HEMATOLOGY

HEMATOPOIESIS
Hematopoiesis
▪ Hematopoiesis – continuous, regulated process of blood cell production that includes cell renewal,
proliferation, differentiation, and maturation.
▪ Types:
 Primitive hematopoiesis
 Definitive hematopoiesis
▪ Site of hematopoiesis:
 ribs  vertebrae
 sternum  pelvic bones
 skull  proximal ends of the long bones
 scapula
Phases of Hematopoiesis
Mesoblastic phase Hepatic phase Myeloid phase
Site of hematopoiesis Yolk sac Liver (main), spleen Bone marrow
Hemoglobin present Gower I, Gower II, Hb F (major), Hb A1, Hb A2 Hb A1 (major), Hb A2,
Portland Hb F
Blood cells produced Primitive erythroblasts Erythroblasts All blood cells
Granulocytes Monocytes
Megakaryocytes
th
Duration (start-end) Start: 19-20 day Start: 5th to 7th week Start: 5th month of
th st nd
End: 8-12 week End: 1 to 2 week (after gestation
birth) *Lifetime

Note:
✓ In extramedullary hematopoiesis, the spleen, liver, and the lymph nodes revert back to produce
immature blood cells in certain abnormal conditions where the bone marrow cannot produce sufficient
number of hematopoietic cells
✓ Hepatomegaly and splenomegaly are frequently noted on physical examination
Hematopoietic hormones
▪ Erythropoietin
− produced by the kidneys (90%) and the liver (10%)
− Functions:
 prevents the apoptosis of erythroid precursors
 Stimulates Hb synthesis
 Serves as differentiation factor causing the CFU-E to differentiate into pronormoblasts
▪ Thrombopoietin
− Also known as mpL kit ligand
− Synthesized by the liver
Note: the primary source of erythropoietin among the newborns is the liver

Adult Hematopoietic tissue

▪ Bone marrow
 Red marrow
− hematopoietically active marrow
− consists of developing blood cells and their progenitors
 Yellow marrow
− hematopoietically inactive
− composed primarily of adipocytes
− under physiologic stress, the yellow marrow will revert back to active marrow
Retrogression: the process of replacing the active marrow by adipocytes

▪ Liver - the primary site of hematopoiesis during the hepatic phase of hematopoiesis
▪ Spleen
 Responsible for splenic culling/pitting of RBCs
 Removes senescent RBCs
 Stores 1/3 of platelets
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RBC STUDIES
Erythropoiesis
▪ Refers to RBC production
▪ Occurs in distinct anatomical sites called erythropoietic islands where each island consists of a
macrophage surrounded by a cluster of erythroblasts (suckling pig phenomenon)
▪ Tissue hypoxia:
− a condition where oxygen content decreases within the tissues
− primary stimulus for the production of RBCs
− produces a dramatic increase in the production of EPO

Nomenclature for naming of Erythroid Precursors

Normoblastic Erythroblastic Rubriblastic
Pronormoblast Proerythroblast Rubriblast
Basophilic normoblast Basophilic erythroblast Prorubricyte
Polychromatophilic normoblast Polychromatophilic erythroblast Rubricyte
Orthochromic normoblast Orthochromic erythroblast Metarubricyte
Polychromatophilic erythrocyte/Reticulocyte
Erythrocyte
RBC Maturation Series
▪ Earliest recognizable RBC precursor
▪ N:C ratio is 8:1
Pronormoblast ▪ Fine and uniform chromatin pattern
▪ It takes approximately 3 days for the pronormoblast to develop
into orthochromic normoblast
▪ N:C ratio is 4:1
▪ Nuclear chromatin becomes more clumped
Basophilic normoblast ▪ Last stage with nucleolus (last stage of RNA synthesis)
▪ Note: Hb is produced in this stage but not detected in light
microscopy but in EM (Rodak)
▪ Hb production begins during this stage
▪ N:C ratio is 1:1
Polychromatophilic normoblast ▪ Characterized by muddy, light gray appearance of cell due to
variable amounts of pink coloration mixed w/ basophilia
▪ Last stage capable of mitosis
▪ Nucleus is tightly condensed and described as pyknotic
Orthochromic normoblast
▪ Last stage w/ nucleus
▪ Part of this stage occurs in the bone marrow (2 days), and the
later part of this stage takes place in the circulation (1 day)
Polychromatophilic erythrocyte ▪ Anucleate
▪ When stained with supravital stain = reticulocyte
▪ Last stage capable of Hb synthesis
Erythrocyte

Quick summary Notes

Earliest recognizable precursor Pronormoblast/Proerythroblast/Rubriblast
Last stage capable of mitosis Polychromatophilic normoblast/Polychromatophilic
erythroblast/Rubricyte
Last stage with a nucleolus Basophilic normoblast/Prorubricyte
Last stage with nucleus Orthochromic normoblast/Metarubricyte
Last stage that can synthesize Reticulocyte
hemoglobin
Stress or shift retics Caused by increased number of reticulocytes that are prematurely
released from the bone marrow under the stimulus of EPO because of
certain conditions (e.g., hypoxia, acute bleeding)
Polychromatophilia/Reticulocytosis An elevated reticulocyte count accompanied w/ a shortened RBC
survival

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RBC Metabolic Pathway
Embden-Meyerhof pathway ▪ Anaerobic glycolysis
▪ Supplies 90-95% ATP
Hexose Monophosphate Shunt ▪ G6PD and glutathione are generated in this pathway
▪ Purpose: prevents oxidative denaturation of hemoglobin
Methemoglobin Reductase pathway ▪ Maintains the iron present in the Hb in a functional reduced
state (Ferrous iron) for oxygen transport
Rapoport-Luebering shunt ▪ Generates 2,3-DPG

RBC Poikilocytes
Acanthocyte ▪ Alcohol intoxication
Thorny cells ▪ PK deficiency
Spur cell ▪ Congenital abetalipoproteinemia (neuroacanthocytosis)
Spike cell ▪ Severe liver disease (spur cell anemia)
▪ Vitamin E deficiency
▪ Lipid metabolism disorder
Stomatocytes ▪ Hereditary stomatocytosis
Mouth cell ▪ Electrolyte imbalance
▪ Liver disease, alcoholism
▪ Rh null syndrome
▪ Hydroxyurea therapy
Target cells ▪ Hemoglobinopathies (Hb CC, Hb SS, Hb SC)
Codocyte ▪ Liver disease (flattened target surface)
Mexican hat cell
Platycyte
Leptocyte
Greek helmet cell
Bull’s eye cell
Ovalocytes/Elliptocytes Egg shape:
▪ MDS
▪ Thalassemia
▪ Megaloblastic process
Pencil shape:
▪ IDA
▪ Hereditary elliptocytosis
▪ Idiopathic myelofibrosis
Spherocytes ▪ associated with hemolytic process
Bronze cell ▪ ABO HDN
▪ Immune hemolytic anemia
▪ Hereditary spherocytosis
▪ Severe burns
▪ Others: normal aging process, storage phenomenon
Echinocytes ▪ Artifact
▪ Seen in old specimen
(Evenly distributed uniformly sized
blunt)
Burr cells ▪ Associated w/ renal insufficiency
▪ Dehydration
(Irregularly sized and unevenly ▪ Azotemia
spaced spicules)
Schistocytes ▪ Hallmark of hemolytic anemia
Schizocytes ▪ MAHA (DIC, TTP, HUS)
Helmet cells ▪ Exposure of RBCs to heat or mechanical trauma
Triangular cells ▪ Prosthetic heart valve
Keratocytes ▪ Clostridial infection
Keratocytes ▪ Removal of Heinz bodies
Bite cell ▪ Seen in G6PD deficiency
Degmacyte
Horned cell
Semilunar bodies ▪ Overt hemolysis
▪ Associated with malaria
Teardrop cells ▪ Myelofibrosis w/ myeloid metaplasia
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Dacryocytes ▪ Myelophthisic anemia
▪ Pernicious anemia
▪ Beta-Thalassemia
▪ Hypersplenism
Pyropoikilocytes ▪ Severe burns
▪ Hereditary pyropoikilocytosis
▪ NOTE: fragments at 45’C while a normal RBC fragments at
49’C
Drepanocyte ▪ Sickle cell anemia
Sickle cell ▪ Hb SC disease
Folded cell ▪ Hb C disease
Biscuit cell ▪ Hb SC disease

RBC Inclusions
Inclusion Characteristic Composition Clinical Significance
Howell-Jolly bodies Small round reddish-blue Accelerated or abnormal
fragments of nucleus erythropoiesis
DNA
Megaloblastic anemia
(+) Feulgen stain Alcoholism
Cabot rings Reddish violet, thin Megaloblastic anemia
ringlike, figure of eight, Lead poisoning
Mitotic spindle
loop-shaped appearance Homozygous thalassemia
Severe anemia
Pappenheimer bodies Small faint basophilic Refractory anemia
(Wright’s stain) coccoid bodies near the Sideroblastic anemia
periphery of RBCs Iron overload (hemosiderosis,
Siderotic granules hemochromatosis)
Iron
(Prussian blue) NOTE: may resemble Thalassemia
basophilic stippling and Hemoglobinopathies
must be differentiated by
Prussian blue stain
Basophilic stippling Multiple, uniform, evenly Fine:
distributed dark blue ▪ Megaloblastic anemia
granules ▪ Thalassemia
▪ Hemoglobinopathies
RNA ▪ Alcoholism
▪ Pyrimidine-5-
nucleotidase deficiency
Coarse:
▪ Lead poisoning (PICA)
Heinz bodies Single or multiple G6PD deficiency (favism)
purplish inclusions on the Naphthalene ball ingestion
RBC periphery Hemoglobinopathies
Precipitated Hb Thalassemia major
Supravital stains: CV, BCB Sulfonamides
Hb Koln and Hb Zurich
Post-splenectomy
Hb H inclusions Small-greenish blue Hb H disease
“Pitted gold ball
appearance” Hb H inclusion

Supravital stain: BCB

Hb SC crystal Fingerlike or quartz like Hb SC disease
crystal of dense Hb
Hb SC crystal
protruding from the RBC
membrane
Hb C crystal Hexagonal crystal of Hb C disease
Hb C crystal
dense Hb
Malarial inclusions Also known as Malaria
Plasmodium spp.
hemozoin/hematin
Babesia inclusion Resembles maltese Babesiosis/Piroplasmosis
Babesia spp.
cross/tetrads
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RBC Destruction
▪ Extravascular hemolysis
− Macrophage-mediated hemolysis
− Occurs in the spleen
− 90% of RBCs are destroyed
▪ Intravascular hemolysis
− Mechanical hemolysis
− Occurs within the lumen of blood vessels
− Extremely damaged cells are being destroyed within the circulation before they reach the liver
or the spleen
− 10% of RBCs are destroyed
▪ Splenic culling
− Removal of senescent and damaged red blood cells in the spleen
▪ Splenic pitting
− Removal of RBC inclusion bodies in the spleen

NOTE: when red blood cells are destroyed, only

the iron and globin chains are recycled for reuse

HEMOGLOBIN STUDIES
Hemoglobin
▪ Function: to transport oxygen to the tissue and carbon dioxide from tissues to the lungs
▪ Composition:
o 4 heme – consists of protoporphyrin IX and ferrous iron
o 4 globin – consists of 2 identical pairs of unlike polypeptide chains
NOTE:
✓ every heme group is capable of carrying 1 mole of oxygen, therefore each Hb
molecule is able to transport 4 moles of oxygen
✓ 1 gram Hb = can carry 1.34 mL of oxygen
✓ 1 gram Hb = can carry 3.47 mg of iron
✓ there are 4 pyrrole rings for every 1 molecule of iron

▪ Heme synthesis: occurs in the mitochondria

▪ Globin synthesis: occurs in the ribosomes
Genetic Inheritance
Globin Chain Number of amino acids Chromosome
Alpha 141
16
Zeta
Beta
Gamma 146
11
Delta
Epsilon
Normal Hb Variants
Hemoglobin Globin chains Normal values Notes
A1 2 alpha, 2 beta Predominant Hb among the
>95%
adults
A2 2 alpha, 2 delta <3% -
F 2 alpha, 2 gamma Predominant Hb during the
1-2% hepatic phase
Major Hb of newborn
Gower I 2 zeta, 2 epsilon -
Gower II 2 alpha, 2 epsilon - Embryonal hemoglobin
Portland 2 zeta, 2 gamma -

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Abnormal Hb Variants
Carboxyhemoglobin Methemoglobin Sulfhemoglobin
Color imparted Cherry red Chocolate brown Mauve lavender
Reversible YES YES NO
Content Hb bounded with CO Hb that contains iron in Hb bounded with sulfur
oxidized or ferric state
(Fe3+)
Notes The affinity of Cannot transport Once formed, SulfHb will stay in
hemoglobin to CO is 200 oxygen the RBC during its entire 120-
times greater than day lifespan
oxygen
Formation is caused by oxidizing
drugs (acetanilid, phenacetin,
and sulfonamides)

Associated with C. perfringens

infection

Oxygen Dissociation Curve

▪ Graphically describe the relationship between oxygen content and partial pressure of oxygen
▪ Shape of the curve: sigmoid (affected by 2,3-DPG and oxygen)
▪ Oxygen affinity:
o Increase in oxygen affinity – hemoglobin has increased affinity for oxygen (Hb won’t let go its
oxygen)
o Decrease in oxygen affinity – hemoglobin has decreased affinity for oxygen (Hb releases oxygen)
▪ Shift to the left – increased oxygen affinity
▪ Shift to the right – decreased oxygen affinity
▪ Bohr effect – hemoglobin’s affinity for oxygen is influenced by pH
▪ Haldane effect – hemoglobin’s affinity for oxygen is influenced by carbon dioxide
Factors affecting the Oxyhemoglobin Dissociation Curve
Carbon Dioxide Increased: shift to the R
Decreased: shift to the L
pH Increased: shift to the L
Decreased: shift to the R
2,3-DPG Increased: shift to the R
Decreased: shift to the L
Exercise Increased: shift to the R
Temperature Increased: shift to the R
Decreased: shift to the L
Hb Hb F: shift to the L
Hb Chesapeake: shift to the L
Hb Kansas: shift to the R
Iron Metabolism
▪ Together with protoporphrin IX, iron is important for the synthesis of heme
▪ Ferrous iron – functional, absorbable form in the intestine
▪ Normal adult iron level – approximately 400 mg (60% in the circulation; 40% in the storage form)
▪ Storage form of iron
o Ferritin – short-term storage form of iron
o Hemosiderin – long-term storage form of iron
▪ Transferrin
o Also known as siderophilin
o Transport protein of iron
▪ Hepcidin
o Produced by the liver
o Negative regulator of intestinal iron absorption
o Suppresses the release of iron from macrophage
o Plays an important role in anemia of chronic inflammation

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▪ Storage site of iron in the body
o Liver (major)
o Bone marrow
o Spleen

WBC STUDIES
White blood cells:
▪ Main function: primary defense against foreign invaders such as bacteria, viruses and other foreign
antigens
▪ Compartments in the body: bone marrow, peripheral blood, tissues
▪ Nuclear chromatin pattern: most valuable and reliable criterion for deciding whether a cell is mature or
immature
Granulocytes Neutrophil
Basophil
According to granularity Eosinophil
Agranulocytes Monocyte
Lymphocyte
Polymorphonuclear cells Neutrophil
Basophil
According to segmentation Eosinophil
Mononuclear cells Monocyte
Lymphocyte
Phagocytes Neutrophil
Monocyte
According to function
Eosinophil
Immunocytes Lymphocyte

