Review Notes On Hematology

HEMATOLOGY
Notes Compiled by: Renz Louie Galanto

Notes Prepared by: John Alvin O. Reyes, RMT
REVIEW NOTES ON HEMATOLOGY 1
OUTLINE
• Blood • Granulocytes
• Safety in the Hematology Laboratory • Mononuclear Cells
o Standard Precaution • Leukocyte Classification
o Handwashing • Common Diseases that Increase WBCs
o Disinfection ▪ Red Blood Cell Indices and RDW
o Sharp Disposal • Mean Corpuscular Volume (MCV)
• Specimen Collection • Mean Cell Hemoglobin Concentration (MCHC)
o Venipuncture • Mean Corpuscular Hemoglobin (MCH)
▪ Order of Draw ▪ Red Cell Distribution Width
▪ Tube Color and Anticoagulant/Additive o Procedure for Examination of the Stained Blood Smear
▪ Preferred Concentration of Anticoagulants ▪ Types of Films
▪ Inversions • Manual Wedge Technique
o Equipments in Venipuncture • Coverslip Technique
▪ Torniquet ▪ Staining of Peripheral Blood Films
▪ Collection Tubes • Romanowsky Stain
▪ Needles
• Problems Encountered in Staining
▪ Antiseptics
• Staining Characteristics of Cells on Wright
o Veins for Routine Venipuncture
Giemsa Stain
▪ Median Cubital Vein
• Methods of Examination
▪ Cephalic Vein
o Platelet Count
▪ Basilic Vein
▪ Specimen
o Skin Puncture
▪ Methods
▪ Capillary Blood
▪ Collection Sites • Phase Microscopy
▪ Skin Puncture Procedure • Tonkantin Method
• Physiologic Factors affecting Test Results • Special Hematology Procedures
o Posture o Reticulocyte Count
o Diurnal Rhythm ▪ Procedure
o Exercise ▪ Principle
o Stress o Absolute Reticulocyte
o Diet o Corrected Reticulocyte Count
o Smoking o Reticulocyte Production Index
• Routine Hematology Procedures o Erythrocyte Sedimentation Rate
o Complete Blood Count ▪ Factors that affect the ESR
▪ Hemoglobin • Erythrocytes
• Methods • Plasma Composition
• Abnormal Hemoglobin Pigments • Mechanical/Technical Factors
▪ Hematocrit ▪ Stages
• Methods ▪ Sources of Error
▪ RBC Count o Eosinophil Count
• Principle ▪ Variations
▪ Methods
• Reagents and Equipments
• Direct Method
• Specimen
• Indirect Method
• Computation
o Sickle Cell Tests
▪ WBC Count
▪ Sodium Metabisulfite Method
• Principle
▪ Sodium Dithionite Test/Solubility Test
• Procedure ▪ Hemocard Hb A and S Procedure
• Reagents and Equipments ▪ Sugar Water Screening Test
• Specimen o Sucrose Hemolysis Test
• Computation o Acid Serum Test
• Corrected WBC Count ▪ Ham’s Method
• Dilution o Osmotic Fragility Test
• Common Dilutions and Counting Areas ▪ Method
▪ Differential Count o Lupus Erythematosus Preparation
•
REVIEW NOTES ON HEMATOLOGY
OUTLINE
▪ Principle • Methemoglobin Reductase Pathway
o Malarial Smear Preparation • Luebering-Rapaport Shunt
o Capillary Fragility Test ▪ RBC Membrane
▪ Methods • RBC Deformability
o Bleeding Time • RBC Elasticity
▪ Methods • RBC Membrane Proteins
• Modified Duke Method • Osmotic Balance and Permeability
• Ivy Method ▪ Erythrocyte Destruction
• Standardized Simplate Test • Macrophage-mediated hemolysis (Normal
o Lee and White Coagulation Time Extravascular Hemolysis)
▪ Procedure • Mechanical hemolysis or intravascular
o Special Histochemical Stains hemolysis
▪ Prussian Blue Reaction • Excessive extravascular hemolysis
▪ Leukocyte Alkaline Phosphatase • Excessive intravascular hemolysis
• Specimen ▪ Hemoglobin Metabolism
▪ Myeloperoxidase Stain • Structure
▪ Sudan Black B
• Synthesis
▪ Periodic Acid Schiff Stain
▪ Normal Human Hemoglobins
▪ Chloroacetate Esterase
▪ Normal Hemoglobin Concentration in Adults
▪ Nonspecific Esterase
• Function
▪ Tartrate-resistant ACP
• Oxygen Dissociation Curve
▪ Toluidine Blue
o Leukopoiesis
▪ Nitroblue Tetrazolium Neutrophil Reduction Test
▪ Neutrophil Maturation
▪ Terminal Deoxyribonucleotidyl Transferase
• Hematopoiesis • Myeloblast
o Stages of Hematopoiesis • Promyelocyte
▪ Mesoblastic Phase (Yolk Sac Phase) • Neutrophilic myelocyte
▪ Hepatic Phase • Neutrophilic metamyelocyte
▪ Medullary (Myeloid) Phase • Neutrophilic band
o Adult Hematopoietic Tissue • Polymorphonuclear neutrophil
▪ Bone Marrow • Neutrophil Pools
• Two Types ▪ Eosinophil Maturation
▪ Liver ▪ Basophil Maturation
▪ Spleen ▪ Monocyte Maturation
▪ Lymph Nodes • Monoblast
▪ Thymus • Promonocyte
o Stem Cell Cycle Kinetics and Cytokines • Monocyte
▪ Terminologies • Macrophase-tissue Monocyte
▪ Stem Cell Theory ▪ Lymphocyte Maturation
▪ Kinetics • Lymphoblast
• Cell Cycle • Prolymphocyte
▪ Cytokines and Growth Factors • Lymphocyte
o Overview of Hematopoiesis ▪ WBC Maturation and Lifespan
o Erythropoiesis o Platelet Production, Structure and Function
▪ Erythrocytic Nomenclature ▪ General Characteristics of Platelets
▪ RBC Developmental Stages ▪ Megakaryocytopoesis
• Pronormoblast (rubriblast) • Stages
• Basophilic normoblast (prorubricyte) ▪ Platelet Structure
• Polychromatic normoblast (rubricyte) • Peripheral zone
• Orthochromic normoblast (metarubricyte) • Sol-gel zone
• Polychromatophilic erythrocyte (reticulocyte) • Organelle zone
• Erythrocyte • Membranous system
▪ Erythrokinetics ▪ Platelet Function
▪ Erythrocyte Metabolism • Adhesion
• Embden-Meyerhof Pathway • Aggregation
• Hexose Monophosphate Pathway • Secretion
BLOOD
● The average human possesses 5L of blood
● 7 – 8% of the body weight
● Transports oxygen from lungs to tissues
● Clears carbon dioxide (CO2)
SAFETY IN THE HEMATOLOGY

