a component of
hemoglobin)
Beta
 A condition in which the body
does not produce enough beta
globin.
ALPHA THALASSEMIA
 In contrast to b-thalassemia, with
most cases caused by point
mutations, the major cause of a-
thalassemia is deletions that
remove one or both a-globulin
genes from the affected
chromosome 16.
 a-thalassemias can be classifi ed
into four types on the basis of
genotype and the total number of
abnormal genes that result:
1. Silent carrier state (one inactive a
gene)
2. a-thalassemia trait (two inactive a
genes)
3. Hb H disease (three inactive a
genes)
4. Hydrops fetalis with Hb Bart (four
inactive a genes)
The most common mutations in a-
thalassemia are deletionsinvolving the
a1- and/or a2- globin genes.
The a-thalassemias are divided into
two haplotypes:
 a0-thalassemia
 a1-thalassemia
Clinical Signs and symptoms
 Four clinical syndromes are
present in a-thalassemia,
depending on the gene number,
cis or trans pairing, and the
amount of a chains produced.The
four syndromes are:
1. Silent carrier state
2. a-thalassemia minor
3. Hb H disease
4. Hb Bart hydrops fetalis syndrome
a-Thalassemia Minor
 Deletion of two a-globin genes is
the major cause of a-thalassemia
minor.
 It exists in two forms:
homozygous a1 (– a/– a) or het-
erozygous a0 (– –/aa).
BETA THALASSEMIA
 Also known as “Cooley’s Anemia”
 b-thalassemia is one of the most
common single-gene disorders.
 Most mutations that cause b-
thalassemia are point mutations,
and rarely by deletions, in
functionally important regions of
the b-globin gene.
 include all the disorders of
reduced globin chain production
59
 arising from the b-globin gene
cluster on chromosome 11.
 The b-thalassemias affect mainly
the b chain production but also
may involve the d, Gg, Ag, and e
chains.
Clinical Signs and symptoms
• b-thalassemia silent carrier
(heterozygous state) with no
hematologic abnormalities or
clinical symptoms.
 b-thalassemia minor
(heterozygous state) with mild
hemolytic anemia, microcytic/
hypochromic RBCs, and no
clinical symptoms.
 b-thalassemia major
(homozygous or compound
heterozygous state) with severe
hemolytic anemia,
microcytic/hypochromic RBCs,
severe clinical symptoms, and
transfusion dependence
 b-thalassemia intermedia with
mild to moderate
hemolyticanemia,
microcytic/hypochromic RBCs,
moderate clinical symptoms, and
transfusion independence
b-Thalassemia Minor
 b-thalassemia minor (also called
b-thalassemia trait) results when
one b-globin gene is affected by a
mutation that decreases or
abolishes its expression, whereas
the other b-globin gene is normal
(heterozygous state).
 It usually presents as a mild,
asymptomatic anemia with
hemoglobin ranging from 12.4 to
14.2 g/dL in affected men and
10.8 to 12.8 g/dL in affected
women.
 The RBC count is within the
reference interval or slightly
elevated.
b-Thalassemia Major
 b-Thalassemia major is
characterized by a severe anemia
that requires regular transfusion
therapy.
 It is usually diagnosedbetween 6
months and 2 years of age (after
completion of the g to b switch)
when the child’s Hb A level does
not increase as expected.
 In untreated b-thalassemia major,
the hemoglobin level can fall as
low as 3 to 4 g/dL.
 The MCV ranges from 50 to 70 fL.
b-Thalassemia Intermedia
 Thalassemia intermedia is a term
used to describe anemia that is
more severe than b-thalassemia
minor but does not require regular
transfusions to maintain
hemoglobin level and quality of
life (transfusion-independent).
THALASSEMIA ASSOCIATED
WITH STRUCTURAL
HEMOGLOBIN VARIANTS
Hemoglobin S-Thalassemia
 Sickle cell anemia (Hb SS)- a-
thalassemia is a genetic
abnormality due to the
coinheritance of two abnormal b-
globin genes for Hb S and an a-
thalassemia haplotype.
Hemoglobin C-Thalassemia
 Hb C-b-thalassemia produces
moderately severe hemolysis,
splenomegaly, hypochromia,
microcytosis, and numerous
target cells. The hemoglobin
electrophoresis pattern varies,
depending on the type of b-
thalassemia gene defect, with
higher Hb C concentrations in
patients when there is minimal or
no b chain production.
Hemoglobin E-Thalassemia
60
  Hb E-b-thalassemia is a  Deletion of one or more globin
significant concern in Southeast