Granulopoiesis
▪ Earliest recognizable blast
▪ No visible granules
Myeloblast
▪ The nucleus is made up of a smooth, delicate, uniformly
distributed chromatin pattern (lacy chromatin pattern)
▪ First appearance of primary granules (azurophilic/non-specific
Promyelocyte
granules)
▪ Appearance of secondary or specific granules
Myelocyte
▪ Dawn of neutrophilia
▪ Also known as Juvenile cells
▪ Appearance of tertiary granules
Metamyelocyte
▪ Nucleus: kidney bean or peanut shaped
▪ Indentation of the nucleus: <50% of the width of the nucleus
▪ Also known as Stab cell/Staff cell
▪ Appearance of secretory granules
▪ Nucleus: sausage-shaped
Band cell ▪ Indentation of the nucleus: >50% of the width of the nucleus
▪ First immature WBC to be released in the circulation
▪ Youngest cell in the granulocytic series to normally appear in
the peripheral blood

Neutrophil
▪ Most common WBC in normal peripheral blood
▪ Two forms in the peripheral blood: Segmenters and Bands
▪ Lifespan:
o 9-10 days (Steininger)
o 5 days (Brown)
▪ Ferrata cell:
o Tissue neutrophil
o Associated with subacute bacterial endocarditis
▪ Barr body:
o Described as drumstick

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o Represents the second X chromosome in females and may be seen in 2-3% of the neutrophils in
females
▪ Neutrophil Pool:
o Pools of neutrophils in the Bone marrow:
 Mitotic/Proliferating pool
− Myeloblasts, Promyelocytes, Myelocytes
− Cells undergoing cell division
 Storage/Maturation pool
− Metamyelocytes, Bands, Segmented neutrophils
− Cells no longer undergoing cell division but progressively maturing
o Pools of neutrophils in the circulation:
 Circulating pool (50%)
 Marginating pool (50%)
Neutrophil Granules
Primary granules Azurophilic/nonspecific granules
Produced by Promyelocytes
▪ MPO
▪ Cathepsins
▪ Acid beta-glycerophosphatase
▪ Defensins
▪ Elastase
▪ Proteinase-3
Secondary granules Specific granules
Produced by Myelocyte
▪ Lactoferrin
▪ Collagenase
▪ Gelatinase
▪ beta-2 microglobulin
▪ Lipocalin
Tertiary granules Produced by metamyelocyte
▪ Gelatinase
▪ Collagenase
▪ Lysozyme
▪ Acetyltransferase
▪ Beta-2 microglobulin
Secretory granules Also known as secretory vesicles
Produced by Band cell
▪ ALP
▪ Vesicle-associated membrane-2
▪ CD13, CD10, CD14, CD16, CD18

Eosinophil
▪ Plays a major role in defense against parasitic invasion and in hypersensitivity reaction
▪ Important products: MBP and Charcot-Leyden crystals
▪ Major Basic Protein – an arginine-rich protein that plays an important role in the eosinophil’s ability to
damage parasites
▪ Charcot-Leyden crystals:
− Hexagonal pyramidal crystals
− Found in the nasal mucus of patients with allergic asthma, pleural fluid of patients w/ pulmonary
eosinophilic infiltrates, and stool of patients w/ parasitic infections
Basophil
▪ Mediator of Immediate hypersensitivity reaction
▪ Basophils and mast cells have specific receptor for IgE
▪ Important products: Histamine and Heparin
Monocyte
▪ Main function: phagocytosis
▪ Slightly immature cells whose ultimate goal is to enter the tissues and mature into macrophages
▪ General appearance:
o Size: 15-20 um
o Chromatin pattern: lacelike
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o Nucleus: horseshoe or tulip shape
o Cytoplasm: blue-gray with fine azure granules often referred to as azure dust or a ground glass
appearance
▪ Maturation series: monoblast → promonocyte → monocyte → macrophage
▪ Important notes:
o There is no storage pool of monocytes in the bone marrow
o Proliferation in the bone marrow: 55 hours
o A mature monocyte spends about 12 hours in the peripheral blood before going to the tissues
o The marginal pool of monocyte in the peripheral blood is 3.5 times greater than the circulating
pool

Lymphocyte
▪ Main function: immune response
▪ Serves as a “marker cell” for estimating the size of surrounding cells
▪ T-lymphocytes – most small lymphocytes
▪ B-lymphocytes – most large lymphocytes
▪ NK cells – third population of lymphocytes
▪ Lifespan: several months to years
Plasma cells
▪ Mononuclear cells with round or oval and smooth or irregular margins
▪ The eccentrically located nucleus is composed of blocks of heterochromatin resembling a tortoise shell
▪ Nucleus exhibits a cartwheel pattern
▪ The area next to the nucleus containing the Golgi apparatus is unstained (Hof)
▪ Russel bodies: located on the cytoplasm that may contain round, discrete globules that appear pale-
clue, or occasionally red which contains immunoglobulins
▪ Morula cell/grape cell/Mott cells – cluster of Russel bodies
RBC DISORDERS
Polycythemia
▪ Associated with increased RBC count, hemoglobin, hematocrit
▪ May be classified as relative or absolute
NOTE: A hematocrit value of >52% in men and >50% in
women is often diagnostic of Polycythemia

ABSOLUTE POLYCYTHEMIA
Refers to true increase in red cell mass
▪ Primary Polycythemia – due to bone marrow defect
▪ Secondary Polycythemia – due to kidney defect
Polycythemia Vera
▪ Chronic myeloproliferative disorder
▪ Due to mutation of JAK2 gene
▪ Pancytosis: absolute increased in RBC, WBC, platelets
▪ Panhyperplasia: the bone marrow is hypercellular showing
an overall increase
▪ Characterized by hyperviscous blood (due to increased RBCs)
Absolute Primary Polycythemia
▪ Prone to IDA (therapeutic phlebotomy)
▪ Hallmark of PV: plethora
Clinical features:
▪ ESR is decreased
▪ EPO is decreased
▪ LAP is increased
▪ Dacryocyte in PBS is a common finding
▪ Due to increased level of EPO
▪ This may occur as a normal response to tissue hypoxia or as
a result of inappropriate EPO production
Absolute Secondary Polycythemia
Causes:
▪ Residence at high altitudes
▪ Chronic pulmonary disease

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▪ CHF
▪ Heavy smoking
▪ Methemoglobinemia
▪ Tumor of the kidneys
RELATIVE POLYCYTHEMIA
▪ Due to decrease in the fluid (plasma) portion of the blood that gives the appearance of an increased
red cell mass in relation to total blood volume rather than a true increase in red cell mass
▪ NOT a hematologic disorder
▪ Actual number of RBC in the blood is not increased, but the number of cells per unit volume of blood
is increased
1. Dehydration secondary to diarrhea, vomiting, excessive
sweating, burns, anaphylaxis, and diuretics
2. Anxiety and stress
3. Tobacco smoking
Causes 4. Gaisbock’s syndrome
− also known as Spurious polycythemia or Stress
syndrome
− Associated with smoking, CVD, hypertension, and
diuretic therapy
Anemia
 A decrease in red blood cells, hemoglobin, and hematocrit below the reference range for healthy
individuals of the same age, sex, and race, under similar environmental conditions
Mechanisms of Anemia
A. Due to Production
▪ Ineffective erythropoiesis
▪ Insufficient erythropoiesis
B. Due to Destruction
▪ Intrinsic defects in the RBC membrane, enzyme, or hemoglobin
▪ Extrinsic causes such as antibody-mediated process, mechanical fragmentation, or infection-
related
Test for Accelerated RBC destruction
1. Lactate dehydrogenase
2. Indirect bilirubin
3. Chromium Radioisotope: the reference method for RBC survival studies by ICSH
Laboratory Diagnosis of Anemia
1. CBC and RBC indices
2. Reticulocyte count – serves as an important tool to assess the bone marrow’s ability to increase RBC
production in response to anemia
3. Peripheral Blood Smear Examination
4. Bone Marrow Examination – indicated for a patient with an unexplained anemia, fever of unknown
origin, or suspected hematologic malignancy
5. H/H – widely used tests for anemia

ANEMIA OF BONE MARROW FAILURE

Aplastic Anemia ▪ A condition in which there is a peripheral blood pancytopenia
▪ Pancytopenia:  RBC, WBC, Platelets, Reticulocytes
▪ Lymphocytes are the predominant cell in the peripheral blood
(less affected)
Clinical Features:
▪ Bleeding
▪ Infection
▪ Anemia
▪ No splenomegaly
▪ No lymphadenopathy
Causes ▪ Genetic defect
▪ Ionizing radiation
▪ Chemicals
▪ Parvovirus B19
▪ Benzene
▪ Chloramphenicol (most common cause)
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▪ Trinitrotoluene
▪ Arsenic
Hereditary ▪ Fanconi Anemia
▪ Diamond-Blackfan anemia
Acquired ▪ Chronic Kidney Disease
▪ Myelophthisic anemia
Fanconi Anemia
Description ▪ Also known as Congenital Aplastic Anemia
▪ Autosomal recessive
▪ Pancytopenia
▪ Normocytic anemia
Signs and Symptoms ▪ Low birth weight (<2,500 gram)
▪ Skin hyperpigmentation (café au lait spots)
▪ Short stature
▪ Renal malformations
▪ Microcephaly
▪ Mental retardation
▪ Hypogonadism
▪ Strabismus
Diamond-Blackfan Anemia
▪ Also known as Congenital Pure Red Cell Aplasia
▪ Defective/reduced CFU-E
▪ Caused by a mutation in RPS19 gene; idiopathic (Steininger)
▪ Normocytic anemia w/ normal leukocyte and platelet count and a marked decrease in marrow
erythroblasts
Myelophthisic Anemia
▪ Also known as leucoerythroblastic anemia
▪ Common finding in patients with carcinoma
▪ Results when the bone marrow is replaced by abnormal cells such as metastatic tumor cells, leukemic
cells, fibroblasts, and inflammatory cells (found in miliary TB and fungal infections)
▪ Myelophthisis = invasion of abnormal cells
Lab Picture:
▪ Normocytic anemia
▪  Reticulocyte, Teardrop cells, nRBCs, immature myeloid cells in the peripheral blood, presence of
abnormal cells in the bone marrow
Anemia of Chronic Kidney Disease
▪ Anemia is due to inadequate production of EPO by the kidneys
▪  EPO, presence of Burr cells (Uremia)

ANEMIA OF ABNORMAL NUCLEAR DEVELOPMENT

▪ Impaired DNA synthesis affects all rapidly dividing cells of the body, including the skin, GIT, and bone
marrow
▪ Vitamin B12 and Folate are essential in DNA synthesis
▪ Deficiencies of either vitamin impair DNA replication, halt cell division, and increase apoptosis, which
results in ineffective erythropoiesis and megaloblastic morphology
Megaloblastic Anemia:

Vitamin B12 Deficiency

▪ Dietary source: meat
▪ The liver stores adequate amount of Vitamin B12 for several years if no more is ingested
▪ Intrinsic factor: forms a protective complex with Vitamin B12 that is transported down the GIT
▪ Vitamin B12 is maximally absorbed in the ileum
Causes:
▪ Inadequate intake
▪ Increased need
▪ Impaired absorption
Pernicious Anemia ▪ Autoimmune disorder characterized by impaired absorption of vitamin
B12 due to lack of IF
▪ Most common form of Vitamin B12 deficiency
▪ More common in people with blood type A

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D. latum infection ▪ The organism has the ability to split vitamin B12 from IF, rendering the
vitamin unavailable for host absorption
Blind loop syndrome ▪ Portions of the intestines becomes stenotic as a result of surgery or
inflammation
▪ These sites can become overgrown with intestinal bacteria that compete
effectively with the host for available vitamin B12
Imerslund-Grasbeck ▪ Causes Vitamin B12 malabsorption
syndrome ▪ Not related to IF deficiency/defect
▪ Defect in cubilin/amnionless receptor (Henry’s)
Schilling Test (Classical Test)
Principle ▪ Provides a measure of body’s ability to secrete viable IF and absorb orally
administered 57Co-labeled B12 in the ileum
Specimen requirement ▪ Fasting specimen
▪ A 24-hour urine collection is begun immediately upon administration of
the labeled B12 by mouth
Interpretation Phase 1 (radiolabeled B12 w/o IF)
▪ >7% of labeled B12 is excreted = Dietary B12 deficiency
▪ <7% of labeled B12 is excreted = Proceed to Phase 2

Phase 2 (radiolabeled B12 w/IF)

▪ >7% of labeled B12 is excreted = Pernicious anemia
▪ <7% of labeled B12 is excreted = Malabsorption syndrome
(Tropical Sprue, D. latum, Blind Loop syndrome)
Folate Deficiency
Sources of Folate: green leafy vegetables
Causes:
▪ Inadequate intake
▪ Increased need
▪ Impaired absorption
▪ Excessive loss due to renal dialysis
▪ Alcohol (alcohol interferes with folate metabolism)
Macrocytic Non-Megaloblastic Anemia
▪ Lacks hypersegmented neutrophil and oval macrocytes in the peripheral blood and megaloblasts in
the bone marrow
▪ Physiologic cause: newborn
▪ Pathologic cause: liver disease, chronic alcoholism, bone marrow failure
Differential Tests for Folate and Vitamin B12 Deficiency
Tests Folate deficiency Vitamin B12 deficiency

Pancytopenia ( RBC, WBC, Platelet count)

 MCV, MCH
CBC
 RDW
 Reticulocyte count
Screening Tests

Macroovalocytosis
Teardrop cells
PBS examination
nRBCs
Hypersegmented neutrophils
Presence of megaloblasts
Bone marrow examination  M:E ratio
Hypercellular bone marrow
Autoantibodies
- (+) anti-parietal cells, anti-IF
Specific Tests