LABORATORY
STANDARD PRECAUTION___________
● Treat all specimens such as blood, body fluids and unfixed tissues
to be potentially infectious
HANDWASHING____________________
● Most effective way of breaking the chain of infection Red Clot activator Serum/chemistry, Silica clot
● Process (plastic) serology activator
o Wet hands and wrists thoroughly under running water Lavender K3 EDTA in liquid Whole Chelates/bi
o Apply germicidal soap and rub hands vigorously for at least (glass) form blood/hematology nds calcium
15 seconds Lavender Spray dried K2 EDTA Whole Chelates/bi
o Rinse hands in a downward flow (plastic) blood/hematology nds calcium
o Dry hands with a paper towel Pink Spray dried K2 EDTA Whole Chelates/bi
o Use the paper towel to turn off the faucets blood/hematology nds calcium
White EDTA and gel Plasma/molecular Chelates/bi
diagnostics nds calcium
DISINFECTION_____________________ Light Na citrate Plasma/ Chelates/bi
● The process of destroying pathogenic microorganisms in
blue coagulation nds calcium
inanimate objects
Black Na citrate Plasma/ESR Chelates/bi
● An appropriate disinfectant is a household bleach (sodium
nds calcium
hypochlorite) used in a 1:10 volume/volume dilution which is
Light Lithium heparin and Plasma/chemistry Inhibits
prepared daily
green / gel thrombin
● Other solutions include phenol-based disinfectants (Amphyl),
Tuberculoidal disinfectants, and 70% ethanol black
Green Sodium heparin, Plasma/chemistry Inhibits
lithium heparin thrombin
SHARPS DISPOSAL_________________ Royal Sodium heparin, K2 Plasma/chemistry/ Heparin
● Needles, blades, pipettes, syringes with needle, and glass slides blue EDTA toxicology inhibits
thrombin,
must be placed in a puncture-resistant container that is
EDTA binds
appropriately labelled with the universal biohazard symbol
calcium
Gray Sodium fluoride / Plasma/glucose Inhibits
SPECIMEN COLLECTION potassium oxalate testing glycolysis
Yellow Sodium Serum Inhibits
polyanetholesulfonate (sterile)/blood complement
VENIPUNCTURE_________________ culture , phagocytes
● The process of obtaining blood from a vein and certain
antibiotics
ORDER OF DRAW Yellow Acid citrate dextrose Plasma/blood WBC
bank/HLA preservative
● 1. Blood culture or sterile tube (yellow)
● 2. Coagulation tube/Na citrate tube (light blue) phenotyping and
● 3. Serum tube with or without clot activator or gel (red, gold, or paternity testing
red-gray marbled stopper) Tan Sodium heparin Plasma/lead Inhibits
● 4. Heparin tube (green/light green) (glass) testing thrombin
● 5. EDTA tubes (lavender stopper) Tan K2 EDTA Plasma/lead Chelates/bi
● 6. Oxalate/fluoride tubes (gray) (plastic) testing nds calcium
Yellow / Thrombin Serum/chemistry Clot
TUBE COLOR AND ANTICOAGULANT/ADDITIVE gray and activator
orange
Red / Silica clot activator, Serum/chemistry Silica clot
COLOR ANTICOAGULANT SPECIMEN MECHANI gray and separation gel activator
/ ADDITIVE TYPE / USE SM OF gold
ACTION
Red None Serum/chemistry, N/A
(glass) serology
PREFERRED CONCENTRATION OF ANTICOAGULANTS USED. LEG, ANKLE, AND FOOT VEINS MAY BE USED
BUT NOT WITHOUT THE PERMISSION OF A PHYSICIAN
OXALATE 1 – 2 mg/mL of blood MEDIAN CUBITAL VEIN
CITRATE 3.2 – 2.8 g/dL (0.105 or 0.129 M)
EDTA 1.2 mg/mL of blood / 1.5 mg/mL (steininger) ● Most preferred vein
FLUORIDE 10 mg/mL of blood ● Largest, closest to the surface and well anchored
HEPARIN 0.2 mg/mL of blood
CEPHALIC VEIN
INVERSIONS ● 2nd option, less anchored, however it
● Easiest vein to palpate in obese patients
8 YELLOW (SPS, ACD), ORANGE (THROMBIN),
GREEN (HEPARIN), LAVENDER AND PINK (EDTA), BASILIC VEIN
GRAY ● Least anchored
5 RED (PLASTIC), GOLD ● Close to median cutaneous nerve and brachial artery
3-4 LIGHT BLUE
SKIN PUNCTURE____________________
EQUIPMENTS IN VENIPUNCTURE___ ● Performed in:
o 1. Newborns, pediatric patients below 1 y/o
TORNIQUET o 2. Adults who are severely burned
o 3. Elderly patients
● Should be applied 3 – 4 inches (7.5 – 10 cm) above the
● NOTE: Punctures should not be more than 2 mm deep because of
venipuncture site and left no longer than 1 minute
the risk of bone injury or possible infection (osteomyelitis)
o Prolonged torniquet application = hemoconcentration
● Torniquet Application
CAPILLARY BLOOD
o Cross the right side of the torniquet over the left side OR
o Place tension on the torniquet, cross one side over the other, ● A mixture of arterial blood, venous blood and tissue fluid
and slip a small loop under one side of the torniquet ● Capillary blood values as compared to venous blood:
o Lower RBC count
COLLECTION TUBES o Lower hematocrit
o Lower hemoglobin
● Most common means of collecting blood specimens is through
o Lower platelet count – activation of platelet adhesion;
the use of an evacuated tube system
platelet adheres to capillary wall
● ADDITIVES IN COLLECTION TUBES
o Higher glucose and WBC count – due to stress
o Antiglycolytic agent: inhibits the use of glucose by blood
cells, recommended if a delay in testing is expected for
COLLECTION SITES
glucose (E.g., NaF and lithium iodoacetate)
o Anticoagulant: prevents blood from clotting (E.g., EDTA, ● Lateral side of the plantar surface of the heel – children
Potassium oxalate) ● Third or fourth finger – older children or adult
o Clot activator: enhances the clotting mechanism by
providing an increased surface area for platelet activation
(glass or silica) and a clotting factor such as thrombin
o Separator gel: inert material that goes a temporary change in
viscosity during the centrifugation process, provides a
separation barrier between the serum or plasma and the cells
NEEDLES
● Routinely 19-, 20- and 21-gauge needles are used
● NOTE: THE MOST COMMON NEEDLE SIZE FOR ADULT
VENIPUNCTURE IS 21 GAUGE WITH A LENGTH OF 1
INCH. THE ADVANTAGE OF USING A 1-INCH NEEDLE IS
THAT IT PROVIDES BETTER CONTROL. SKIN PUNCTURE PROCEDURE
ANTISEPTICS ● 1. Blood gases