Asia and Eastern India owing to
the high prevalence of both
genetic mutations.Hb E is due to
a point mutation that inserts a
splice site in the b-globin gene,
and results in decreased
production of Hb E.3 In the
homozygous state (Hb EE) the
clinical symptoms are similar to a
mild b-thalassemia.
GENETIC DEFECTS CAUSING
THALASSEMIA
 Types of genetic defects that
cause a decrease or absent
production of a particular globin
chain include single nucleotide(or
point) mutations, small insertions
or deletions, or largedeletions.
The mechanisms by which these
mutations interfere with globin
chain production include:
 Reduced or absent
transcription of messenger
ribonucleic acid (mRNA) due to
mutations in the promoter region
or initiation codon of a globin
gene, as well as mutations in
polyadenylation sites that
decrease mRNA stability
 mRNA processing errors due to
mutations that add or remove
splice sites resulting in no globin
chain or altered globin chain
production.
 Translation errors due to
mutations that change the codon
reading frame (frameshift
mutations), substitute an incorrect
amino acid codon (missense
mutations), add a stop codon
causing premature chain
termination (nonsense mutations),
or remove a stop codon, which
results in an elongated and
unstable mRNA that produces a
dysfunctional globin chain
genes resulting in the lack of
production of the corresponding
globin chains .
Laboratory Methods
• Complete Blood Count with
Peripheral Blood Film
 Reticulocyte Count
 Supravital Staining
 Molecular Genetic Testing
 Assessment of Normal and
Variant Hemoglobins
DIAGNOSIS OF THALASSEMIA
 History and Physical Examination
 Individual and family histories are
paramount in the diagnosis of
thalassemia. The ethnic
background of the individual
should be investigated because of
the increased prevalence of
specific gene mutations in certain
populations. In the clinical
examination, findings that suggest
thalassemia include pallor (due to
the anemia); jaundice (due to the
hemolysis); splenomegaly
(caused by sequestration of the
abnormal RBCs, excessive
extravascular hemolysis, and
some extramedullary
erythropoiesis); and skeletal
deformities (due to the massive
expansion of the bone marrow
cavities). These findings are
particularly prominent in untreated
or partially treated b-thalassemia
major.
61
LEUKEMIA
LEUKEMIA
Acute Leukemias
 Leukemia is derived from the
ancient Greek words leukos
(leykóç), meaning “white,” and
haima (aἷma), meaning “blood.”
 Acute leukemia refers to the
rapid, clonal proliferation in the
bone marrow of lymphoid or
myeloid progenitor cells.
 Leukemia is currently believed to
be a stepwise progression of
mutations or “multiple hits”
 French-American-British (FAB)
classification of the acute
leukemias was devised in the
1970s
 Morphologic examination along
with cytochemical stains to
distinguish lymphoblasts from
myeloblasts
 The use of cytochemical stains
continues to be a useful adjunct
for differentiation of hematopoietic
diseases.
 For most acute leukemias,
causes directly related to the
development of the malignancy
are unknown, but a few
exceptions exist. Some known
causes include environmental
toxins, certain viruses,
previous chemotherapy, and
familial predisposition.
ACUTE LYMPHOBLASTIC
LEUKEMIA (ALL)
 Acute lymphoblastic leukemia
(ALL) is a malignant disorder that
originates in a single B- or T-
lymphocyte progenitor.
 Proliferation and accumulation of
clonal blast cells in the marrow
result in suppression of
hematopoiesis and, thereafter,
anemia, thrombocytopenia, and
neutropenia.
 Lymphoblasts can accumulate in
various extra medullary sites,
especially the meninges, gonads,
thymus, liver, spleen, and lymph
nodes.
 ALL has many subtypes and can
be classified by immunologic,
cytogenetic, and molecular
genetic methods.
 Cases of childhood ALL having
a hyperdiploid karyotype
respond well to extended
treatment with methotrexate and
mercaptopurine.
 Adults whose leukemic cells
contain the Philadelphia
chromosome and BCR-ABL1
fusion benefit from intensive
treatment that includes a tyrosine
kinase inhibitor and
transplantation of allogeneic
hematopoietic stem cells.
CHRONIC LYMPHOCYTIC
LEUKEMIA
 Chronic lymphocytic leukemia