Gastric analysis
NORMAL Achlorhydria
Stool analysis
- (+) D. latum eggs

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Anemia of Iron and Heme Metabolism
Mechanisms:
1. Deficiency of raw material (e.g., iron)
2. Defective release of stored iron from macrophages (Anemia of Chronic Inflammation)
3. Defective utilization of iron within the erythroblast (SDA, lead poisoning)
Tests used to differentiate iron metabolism disorders:
1. Serum Ferritin
2. Serum Iron
3. FEP
4. TIBC
5. Transferrin saturation
6. Zinc Erythrocyte Porphyrin
▪ Reflects the body’s tissue iron stores
▪ A good indicator of iron storage status
Serum Ferritin ▪ First laboratory test to become abnormal when iron stores begin
to decrease
▪ Decreased only in IDA
▪ Helpful in cases where the diagnosis is not obvious from other
Serum Iron
laboratory tests
▪ Normally, red cells produced slightly more protoporphyrin than is
needed
FEP
▪ However, when iron is deficient, protoporphyrin levels builds up
in RBCs than the normal level
▪ Indirect measurement of transferrin concentration
TIBC ▪ Measures the binding site
▪ ( Anemia of chronic inflammation;  IDA)
▪ Obtained through the measurements of serum iron and TIBC
Transferrin saturation
▪ %Transferrin saturation = serum iron/TIBC x 100
▪ Measures unused protoporphyrin
Zinc Erythrocyte Porphyrin Test
▪ Increased in IDA, Lead poisoning, and Porphyria
Iron Deficiency Anemia
Description ▪ Most common form of anemia
▪ The individual may not exhibit signs or symptoms until the
appearance of Frank anemia
▪ Microcytic, hypochromic anemia
▪  Iron
▪  TIBC and FEP
▪ Normal Reticulocyte count
▪  Reticulocytes after iron therapy
Causes ▪ Inadequate intake
▪ Increased body demand (pregnancy, growing children)
▪ Impaired absorption
▪ Chronic blood loss due to infection (Hookworm infection)
▪ Marching anemia: develops when RBCs are hemolyzed by foot-
pounding trauma and iron is lost (hemoglobinuria is a common
finding)
Stages of iron loss ▪ Stage 1 – progressive loss of storage iron
▪ Stage 2 – exhaustion of the storage pool of iron
▪ Stage 3 – Frank anemia (storage pool and circulatory iron is
depleted)
Characteristic Symptoms ▪ Glossitis
▪ Angular cheilosis – inflamed cracks at the corner of the mouth
▪ Koilonychia – spooning of the fingernails
▪ Pica
▪ Pagophagia
Laboratory findings ▪ Decreased: RBC count, H/H, MCV, MCH, MCHC
▪ Increased RDW (anisocytosis)
▪
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HEMATOLOGY
Sideroblastic Anemia
Description ▪ Sideroblasts are iron-containing normoblasts found in a normal
bone marrow
▪ The iron deposits are identified using Prussian blue stain, and the
resulting abnormal cells are identified as ringed sideroblasts
▪ Caused by: defective iron loading (accumulation of erythroid
precursor in the mitochondria) due to deficiency of ALA
synthetase
Types Hereditary:
▪ Hereditary Sideroblastic Anemia
Acquired:
▪ Refractory Anemia w/ Ringed Sideroblasts
▪ Idiopathic Acquired Sideroblastic Anemia
▪ Primary Idiopathic Sideroblastic Anemia
▪ Secondary Sideroblastic Anemia
Hereditary Sideroblastic Anemia ▪ Severe anemia (Hct = <20%)
▪ Dimorphic population of RBCs:
✓ Normocytic, normochromic
✓ Microcytic, hypochromic
▪ (+) Target cells and basophilic stippling
▪  iron and % transferrin saturation
Primary Idiopathic SDA ▪ More common
▪ Moderate anemia (Hct = 25-30%)
▪ (+) normocytes and macrocytes w/ few microcytes
▪ Erythroid hyperplasia w/ ringed sideroblasts in all stages of
development
Secondary SDA ▪ Due to toxins and drugs that interfere w/ heme synthesis
▪ Alcoholism, lead poisoning, TB drugs, and chloramphenicol
Anemia of Chronic Inflammation
Description ▪ Second most common anemia
▪ Associated with infections, inflammatory, or malignant diseases
of more than 1 or 2 months of duration
▪ Most common anemia among hospitalized patients
▪ Iron appears to be trapped in macrophages, therefore iron is not
made available for reutilization in normoblasts
▪ The anemia is often corrected when the primary disease is
resolved
▪  Transferrin (due to being a negative APR)
Causes ▪ Tuberculosis
▪ Lung abscess
▪ Bacterial endocarditis
▪ Neoplasms
▪ RA
▪ Rheumatic fever
▪ SLE
▪ Chronic liver disease
Hepcidin ▪ Hormone produced by the liver to regulate body iron levels
particularly absorption of iron in the intestine and release of iron
from macrophages
Lactoferrin ▪ Iron-binding protein in the granules of the neutrophils
▪ Prevents phagocytized bacteria from using intracellular iron
▪ During infection and inflammation, inflammation is released into
the plasma
Ferritin ▪ Binds iron
▪ Because developing RBCs do not have a ferritin receptor, this iron
is unavailable for incorporation into hemoglobin
Lead Poisoning
Description ▪ A form of acquired porphyria and acquired Sideroblastic Anemia
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▪
Children may be exposed to lead secondary to ingestion of lead-
containing paint
▪ Also associated with the use of improperly glazed pottery for
cooking or eating
▪ Similar to Sideroblastic anemia (lead inhibits several enzymes
needed in heme biosynthesis)
▪ Anemia, when present in lead poisoning, is most often
normocytic and normochromic; however, with a chronic
exposure to lead, a microcytic hypochromic clinical picture may
be seen
Laboratory finding ▪ (+) Coarse basophilic stippling
Porphyria
Inherited disorder of defective heme synthesis
Accumulation of porphyrin precursors
Hemochromatosis
Rare autosomal recessive disorder common in males
Abnormal iron deposition in the tissues causing “bronze skin pigmentation”
Associated with Bronze diabetes
Normal H/H
 Iron and transferrin saturation
 Transferrin

Iron Studies Result

Iron Deficiency Anemia of Chronic Sideroblastic
Tests Lead Poisoning
Anemia Inflammation Anemia
Serum Ferritin    N
Serum Iron    N
TIBC   /N N
Transferrin  /N  
saturation
BM Iron (Prussian No stainable iron /N  N
Blue reaction)

Anemia of Abnormal Globin Development

Hemoglobinopathies Caused by qualitative structural abnormalities of the
globin chains that result from alteration of genetic
sequence
Thalassemia Quantitative defect/reduction in globin chain
synthesis
High incidence in Mediterranean descent
Sickle Cell Anemia
Description ▪ Homozygous SS = sickle cell anemia
▪ Heterozygous SS = sickle cell trait
▪ Autosomal codominant
▪ When fully oxygenated, hemoglobin S is fully soluble (reversible)
HEMOGLOBINOPATHIES

▪ Sickling occurs when oxygen decreases at the tissue level

▪ Provides resistance against P. falciparum
▪ When oxygen is released from the molecule, a conformational
change occurs, which results in polymerization of Hb molecule
leading to the formation of tactoids or crystals which causes the
cells to become rigid
Sickle cell crises
 Any situation that produces excessive deoxygenation of the RBC may cause painful sickle
cell crises (e.g., infection, dehydration, strenuous exercise, obstetric delivery and high
altitudes)

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Vaso occlusive crises ▪ Occur when rigid sickle cells increase the blood viscosity
▪ Associated with the development of microthrombi, vascular
occlusions, and microinfarction in the joints and extremities as
well as in the major organs, which can cause organ failure
Infectious crises ▪ Primary cause of death of patients with sickle cell anemia
▪ Causative agent: S. pneumoniae (common in children)
▪
Hand-Foot syndrome ▪ Also known as dactylitis
▪ First sites affected by decreased blood flow are the small bones
of hands and feet
Lab Findings ▪ Normocytic, normochromic anemia
▪ (+) Sickle cell, Target cell, Ovalocyte, Schistocyte, Polychromasia,
nRBC
▪  RDW
▪  OFT, ESR
▪  M:E ratio
NOTE ▪ Splenic sequestration occurs when sickle cells become trapped in
the splenic microcirculation.
▪ The spleen enlarges as more cells are trapped leading to
hypovolemia which may cause shock and death
Hemoglobin C disease
Hemoglobin C ▪ 2nd most common Hb variant
Hemoglobin CC ▪ Hb CC tends to crystallize when dehydrated
▪ Note: the cells most vulnerable to intracellular crystallization are
older RBCs because they tend to lose water as they age
Lab Finding ▪  MCHC
▪ Normocytic, normochromic anemia
▪ (+) Target cell, Spherocyte
▪ (+) Hb CC or SC crystal
Alpha Thalassemia
Bart’s Hydrops Fetalis ▪ Caused by deletion of all four alpha globin genes (--/--) resulting
in the production of hemoglobin Barts (γ4)
▪ Hb Barts has high affinity for oxygen
▪ The disorder is lethal; infants are usually stillborn or die within
hours of birth
Hb H Disease ▪ Caused by deletion of 3 out of 4 alpha globin genes (--/-a)
▪ This disorder results from the decreased synthesis of alpha chains
and the resultant formation of the unstable hemoglobin, Hb H
(β4)
▪ At birth = Hb Barts (due to the presence of gamma globin
chains)
THALASSEMIA

▪ Adult = Hb H (due to availability of beta globin and replaces

Barts Hb
Alpha Thalassemia ▪ Also known as alpha thalassemia trait
minor ▪ Caused by deletion of 2 out of 4 alpha globin genes
▪ Heterozygous a0 (--/aa)
▪ Homozygous a+ (a-/a-)
Silent carrier ▪ Also known as heterozygous alpha thalassemia
▪ Caused by deletion of 1 out of 4 alpha globin genes (-a/aa)
▪ Benign, and often discovered only during family studies
Beta Thalassemia
Results to reduced production of beta globin chains → excess production of alpha chain (unstable)
Thalassemia major ▪ Also known as Cooley’s anemia/Mediterranean anemia
▪ Beta globin chain synthesis is impaired
▪  Hb A2
Pathophysiology ▪ With the absence or marked decreased in beta-chain production,
there is an excess production of alpha chains

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▪ Aggregates of alpha chains are unstable and precipitate in the
normoblast or red cell
▪ These excess alpha chains will precipitate
▪ These precipitates are removed by the spleen causing ineffective
erythropoiesis and severe hemolytic anemia
Clinical Features ▪ Marked skeletal deformities with frontal bossing, cheek bone and
jaw protrusion

Hereditary Persistence of Hemoglobin F

Characterized by increased levels of Hb F in adults in the absence of the usual hematologic features of
thalassemia

Enzymopathies
st
G6PD Deficiency 1 most common
Most common RBC enzymopathy
Produced in: HMP
Triggers of hemolysis: antimalarial drugs, fava beans
Classical finding: Heinz bodies

PK Deficiency 2nd most common

Produced: EMP
PK deficiency can lead to hemolytic anemia
Pyrimidine-5-nucleotidase 3rd most common
Deficiency

Hemoglobin Variants
Hemoglobin C Substitution of glutamic acid to lysine at 6th position of the β-chain
Hemoglobin E Substitution of glutamic acid to lysine at 26th position of the β -chain
Hemoglobin Kansas Substitution of asparagine to threonine at 102nd position of the β-chain
Hemoglobin O-Arab Substitution of glutamic acid to lysine at 121st position of the β-chain
Hemoglobin D-Los Angeles Also known as D-Punjab
Substitution of glutamic acid to glycine at 121st position of the β-chain
Hemoglobin C-Harlem Also known as C-Georgetown
Caused by two amino acid substitutions
Substitution of glutamic acid to valine at 6th position and aspartic acid to
asparagine at 73rd position of the β-chain
Hemoglobin G-Philadelphia Substitution of asparagine to lysine at 68th position of the alpha chain
Hemoglobin Chesapeake Substitution of arginine to leucine at 92nd position of the alpha chain
Hemoglobin Constant Spring Addition of 31 amino acids in the alpha chain
Hemoglobin Gun Hill Amino acid deletion
Hemoglobin Koln Unstable hemoglobin

Membrane Defect – Inherited

Hereditary Spherocytosis Protein 4.1 and Spectrin deficiency
(+) Autohemolysis test
 OFT
Hereditary Elliptocytosis Protein 4.1 deficiency
Hereditary Pyropoikilocytosis Spectrin deficiency
Commonly seen in burn patients
Southeast Asian Ovalocytosis Band 3 deficiency
Hereditary Xerocytosis Hemolytic anemia with dehydrated red blood cells
(+) stomatocytes, target cells, macrocytes
Hereditary Stomatocytosis Also known as hydrocytosis
Neuroacanthocytosis Characterized by neurologic impairment and acanthocytes on the PBS
Abetalipoproteinemia Also known as hereditary acanthocytosis
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(Bassen-Kornzweig Syndrome) Due to absence of β-lipoprotein
Characterized by malabsorption of fat, retinitis pigmentosa, neurologic
damage and acanthocytosis
Chorea Acanthocytosis Characterized by chorea, hyperkinesia, cognitive impairment, and
neuropsychiatric symptoms
McLeod syndrome Due to KX gene mutation
Kx substance – precursor for the production of Kell antigen
Membrane Defect – Acquired
Paroxysmal Nocturnal ▪ Also known as Marchiafava-Micheli syndrome
Hemoglobinuria ▪ Hemoglobinuria occurs at night when blood pH falls (acidic)
▪ Complication of PNH may progress to aplastic anemia
▪ Cause: stem cell mutation that results in circulating blood cells
that lack CD55 and CD59
▪ CD55 = DAF
▪ CD59 = MIRL
▪ CD55 and CD59 are complement-inhibiting regulator proteins
Lab Diagnosis:
▪ Screening test: Sugar water test or sucrose hemolysis test
▪ Confirmatory test: Acidified Serum Test (Ham’s test)
▪  LAP
Spur cell anemia Due to severe liver disease that develop a hemolytic anemia with
acanthocyte

Fragmentation Syndrome
Microangiopathic Hemolytic Anemia
 A disease of small blood vessels
 Can be a complication of one of several conditions in which there is a disturbance of the
microvascular environment (DIC, TTP, HUS)
DIC ▪ When there is extensive damage to vessel endothelium or
exposure to compounds to initiate clotting (thromboplastic
substances that encourage coagulation), DIC may follow
▪ As a direct result of fibrin deposition along and across the vessel
lumen, RBCs can be fragmented or destroyed as they are pushed
through the vessel by the action of blood pressure and rapidly
flowing circulation
▪ (+) Schistocyte
▪ (+) D-dimer
TTP ▪ Also known as Moschkovitz syndrome
▪ Due to ADAMTS13 deficiency
▪ (+) Schistocyte
▪ (+) D-dimer
HUS ▪ Most common in children
▪ Involves acute intravascular hemolysis and renal failure
▪ Causative agent: E. coli O157:H7
▪ EHEC produces Shiga-like toxin
▪ (+) Shistocytes, Burr cells, polychromasia
▪  BUN, Creatinine

Acquired Immune Anemia of Increased Destruction

Autoimmune Hemolytic Anemia WAIHA
CAIHA
PCH
Alloimmune Hemolytic Anemia HDN
HTR
DIHA
Paroxysmal Cold Hemoglobinuria Caused by Autoanti-P (biphasic hemolysin)
Diagnostic test: Donath-Landsteiner Test
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Infection
Leishmaniasis
Malaria
Babesiosis
B. bacilliformis

WBC DISORDERS
A. Malignant WBC Disorders
a. Myeloproliferative disorder
b. Myelodysplastic syndrome
c. Leukemia
d. Lymphoma
B. Non-malignant WBC Disorders