● 2. Slides, unless made from EDTA microcollection tubes
● Most common skin cleanser is 70% isopropyl alcohol ● 3. EDTA microcollection tube
● Isopropyl alcohol and iodine for blood culture collection ● 4. Other anticoagulated microcollection tubes (green or gray)
● Benzalkonium chloride or nonalcoholic anti-septic is used for ● 5. Serum microcollection tubes
sample collection for blood alcohol measurements
VEINS FOR ROUTINE VENIPUNCTURE

● The superficial veins of the antecubital fossa are the most
common veins for venipuncture
● NOTE: VEINS ON THE BACK OF THE HAND AND WRIST
MAY BE USED FOR VENIPUNCTURE. HOWEVER, VEINS
ON THE UNDERSIDE OF THE WRIST SHOULD NEVER BE
PHYSIOLOGIC FACTORS AFFECTING TEST RESULTS
POSTURE__________________________ STRESS____________________________
● E.g., Lipids, Enzymes, Proteins ● E.g., WBC count, acid base balance
DIURNAL RHYTHM_________________ DIET_______________________________

● E.g., Cortisol, ACTH, Fe, Eosinophils ● E.g., Glucose, sodium, CBC
EXERCISE_________________________ SMOKING__________________________
● E.g., Creatine, protein, CK, AST, LD, Platelet and WBC count ● E.g., WBC Count
ROUTINE HEMATOLOGY PROCEDURES

▪ Methemoglobin then combines with potassium
COMPLETE BLOOD COUNT_________ cyanide to form the stable pigment
● Consists of WBC count, RBC count, Hb, Hct and WBC cyanmethemoglobin (HiCN)
Differential ▪ The color intensity of this mixture is measured in a
● Also included are the RBC indices spectrophotometer at a wavelength of 540nm
● Platelet count is not a part of CBC ▪ The optical density of the solution is proportional to
the concentration of hemoglobin, all forms of
HEMOGLOBIN hemoglobin are measured except sulfhemoglobin
o Sources of Error
● Comprised of heme (iron + protoporphyrin) and 4 globin chains High WBC count: Hemoglobin S Lipemic blood
● Measurement of hemoglobin is one of the several tests used to Greater than 20 x and hemoglobin ● Can cause
diagnose and follow treatment of anemia 109/L C turbidity
● 15 – 20 g/dL – at birth ● Can cause and falsely
● 12 – 16 g/dL – adult women High PLT count: turbidity high result
● 13 – 18 g/dL – men Greater than 700 x and falsely
● Hemoglobin fluctuations: 109/L high result
o Diurnal variation ● Can cause
o Position/Posture turbidity and a
o Strenous muscular activity falsely high
o Smokers result
o High altitude Remedy: centrifuge Remedy: dilute Remedy: add
the mixture and use mixture 1:2 with 0.01 ml of
the supernatant water then patient’s plasma
multiply results to 5.0 mL of
by 2 HiCn reagent
and use this
mixture as the
blank
● Blood Oxygen Capacity (Gasometric/Van Slyke Method)

o Measures functional hemoglobin only
o Based on the fact that 1g of Hb carries 1.34 mL of oxygen
● Blood Iron Content
METHODS o Hemoglobin iron content = 100g of Hb = 0.347 g of Fe++
● Copper Sulfate Method (Gravimetric method)
● Cyanmethemoglobin Method o Used for blood donor screening
o Reagents: o The density of the drop of the blood is directly proportional
▪ Potassium cyanide - donates cyanide to hemoglobin to the amount of Hb
▪ Potassium ferricyanide (modified Drabkin’s reagent) o If the hemoglobin is equal to or more than 12.5 g/dL the drop
- converts Fe2+ to Fe3+ of blood will sink within 15 seconds and the donor is
▪ KH2PO4 - replaced the sodium bicarbonate (in the accepted
original drabkin’s reagent), shortens the reaction time o The specific gravity of the copper sulfate solution is 1.053
from 15 minutes to 3 minutes o The drop of blood should be added from a height of about 1
▪ Nonionic detergent – decreases the amount of turbidity cm
resulting from abnormal proteins and improves RBC ● Colorimetric Method
lysis o Acid hematin (Sahli’s Method)
o Sample: EDTA whole blood or capillary ▪ Reagent: 0.1. N HCl
o Principle ▪ A comparator block is used to compare the brownish-
▪ Potassium ferricyanide converts the hemoglobin iron yellow color of the resulting solution
from the Fe++ to Fe+++ to form methemoglobin (Hi = o Alkali hematin
hemiglobin) ▪ Reagent: 0.1 N NaOH
● Cannot lyse fetal RBCs (HbF is alkali-resistant)
▪ HbF is alkali resistant and therefore it can’t be used for ▪ Overanticoagulation
Hb determination of newborns ▪ TRAPPED PLASMA
● After centrifugation, a small amount of plasma
ABNORMAL HEMOGLOBIN PIGMENTS remains in the PCV, and is usually expressed as
a percentage of the RBC column
● Carboxyhemoglobin ● Causes spun hematocrit results to be 1-3% higher
o Formed by combination of hemoglobin with carbon than electronic cell counter
monoxide ● Increased amount of trapped plasma is found in:
o Unable to transport oxygen o Macrocytic anemia
o Affinity for carbon monoxide is 200x greater than oxygen o Spherocytosis
o The formation is reversible o Thalassemia
o Has a brilliant cherry – red color o Hypochromic anemia
o Peak absorbance at 576 nm o Sickle cell anemia
● Methemoglobin ● Macrohematocrit method
o Ferrous ion has been oxidized to ferric state o Time consuming
o Incapable of transporting oxygen molecule o Requires large amount of blood
o Reversible o Contains higher amount of trapped plasma
o Most cases are acquired primarily due to exposure to certain o Equipment
drugs and chemicals (nitrates, nitrites, quinones, chlorates) ▪ Wintrobe tube
o Can cause chocolate brown discoloration of the blood ● 115 mm long
o Peak absorbance at 630 nm ● 3 mm internal bore
● Sulfhemoglobin ● Macrohematocrit: use right calibration
o Not normally found in the blood ● ESR: use left calibration
o Formation is irreversible, it remains for life in the red blood o Rule of Three
cell of the carrier ▪ Applies to specimens that have normocytic,
o It is thought to be formed by the action sulfonamides and normochromic erythrocytes
aromatic amines ▪ RBC x 3 = Hb
o Can combine with carbon monoxide to form ▪ Hb x 3 = Hct +/- 3
carboxyhemoglobin ▪ 1Hct= 0.34g Hb per 100 mL of WB
o Causes a mauve-hemoglobin discoloration of the blood ▪ 1 Hct= 107,000 RBCs/cumm
o Peak absorbance at 618 nm
RBC COUNT
HEMATOCRIT
● 5.0 – 6.5 x 1012/L – N.V. for newborns
● Hematocrit is the volume of packed RBCs that occupies a given ● 3.6 – 5.6 x 1012/L – N.V. for females
volume of whole blood ● 4.2 – 6.0 x 1012/L – N.V. for males
● It is either reported as a percentage (%) or in liters per liter (L/L) ● Highest in the morning and lowest in the evening
● 45-60% - at birth ● Increased in polycythemia vera and in patients who live in places
● 36-48% - Females at a high altitude
● 40-55% - Males
● Fluctuations in hematocrit levels are the same with hemoglobin PRINCIPLE
METHODS ● Whole blood is diluted with isotonic diluting fluid to facilitate