is a malignancy of mature B cells
characterized by progressive
lymphocytosis,
lymphadenopathy,
splenomegaly, and cytopenias.
 The progressive accumulation of
leukemic B cells is a
consequence of defective
apoptosis and survival signals
derived from the
microenvironment.
 Progressive disease results in
dysregulation of the cellular and
humoral components of the
effector immune system with a
resultant increase in the incidence
of infectious complications,
which constitutes the leading
cause of morbidity and mortality
in this disease.
 Significant therapeutic advances
have been realized, especially
with the development of well
tolerated targeted antibodies
and kinase inhibitors.
 Although not curative, these
therapies have resulted in
significant improvements in
62
 patient outcomes with substantial
increases in progression-free and
overall survival intervals.
ACUTE MYELOID LEUKEMIA
 AML is the most common
type of leukemia in adults,
and the incidence increases
with age.
 AML is less common in
children.
SUBTYPES OF ACUTE
MYELOID LEUKEMIA AND
RELATED PRECURSOR
NEOPLASMS
➢ AML with Recurrent Genetic
Abnormalities
➢ Acute Myeloid Leukemia with
Myelodysplasia Related Changes
➢ Therapy-Related Myeloid
Neoplasms
➢ Acute Myeloid Leukemia, Not
Otherwise Specified
• Acute Myeloid Leukemia with
Minimal Differentiation.
• Acute Myeloid Leukemia
without Maturation.
• Acute Myeloid Leukemia with
Maturation.
• Acute Myelomonocytic
Leukemia.
• Acute Monopolistic and
Monocytic Leukemias
• Acute Erythroid Leukemia.
• Acute Megakaryoblastic
Leukemia
➢ Myeloid Sarcoma
➢ Myeloid Proliferations Related to
Down syndrome
➢ Blastic Plasmacytoid Dendritic
Cell Neoplasm
ACUTE LEUKEMIAS OF
AMBIGUOUS
LINEAGE
 Include leukemia in which there is
no clear evidence of
differentiation along a single cell
line and are commonly referred to
as acute undifferentiated
leukemias (AULs).
 ALAL that demonstrate a
multiplicity of antigens where it is
not possible to determine a
specific lineage are called mixed
phenotype acute leukemias
(MPALs).
STAINING
 Cytochemical reactions may be
enzymatic (uses Fresh smears)
or nonenzymatic (performed on
specimens that have been stored
at room temperature)
 Myeloperoxidase (MPO) stains
primary granules and is useful in
differentiating granulocytic from
lymphoid cells.
 Sudan Black B (SBB) stains
lipids and results parallel those
with the MPO stain.
 Esterases help differentiate
granulocytes and their precursors
from cells of monocytic origin.
 Butyrate esterase testing gives
positive results in monocytes but
not in granulocyte precursors,
whereas naphthol AS-D
chloroacetate esterase stains
granulocyte precursors.
 a-Naphthyl butyrate esterase
positivity in cells of monocytic
origin from a patient with acute
monoblastic/monocytic leukemia.
MATURE LYMPHOID
NEOPLASMS
 Lymphomas are neoplasms of the
lymphoid system.
 The diagnosis is based on a
combination of biologic features
such as morphology,
immunophenotype and
molecular genetic
characteristics, and clinical
information.
 Most lymphomas develop in
previously healthy individuals.
The strongest risk factor for
63
 development of
lymphoproliferative disorder is
altered immune function as seen
in immunocompromised patients
or individuals with autoimmune
diseases
CHRONIC LYMPHOCYTIC
LEUKEMIA/SMALL
LYMPHOCYTIC LYMPHOMA
Mature B cell Lymphomas
 Mantle Cell Lymphoma is a
lymphoproliferative disorder
characterized by medium-sized
lymphoid cells with irregular
nuclear outlines derived from the
follicular mantle zone
 Follicular lymphoma originates
from germinal center B cells and
in most cases recapitulates
follicular architecture.
 Extranodal Marginal Zone
Lymphoma of Mucosa-
Associated Lymphoid Tissue
the neoplastic proliferation is
usually heterogeneous,
encompassing small and
medium-sized lymphocytes,
plasma cells, and scattered large
lymphoid cells. MALT lymphoma
is frequently associated with
autoimmune conditions.
 Plasma cell neoplasms are
characterized by a monoclonal
proliferation of terminally
differentiated B cells.
 Diffuse Large B cell Lymphoma
-The defining feature of DLBCL is
the large cell size. In contrast to
the neoplastic cells in the
lymphoproliferative disorders