MYELOPROLIFERATIVE DISORDERS
Description Also known as Chronic Myeloproliferative Disorders
NOTE: Splenomegaly is a common finding (extramedullary hematopoiesis)
Examples Polycythemia vera
Essential Thrombocythemia
Primary Myelofibrosis
CML
Essential Thrombocythemia
Description ▪ Also known as Primary Thrombocytosis, Idiopathic Thrombocytosis
▪ Characterized by a thrombocytosis of 1000 x 109/L with spontaneous
aggregation of functionally abnormal platelets
▪ Must be differentiated from secondary or reactive thrombocytosis
Gene abnormality ▪ Due to mutation of JAK2 gene
Lab Findings ▪ Markedly increased platelet count
▪ Bone marrow examination: megakaryocytes stick together
(Glued together appearance)
Primary Myelofibrosis
Description ▪ Characterized by fibrosis and granulocytic hyperplasia of the bone
marrow, with granulocytic and megakaryocytic proliferation in the liver
and spleen
▪ Hepatomegaly and splenomegaly are common (extramedullary
hematopoiesis)
Gene abnormality ▪ Due to mutation of JAK2 gene
Lab Findings ▪ Normocytic, normochromic anemia
▪  Reticulocyte count
▪  WBC
▪ PBS: Teardrop cell, Polychromatophilia, NRBC
▪ Bone marrow examination: “dry tap” → presence of fibrotic tissues
Chronic Myelogenous Leukemia
Description ▪ A stem cell disorder affecting the granulocytic, monocytic, erythrocytic,
and megakaryocytic cell lines
▪ Common in adults (between ages of 30 and 50)
▪ A WBC count of 50,000-300,000/uL is often diagnostic of CML
▪ Cause: translocation between the long arms of chromosome 9 and 22
▪ Philadelphia chromosome: indicates good prognosis
▪ Must be differentiated from Leukemoid reaction using LAP test
Lab Findings ▪  RBC, WBC, and Platelet count
▪ BM Examination: hypercellular bone marrow
▪  M:E ratio
▪  LAP

Myelodysplastic Syndrome
Description Formerly known as Refractory anemia
Caused by proliferation of abnormal stem cells
Lab Findings Poikilocytosis, Basophilic stippling, Howell-Jolly bodies, and Siderocytes

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French-American-British 1. RARS
Classification of MDS 2. RAEB-t
3. RAEB
4. CMML

Leukemia
 An abnormal, uncontrolled proliferation and accumulation of one or more of the hematopoietic cells
 A disease of the blood forming tissues and the bone marrow
Classification of Leukemia
Based on duration of the untreated disease ▪ Acute leukemia: several days to 6 months
▪ Subacute leukemia: 2 to 6 months
▪ Chronic leukemia: 1-2 years
Based on number of WBC present ▪ Leukemic leukemia: >15,000/uL
▪ Subleukemic leukemia: <15,000/uL w/ immature WBCs
▪ Aleukemic leukemia: <15,000/uL
Based on WBC type involved ▪ Acute leukemia: predominance of blasts
▪ Chronic leukemia: predominance of mature WBCs
Acute Lymphoblastic Leukemia (ALL)
L1 Small cell, homogenous
Most common acute leukemia in children
Best prognosis
L2 Large cell, heterogenous
L3 Burkitt type
Large lymphocytes w/ basophilic cytoplasm and numerous
vacuoles
Poor prognosis
Acute Myeloblastic Leukemia (AML)
M0 Acute myeloblastic leukemia, minimally differentiated
M1 Acute myeloblastic leukemia without maturation
M2 Acute myeloblastic leukemia with maturation
M3 Acute promyelocytic leukemia
Characterized by the presence of bowtie/butterfly appearance
of nucleus
DIC is common
(+) FSP
(+) Faggot cells
PT and APTT: prolonged
 Fibrinogen

M4 Acute myelomonocytic leukemia

Also known as Naegeli monocytic leukemia
M5 Acute monocytic leukemia
Also known as Schilling leukemia
M5a:
▪ Poorly differentiated leukemia
▪ Nucleus: lacy chromatin w/ nucleoli
M5b
▪ Well differentiated leukemia
▪ Nucleus: cerebriform shape with nucleoli
▪ Presence of all stages of monocytes in the peripheral
blood
M6 Erythroleukemia
Also known as Di Guglielmo’s syndrome
Predominance of myeloblasts and erythroblasts in the
peripheral blood
M7 Acute megakaryocytic leukemia
Predominance of megakaryocytes
(+) PAS, ACP, platelet peroxidase
AML Stain Reaction
MPO M1, M2, M3, M4
SBB M1, M2, M3, M4

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HEMATOLOGY
Specific esterase (Naphthyl AS-D M1, M2, M3, M4
chloroacetate)
Nonspecific esterase (Naphthyl Acetate M4, M5, M6
and Butyrate)
PAS M5, M6, M7
Differential Cytochemical Test for ALL vs. AML
ALL AML
MPO - +
SBB - +
Terminal deoxynucleotidyl transferase + -
PAS + -
Oil Red O + -
Chronic Lymphocytic Leukemia
Most affected lymphocytes are B-cells
Characterized by fragile lymphocytes
Smudge cells (formed during film preparation, thumbprint appearance)
(+) PAS
Chronic Myelocytic Leukemia
Description ▪ A stem cell disorder affecting the granulocytic, monocytic, erythrocytic,
and megakaryocytic cell lines
▪ Common in adults (between ages of 30 and 50)
▪ A WBC count of 50,000-300,000/uL is often diagnostic of CML
▪ Cause: translocation between the long arms of chromosome 9 and 22
▪ Philadelphia chromosome: indicates good prognosis
▪ Must be differentiated from Leukemoid reaction using LAP test
Lab Findings ▪  RBC, WBC, and Platelet count
▪ BM Examination: hypercellular bone marrow
▪  M:E ratio
▪  LAP
▪ (+) Basket cells = nuclear remnants of granulocytic cells w/ netlike
chromatin pattern
Hairy Cell Leukemia
Also known as Leukemic Reticuloendotheliosis
Hairy cell: lymphocyte w/ hairlike projections around the outer border
(+) TRAP

Lymphoma
 A group of malignant tumors of the lymphoid tissue
 Usually, blood and bone marrow are not involved
 Classification: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Miscellaneous lymphoma

Hodgkin’s Lymphoma
Starts in one lymph node group and spreads in a predictable fashion to adjacent lymph nodes (unifocal)
Definitive diagnostic test: Lymph node biopsy
▪ Rye Classification = classifies the different types of Hodgkin’s lymphoma
▪ Ann Arbor staging system = used to formulate the treatment plan
Types of Hodgkin’s Lymphoma:
a. Nodular lymphocyte-predominant Hodgkin lymphoma
− presence of popcorn cell
b. Classical Hodgkin lymphoma
− presence of Reed-Sternberg cell
− types:
✓ Lymphocytic predominant (best prognosis)
✓ Lymphocyte depleted (worst prognosis)
✓ Mixed cellularity
✓ Nodular sclerosis (most common type)
Non-Hodgkin’s Lymphoma
Spreads in a much less predictable way
Classified under Rappaport system (replaced by NCI)
Types:
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✓ Well differentiated lymphocytic lymphoma
✓ Poorly-differentiated lymphocytic lymphoma
✓ Histiocytic lymphoma
✓ Mixed histiocytic-lymphocytic lymphoma
Miscellaneous Lymphoma
Mycosis fungoides ▪ Caused by neoplastic T-cells that migrate into the skin
▪ Affects primarily the T-cells
▪ Classic symptoms: pruritus
▪ Pautrier's microabscesses = cluster of lymphocytes in the
epidermis
▪ NOTE: fungal infection is not present
Sezary syndrome ▪ Leukemic phase of mycosis fungoides
▪ (+) Sezary cells
▪ Prognosis is worst at this stage

PLASMA CELL DYSCRASIAS

Multiple Myeloma ▪ Also known as Kahler’s disease
▪ Caused by excessive production of IgG
▪ The homogenous protein synthesized by the abnormal
clone may be complete immunoglobulins or light chains
(kappa and lambda)
▪ Idiopathic
▪ Clonal proliferation begins in the bone marrow and
multiple tumors appear as patchy infiltrates in skeletal
structures producing osteoporosis and lytic bone disease
▪ As the neoplastic mass grows, pathologic bone fractures
and vertebral collapse may occur
Lab Findings:
▪ (+) Bence Jones proteins in urine
▪ PBS: Rouleaux formation, Dutcher bodies, Russell bodies
Waldenstrom’s Macroglobulinemia ▪ Caused by excessive production of IgM
▪ Monoclonal IgM may exhibit cryoglobulin activity
demonstrated by precipitation or gel formation during
refrigeration at 4’C and dissolve when heated
▪ May result to renal damage caused by deposition of IgM
complexes
▪  ESR

NON-MALIGNANT WBC DISORDERS

Leukemoid Reaction ▪ Characterized by a WBC count of greater than 50,000/uL
▪ Resembles CML
▪ Differential test: LAP score
▪  LAP
Leukoerythroblastic Reaction ▪ Also known as Leukoerythroblastic anemia
▪ (+) NRBCs and immature neutrophils in peripheral blood smear
▪ Caused by space-occupying disturbances of the bone marrow
Causes:
▪ Myelofibrosis w/ myeloid metaplasia
▪ Metastatic carcinoma
▪ Leukemia
▪ Multiple myeloma
▪ Gaucher disease
Pelger-Huet anomaly ▪ Hyposegmented neutrophils (bilobed)
▪ The bilobed nuclei are commonly described as “pince-nez
spectacles” or peanut or dumbbell shape
▪ Homozygous = round nucleus
▪ Heterozygous = pince-nez nucleus (more common)
Undritz anomaly ▪ Also known as hereditary hypersegmentation of neutrophils
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May-Hegglin anomaly ▪ Characterized by leukopenia, thrombocytopenia, giant platelets,
and presence of gray-blue spindle-shaped inclusions in the
cytoplasm of granulocytes and monocytes
▪ NOTE: the cytoplasmic inclusion resembles Dohle bodies
Alder-Reilly anomaly ▪ Characterized by the presence of abnormally large azurophilic
granules resembling severe toxic granulation in the cytoplasm of
granulocyte, lymphocyte and monocyte
▪ Associated with mucopolysaccharidoses
Job’s syndrome ▪ Random movement of phagocytes is normal, but chemotaxis
(directional motility) is impaired
▪ As a result, bacteria have more time to multiply in the tissues
Lazy Leukocyte syndrome ▪ Both random and directed movement of the phagocytes are
impaired
Chediak Higashi anomaly ▪ Presence of giant cytoplasmic granules in the phagocytes and
lymphocytes
Chronic Granulomatous Disease ▪ Inability of phagocytes to produce superoxide and ROS
▪ Due to NADPH oxidase
▪ Diagnostic test: (-) NBT dye test
Myeloperoxidase deficiency ▪ Also known as Alius-Grignaschi Anomaly
▪ Most common form of neutrophil abnormality
▪ Function abnormality is not severe
NON-MALIGNANT WBC ANOMALIES
LE cell Neutrophil that contains a large spherical SLE
body in its cytoplasm
Tart cell Formed during LE cell preparation Unknown
(May be confused with LE cell)
Gaucher cell Crumpled tissue paper appearance Gaucher disease
Foam cell Macrophage with swollen cytoplasm Niemann-Pick disease
composed of numerous small, uniform lipid
droplets
Morula cell Abnormal plasma cell with immunoglobulin Plasma cell myeloma
trapped in endoplasmic reticulum
Basket cell Nuclear remnants of granulocytic cells with CML
netlike chromatin pattern
Formed during blood film preparation
Smudge cell Nuclear remnants of lymphocytes CLL
Formed during blood film preparation
Thumbprint appearance
Reed-Sternberg cell Large lymphoid cells with a bilobed nucleus Hodgkin’s lymphoma
with prominent eosinophilic nucleoli and
abundant cytoplasm (owl’s eye appearance)
Popcorn cell Large lymphoid cells with abundant cytoplasm Nodular Lymphocyte
and vesicular multilobed nuclei Predominant-Hodgkin’s
lymphoma
Flame cell Plasma cell with abundant cytoplasm with a IgA myeloma
reddish tinge of ribosomal protein
Hairy cell Lymphocyte with hairlike projections around HCL
the outer border
Faggot cell Contains bundles of Auer rods in the AML M3/APL
cytoplasm
Downey cells Also known as Reactive lymphocytes, atypical Non-malignant reactive
lymphocytes, stress lymphocytes, virocytes, disorders
variant lymphocytes, transformed
lymphocytes
Types of Downey cells:
✓ Type 1 – Turk’s cell (also known as plasmacytoid lymphocyte, Turk’s irritation cell)
✓ Type 2 – Infectious mononucleosis cell (Flared skirt appearance)

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HEMATOLOGY
✓ Type 3 – Transformed/Reticular lymphocytes
WBC INCLUSIONS
Dohle bodies ▪ Pale blue, round or elongated bodies
▪ Consists of rRNA
▪ Found in neutrophils
▪ Resembles May-Hegglin bodies
▪ Associated with pregnancy, infection, poisoning, burn and
surgery
May-Hegglin bodies ▪ Larger than Dohle bodies
▪ Consists of mRNA
▪ Found in granulocytes and monocytes
▪ Associated with May-Hegglin Anomaly
Alder-Reilly bodies ▪ Heavy, coarse, blue-black granulation of the leukocytes
▪ Resembles toxic granules
▪ Associated with Hurler’s and Hunter’s syndrome and Alder-Reilly
Anomaly
Toxic granules ▪ Dark blue-black cytoplasmic granules in the neutrophil
▪ Composed of primary granules
▪ Associated with severe infection
Vacuolation ▪ Results when the degenerating cytoplasm begins to acquire holes
or as the result of active phagocytosis
▪ Associated with infection
Auer rods ▪ Rod-like bodies representing aggregated primary granules that
stain a reddish purple
▪ Associated with AML

SPECIMEN COLLECTION
Methods of Collection
 Macrosampling
o Venipuncture
▪ Preferred sites: Median cubital vein, cephalic vein, basilic vein
▪ Alternative sites: Ventral forearm, Wrist area, back of the hand, Ankle vein, foot
✓ NOTE: if ankle or foot must be used, the attending physician or nurse must be
consulted first because these sites cannot be used in patients with certain clinical
conditions (uncontrolled diabetes, hemoglobinopathies)
▪ Angle of the needle: 15-30 degree (25 = average)
▪ Torniquet application: 3-4 inches above the venipuncture site
▪ Duration of torniquet application: less than 1 minute
▪ Factors to consider when selecting site:
✓ Hematoma
✓ Burns
✓ Scars
✓ Edema
✓ Site on which a mastectomy was performed
✓ Site with IV infusion
→ Specimens should be drawn from the opposite arm
→ If IV infusion may be running in both arms, blood should be drawn
below the IV line after the IV fluids have been stopped for 2-5 minutes
→ A small amount of blood should be discarded before specimen
collection because the specimen is diluted with IV fluid
o Arterial Puncture
▪ Modified Allen test – used to determine whether the ulnar artery can provide circulation
to the hand after the radial artery puncture
▪ Preferred sites: brachial artery, femoral artery, radial artery, inguinal artery
▪ Angle of the needle:
✓ Femoral artery: 90 degrees
✓ Radial and brachial artery: 45-60 degree
▪ Performed without the application of torniquet
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HEMATOLOGY
▪ Anticoagulant: 0.05 mL of heparin per mL of blood
 Microsampling
o The method of collection for pediatric patients, geriatric patients, obese, patients with
thrombotic tendencies, severe burns, and for POCT
o Preferred sites:
▪ Infants – medial or lateral plantar heel surface of the big toe
▪ Adults – 3rd or 4th finger; margins of ear lobe
o Sites to be avoided:
▪ Infants – central arch area of the heel and fingers
▪ Adults – thumb, index finger, 5th finger, on the site of mastectomy, edematous, or
previous puncture site
o Length of lancet: 1.75 mm (<2 mm)
 Defibrination
− Special hematology collection procedure
− Involves the removal of fibrin from a whole blood specimen
− Whole blood is added to an Erlenmeyer flask containing glass beads or paper clips
− The flask is rotated for about 10 minutes until the beads or paper clips are covered w/ fibrin and
no longer make a rattling noise
− Tests that require defibrination:
o OFT
o Autohemolysis test
o Ham’s acidified serum test
Materials Needed
 Needles
o Gauge 21 – routine venipuncture
o Gauge 23 – pediatric patients
o Gauge 25 – for small veins
 Evacuated tube
o For adult specimens: 5-7 mL of evacuated tube
o For pediatric specimens: 2-3 mL of evacuated tube
 Torniquet
o Rubber
o Velcro
o Seraket – the phlebotomist can partially release venous pressure, thus preventing
hemoconcentration w/o removing the torniquet
o Blood pressure cuff: 40-60 mmHg
o NOTE: tests that does not require torniquet application = calcium and ABG