counting and prevent the lysis of RBCs
● Microhematocrit method
o Reagents and Equipment REAGENTS AND EQUIPMENTS
▪ Microhematocrit tube – 75 mm long with an internal
bore of 1.2 mm can hold 0.05 ml of blood ● Pipets
▪ 2 types o RBC unopette (1:200 dilution)
● Red band – contains heparin (spray-dried), to be o 20 uL pipet
used for samples that are non-anticoagulated o Thoma red count pipet
● Blue band – plain tubes, to be used for samples
that are anticoagulated
▪ Clay-like sealing compound
▪ Microhematocrit centrifuge capable of 10,000 –
15,000 RCF
▪ Microhematocrit tube reader
o Specimen
▪ K2-EDTA whole blood is preferred
▪ K3-EDTA causes a 2-3% decrease in the hematocrit
due to shrinkage of the RBCs
o Sources of Error ● RBC count diluting fluid
▪ Incomplete sealing (less than 4-6 mm), leads to falsely o Gower, Eagle, NSS, TOISON, STRONG, BETHEL,
low results HAYEM, DACIE
▪ Inadequate centrifugation (shorter than 5 minutes), ● Hemocytometer
leads to a falsely increased result o Levy chamber with Improved-Neubauer Ruling
▪ Allowing the tube to stand longer than several minutes ▪ Consist of two identically ruled platforms
leads to falsely increased result ▪ The space between the top of the platform and the
cover glass over it is 0.1 mm
▪ Each of the two platforms are composed of nine large
squares which measure 1mm wide and 1mm long;
▪ Therefore, the entire rules are is 9 mm2
▪ The volume of one entire platform is 0.9 uL (3mm x ● White count diluting fluids
3mm x 0.1mm) o 2% Acetic acid, 1% HCl, Turk’s diluting fluid
▪ The volume of one large square is 0.1 uL ● Improved Neubauer Counting Chamber
▪ The large middle square containing 25 smaller squares
is used for RBC count SPECIMEN
▪ The volume of each 25 smaller square is 0.004 uL, for
a total volume of 0.02 uL five small squares ● EDTA whole blood or capillary blood
▪ The four large corner squares, each of which is divided COMPUTATION
into 16 smaller squares, and are used for counting
WBC ● WBC count = # of WBCs counted x VCF x Dilution Factor
CORRECTED WBC COUNT
● Performed when 5 or more nucleated RBCs are present in the PBS