discussed so far, DLBCL cells are
significantly larger than normal
lymphocytes.
 Burkitt lymphoma is
characterized by medium- sized,
highly proliferating lymphoid cells
with basophilic vacuolated
cytoplasm.
CHRONIC LYMPHOCYTIC
LEUKEMIA (CLL) AND SMALL
LYMPHOCYTIC LYMPHOMA
(SLL)
- are characterized by
accumulation of small lymphoid
cells in peripheral blood, bone
marrow, and lymphoid organs.
 The exact cell of origin in
CLL/SLL is not known, however
gene expression profiling has
shown that antigen-experienced
B cells, both memory B cells
and marginal zone B cells, are
likely candidates
 In CLL, bone marrow and
peripheral blood films show
small lymphoid cells with a
characteristically coarse
chromatin (“soccer-ball”
pattern), absent or inconspicuous
nucleoli, and scant cytoplasm
 CLL is diagnosed based on a
sustained increase in the
monoclonal B lymphocytes with
CLL immunophenotype which is
equal or greater than 5000/uL.
 The CLL immunophenotype
includes an expression of CD19,
CD20, and CD23, with aberrant
expression of CD5
PROLYMPHOCYTIC LEUKEMIA
 Prolymphocytic leukemia (PLL)
is a rare mature lymphoid
leukemia that can be derived from
B or T cells.
 Both B cell and T cell types
involve peripheral blood, bone
marrow, and spleen.
 Lymph node involvement is more
commonly seen in T cell PLL.
 This lymphoproliferative disease
is distinct from CLL, and its
diagnosis requires that more than
55% of circulating lymphoid cells
have the morphology of a
prolymphocyte.
 The pathognomonic cell of B
cell PLL is a prolymphocyte of
medium size with round
nucleus, moderately abundant
cytoplasm, and distinct
64
 “punched-out” nucleolus.
 The cell size (twice that of a
normal lymphocyte) and
prominent central nucleolus allow
PLL to be distinguished from
CLL/SLL.
 PLL is a disease of the elderly
(mean age of presentation is 70
years).
 Overall prognosis is poor
(median survival is 3 years for B
cell PLL), partly due to the high
incidence of mutation in the tumor
suppressor gene TP53 and the
unavailability of targeted therapy.
HAIRY CELL LEUKEMIA
 Hairy cell leukemia is
characterized by small B
lymphocytes with abundant
cytoplasm and fine (“hairy”)
cytoplasmic projections. T
 The postulated cell of origin is
the peripheral B cell of post–
germinal center stage (memory B
cell).
 Found predominantly in bone
marrow and the red pulp of the
spleen.
 A low number of neoplastic B
cells are seen in peripheral blood.
 Lymph node involvement is rare
 Typical cases of hairy cell
leukemia show strong positivity
for B cell markers (CD19, CD20,
CD22)
 Rare lymphoproliferative disorder
occurring in middle-aged
individuals (median age, 55
years).
 The presenting signs include
splenomegaly and
pancytopenia.
 Durable remissions can be
achieved using purine
analogues.
 Conventional lymphoma
therapy is not effective.
MATURE T CELL AND
NATURAL KILLER CELL
LYMPHOMAS
 Mycosis Fungoides and Sézary
Syndrome Mycosis fungoides is
the most common cutaneous
lymphoma. It is composed of
small to medium-sized lymphoid
cells with irregular nuclear
outlines. The expression of pan–T
cell markers CD3, CD5, and CD2
is seen along with CD4 antigen.
An important feature, rarely seen
in benign lymphoid infiltrates, is
the absence of CD7 antigen.
 Peripheral T cell lymphoma,
unspecified, comprises a
morphologically heterogeneous
group of lymphomas with mature
T cell phenotype.
 Anaplastic Large Cell
Lymphoma- Although
considerable morphologic
variability can be seen, a typical
case of anaplastic large cell
lymphoma is characterized by
large atypical cells with
pleomorphic nuclei and abundant
cytoplasm.
HODGKIN LYMPHOMA
 Nodular lymphocyte-
predominant Hodgkin
lymphoma is a B cell neoplasm
composed of relatively rare
neoplastic cells
(lymphocytic/histiocytic or
“popcorn” cells) scattered within
nodules of reactive lymphocytes.
CLASSICAL HODGKIN
LYMPHOMA comprises a
heterogeneous group of lymphoid
neoplasms derived from the germinal
center. It is characterized by the
presence of relatively few diagnostic
neoplastic cells, Reed-Sternberg cells,
in a rich reactive background.
65
STUDY
BLOOD COLLECTION
1.) The first vein of choice to select
for venipuncture
a. Cephalic Vein