Anticoagulants used in Hematology

 EDTA
− Most commonly used in Hematology
− Chelates calcium
− Optimal concentration: 1.5 mg/mL of blood
− Blood to anticoagulant ratio = 4:1
− Inversion: 8 times
− K2EDTA = spray-dried
− K3EDTA = liquid (causes 1-2% dilution)
− ADVANTAGE:
o preserves cellular morphology when the blood smears are made within 2 hours of
collection
o may be used for preparation of blood films within 2-3 hours of blood collection
− DISADVANTAGE: excessive EDTA induces RBC shrinkage causing the hematocrit and ESR to be
falsely decreased
− Effects of Excessive EDTA:
o  Hematocrit
o  ESR
o  MCV
o  MCHC
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o  Platelet count (due to platelet satellitism phenomenon)
o WBC Morphology – cytoplasmic vacuolization of neutrophils and monocytes
 Citrate
o Light Blue top
− Inhibits blood clotting by binding the calcium in a soluble complex
− Blood to anticoagulant ratio = 9:1
− Additive: 3.2% Na Citrate (0.109 M)
− Inversion: 4 times
− For coagulation studies (PT, APTT)
o Black top
− Additive: 3.8% buffered Na Citrate
− Blood to anticoagulant ratio = 4:1
− Inversion: 4 times
− For ESR
 Heparin
− A mucopolysaccharide (mucoitin polysulfuric acid) that inhibits coagulation by inactivation of
thrombin
− Heparin accelerates the action of antithrombin III, neutralizing thrombin, and preventing the
formation of fibrin
− Both an in vivo and in vitro anticoagulant
− The only anticoagulant that has the least effect in different tests
− Forms: Lithium heparin and Sodium heparin
− Optimal concentration = 15-20 U/mL of blood
− Anticoagulant of choice for: Osmotic Fragility Test, Serum Electrolytes
− DISADVANTAGE:
o Not used in blood film preparation because it causes morphologic distortion of platelets
and WBCs
o Can interfere in some immunoassays
o It causes bluish coloration of the background on blood films stained with Romanowsky
stain because of its pH
o Should never be used for coagulation studies because of its inhibitory effect on
thrombin
o Expensive
 Oxalate
− Chelates calcium
− Anticoagulant: oxalate
− Antiglycolytic/preservative: Na Fluoride
− Inhibits glycolysis for 3 days by forming a complex with magnesium, and inhibiting the
magnesium-dependent enzyme, enolase.
− Used in ESR (Wintrobe method)
− Ammonium oxalate and Potassium oxalate does not shrink RBCs
− Inversion: 8 times
− Optimal concentration: 1-2 mg/mL of blood

Order of Draw
Open and Closed System Order of Draw
1. SPS Yellow top Blood culture
2. Citrate Light blue top Coagulation studies
3. Plain tube Red top/SST Chemistry
4. Heparin Green top Electrolytes, OFT
5. EDTA Lavender top CBC
6. Oxalate Gray top Glucose determination
Catheter Line Order of Draw
Draw 3-5 mL in a syringe Discard
Blood culture tube Yellow top
Anticoagulated tubes Lavender top, green top, Light blue top, etc.
Plain tube Red, Yellow, Gold top

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Capillary Puncture Order of Draw
1. Blood gas analysis
2. Slides (unless made from a specimen in EDTA microcollection tube)
3. EDTA microcollection tube
4. Other microcollection tube w/ anticoagulant
5. Serum microcollection tube
Physiologic Factors
 Posture
 Diurnal variation
 Exercise
 Stress
 Diet
 Smoking
Complications in Blood Collection
1. Ecchymosis (Bruise)
 Most common complication
 Cause by leakage of small amount of fluid around the tissue
2. Syncope (Fainting)
 Second most common complication
 If the patient begins to faint, the phlebotomist should remove the needle immediately
3. Failure to draw blood
 Due to improper needle positioning
 Number of attempts: _____
Factors to Consider
✓ The patient’s arm should be kept straight and fully extended and may be elevated above the heart
✓ Do not bend the patient’s arm at the elbow, as this reopens the wound and may result in hemorrhage
into surrounding tissues
✓ Before leaving the patient, be certain that the bleeding has stopped
✓ If bleeding does not cease, attending personnel must be notified.
ROUTINE HEMATOLOGY PROCEDURES
Smear Preparation
 smears using blood anticoagulated w/ EDTA should be made within 2-3 hours of blood collection
Types of Blood Smear According to Method
 Cover Glass Smear
− Also known as Ehrlich’s method
− Cover glass to cover glass
− Advantage: even distribution of WBC
− Disadvantage: time consuming, difficult to master, harder to label, easily broken
 Glass slide-coverslip method
− Also known as Beacom’s method
 Wedge smear
− Glass slide to glass slide
− Also known as push smear method
− Smear should occupy 1/2 to 2/3 of the slide
− Distance of drop of blood from the labeled end: 1 cm
− Angle between the spreader slide and the stationary slide: 30-45 degree
− Size of drop of blood: 2-3 mm in diameter
− Causes of poor blood smear: too large or too small drop of blood
− Size of smear adjustment:
o Thick smear: increased angle
o Thin smear: decreased angle
− ADVANTAGE:
o Easier to master
o Slides are not easily broken
o Least expensive
o Tendency of larger cells to settle at the slide edges and the feathered end makes it
easier to find abnormal cells

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− DISADVANTAGE:
o Poor distribution of nucleated cells
o Greater trauma to the cells during film preparation, this may lead to a large number of
smudge or basket cells

Characteristics of an Ideal Blood Smear

✓ Gradual transition from thick to thin area
✓ Smear occupies 1/2 to 2/3 only of slide
✓ No overlapping cells
✓ Should have a feathery edge
Problems encountered in Wedge Smear
Gritty appearance of feathered
Too thick smear Too thin smear
end
Too large drop of blood Too small drop of blood Increased nucleated cells (WBCs)
Too fast spreading Too slow spreading Too slow spreading
Too high angle Too low angle Delay in spreading
 Spun smear (Automated method)
− Prepared using Hemaspinner
− ADVANTAGE:
o WBCs are evenly distributed
o RBCs are free of distortion

Types of Blood Smear According to Specific Purpose

 Buffy coat smear
− For patients with WBC less than 1,000/uL
− It concentrates NRBCs
 Thick and thin blood smear
− For malarial parasite
Staining Techniques
 Supravital stain
o BCB
o NMB
o CV
 Romanowsky stain (routinely used to stain peripheral blood and bone marrow smears)
o Wright’s
o Giemsa
o Wright-Giemsa
o May-Grunwald
o Jenner
o Leishmann
 Wright’s stain
− Most commonly used stain
− Fixative: methanol
− Acidic stain: Eosin
✓ Has an affinity for basic substance (negatively charge)
✓ Basic substance: Hemoglobin
− Basic stain: Methylene blue
✓ Has an affinity for acidic substance (positively charge)
✓ Acidic substance: DNA, RNA
 Iron stain – Prussian blue (Perl’s reaction)

Criteria for a Good Stain (Wright’s)

✓ RBC – salmon pink
✓ WBC – purple-blue nuclei
✓ Eosinophils – orange granules (excellent pH meter)
✓ Neutrophils – pinkish tan cytoplasm
✓ Monocytes – gray ground-glass cytoplasm
✓ Platelets – purple-blue to lilac cytoplasm

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Causes of Color Deviation
TOO REDDISH TOO BLUISH
Too acidic buffer of stain Too alkaline buffer
Insufficient staining time Excessive staining
Excessive washing Inadequate washing
Very thin smear Short drying period
Contaminants in wash water (e.g., chlorine) Wash water too alkaline
Exposure of buffer or stain to acid fumes Thick smear
Old stain (methanol undergoes oxidation) Old smear (dried plasma produces blue background)
Protein abnormality
Heparin is used
Very high WBC count with many blasts
Low hematocrit
Methods of Counting
 Cross-sectional or Crenellation
− WBCs are counted in consecutive fields as the blood film is moved from side to side
− Counting should begin in the thin area of the smear where the RBCs are slightly overlapping and
proceed into the thicker area
 Longitudinal method
− WBCs are counted in consecutive fields from the tail toward the head of the smear
− Ideal method for thin smear
 Battlement method
− Uses a pattern of consecutive fields beginning near the tail on a horizontal edge
Complete Blood Count
 Consists of RBC count, hemoglobin, hematocrit, RBC indices, platelet count, WBC count, and WBC
differential count
 Screening procedure for the diagnosis of various disease
 Considered as the primary screening test for the diagnosis of a certain disease

Hemocytometry
 The Improved Neubauer Counting Chamber
− Consists of 2 identically ruled platforms with a raised ridge on both sides of the 2 platforms on
which a cover glass is placed
− The space between the top of the platform and the cover glass over it is 0.1 mm (depth)
− Each of the 2 platforms contains a ruled area composed of 9 large squares of equal size
− Each large square is 1 mm wide and 1 mm long
− Therefore, the entire ruled area is 9mm2 (3 mm wide and 3 mm long)
− The volume of the entire ruled area on one platform is 0.9 uL (width x length x depth)
− The volume of 1 square is 0.1 uL
Thoma Pipette
RBC Pipette WBC Pipette
Bulb size Larger Smaller
Bead color Red White
Volume 100 10
Dilution 1:200 1:20
Dilution range 100-1000 10-100

RBC COUNT
Counting chamber ▪ The 5 small squares are the areas to be counted for the RBC
▪ The large central square contains 25 smaller squares
▪ The large central square has a volume of 0.1 uL
▪ The volume of each of the 25 smaller squares is 0.004 uL, for a total
volume of 0.02 uL for 5 small squares
Thoma Pipette ▪ Has 0.5 and 101 marks
RBC Diluting Fluid ▪ NSS (Eagle’s fluid)
− For emergency use only
− Ideal for excessive rouleaux formation and autoagglutination
▪ Hayem’s fluid
− Initiates mold formation and rouleaux formation
− Can be stored for a longer period with no corrosive effect
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▪ Gower’s solution
▪ Toisson’s fluid – initiates mold formation; with methyl violet
▪ Dacie’s fluid (Formol-Citrate)
− BEST RBC diluting fluid
▪ 3.8% Na Citrate
Computation RBC count = no. of RBC counted x VCF x DF
VCF 1 cumm / volume of 1 square used x no. of squares used
DF Volume of fluid in the bulb / volume of sample used
WBC COUNT
Counting chamber ▪ The 4 corner large WBC squares are the areas to be counted for WBC
▪ These 4 large squares are divided into 16 small WBC square
Thoma Pipette ▪ Has 0.5 and 11 marks
WBC Diluting fluid ▪ 2-3% Glacial Acetic acid
▪ 1% HCl
▪ Turk’s solution (w/ methyl violet)
Computation WBC count = no. of WBCs counted x DF x VCF
CORRECTED WBC COUNT
Corrected WBC count is done if 5 or more NRBCs per 100 WBC are present
Formula: Uncorrected WBC count / 100 + no. of NRBCs x 100

WBC Differential Count

Differential Count Normal Values
Neutrophil 51-67%
Lymphocytes 25-33%
Monocytes 2-6%
Eosinophils 1-4%
Basophils 0-1%

 100-cell differential
− The relative number of the various types of leukocytes present in the peripheral blood smear,
expressed in percentage (%)
 50-cell differential
− May be performed when the WBC count is <1 x 109/L
− Alternative method: buffy coat smear
− NOTE: a notation must be made on the report that 50 WBCs were counted
 200-cell differential
− May be performed when the differential shows an abnormal distribution of cell types
− The results are then averaged (divided by 2)
− Criteria:
o >10% eosinophils
o >2% basophils
o >11% monocytes
o more lymphocytes are present than neutrophils (except in children)
− NOTE: a notation must be made on the report that 200 WBCs were counted
Absolute WBC Count
 Gives the number of specific WBC type per uL of blood
 Absolute count = WBC differential count x WBC count
Absolute Count Normal Values
Neutrophil 1,600-7,260/uL
Lymphocytes 960-4,400/uL
Monocytes 180-880/uL
Eosinophils 45-440/uL
Basophils 45-110/uL

Hemoglobin Measurement
 Cyanmethemoglobin method
o HiCN is the most stable among various hemoglobin pigment
o Most accurate method
o Drabkin’s reagent:

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▪ KCN: converts Hi → HiCN
▪ (K3Fe[CN]6): oxidizes Ferrous iron to Ferric iron
▪ Non-ionized detergent (Sterox or Triton): liberates hemoglobin
▪ KH2PO4 (NaCO3: original Drabkin’s) → shorten the time in converting hemoglobin to
HiCN from 10 minutes to 3 minutes
o Physiologic errors:
▪ Lipemic blood – use patient blank
▪ High WBC count (>30,000/uL) – centrifuge, use the supernatant
▪ Abnormal Hb – dilute sample 1:1
o NOTE: HiCN can measure all forms of hemoglobin except Sulfhemoglobin
 Chemical method
o Indirect method of hemoglobin determination based on the iron contents of blood
▪ Wong’s method
▪ Kennedy method
▪ Assendelft method
 Gasometric method
o Indirect method of hemoglobin determination based on the oxygen content of blood
▪ Van Slyke’s method
 Gravimetric method
o Based on specific gravity
o Based from the fact that specific gravity of blood is the ratio of the weight of a volume of blood
to the weight of the same volume of water at room temperature
o Uses a standard copper sulfate solution, 40 copper sulfate solution to be exact (reagent) with
specific gravity of 1.035 to 1.075 at interval of .001
o Also known as Copper Sulfate method

 Clinical Significance:
o Increased: Polycythemia, dehydration (burns, diarrhea)
o Decreased: all types of anemia, leukemia
o NOTES:
▪ After 50 years of age = slightly decrease
▪ Hemoglobin is lower if lying down
▪ Increased in smokers
▪ Increased among males
▪ Increased in high altitude
Hematocrit
 Also known as Packed Cell Volume (PCV)
 Expressed as percentage of total whole blood volume
 Prone to parallax error (an object being seen in a different position)
 Sources of Errors:
o Increased Hct:
▪ Including the buffy coat in the reading
▪ Inadequate centrifugation
▪ Allowing the sample to stand too long after centrifugation
▪ Trapped plasma
o Decreased Hct:
▪ Excess anticoagulant
▪ Improper sealing of the capillary tube
 Methods:
o Macrohematocrit method (Wintrobe)
o Microhematocrit method (Spun hematocrit)