● Formula: Corrected WBC ct. = (Uncorrected WBC count x 100%)
(100 + # of nRBCs per 100 WBC)
● Sample Problem
o A total of 122 white blood cells were counted on one chamber
of the hemocytometer using four WBC squares, and 116 were
counted on the second chamber also using 4 WBC squares.
Blood was aspirated up to the 0.5 mark and diluted up to the 11
mark on the WBC Thoma pipet.
▪ Compute for the WBC count
SPECIMEN ▪ Compute for the corrected WBC count if 5 nRBCs were
present per 100 WBCs
● EDTA whole blood or capillary blood
DILUTION
COMPUTATION
Normal WBC count 1:10 or 1:20
● RBC/L = #cells in five squares x VCF x Dilution Factor If WBC is >30 x 109/L 1:101 (0.02 mL blood + 2.0 mL
● Sample Problem
diluent)
o Blood was aspirated up to .5 mark of the RBC thoma pipet
1:100 (aspirate blood up to 1
and diluted up to the 101 mark. 400 cells were counted on
the first platform using 5 RBC squares, and 415 cells were mark and dilute up to 101 mark
counted on the second platform using the same technique in in the RBC Thoma pipet)
the first chamber. Compute for the RBC count. If WBC is 100 – 300 x 109/L 1:201 (0.02 mL blood + 4 mL
diluent)
WBC COUNT 1:200 (aspirate blood in RBC
● 4.0 – 11.0 x 109/L – N.V. for adults thoma pipet up to the 0.5 mark
● 10.0 – 30.0 x 109/L – N.V. for newborns and dilute up to 101)
● 6.0 – 17.0 x 109/L – N.V. at 1 year of age If WBC is below 3.0 x 109/L 1:11 (0.02 mL blood + 0.2 mL
● Higher in the afternoon
diluent)
● Lower in patients who are exposed to radiation or patient’s
undergoing certain drug therapy 1:10 (aspirate blood up to 1
mark and dilute up to 11 mark
PRINCIPLE in WBC Thoma pipette)
● The diluting fluid is a weak acid which lyses the RBCs to facilitate COMMON DILUTIONS AND COUNTING AREA
WBC counting
CELLS DILUENT DILUTION OBJECTIVE AREA
PROCEDURE WBC 1% ammonium 1:20 or 10x 4mm2
oxalate, 3% 1:100 or 9
● Allow the dilution to sit for 10 minutes to ensure that the red blood acetic acid, 1% mm2
cells (RBCs) have lysed.
HCl
● Leukocyte counts should be performed within 3 hours of dilution
RBC ISOTONIC 1:100 40x 0.2
REAGENTS AND EQUIPMENTS SALINE mm2
PLATELETS 1% ammonium 1:100 40x, phase 1 mm2
● Pipets oxalate
o WBC Unopette (1:20 dilution)
o 20 uL pipet DIFFERENTIAL COUNT
o WBC Thoma pipet
% Absolute Number (x 109/L)
Neutrophil (35-71%) 1.5 – 7.4
Band (0-6%) 0.0 – 0.7 o Cytoplasm: Abundant cytoplasm with gray-blue containing
Lymphocyte (24-44%) 1.0 – 4.4 indistinct granules giving it a “ground glass appearance”
● Lymphocyte
Monocyte (1-10%) 0.1 – 1.0
o Size: Small (6-8 um), medium to large (8-12 um)
Eosinophil (0-4%) 0.0 – 0.4 o Nucleus: Deep purple, compact, densely packed clumps,
Basophil (0-2%) 0.0 – 0.2 may be round oval, or indented
o Cytoplasm: Stains pale to bright sky blue, may contain a few
NOTE: prominent reddish (azurophilic) granules
1. Absolute count is preferred ▪ Robin’s egg blue color
2. If the differential count shows the presence of immature
granulocytes, this is termed shift to the left and may be found in LEUKOCYTE CLASSIFICATION
disorders such as leukemias and bacterial infections
3. A shift to the right refers to an increased number of hyper Granulocytes Neutrophil, Eosinophil, Basophil
segmented neutrophils Non-Granulocytes Monocyte, Lymphocyte
Polymorphonuclear Neutrophil, Eosinophil, Basophil
IF the differential count shows the ff: Mononuclear Monocyte, Lymphocyte
A. Over 10% eosinophils Phagocytes Neutrophil, Eosinophil, Basophil, Monocyte
B. Over 2% basophils Immunocyte Lymphocyte
C. Over 11% monocytes
D. More lymphocytes than neutrophils except in children, a 200-cell COMMON DISEASES THAT INCREASE WBCs
differential count may be performed. The results are averaged and
noted in the report that 200 WBCs were counted NEUTROPHILIA NEUTROPENIA
Appendicitis Decreased neutrophil production
GRANULOCYTES
Myelogenous leukemia ● Inherited stem cell disorder
● Neutrophil, Segmented Bacterial infection ● Acquired stem cell disorder (benzene
o Size: 10 – 15 um poisoning)
o Nucleus: Segmented into 2 – 5 lobes (2 – 4 lobes) Increased neutrophil destruction
o Cytoplasm: Stains light pink, grainy appearance due to the ● Certain bacteria
presence of secondary granules and a few primary granules ● Viral
(purple) Immune reactions
o Other names: Seg., polymorphonuclear neutrophil, poly, ● Autoimmune
PMN ● Isoimmune
● Neutrophil, Band ● Drug-induced
o Size: 10 – 15 um Sequestration
o Nucleus: elongated, curved, sausage shape EOSINOPHILIA EOSINOPENIA
▪ Pinkish than purplish due to secondary granules Allergies (asthma, hay fever, Decreased production
(pinkish (+) secondary granules) psoriasis eczema) Acute bacterial infections
o Cytoplasm: Identical to segmented neutrophil Scarlet fever ACTH administration
o Other name: Nonsegmented neutrophil, neutrophil staff or Parasitic infections
stab
● Eosinophil MONOCYTOSIS MONOCYTOPENIA
o Size: 12 – 17 um Brucellosis Glucocorticoids
o Nucleus: Dark purple, band shaped or segmented with only Tuberculosis Overwhelming infections that also cause
two lobes Subacute Bacterial neutropenia
▪ Bilobed Endocarditis
o Cytoplasm: Contains large, spherical granules that stain Typhoid
orange – pink
Rickettsial infections
▪ Secondary granules contain MBP, and is basic (attracts
acidic dye) Hodgkin’s disease
o Other name: Acidophil (affinity for the acidic dye or eosin) Gaucher disease
o Major basic protein (MBP) = has anti-helminthic properties LYMPHOCYTOSIS LYMPHOCYTOPENIA
● Basophil Viral infections Long-term drug therapy
o Size: 10 – 14 um Whooping cough Immunodeficiency
o Nucleus: Light to purple staining, usually difficult to see due
Infectious mononucleosis
to overlying granules
o Cytoplasm: Densely stained, dark violet granules Lymphocytic leukemia
o Contains histamine, heparan, chondroitin sulfate BASOPHILIA BASOPENIA
o Acidic: attracts basic dye Immediate hypersensitivity Stress
o Bilobed most of the time reactions Hyperthyroidism
Hypothyroidism Increased glucocorticoid levels
MONONUCLEAR CELLS
RED BLOOD CELL INDICES AND RDW
● Monocyte
o Size: 12 – 20 um
o Nucleus: Round, horseshoe - shaped or lobulated, usually MEAN CORPUSCULAR VOLUME (MCV)
folded or with convolutions
● Indicates the average volume of RBCs in femtoliters (fL)
● Formula: MCV= Hct x 10 o Pressure
RBC ct o Angle
● Reference Range: <80 fL – MICROCYTIC, 80 – 100 fL – o Size of Blood Drop
NORMOCYTIC, >100 fL - MACROCYTIC o Speed
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
● An expression of the average concentration of hemoglobin in red