QUESTIONS
b. Median vein
c. Basilic vein
d. Antecubital fossa

2.) Method of blood collection using

a double sided needle whereby the
ANEMIA needle is attached to a holder or
adapter
Q1: Anemia is a common condition. a. Syringe method
What happens when a person has b. Butterfly method
anemia? c. ETS
a. The body produces too much iron d. Winged blood collection
b. The blood does not have enough
red blood cells 3. What is the total blood volume?
c. The blood becomes thick a. 5-6 L
d. Too many white blood cells are b. 4-5 L
produced c. 7-8 L
d. 3-6 L
Q2: Iron-deficiency anemia can
cause pica, a rare condition in which 4.) EDTA stands for?
a person craves eating nonfood a. Ethylenediaminetetraacetic acid
items. Which of these would he or b. Ethylenediaminotetraacetic acid
she eat? c. Ethylenediaminetetracetic acid
a. Ice d. Ethyldiaminetetraacetic acid
b. Soil
5.) Most common and preferred
c. Clay
anticoagulant for coagulation
d. Any of the above
studies
Q3: Deficient DNA synthesis due to
a. EDTA
vitamin B12 or folate deficiency
b. Heparin
a. Thalassemia
c. Sodium Citrate
b. Megaloblastic anemia
d. Oxalates
c. Sideroblastic anemia
d. Aplastic anemia
Q4: To detect the presence of
anemia, the medical laboratory
professional performs a complete HEMATOPOIESIS
blood count (CBC) using an
automated hematology analyzer to
determine
a. RBC count
b. Hemoglobin concentration
c. Hematocrit
d. AOTA

66
 unnecessary treatment of
HEMOGLOBINOPATHIES misdiagnosed iron deficiency.
AND THALASSEMIA 8. Any other medical conditions that
can be caused by or related to
1. What is the percentage of
thalassemia.
hemoglobinopathies result from
mutations in the globin of genes? 9. Ineffective erythropoiesis
A. 70 predominates in _____ thalassemia?
B. 95 A.Beta Thalassemia
C. 80 B.Alpha Thalassemia
D. 85 C.Both of them
2. It is a variant of the sickle cell 10. This form of beta thalassemia is
anemia mutation. The same amino severe and transfusion dependent
acid, Glutamate (6th position in the A. Minor beta thalassemia
beta chain) is mutated to Valine in B. Major beta thalassemia
HbS and to Lysine in HbC. Causes C. Intermedia beta thalassemia
hemolytic anemia D. Silent carrier beta thalassemia
A. HbSS
B. HbC
C. HbSA ANSWER KEY
D. HbH 1. B. 95%
2. HbC
3.Hemoglobinopathies can only be 3. False. It can be both acquired and
acquired and not inherited. True or inherited.
False? 4. It happens in the post-capillary
venules where RBCs get stuck
4. Where does sickling happens?
5. Sickle Cell Anemia
5. It is the most common red sickling 6. True. A procedure called a bone
diseases. Its Point mutation, marrow transplant is the only cure
homozygous recessive disordre, in currently available for
the Beta-globin gene. Mutated beta- thalassemia. However, as this
globin is designated beta-s, reffers procedure can be risky, it is often
to Hb S. Primarily in african- reserved for people with the most
american population (1/500 severe problems from
newborns). Symptoms progress thalassemia.
when Hb F has been replaced with 7. Alpha thalassemia minor
the Hb S during infant period. 8. Iron overload can cause
A. Sickle-c disease complications with the major
B. Sickle b-thalassemia organs. 
C. Sickle Cell Trait 9. A. Beta Thalassemia
D.Sickle Cell Anemia 10. B. Major beta thalassemia

6.Bone marrow transplant is the only

cure for Thalassemias. True or false?