MICROHEMATOCRIT METHOD
Capillary tube Length: 75 mm
Volume: 0.05 mL
Length of sealant: 4-6 mm
Inner bore: 1.2 mm in diameter
Should be filled with whole blood approximately 2/3
of the tube
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Capillary tube types Red band
Blue band
Centrifugation 10,000-15,000 x g for 5 minutes
MACROHEMATOCRIT METHOD
Wintrobe tube Length: 115 mm
Graduations: red and white markings
Diameter: 3 mm
Centrifugation 3,000 RPM for 30 minutes
Formula Height of PCV / Height of whole blood x 100
Disadvantage Time consuming
Requires large amount of blood
Contains large amount of trapped plasma

RBC Indices
 MCV (Mean Cell Volume)
o Indicates the average volume of RBCs in femtoliters (10-12L)
o MCV = Hct (%) / RBC count x 10
o MCV = Hct (L/L) / RBC count x 1000
o Normal value: 80-96 fL
o MCV Values:
▪ >100 fL (Macrocytic)
▪ 80-100 fL (Normocytic)
▪ <80 fL (Microcytic)
 MCH (Mean Cell Hemoglobin)
o Indicates the average weight of hemoglobin in the RBC
o Expressed in picogram (10-15L)
o Calculated from hemoglobin and RBC count
o Least valuable in the diagnosis of anemia
o MCH = Hgb (g/L) / RBC count
o MCH = Hgb (g/dL) / RBC count x 10
o Normal values: 27-33 pg
 MCHC (Mean Cell Hemoglobin Concentration)
o Ratio of the weight of the hemoglobin to the volume of RBC and expressed as percentage or in
g/dL
o Expression of the average concentration of hemoglobin in the RBC
o Reflects the RBC staining intensity and amount of central pallor
o Calculated from hemoglobin and hematocrit
o MCHC = Hgb / Hct (%) x 100
o MCHC = Hgb / Hct (L/L)
o Normal values: 32-36 g/dL or 320-360 g/L
o MCHC values:
▪ >36% (hyperchromic RBCs)
▪ 32-36% (normochromic RBCs)
▪ <32% (hypochromic RBCs)
 RDW (Red Cell Distribution Width)
o Fourth RBC index
o Expresses the degree of variation in RBC volume/size
o Routinely reported in automated cell counters
o Normal value: 11.5-14.5
o Clinical significance:
▪ Increased in IDA
▪ Normal in Thalassemia
Erythrocyte Sedimentation Rate
 Directly proportional to the red cell mass and inversely proportional to plasma viscosity
 Nonspecific test used to detect and monitor and inflammatory process
 Replaced by CRP
 ESR is affected by 3 factors: RBC, Plasma composition, and mechanical factors
 NOTE: a tilt of 3° can cause errors up to 30%
 Plasma composition: single most important factor determining the ESR
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 Methods of ESR
Wintrobe Westergren
Anticoagulant Double oxalate/Paul-Heller’s 3.8% Na Citrate
solution/Ammonium Potassium EDTA (mod. Westergren)
Oxalate
Tubes Length: 115 mm Length: 300 mm
Normal value Male: 0-9 mm/hr Male: 0-10 mm/hr
Female: 0-20 mm/hr Female: 0-15 mm/hr
NOTE: ESR needs to be corrected for anemia when ESR is high but hematocrit is low

 Stages of ESR
o Lag phase (10 minutes)
▪ Initial rouleaux formation
o Decantation phase (40 minutes)
▪ Period of rapid settling
o Period of final settling (10 minutes)
 Factors in ESR
o Intrinsic factors: RBC and Plasma
o Extrinsic factors: Mechanical and Technical Factors
INTRINSIC FACTORS EXTRINSIC FACTORS
Faster ESR ( ESR): Faster ESR ( ESR):
▪ Macrocyte ▪ Longer tube
▪ Less viscous plasma ▪ Wider tube
▪ Alpha globulins ▪ Slightly inclined tube
▪ Beta globulins ▪ Increased temperature
▪ Hyperalbuminemia ▪ Presence of bubbles
▪ Increased fibrinogen ▪ Less blood in tube
Slower ESR ( ESR): Slower ESR ( ESR):
▪ Polycythemia ▪ Decreased temperature
▪ Microcyte ▪ Excess anticoagulant
▪ More viscous plasma
▪ Hypoalbuminemia
▪ Poikilocytosis

 Clinical Significance
o Markedly increased ESR
▪ Multiple myeloma
▪ Waldenstrom’s Macroglobulinemia
▪ Malignant Lymphoma
▪ Acute and severe bacterial infections
▪ Collagen disease
▪ Sarcoma
▪ Severe renal disease
o Moderately increased ESR
▪ Rheumatic fever
▪ Rheumatoid arthritis
▪ Myocardial infarction
▪ Hyperthyroidism
▪ Hypothyroidism
▪ Tuberculosis
o Decreased ESR
▪ Poikilocytosis
▪ Sickle cell anemia
▪ Hemolytic anemia
▪ Severe IDA
▪ Thalassemia
Zeta Sedimentation Rate
 ZSR is dependent on fibrinogen and gamma globulin
 Represents the percentage of sedimented RBCs
 To obtain the ZSR, the value is compared with the patient’s hematocrit
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 ZSR (%) = Hct / Zetacrit x 100
 Normal value: 40-51%
 ADVANTAGE:
o Requires small amount of sample
o Not affected by anemia
o Reference range for both male and female is the same
Reticulocyte Count
 Marker of effective RBC production of bone marrow in response to hemolysis and loss of RBC
 Reticulocytes: young RBCs containing residual RNA and cannot be seen using Wright’s stain (requires
supravital staining)
 In the bone marrow, it spends approximately 2-3 days maturing and is then released into the blood
where it spends 1 day in the circulation before it undergoes complete maturation
 Indication of increased red cell demand:
o Polychromatophilia/Shift cells
o Poor man’s bone marrow aspirate (reflects BM activity)
 Supravital stains: BCB, NMB (recommended by NCCLS)

Computation:
Reticulocytes (%) = no. of retics / 1000 RBCs x 100

Normal Value: 0.5% to 1.5%

 Clinical Significance:
o Increased Retic count:
▪ Acute and chronic blood loss
▪ Newborn (physiologic)
▪ Sideroblastic anemia
▪ Hemolytic anemia
▪ IDA
▪ Thalassemia
o Decreased Retic count
▪ Bone marrow failure (aplastic anemia)
▪ Megaloblastic anemia
▪ Parvovirus B19 infection
Absolute Reticulocyte Count (ARC)
 Reflects the actual number of reticulocytes

Computation:
ARC = Retic count (%) x RBC count

Normal Value: 25 to 75 x 106/uL

Corrected Reticulocyte Count (CRC)

 Also known as Reticulocyte Index or Hematocrit Correction
 corrects the observed retic count to a normal hematocrit of 0.45

Computation:
CRC = Retic count (%) x Hct/0.45

Normal Value: 1%

Reticulocyte Production Index (RPI)

 Indicator of the rate of RBC production increase above normal in anemia
 Also known as Shift Correction
 Clinically more useful than CRC

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 Hematocrit and Maturation Time
HEMATOCRIT MATURATION TIME (DAYS)
0.45 1.0
0.35 1.5
0.25 2.0
0.15 2.5
 RPI Formula = Corrected Retic count / Maturation time
 RPI Interpretation
RPI = 1 Normal, only in the absence of anemia
RPI <1 Presence of anemia due to:
Insufficient erythropoiesis
Ineffective erythropoiesis
RPI >2 Bone marrow is responding
RPI >3 Hemolytic process, because reticulocyte is more than 3 times increased

Eosinophil and Basophil Count

 Eosinophil count
o Diluting fluid:
▪ Phyloxine diluting fluid
▪ Pilot’s solution
o Stain: Randolph’s stain
 Basophil count
o Stain: Cooper and Cruickshank

Estimates

✓ For each platelet, there are 10 to 40 RBCs in normal peripheral blood smear
✓ For every 100 RBCs, there should be 3 to 10 platelets
✓ For every 200 RBCs, there should be 5 to 20 platelets
✓ In every 1000 RBCs, there is 1 WBC

Grading and Reporting

A. Degree of Hypochromia
Normal Area of pallor is 1/3 of the cell diameter
1+ Area of pallor is 1/2 of the cell diameter
2+ Area of pallor is 2/3 of the cell diameter
3+ Area of pallor is 3/4 of the cell diameter
4+ Thin rim of hemoglobin

B. Polychromasia
1+ 1-3 polychromatic cells are found per field
2+ 3-5 polychromatic cells are found per field
3+ >5 polychromatic cells are found per field

C. Rouleaux formation
Slight If 1-2 RBC chains are found per field
Moderate If 3-4 RBC chains are found per field
Marked If 5 or more RBC chains are found per field

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RBC Morphology Grading and Reporting
Morphology Grading and Reporting
Bizarre-shaped RBC
Elliptocyte
Burr cell
1+ = 3-10/OIF
Ovalocyte
2+ = 11-20/OIF
Target cell
3+ = >20/OIF
Poikilocytosis
Stomatocytes

Helmet cell
Acanthocyte
Spherocyte 1+ = 1-5/OIF
Schistocyte 2+ = 6-10/OIF
Polychromatophilia 3+ = >10/OIF
Teardrop cell

Basophilic stippling
Howell-Jolly bodies
POSITIVE ONLY
Sickle cell
Pappenheimer bodies
NOTE: Poikilocytosis is graded when there is more than 10%/hpf
Reference: Brown, B.

SPECIAL HEMATOLOGY PROCEDURES

A. Bone marrow examination
 Sites for BM Examination
 Posterior superior iliac crest – preferred site for adults
 Sternum
 Spinous process of the lumbar vertebrae (rarely used)
 Tibia – for children <2 years old
 Bone Marrow Biopsy
 To demonstrate the bone marrow architecture: the spatial relationship of hematologic
cells to fat, connective tissue, and bony stroma
 Used to estimate cellularity
 Uses Jamshidi needle
 Bone Marrow Aspiration
 To identify the types and proportions of hematologic cells and to look for morphologic
variance
 Uses University of Illinois sternal needle
 NOTES
 M:E Ratio
− the relative proportions of the two principal bone marrow cell lines (Myeloid
and Erythroid)
− Normal M:E ratio = 3:1-4:1
−  M:E ratio
• Infection
• CML
• Erythroid hypoplasia
−  M:E ratio
• Normoblastic hyperplasia
• Depression of leukopoiesis
 Dry tap – the inability to aspirate marrow into the syringe
B. Leukocyte Alkaline Phosphatase
 Principle
 Used to differentiate Leukemoid reaction from CML
 Neutrophil are the only WBC that normally contain various amounts of ALP
 The degree of reactivity is determined by scoring each of 100 neutrophils according to
the stain intensity
 Scoring method = Kaplow score
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 Grading:
0 No staining
1+ Faint and diffuse staining
Pale, with a moderate amount of
2+
blue staining
3+ Strong blue precipitated staining
Deep blue or brilliant staining w/
4+
no visible cytoplasm
 Specimen requirement
 Fresh capillary blood
 HEPARIN (because EDTA has an inhibitory effect on the staining reaction
 Normal value: 30-185
 Clinical significance:
 INCREASED LAP
▪ Multiple myeloma
▪ Aplastic anemia
▪ Leukemoid reaction
▪ Intoxication
▪ Polycythemia vera
▪ Pregnancy (3rd trimester)
 DECREASED LAP
▪ CML
▪ PNH
▪ Sickle cell anemia
C. Tests for Hemoglobin S
a. Dithionite Tube Test
 Screening test for the detection of sickling hemoglobin
 NOT specific for Hb S
 Principle:
 RBCs are lysed by saponin, allowing Hb to liberate
 Sodium dithionite binds and removes oxygen from the test environment
 Hb S polymerizes in the resulting deoxygenated state and forms a precipitate in
a high-molarity phosphate buffer solution
 These precipitates consist of tactoids (liquid crystals), which deflects light, and
make the solution turbid
 (+) result: turbidity
 False (+) results: Hb C-Georgetown and Hb C-Harlem, hyperlipidemia,
hypergammaglobulinemia
b. Sodium Metabisulfite Method
 Whole blood is mixed with 2% Sodium Metabisulfite, which is a reducing agent
(deoxygenates the hemoglobin)
 Sample is examined using HPO
 (+) result: presence of sickle cells of holly-leaf form (may be seen in sickle cells trait)
c. Other Test:
✓ Scriver and Waugh
✓ Daland and Da Silva
✓ Sherman’s test
D. LE Preparation
 LE cell are neutrophils that contains a large spherical body in its cytoplasm
 LE factor is a gamma globulin that acts as an antibody to nuclear proteins
 In vitro, this can be induced to the produce the classic LE cell
 Replaced by ANA
 Factors to be considered to produce the classic LE cell:
o LE factor (if present, it causes nuclear lysis)
o Extruded cell nuclei
o Phagocytic neutrophilic leukocytes
E. Quantitation of Hb F
a. Alkali Denaturation Test: patient sample + KOH
b. Kleihauer-Betke Acid Elution Test:
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 For detection of fetal red cells in maternal circulation
 Standard test for quantitating fetomaternal hemorrhage
 Principle: Hb F resists acid elution
 Maternal red cells = appears as ghost cells
 Fetal red cells = appears as rose pink in color
F. Heinz body staining
 Demonstrated by supravital staining (CV, BCB)
G. Schumm Test
 Test for methemalbumin
 Spectroscopic detection
H. Tests for Hereditary Spherocytosis
a. Osmotic Fragility Test
 Reflects the shape of erythrocytes
 Measures the ability of the RBCs to take up fluid without lysing
 The primary factor affecting the OFT is the shape of the RBC which depends on the
volume, surface area, and functional state of the RBC membrane
 The larger the amount of RBC membrane (surface area) in relation to the size of the cell,
the more fluid the cell is capable of absorbing before rupturing
 Spherocyte has the smallest surface area for its volume, ruptures the most quickly, and
has increased fragility
 Anticoagulant used: heparin
 Clinical Significance:
o INCREASED OFT
▪ Spherocytosis
▪ Hemolytic anemia
▪ Old RBCs
o DECREASED OFT
▪ Target cell
▪ Liver disease
▪ Sickle cell
▪ IDA
▪ Thalassemia
▪ Polycythemia vera
▪ Reticulocytosis
b. Autohemolysis Test
 Patient’s RBC and serum are incubated for 48 hours, with and without glucose
 Principle: glucose catabolism provides the ATP to drive the cation pump to help
maintain the osmotic balance in the RBCs
 NOTE: sensitivity is similar to EOFT
 Normal values:
o w/o glucose = <5% at the end of incubation
o w/ glucose = <1% at the end of incubation
 Increased Autohemolysis test: Hereditary spherocytosis
c. Hypertonic Cryohemolysis
 This test is based on the fact that cells from hereditary spherocytosis are particularly
sensitive at 0°C in hypertonic solutions
 Increased: Hereditary spherocytosis, Southeast Asian Ovalocytosis
d. EMA binding (Eosin-5’-maleimide) Test
 Proposed as a more sensitive alternative confirmatory test of Hereditary Spherocytosis
 EMA is a fluorescent dye that specifically binds to transmembrane proteins band 3, Rh,
RhAg, and CD47
 When measured in flow cytometer, specimens from HS show a lower mean fluorescence
intensity (MFI) than normal RBCs and spherocytes caused by immune-mediated
hemolysis
 (+) result: Hereditary Spherocytosis, Southeast Asian Ovalocytosis, Hereditary
Pyropoikilocytosis
 Advantages: suitable for low volume such as pediatric specimen, specimens are
acceptable up to 7 days