blood cells COVERSLIP TECHNIQUE
● Formula MCHC = Hb x 100
Hct ● The only advantage is the even distribution of leukocytes
● <32 g/dL – HYPOCHROMIC, 32 – 36 g/dL – ● Rarely used
NORMOCHROMIC, >36 g/dL – ● Requires a small drop of blood be placed on a 22mm x 22mm
SPHEROCYTIC/HYPERCHROMIC coverslip
● Another coverslip is drawn across to produce 2 blood films
MEAN CORPUSCULAR HEMOGLOBIN (MCH)
STAINING OF PERIPHERAL BLOOD FILMS
● Indicates the average weight of hemoglobin in the red blood cells
● Not considered in the classification of anemias ROMANOWSKY STAIN
● Formula: MCH = Hb x 10
RBC ct ● Pure Wright stain, Wright-Giemsa stain
● Reference Range: 28 – 32 pg ● Considered as polychrome stains because they contain methylene
blue and eosin
RED CELL DISTRIBUTION WIDTH ● Free methylene blue is basic (positively charged) and stains acidic
● Determined from the RBC histogram; coefficient of the variation components such as RNA
of the MCV ● Free eosin is acidic (negatively charged) and stains basic
● Directly proportional to the variation in RBC size (anisocytosis) components such as hemoglobin and eosinophilic granules
● Reference range: 11.5 – 14.5%
● High RDW: post-transfusion, post-treatment (Fe supplements, PROBLEMS ENCOUNTERED IN STAINING
vit. B12, or folic acid therapy), idiopathic sideroblastic anemia,
presence of two deficiencies (iron and folic acid deficiency) EXCESSIVELY BLUE STAIN EXCESSIVELY PINK STAIN
● Thick films ● Insufficient staining time
MCV (fL) MCHC RBC Morphology Condition/Disease ● Prolonged staining time ● Prolonged washing time
<80 <32 Microcytic; IDA, Thalassemia, ● Inadequate washing ● Mounting the coverslip before the
● Too high alkalinity of buffer slide is dry
hypochromic sideroblastic anemia,
● High acidity of the stain
defective iron use, chronic
infection/inflammation STAINING CHARACTERISTICS OF CELLS ON WRIGHT
80 – 100 32 – Normocytic; Hemolytic anemia, aplastic GIEMSA STAIN
36 normochromic anemia, acute blood loss
anemia ● RBC: pink to orange
● Reticulocytes: pinkish gray
>100 32 - 36 Macrocytic; Liver disease,
● Neutrophils: Dark purple nuclei, light pink cytoplasm with lilac
normochromic myelodysplasias, granules
megaloblastic anemia ● Lymphocytes: dark purple nuclei, varying shades of blue
cytoplasm
PROCEDURE FOR EXAMINATION OF ● Monocyte: lighter purple nucleus, gray blue cytoplasm
● Eosinophil: bright orange granules
THE STAINED BLOOD SMEAR_______ ● Basophils: dark blue-black granules
● Platelets: Violet to purple
TYPES OF FILMS
METHODS OF EXAMINATION
MANUAL WEDGE TECHNIQUE ● Cross-sectional or crenellation: slide is moved side to side
● Longitudinal method: slide is moved tail towards the head
● Most convenient and most commonly used ● Battlement method: uses a pattern of consecutive fields beginning
● Makes use of 2 clean glass slides near the tail on a horizontal edge: count three consecutive
● The size of the blood drop must be 2 – 5 mm horizontal edge fields, count two fields towards the center of the
● A drop of blood that is too large causes a long and thick smear, smear, count two fields horizontally, count two fields vertically
and a too small of a blood drop causes a short and thin smear to the edge
● The angle must be 30 – 45 degrees o “Serpentine/Track” pattern: most preferred
o 25 – 40 degrees: alternative answer (from Steinenger)
● Qualities of a Properly Made Wedge Smear
o The film is 2/3 to ¾ of the slide PLATELET COUNT_________________
o The film is finger shaped not bullet shaped ● Important in helping to diagnose bleeding disorders
o Without holes or irregularities ● Normal range: 150,000 – 450,000/uL (150 – 450 x 109/L)
● Factors that Affect the Thickness of a Wedge Smear
● Thrombocytosis: Polycythemia vera, idiopathic ● Whole blood is diluted with 1% ammonium oxalate, which
thrombocythemia, chronic myelogenous leukemia, splenectomy hemolyzes red cells
● Thrombocytopenia: Thrombocytopenia purpura, aplastic anemia, ● Platelets are counted using phase contrast microscope
acute leukemia, Gaucher’s disease, pernicious anemia ● EDTA can cause platelet satellitosis, this can be corrected using
sodium citrate as the anticoagulant and multiply the platelet count
SPECIMEN by 1.1
TONKANTIN METHOD
● EDTA decreases platelet clumping but the MPV will increase
during the first hour in the tube ● Uses a light microscope
● Capillary blood ● Rees and Ecker diluent
o Brilliant Cresyl Blue – stain
METHODS
o Sodium Citrate – anticoagulant
● NOTE: Place the charged hemocytometer in a moist chamber for o Formaldehyde – preservative
15 minutes to allow the platelets to settle o Distilled H2O
● Platelets appear as small, round, oval or elongated particles that
PHASE CONTRAST are highly refractile and stain a light bluish color
● “Bretcher – Cronkite Method”

● Reference method
SPECIAL HEMATOLOGY PROCEDURES
RETICULOCYTE COUNT____________ ● ARC = 2 x (2.20 x 1012) = 44 x 109/L