7. It is generally asymptomatic with

mild hypochromic microcytic
anemia. Pregnant women at risk for
hemoglibin Bart's hydrops fetalis.
Important to identify to prevent

67
 myeloid cells.
LEUKEMIA
2. Answer: B
Rationale: a-Naphthyl butyrate esterase
1. SBB stains which of the following positivity in cells of monocytic origin
component of cells? from a patient with acute
a. Glycogen monoblastic/monocytic leukemia.
b. Lipids
c. Structural proteins 3. Answer: B
d. Enzymes Rationale: The expression of pan–T cell
markers CD3, CD5, and CD2 is seen
2. The cytochemical stain a-naphthyl along with CD4 antigen. An important
butyrate is a nonspecific esterase feature, rarely seen in benign lymphoid
stain that shows diffuse positivity in infiltrates, is the absence of CD7
cells of which lineage? antigen.
a. Erythroid
b. Monocytic 4. Answer: B
c. Granulocytic Rationale: Nodular lymphocyte-
d. Lymphoid predominant Hodgkin lymphoma is a B
cell neoplasm composed of relatively
3. The immunophenotype of mycosis rare neoplastic cells
fungoides is: (lymphocytic/histiocytic or “popcorn”
a. The normal T cell immunophenotype cells) scattered within nodules of
b. An abnormal T cell reactive lymphocytes, while Mature B
immunophenotype with expression of cell lymphomas are derived from
CD4 and loss of CD7 antigen various stages of B cell differentiation.
c. A mix of CD41 and CD81 T cells Although they show significant
d. An abnormal T cell morphologic and immunophenotypic
immunophenotyped. Mycosis fungoides heterogeneity, all B cell lymphomas
with expression of CD8 and loss of CD7 produce monoclonal light chain
antigen immunoglobulins, clonal
immunoglobulin gene rearrangements,
4. What is the major morphologic or both.
difference between Hodgkin
lymphoma and other B cell
lymphomas?
REFERENCES
a. The extent of the lymph node Anemia
involvement -
b. The presence of numerous reactive
lymphocytes and only a few malignant Blood Collection
cells in Hodgkin lymphoma - M. Keohane, L. Smith, J.
c. The presence of numerous tingible- Walenga (2015). Rodak’s
body macrophages in Hodgkin Hematology Clinical Principles
lymphoma and Applications 5th edition
d. The preservation of normal lymph - B. Brown. Hematology:
node architecture in Hodgkin lymphoma Principles and Procedures 5th
edition
ANSWER KEY - M.L. Turgeon (2012). Clinical
1. Answer: B Hematology Theory and
Rationale: SBB stains cellular lipids. Procedures 5 edition
th

The staining pattern is quite similar to - B. Thimesch (2018).

that of MPO. SBB staining is possibly a Phlebotomy A how-to guide for
little more sensitive for the early drawing blood

68
 - https://www.interiorhealth.ca/
sites/Partners/LabServices/
SampleProtocols/Documents/
Capillary%20Order%20of
%20Draw%20Quick
%20chart.pdf

Hematopoiesis
- Reference: RODAK’S
HEMATOLOGY Clinical
Principles and Applications.

Hemoglobinopathies/Thalassemia
- Turgeon, M.L. Clinical
Hematology Theory and
Procedures 5 Edition. 2012.
th

Pages 210-227
- Rodak, B., Fritsma, G.,
Keohane.,; Hematology: Clinical
Principles and Applications; 4th
edition; page 391- 403

Leukemia
- RODAK’S Hematology
CLINICAL PRINCIPLES AND
APPLICATIONS Elaine M.
Keohane et.al Chapters 35 and
36 page: 604-639
- WILLIAM’S Hematology 9th
Edition Kenneth Kaushansky
et.al Part XI page: 1505-1527

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