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 Disadvantage: standardization

Summary of Specific and Diagnostic Tests in Hematology

Sugar Water Test/Sucrose Hemolysis Test Screening test for Paroxysmal Nocturnal Hemoglobinuria
Ham’s acidified serum Confirmatory test for Paroxysmal Nocturnal Hemoglobinuria
Autohemolysis test Hereditary Spherocytosis
Isopropanol Precipitation Test Unstable hemoglobin
Heat Denaturation test Unstable hemoglobin
Sodium metabisulfite Screening test for Hb S
Dithionite tube test Screening test for Hb S
Donath-Landsteiner Test For the demonstration of biphasic hemolysin seen in PCH
LAP Differential test for CML and Leukemoid reaction
Alkali-Denaturation test (Singer) Quantitative test for Hb F
Kleihauer-Betke Test Quantitative test for Hb F
Fluorescent Spot Test Screening test for G6PD deficiency and PK deficiency
Red cell zinc protoporphyrin test Screening procedure for Lead Poisoning
Ascorbate-Cyanide Test Screening test for G6PD deficiency
Schumm test Test for Methemalbumin

HEMOSTASIS
Hemostasis
 Derived from the Greek word meaning “the stoppage of blood flow”
Hemostatic Components
 Extravascular – involves the tissue surrounding a vessel, which become involved in hemostasis when a
local vessel is injured
 Vascular – involves the vessels through which blood flows
 Intravascular
o The key components in intravascular hemostasis are platelets and procoagulants in the plasma
o These components are involved in coagulation and fibrinolysis
Steps in Primary and Secondary Hemostasis
1. Vasoconstriction
2. Platelet adhesion
→ When vascular injury occurs, platelets come in contact with subendothelium and adhere to
portions of it
→ Platelet adhesion occurs due to the presence of von Willebrand factor being deposited on
injured tissues
→ vWF binds to Gp Ib-IX complex on the platelet membrane
3. Platelet activation and secretion
→ Following activation, the platelet undergoes a shape change caused by contraction of
microtubules
→ The platelet changes from disk-shape to a spherical shape with the extrusion of numerous
pseudopods
→ During activation, ADP and Calcium ion activates phospholipase A2
→ Phospholipase A2 converts membrane phospholipid to arachidonic acid
→ Arachidonic acid is converted by cyclooxygenase into prostaglandin endoperoxide
→ In the platelets, prostaglandin is converted by thromboxane synthetase into thromboxane A2
→ Thromboxane A2 causes the release of calcium ions and promotes platelet aggregation and
vasoconstriction
→ Important note:
o Aspirin acetylation inactivates cyclooxygenase, blocking thromboxane A2 production
and impairs platelet aggregation
4. Platelet aggregation
→ Initiated by Gp IIb-IIIa complex
5. Fibrin plug formation
→ Fibrin clot is stabilized by coagulation factor XIII

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Blood Vessel Products
Substance Action Hemostatic Role
Prostacyclin (PGI2) ▪ Inhibits platelet activation Anticoagulant
▪ Stimulates vasodilation Reduces blood flow rate
Adenosine ▪ Stimulates vasodilation Reduces blood flow rate
Thrombomodulin ▪ Endothelial receptor for thrombin Anticoagulant
▪ Binds and inactivates thrombin and enhances Fibrinolytic
anticoagulant and fibrinolytic action of Protein
C
Heparan sulfate ▪ Coats the endothelial cell surface and weakly Anticoagulant
enhances the activity of antithrombin-III
tPA ▪ Converts plasminogen to plasmin which plays Fibrinolytic
an important role in fibrinolysis
▪ NOTE: released only on appropriate stimulus,
such as vessel injury, to prevent excessive clot
formation at the site of tissue injury
von Willebrand factor ▪ Secreted by endothelium Coagulation
▪ Required for platelet adhesion
von Willebrand Factor
✓ Synthesized by endothelial cells, and megakaryocytes
✓ Stored in endothelial cells (Weibel-Palade bodies) and platelets
✓ VIII/vWF: entire molecule as it circulates in the plasma
✓ VIII:vWF – portion of molecule responsible for binding to endothelium and supporting normal platelet
adhesion and function
✓ VIII:C – portion of molecule participating in intrinsic pathway

Platelet General Characteristics

 Considered as NOT true cells because they are cytoplasmic fragments of megakaryocytes
 Function in primary (adhesion, secretion, aggregation) and secondary hemostasis (coagulation)
 Mean platelet volume: approximately 7 fL
 Average lifespan: 8-12 days
 Morphologic shape:
o Discoid (inactive)
o Spherical w/ pseudopod (activated)
 Reticulated platelets = newly released platelets that have residual RNA
 Distribution:
o Circulation: 2/3
o Spleen: 1/3
 Roles in hemostasis:
o Adhesion, aggregation, secretion of granules
o Promote coagulation
o Induce clot retraction
 Clot Retraction:
o Interaction of Gp IIb/IIIa (required for clot retraction)
o Delayed or incomplete retraction: thrombocytopenia, Glanzmann’s thrombasthenia
 Important Notes:
✓ Endomitosis refers to multiple mitotic division without cell division, resulting to giant
multinucleated or polyploid cells
✓ Thrombopoietin – stimulates platelet production, produced by the liver
✓ There are 3,000-4,000 platelets produced by each megakaryocyte
✓ Platelet shedding refers to the release of platelets into the bone marrow sinus
✓ Demarcating membrane system functions as the future membrane system of platelets
Platelet Structure
▪ Peripheral zone
− platelet’s outer membrane
− consists of glycocalyx, plasma membrane, and submembrane area
− the glycocalyx provides surface to which some coagulation factors may adhere
▪ Submembrane area
− links the membrane and the inner cell body
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▪ Sol-gel zone
− the cytoskeleton structure of platelets
− maintains the shape of platelets and structural support
− consists of microtubules and microfilaments
− influences communication of the organelles with the platelet’s external surroundings
▪ Organelle zone
− consists of granules, lysosomes and mitochondria
− serves as metabolic center to influence platelet function in response to exogenous stimuli such
as hypercoagulation, viruses and foreign bodies

Platelet Granules
▪ Alpha granules
o HMWK o Platelet-derived growth factor
o Fibrinogen o Beta-thrombomodulin
o Factor V o Plasminogen
o von Willebrand factor o Alpha-2 antiplasmin
o Platelet factor 4 o C1 esterase inhibitor
o Thrombospondin
o
▪ Dense granules
o ADP
o ATP
o Calcium
o Magnesium
o Serotonin
Coagulation Factors
I Fibrinogen -
II Prothrombin Prethrombin
III Tissue factor Tissue thromboplastin
IV Calcium ions -
Labile factor
V Proaccelerin
Accelerator globulin
Stable factor
VII Proconvertin
Serum Prothrombin Conversion Accelerator
Antihemophilic globulin
VIII (VIII:C) Antihemophilic factor Antihemophilic factor A
Platelet cofactor 1
Christmas factor
IX Plasma thromboplastic component Antihemophilic factor B
Platelet cofactor 2
Stuart factor
X Stuart-Prower factor Prower factor
Autoprothrombin III
XI Plasma thromboplastin antecedent Antihemophilic factor C
Glass factor
XII Hageman factor
Contact factor
Laki-Lorand factor
Fibrinase
XIII Fibrin Stabilizing factor
Plasma transglutaminase
Fibrinoligase
- Prekallikrein Fletcher factor
Fitzgerald factor
Williams factor
- HMWK
Flaujeac factor
Contact activation cofactor

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Important Notes
✓ Labile factors: V and VIII
✓ Activated by cold temperature: VII and XI
✓ Fibrinogen is the most abundant clotting factor with a normal value of 200-400 mg/dL
✓ Serine protease: II, X, VII, IX, XII, XI, and PK
✓ Prothrombinase: Xa-Va
✓ Tenase:
▪ Intrinsic: VIII-IX
▪ Extrinsic: VII-III-IV
✓ Thrombin (IIa): central regulatory component of coagulation (can inhibit or accelerate coagulation)

Clotting Factors Classification

According to Pathway ▪ Extrinsic pathway: III and VII
▪ Intrinsic pathway: XII, XI, IX, VIII
▪ Common pathway: I, II, V, X
According to Properties ▪ Fibrinogen group: I, V, VIII, XIII
▪ Prothrombin group (Vitamin K dependent): II, VII, IX, X
▪ Contact group: XI, XII, PK, HMWK

Coagulation Pathways
 Extrinsic pathway – activated by the release of tissue thromboplastin (Factor III) into the plasma from
the injured cells
 Intrinsic pathway – activated when a vessel is injured, exposing the subendothelial basement membrane
and collagen promoting coagulation
 Common pathway – begins with the activation of Factor X to Factor Xa
Clotting Factors
Tissue factor (thromboplastin) ▪ A transmembrane receptor for Factor VIIa
▪ Tissue thromboplastin = mixture of tissue factor and
phospholipid
▪ Found on extravascular cells such as fibroblasts and smooth
muscle cells (not found in endothelial cells under normal
conditions)
▪ High levels are found in brain, lung, heart, kidneys, and testes
Vitamin K ▪ Produced by B. fragilis and E. coli
▪ Found in green leafy vegetables
▪ Catalyzes an essential post-translational modification of the
prothrombin group proteins
▪ Vitamin K dependent factors:
o II, VII, IX, X
o Protein C, S, Z
von Willebrand Factor ▪ Participates in platelet adhesion and transports Factor VIII
▪ Large multimeric glycoprotein
▪ Composed of multiple subunits of 240,000 Da each
▪ The subunits are produced by endothelial cells and
megakaryocytes, where they combine to form multimers that
range from 600,000 to 20,000,000 Da
▪ Once released into the plasma, they are normally degraded into
small-multimers by vWF-cleaving protease called ADAMTS13 (a
disintegrin-like metalloprotease with a thrombospondin type 1
motif, member 13)
▪ TTP = associated with abnormally large vWF
Calcium ▪ Required for the assembly of coagulation complexes
Thrombomodulin ▪ Expressed by vascular endothelial cells
▪ Cofactor of thrombin
▪ Thrombomodulin + Thrombin = activates Protein C
▪ Protein C: a coagulation inhibitory protein, and thrombin
activatable fibrinolysis inhibitor (TAFI)
▪ Once thrombin is bound to thrombomodulin, it loses its
procoagulant ability to activate factors V and VII, and through
the activation of Protein C, leads to the destruction of factors V
and VII, thus suppressing further generation of thrombin
Thrombin ▪ Considered as the key protease of coagulation pathway
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▪ Primary function is to cleave fibrinopeptidases A and B from the
alpha and beta chains of fibrinogen molecule, triggering
spontaneous polymerization
▪ Other functions:
✓ Activates cofactors V, VIII, and IX
✓ Activates factor XIII
✓ Initiates platelet aggregation
✓ Activates TAFI to suppress fibrinolysis

Regulation of Coagulation
Tissue factor pathway inhibitor Principal regulator of tissue factor pathway
Protein C Thrombin + Thrombomodulin → Protein C → Activated Protein C-
Protein S → inactivates factor Va and VIIIa
Antithrombin and other serine Antithrombin
protease inhibitors (Serpins) Heparin cofactor II
Protein Z-dependent protease inhibitor (ZPI)
Protein C inhibitor
a1-antitrypsin
a2-macroglobulin
a2-antiplasmin
PAI-1
Antithrombin III Inhibits thrombin
Synthesized by the liver
SERPIN
Heparin cofactor II Inactivates thrombin
SERPIN
Protein Z-dependent protease Potent inhibitor of factor Xa
inhibitor (ZPI)

Fibrinolytic System
Fibrinolysis Final stage of coagulation
Dependent on plasmin
Plasmin Destroys fibrinogen, fibrin, factor V, and factor VIII
Not normally present in the blood in an active form
Plasminogen Zymogen of plasmin
Normally present in the plasma
Homologous to Lp (a)
Tissue plasminogen activator Released in vivo by endothelial cell damage
Activates plasminogen
Urokinase plasminogen activator Activates plasmin
May be administered to a patient to activate plasminogen
Streptokinase Activates plasminogen
FDP/FSP Early degradation products: X, Y
Late degradation products: D (D-dimer) and E
D-dimer Indicates fibrin degradation products
Marker of thrombosis and fibrinolysis
Fragment X, Y, E Produced by digestion of either fibrin or fibrinogen by plasmin

COAGULATION DISORDERS
Glossary
▪ Petechiae
o Purplish red, pinpoint hemorrhagic spots caused by loss of capillary ability to withstand normal
blood pressure and trauma
o Size: <3 mm
▪ Purpura
o Produced by hemorrhage of blood into small areas of skin, mucous membrane, and other
tissues
o Size: <1 cm
▪ Ecchymosis

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o Form of purpura in which blood escapes into large areas of the skin or mucous membrane but
not into deep tissues
o Size: >3 cm

VASCULAR DISORDERS
Ehlers-Danlos Syndrome ▪ Autosomal Dominant
▪ Characterized by hyperextensible joints and
hyperelastic skin
Pseudoxanthoma elasticum ▪ Autosomal Recessive
▪ The connective tissue elastic fibers in small
arteries are calcified and structurally abnormal
▪ Autosomal Dominant
HEREDITARY

Hereditary hemorrhagic telangiectasia

▪ Also known as Rendu-Osler-Weber syndrome
▪ Characterized by vascular malformations and
skin lesions called telangiectasias
Congenital hemangioma- ▪ Also known as Kasabach-Merritt syndrome
thrombocytopenia syndrome ▪ Associated with tumors composed of vessels
that commonly swell and bleed at the surface
▪ Formation of fibrin clots, platelet consumption,
and RBC destruction secondary to vascular
obstruction occur at the site of tumor
Vitamin C deficiency ▪ Also known as Scurvy
▪ Vitamin C is required for the formation of intact
structure of the vascular basement membrane
▪ Gingival bleeding and hemorrhage into
subcutaneous tissues and muscles are common
finding
Senile Purpura ▪ Acquired and chronic disorder of the elderly
ACQUIRED

causing abnormalities in connective tissues

▪ The aging process brings about a degeneration
of collagen, elastin, and subcutaneous fat
▪ Common in elderly men
Henoch-Schonlein Purpura ▪ Purpura associated with abdominal pain
secondary to GIT bleeding (Henoch’s purpura)
▪ Abdominal and joint pain related to allergic
purpura
▪ Common in children
QUALITATIVE PLATELET DISORDERS
Adhesion Defect
Bernard-Soulier Syndrome ▪ Caused by lack of expression of Gp Ib/IX
complex on the platelet surface
▪ Characterized by large platelets
▪ Autosomal Recessive
Lab Findings:
▪ Giant platelets
▪ BT: Prolonged
▪ Platelet Aggregation studies: Ristocetin is
abnormal
HEREDITARY

von Willebrand Disease ▪ Lacks vWF

Lab Findings:
▪ APTT, BT, TT, CT = Prolonged
▪ Platelet Aggregation studies: Ristocetin is
abnormal
▪ Treatment of choice: cryoprecipitate, DDAVP
Aggregation Defect
Glanzmann’s Thrombasthenia ▪ Lacks Gp IIb-IIIa
▪ Autosomal Recessive
▪ Clot Retraction: Abnormal
▪ BT: Prolonged
▪ Platelet aggregation studies: ADP, Collagen, and
Epinephrine are abnormal