100
● Reticulocyte is the last immature erythrocyte stage ● Reference Range: 25 x 109/L up to 75 x 109/L are within the
● Normally a reticulocyte spends 2-3 days in the bone marrow and reference range for most populations
1 day in the peripheral blood
● Reticulocytes contain remnants RNA and organelles such as
ribosomes CORRECTED RETICULOCYTE COUNT
● Reticulocyte count is used to assess the erythropoietic activity of ● In specimens with low hematocrit, the percentage of reticulocytes
the bone marrow may be falsely elevated because whole blood contains fewer
● N.V.=0.5 – 1.5% RBCs
● % RC = # of reticulocytes/1000 RBCs ● A correction factor is used, with the average normal hematocrit
10 considered to be 45%
● Decreased in aplastic anemia, and in conditions in which the bone ● CRC = (%) reticulocyte x patient Hct
marrow is not producing red blood cells 45 (constant; assumed to be the
● Increased in hemolytic anemias, individuals with IDA receiving normal average hematocrit)
iron therapy, thalassemia, sideroblastic anemia, and in acute and ● Reference Range: Patients with a hematocrit of 35% are expected
chronic blood loss anemia to have a corrected reticulocyte count of 2%-3%. In patients with
a hematocrit of less than 25%, the count should increase to 3%-
PROCEDURE 5% to compensate for anemia
● 1. Mix equal amounts of blood and new methylene blue stain (2
to 3 drops, or 50 mcl each) and allow to incubate at room RETICULOCYTE PRODUCTION INDEX
temperature for 3 – 10 minutes ● Reticulocytes that are released from the bone marrow prematurely
● 2. Remix the preparation are called shift reticulocytes
● 3. Prepare two wedge films ● Cells shifted to the peripheral blood prematurely stay longer as
● 4. In an area in which cells are close together but not touching, reticulocytes and contribute to reticulocyte count for more than 1
count 1000 RBCs under the OIO, reticulocytes are included in day
the total RBC count (i.e., A reticulocyte counts as both an RBC ● The reticulocyte count is falsely increased because the count no
and a reticulocyte). longer represents the cells maturing in just 1 day
● 5. To improve accuracy, have another laboratorian count the other Patient’s Correction Factor
smear; values should agree within 20% Hct (Maturation Days)
● 6. Calculate the reticulocyte count 40 – 45 1
35 – 39 1.5
PRINCIPLE 25 – 34 2
● Whole blood EDTA sample is stained with a supravital stain, such 15 – 24 2.5
as new methylene blue < 15 3
● Reticulocytes appears as nonnucleated RBCs that contain two or ● RPI = reticulocytes (%) x [Hct (%)/45] or RPI = corrected reticulocyte count
more blue-stained granulofilamentous material Maturation Time Maturation Time
o For example, a patient with a reticulocyte count of 7.8% and
Hct of 30%, and with polychromasia noted, the previous
ABSOLUTE RETICULOCYTE _______ table indicates a maturation time of 2 days. Thus
● The absolute reticulocyte count is the actual number of RPI = 7.8 x [30/45]
reticulocytes in 1L of whole nlood 2
● ARC = (%) reticulocytes x RBC count (x 1012) RPI = 2.6
100 ● Reference Range: RPI > 3 = Adequate bone marrow response
RPI < 2 = Inadequate bone marrow response STAGES
● Lag phase: in the first 10 minutes rouleaux formation occurs and
ERYTHROCYTE SEDIMENTATION RATE sedimentation rate is slight
● A nonspecific measurement used to detect and monitor an ● Decantation phase: Sedimentation occurs for a period
inflammatory response approximately 40 minutes at a more rapid and constant rate
● The settling of RBCs at the bottom of a tube upon standing ● Slow sedimentation: occurs at the last 10 minutes, RBCs
undisturbed for 1 hour accumulate at the bottom of the tube
● ESR is the distance in millimeters that the RBCs fall in 1 hour CATEGORY INCREASED ESR DECREASED ESR
Blood proteins Hypercholesterolemia Hyperalbuminemia
FACTORS THAT AFFECT THE ESR and lipids Hyperfibrinogenemia Hyperglycemia
Hypergammaglobulinemia Hypofibrinogenemia
ERYTHROCYTES Hypoalbuminemia Hypogammaglobulinem
Increased bile salts
● The larger the RBCs, the faster the rate it falls Increased phospholipids
● Normal erythrocytes have a relatively low mass and settle slowly
Red blood cells Anemia Acanthocytosis
● They are negatively charged and repel each other
● Increased Macrocytosis Anisocytosis (marked)
o Rouleaux formation: caused by an alteration in plasma Hemoglobin C
protein concentration, this leads to a larger RBC mass and Microcytosis
faster sedimentation velocity Polycythemia
o Agglutination Sickle cells
o Macrocytes
Spherocytosis
o In severe anemia, the ESR is markedly elevated
Thalassemia
● Decreased
o Sickle cells and spherocytes White blood Leukemia Leukocytosis (marked)
o Anisocytosis and poikilocytosis cells
Drugs Dextran Adrenocorticotropic hormo
PLASMA COMPOSITION Heparin (corticotropin)
Penicillamine Cortisone
● The single most important factor in determining ESR
Procainamide Ethambutol
● Rouleaux and aggregation of the red blood cells are controlled
primarily by the levels of acute phase proteins (fibrinogen, a-1 Theophylline Quinine
globulin, and a-2 globulin) Vitamin A Salicylates
● ESR is directly proportional to RBC mass and inversely Clinical Acute heavy metal poisoning Cachexia
proportional to plasma viscosity conditions Acute bacterial infections Congestive heart failure
● Increased concentration of albumin will tend to lower ESR Collagen vascular diseases Newborn status
Diabetes mellitus
MECHANICAL / TECHNICAL FACTORS
End-stage renal failure
● A tilt of 3o can cause errors up to 30% Gout
● The rack holding the tubes should not be subject to vibration Malignancy
● Setting the rack of sedimentation rate tubes on top of the Menstruation
refrigerator could lead to: Multiple Myeloma
o A falsely decreased ESR because of lower temperatures form Myocardial infarction
air rushing out on opening the refrigerator or freezer
Pregnancy
o A falsely increased ESR attributable to vibrations from
opening and closing the refrigerator doors Rheumatic fever
o A falsely increased ESR because of the head released from Rheumatoid arthritis
the refrigerator motor Syphilis
Temporal arteritis
WESTERGREN WINTROBE Specimen Refrigerated sample not Clotted blood sample
Length: 30 cm (300mm) Length: 115 mm handling returned to room Delay in testing
Bore: 2.55 mm Bore: 3 mm
temperature
Calibration: 0-200 Calibration: scale on the left is used
for ESR (0-100 mm) Technique High room temperature Bubbles in ESR column
NORMAL VALUES: Scale on the right is for Tilted ESR tube Low room temperature
0-15 mm: Women macrohematocrit (100-0) Vibration Narrow ESR column
0-10 mm: Men diameter
0-10 mm: Children NORMAL VALUES:
0-20 mm/hr: Women SOURCES OF ERROR
Specimen: Na citrate whole blood 0-9 mm/hr: Men
(4:1) More sensitive for patients with ● 1. Overanticoagulation with EDTA causes a falsely low ESR
More sensitive for patients with high lower ESR ● 2. If ESR stands more than 60 minutes, the results will be falsely
ESR elevated, if the test is timed for less than 60 minutes, invalidly low
results are obtained
● 3. Temperature
● 4. Tilting of the ESR tube
● 5. Bubbles in the blood causes an invalid result
● 6. Fibrin clots present invalidate results ● The Hb A and Hb S reagents contain monoclonal antibodies
(IgG), which specifically binds to amino acids at or near the sixth
EOSINOPHIL COUNT__________________ position of the globin chain of hemoglobin A and hemoglobin S
● Interpretation:
● N.V. 50 – 350 x 106/L o Positive: Red or Pink Color
● Eosinopenia: hyperadrenalism, (Cushing’s disease), shock, o Negative: Green, White or Gray
ACTH administration o Positive for HbS and HbA is indicative of sickle cell trait
● Eosinophilia: Allergic reactions, parasitic infestations, o Positive for HbS and negative for HbA is indicative of
brucellosis, certain leukemias sickle cell anemia
VARIATIONS SUGAR WATER SCREENING TEST