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Storage Pool Defects
Gray Platelet Syndrome ▪ Lacks alpha granules
▪ Giant platelets
▪ Platelets are gray or blue-gray
▪ Autosomal Dominant
Hermansky-Pudlak Syndrome ▪ Lacks dense granules
▪ Triad of oculocutaneous albinism, bleeding
tendency associated with abnormal platelet
function, and accumulation of ceroid-like
pigment in macrophages
▪ Autosomal Recessive
Chediak-Higashi Anomaly ▪ Lacks dense granules
▪ Characterized by albinism, recurrent infection,
and giant lysosomes
▪ Autosomal Recessive
Wiskott-Aldrich Syndrome ▪ Decreased delta granules
▪ Platelets are small
▪ Triad of thrombocytopenia, recurrent infection
and eczema
▪ X-linked recessive
Thrombocytopenia w/ absent Radii ▪ Platelets have structural defects in delta
granules
▪ Characterized by congenital absence of the
radial bones
▪ Autosomal Recessive
Aspirin intake:
 inhibits the synthesis of cyclooxygenase
 inhibits platelet aggregation
 most common acquired platelet disorder

QUANTITATIVE PLATELET DISORDERS

THROMBOCYTOPENIA
ITP ▪ Autoimmune disorder
PTP ▪ Develops after transfusion of platelet containing blood products
TTP ▪ Also known as Moschowitz syndrome
▪ Characterized by triad of microangiopathic hemolytic anemia,
thrombocytopenia, and neurologic abnormalities
▪ Due to ADAMTS13 deficiency
 Upshaw-Schulman Syndrome
− Inherited TTP
− Severe
− Caused by mutation in the ADAMTS13 gene
 Idiopathic TTP
− Caused by autoantibodies
 Secondary TTP
− triggered by infections, pregnancy, surgery, trauma,
inflammation, and disseminated malignancy
− Trimethoprim, Ticlopidine, Quinine drugs
HUS ▪ Resembles TTP
▪ Common in children
▪ Due to E. coli infection
DIC ▪ Similar to TTP
▪ Thrombi are primarily composed of platelets and fibrinogen
▪ (+) D-dimer
Dilutional ▪ Due to massive blood transfusion (effect is temporary)
▪ Rationale: stored blood contains platelets whose viability is severely
impaired by the effects of storage and temperature. Under these
conditions, the damaged platelets are rapidly sequestered by the RES
of the patient resulting to thrombocytopenia
THROMBOCYTOSIS
Primary Thrombocytosis Due to uncontrolled proliferation of platelets (e.g., PV, ET)

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Secondary Thrombocytosis Splenectomy
(Reactive)

Coagulation Factor Disorders

PK deficiency ▪ Do not demonstrate clinical bleeding
▪ May be vulnerable to thrombotic tendencies
▪ Prolonged APTT
HMWK deficiency ▪ Prolonged APTT
Factor XIII ▪ 5M Urea: Abnormal
Factor XII deficiency ▪ Do not manifest bleeding disorder
▪ May be vulnerable to thrombosis
▪ Prolonged APTT
▪ Confirmatory test: Factor XII assay
Factor XI deficiency ▪ Hemophilia C
▪ Prolonged APTT
Factor X deficiency ▪ Prolonged APTT, PT, Stypven time
Factor IX deficiency ▪ Also known as Hemophilia B, Christmas Disease, Rosenthal syndrome
▪ Prolonged APTT and PT
Factor VIII deficiency ▪ Also known as Hemophilia A or Classic hemophilia or Royal disease
▪ Prolonged APTT
▪ X-linked disorder (males are affected)
Factor VII deficiency ▪ Prolonged PT
Factor V deficiency ▪ Also known as Owren’s disease or Parahemophilia
▪ Prolonged APTT and PT
▪ Factor V assay
Factor II deficiency ▪ Prolonged APTT and PT
Factor I deficiency ▪ Prolonged APTT and PT

Summary of Laboratory Diagnosis of Coagulation Disorders

Disease APTT PT Other findings
Factor I deficiency Prolonged Prolonged Thrombin time
Reptilase time
Factor II deficiency Prolonged Prolonged Substitution test
Factor V deficiency Prolonged Prolonged Substitution test
Factor VII deficiency Normal Prolonged Substitution test
Factor VIII deficiency Prolonged Normal Substitution test
Factor IX deficiency Prolonged Normal Substitution test
Factor X deficiency Prolonged Prolonged Substitution test
Factor XI deficiency Prolonged Normal Substitution test
Factor XII deficiency Prolonged Normal Substitution test
Factor XIII deficiency Normal Normal Duckert’s test
PK deficiency Prolonged Normal -
HMWK deficiency Prolonged Normal -
von Willebrand disease Prolonged Normal -
DIC Prolonged Prolonged  Platelet count
(+) schistocyte
(+) D-dimer
HUS Normal Normal  Platelet count
(+) schistocyte
(+) E. coli O157:H7
 BUN, Creatinine
TTP Normal Normal  Platelet count
(+) schistocyte

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COAGULATION TESTS
Factors
▪ Temperature
o Room temperature: labile factors (V, VIII) will deteriorate for an extended period of time
o Cold temperature: activates factor VII and XI
▪ Hemolysis
o Hemolyzed RBCs will act like tissue thromboplastin in activating plasma clotting factors
▪ Glass surface
o The contact factors (HMWK, PK, XI, XII) will be activated prematurely by contact with glass
o Recommended materials are plastic, polystyrene, or silicone-coated glass
▪ Tissue thromboplastin
o Potent clot-activating substance found in fluids that escape from injured cells and tissue spaces
▪ Short draw = causes falsely prolonged coagulation test

Anticoagulant used in Coagulation Tests

▪ 3.2% Na Citrate
 For coagulation studies
 Contains 0.109 M Na Citrate
 Preserves the labile clotting factors
 Blood to anticoagulant ratio = 9:1
A. Platelet Count
 Anticoagulant
o EDTA
o 3.2% Na Citrate
▪ Can be used if platelet satellitism occurs
▪ The platelet count is multiplied by 1.1
 Materials: Neubauer counting chamber, RBC Thoma pipet
 Dilution: 1:100 or 1:200
 Diluting fluid properties: must preserve platelet integrity while inhibiting their aggregation
 Method:
o Tocantins’ method
▪ Diluting fluid: Rees-Ecker
▪ Stain: BCB
o Brecker-Cronkite method
▪ Phase-contrast microscopy method
▪ Diluting fluid: 1% Ammonium oxalate (hemolyze the RBCs)
 Computation: platelet count = no. of platelets counted x DF x VCF
 Normal value: 150,000 to 450,000/uL
B. Estimation of Platelet
 Scan 10 oil immersion field
 Average number of platelets x 20,000
Platelet Estimate Reporting
0 to 49,000 Markedly decreased
50,000 to 99,000 Moderately decreased
100,000 to 149,000 Slightly decreased
150,000 to 200,000 Low normal
200,000 to 399,000 Normal
400,000 to 599,000 Slightly increased
600,000 to 799,000 Moderately increased
Above 800,000 Markedly increased

C. Clot Retraction Tests

 When coagulation is complete, the clot normally undergoes clot retraction (serum is expressed
from the clot) and the clot becomes denser
 Screening test for platelet function
 Useful index of platelet activity
 NOTE: clot retraction is poor when the platelet count is <100,000/uL
 Responsible for clot retraction: actin, myosin, thrombosthenin
 Abnormal clot retraction:
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o Glanzmann’s Thrombasthenia
o Paraproteinemia (MM) = proteins interfere with fibrin formation
 Methods:
o Hirschboeck method (Qualitative test)
▪ Also known as Castor oil method
▪ (+) result: dimpling or extrusion of droplet-like serum on top portion of the drop
of blood
▪ Normal value: 15 to 45 minutes
▪ Dimpling in less than 15 minutes = thrombotic tendency
▪ Dimpling of more than 45 minutes = Hemorrhagic tendency
o Quantitative test
▪ Stefanini method
▪ McFarlane method
• Normal value: 44 to 67%
D. Tests for Hemostasis
Bleeding Time ▪ Screening test for primary hemostasis
▪ Principle: the time it takes for a standard wound to
stop bleeding
▪ NV: 2 to 4 minutes
Methods:
▪ Duke’s method – earlobe was punctured (not
accurate, obsolete)
▪ Ivy method – uses blood pressure cuff (40 mmHg)
▪ Mielke – standardization of wound
Whole Blood Clotting Time ▪ For primary hemostasis
▪ Principle: when venous blood is put into a foreign
surface, it will form a solid clot
Methods:
▪ Lee and White method
 NV: 5-15 minutes
 Uses 75 x 100 mm test tube
▪ Tilt tube method
 Visual detection of fibrin clot formation
Tourniquet Test ▪ Also known as Capillary Fragility Test
▪ Used to measure capillary fragility
▪ (+) result: formation of petechiae
Grading:
1+ = few petechiae on the anterior part of the forearm
2+ = many petechiae on the anterior part of the forearm
3+ = multiple petechiae over the whole arm and back of
the arm
4+ = confluent petechiae on the arm and back of the hand
Platelet aggregometry ▪ For platelet aggregation in vitro
▪ Reagents used: ADP, Collagen, Epinephrine, Ristocetin
Prothrombin Time ▪ For extrinsic and common pathway
▪ Used to monitor oral anticoagulants (Warfarin,
Coumadin, Coumarin)
▪ NOTE: PT is prolonged if fibrinogen level is <80 mg/dL
▪ PT reagent:
 Thromboplastin (rabbit brain or lung tissue)
 Calcium chloride (CaCl2)
▪ INR
 Calculation made to standardize PT
 It is based on ratio of patient’s PT and normal
mean PT
▪ ISI – assigned by the manufacturer
▪ NV: 10-14 seconds
Activated Partial Thromboplastin ▪ For intrinsic and common pathway
Time ▪ Used to monitor heparin therapy
▪ APTT Reagent:
 Platelet substitute (phospholipid)

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 Activator (Kaolin, Celite, Silica, Ellagic acid)
▪ NV: 20-45 seconds
Stypven Time ▪ Also known as Russell’s viper venom time
▪ For common pathway
▪ Reagent: Russell’s viper venom is obtained from the
snake Vipera russelli
Thrombin Time ▪ For fibrinogen
▪ NOTE: affected by heparin therapy
▪ Sensitive test in detecting heparin inhibition
▪ Prolonged Thrombin time is noted when fibrinogen
level is below 75 to 100 mg/dL
Reptilase Time ▪ For fibrinogen
▪ NOTE: NOT affected by heparin therapy
▪ Reptilase is an enzyme found in the venom of
Bothrops atrox snake
Plasma Recalcification Time ▪ Also known as Plasma Clotting Time
▪ For intrinsic pathway
Duckert’s test ▪ For Factor XIII
▪ Reagent: 5M Urea and 1% Monochloroacetic acid
▪ (+) Factor XIII deficiency: the clot is dissolved in the
presence of 5M Urea
▪ NOTE: a clot that has not stabilized by Factor XIII is
soluble to 5M Urea
Fibrinosticon ▪ Screening test for DIC
▪ The presence of crosslinked D-dimer indicates that a
stable fibrin clot has been lysed
Ethanol Gelation Test ▪ Screening test for DIC
▪ Used to detect fibrin monomers in the plasma
▪ Used to differentiate DIC from primary fibrinolysis
Principle:
▪ During the process of DIC, the level of fibrin monomer
(product of fibrinogen conversion to fibrin) in the
blood increases
▪ NaOH is added in the plasma to increase the pH to
7.70 (if pH is below 7.70, this will cause precipitation
of fibrinogen instead of fibrin monomer)
▪ Ethyl alcohol will cause precipitation of any fibrin
monomers
Protamine Sulfate ▪ Used to detect the presence of fibrin monomers
▪ NOTE: normally, there should be no fibrin monomers
present in the plasma
Euglobulin clot lysis time ▪ Screening test for fibrinolytic activity
▪ Clot lysis in less than 1 hour indicates abnormal
fibrinolytic activity
Platelet neutralization test ▪ Used for the detection of lupus anticoagulant
Substitution Test ▪ Also known as Mixing studies
▪ Used to identify specific factor deficiency
▪ A specific factor deficiency may be identified by
mixing correction reagents with a patient’s plasma
and then performing PT and APTT (1:1 dilution)
SUBSTITUTION TEST
Factor Fresh Aged Adsorbed
APTT PT Aged serum
Deficiency plasma Plasma plasma
I P P C C ✓ X
II P P C C X X
V P P C NC ✓ X
VII N P C C X ✓
VIII P N C NC ✓ X
IX P N C C X ✓
X P P C C X ✓
XI P N C C ✓ ✓
XII P N C C ✓ ✓
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HEMATOLOGY
Important Notes:
✓ Substitution test cannot identify Factor III and XIII deficiency
✓ For Factor XIII deficiency, perform Duckert’s test
✓ Consumed during coagulation: fibrinogen group and prothrombin group
✓ Use of fresh plasma will detect circulating inhibitor
E. Platelet Adhesion and Aggregation Test
Bernard-Soulier syndrome
Test Glanzmann’s Thrombasthenia
von Willebrand disease
ADP, Collagen, Epinephrine NORMAL ABNORMAL
Ristocetin ABNORMAL NORMAL

AUTOMATION
Quality Control and Quality Assurance
 Control – must be similar in properties to, and be analyzed along with patient specimen
 Standard/Calibrator – for calibration purposes
 Reference method – one that is specific for analyte and which quantitates the true concentration of the
analyte
Rule of Three
 Purpose is to check the accuracy of RBC count, Hb and Hct values
 If the values do not agree, the blood smear should be examined for abnormal RBCs
 Applicable only to normocytic, normochromic RBCs
 If the RBCs are found to be abnormal, the automated values cannot be expected to conform to these
results
 Formula: 3 x RBC = hemoglobin; 3 x Hb = Hct (%)

Ohm’s Law
 The magnitude of the voltage pulses produced by cells is directly related to their size, a fact that has
been used on subsequent clinical instruments for direct measurements of cell volume
 Voltage = current x resistance

Electrical Impedance
 Coulter machines
 Manner of reporting: histogram, scattergram
 Principle: blood cells are counted as changes in voltage pulse
 Commercial diluting fluid: Isoton

Light Scattering
 Forward low angle light scatter = measures cell volume and size
 Forward high angle light scatter = measures cell granularity/inclusions

Differential Count
 3-part differential = mononuclear cells, granulocytes, lymphocytes
 5-part differential = basophil, eosinophil, neutrophil, lymphocyte, monocyte
Instrumental Error
 Positive error – caused by bubbles, electrical pulses, aperture plugs (most common error in cell
counting)
 Negative error – excessive RBC lysing
 positive and negative error – improper settings of aperture current or threshold

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