● Lowest in the late morning ● A screening test for Paroxysmal Nocturnal Hemoglobinuria
● Highest during the night ● PNH is an acquired disorder in which RBCs are abnormally
sensitive to normal plasma constituents
METHODS ● PRINCIPLE:
o Whole blood is mixed with a sugar water solution and
DIRECT METHOD incubated at room temperature
o PNH red cells are abnormally susceptible to lysis by
● Similar to the method used for RBC and WBC count complement and under the conditions of this test will show
● FORMULA: Eosinophils/L = Eosinophils counted x DF x VCF hemolysis
● DILUTING FLUIDS: o Hemolysis indicates a positive result
o Phyloxine Diluting fluid: Propeleneglycol, distilled water,
phyloxine, sodium bicarbonate SUCROSE HEMOLYSIS TEST___________
o Pilots solution: Same as phyloxine diluting fluid except that
● A confirmatory test for PNH when the sugar water test is positive
100 units of heparin is added
● PRINCIPLE:
o Randolph;s solution: either uses methylene blue in propylene
o Washed RBCs are incubated in an isotonic sucrose solution
glycol or phyloxinein methylene blue
containing normal ABO compatible serum
● Principle:
o RBCs absorb complement components from serum
o Phyloxine present in the diluting fluid stains the eosinophils
o PNH red cells are more sensitive than normal cells and will
red, sodium carbonate and water help to lyse the WBCs
hemolyze under this condition
(except eosinophils), and propelene glycol lyses RBCs.
o Heparin if present helps prevent clumping of WBCs.
ACID SERUM TEST____________________
INDIRECT METHOD
HAM’S METHOD
● A WBC count is performed on a specimen of blood
● Make two blood smears and stain ● The patient’s RBCs are mixed with normal serum, with the
● Perform a 200-cell differential count on the blood smear patient’s own serum, and with the normal serum inactivated to
● FORMULA: Eosinophils/L = (%) Eosinophil in differential x destroy the complement
WBC/L ● A weak acid is added to the tubes in order to adjust the pH and
maximize hemolysis
● Normal RBCs are also incubated with acidified serum to serve as
SICKLE CELL TESTS__________________ normal control
● Sickle cell anemia and sickle cell trait are caused by hemoglobin S ● INTERPRETATION:
● In the presence of hemoglobin S, the RBCs take on a sickle-like o Normal – no lysis in any tube
shape when the oxygen supply is decreased o PNH – hemolysis of patient’s red cells with non-inactivated,
● In sickle cell anemia, sickling readily occurs at only slightly acidified, normal serum and in the patient’s own non-
reduced oxygen concentration because the concentration of HbS inactivated, acidified serum
is 80 – 100% ● NOTE: When the patient has received blood transfusion, less lysis
● In sickle cell trait, there is 20-40% HbS, sickling only occurs at occurs because of the presence of normal transfused red blood
much lower oxygen concentrations cells
SODIUM METABISULFITE METHOD OSMOTIC FRAGILITY TEST____________

● Sodium metabisulfite deoxygenates hemoglobin ● A test to measure the ability of the red cell to take up diluting fluid
● Hemoglobin S present in the red cell causes the formation of without lysing
sickle shaped red cells ● The primary factor affecting the red the osmotic fragility test is
● Cannot differentiate between sickle cell anemia and trait the shape of the red cell, which, in turn, depends on the volume,
● Interpretation: The sickle cells or holly-leaf forms must come to surface area, and functional state of the RBC membrane
a point/s to be considered positive ● Increased osmotic fragility (less resistant) – spherocytosis,
hemolytic anemia
SODIUM DITHINITE TEST / SOLUBILITY TEST ● Decreased osmotic fragility (more resistant) – splenectomy, liver
disease, sickle – cell anemia, 10A, thalassemia, conditions
● Red blood cells immediately lyse in the presence of saponin
wherein target cells are present
● HbS and other sickling hemoglobins, in reduced state, form liquid
● Reticulocytes have a decreased OFT, while older cells are more
crystals and yield a turbid appearance
fragile
HEMOCARD HB A AND S PROCEDURE
● Hemolyzes the RBCs using a sample conditioner reagent
METHOD o Count petechiae 5 minutes after release
● SANFORD METHOD
o 0.5% saline is used as a diluent BLEEDING TIME______________________
o 12 tubes labeled 14-25 ● Time it takes for a standard wound to stop bleeding
o Number corresponds to the drops of 0.5% saline ● Tests for:
o Equate number of drops to 25 in all tubes o Abnormalities of platelet function and number
o Factor used is 0.02 o vWF deficiency
o N.V. initial hemolysis at tube 22 and complete hemolysis at o Abnormality of vessel wall structure
tube 17
METHODS
Tube # 14 15 16 17 18 19 20 21 22 23 24 25
0.5% 14 15 16 17 18 19 20 21 22 23 24 25
saline MODIFIED DUKE METHOD
Distilled 11 10 9 8 7 6 5 4 3 2 1 0
H20 ● 15 – 20 mm above rounded fatty portion of earlobe
● Uses a no. 11 sterile Bard-Parker surgical blade
● N.V.: < 8 minutes (2 – 4 minutes)
LUPUS ERYTHEMATOSUS PREPARATION
● Antinuclear antibodies occur in the serum of patients with a IVY METHOD
number of disorders (SLE)
● The ANA is also termed as the LE factor and is a component of ● Volar surface of the forearm is used
the globulin fraction ● Use a blood pressure cuff and apply 40 mmHg of pressure
● Puncture twice
PRINCIPLE ● Collect blood using filter paper strips after 2 minutes and again
after every 30 seconds
● In order for the LE factor to lyse the neutrophil nuclei, the cells ● N.V.: 3 – 6 minutes
must first be damaged to allow for liberation of nucleic from the
cells STANDARDIZED SIMPLATE TEST
● Whole blood is mashed for a period of time in order to damage
some of the neutrophils ● Incision: 9 mm x 1 mm deep
● The blood is then incubated to allow for the lysis of the neutrophil ● N.V.: 6 – 10 minutes
nuclei
The whole blood is centrifuged and buffy coat smears are stained
●
and examined
LEE AND WHITE COAGULATION TIME_
● In the past, the Lee and White clotting time was used as a
screening test to measure all stages of intrinsic coagulation system
MALARIAL SMEAR PREPARATION_____ and monitor heparin therapy
● Wright or Giemsa Stained Smear ● It is sensitive only to extreme factor deficiencies and is insensitive
● Specimen of choice is capillary blood to high doses of heparin
● Thick smears are for detection while thin smears are used for
identifying the species PROCEDURE
● Identification requires consideration of three factors
o Appearance of infected RBC ● 1 mL is dispensed into three tubes (3,2,1)
o Appearance of parasites ● Timer is started by the time blood is dispensed at tube 3
o Stages found in the smear ● Incubate at 37oC for 5 minutes using a water bath
● Timing of collection ● Tube 1 is tilted every 30 seconds at an angle of 45o to check for
o Malaria – in between paroxysms clotting
o Trypanosomes – Early acute phase ● The time when tube has clotted is recorded, after 30 seconds tube
o Filaria – depends on species 2 is checked for clotting until a clot has formed
● Repat the same procedure for tube 3
● Clotting time for tube 3 is reported
CAPILLARY FRAGILITY TEST__________ ● N.V.: 5 – 15 minutes
● Tests for capillary abnormality that may be due to a defect in the
capillary walls or to some type of thrombocytopenia SPECIAL HISTOCHEMICAL STAINS_____
● Occasionally abnormal in hemophilia and Vit. K disorders
METHODS PRUSSIAN BLUE REACTION
● 1. Positive pressure test / Rumple – Leede / Torniquet test ● Perl’s reagent (potassium ferricyanide – HCl)
o Blood pressure cuff is applied to the upper arm ● Principle: Prussian blue reagent stains Fe++ a vivid blue or green
o Pressure applied should be between systole and diastole (100 color
mmHg for males; 80 mmHg for females) ● Stains siderotic granules, Pappenheimer bodies and hemosiderin
o After 5 minutes examine for petechiae formation
o First examination site should not be repeated within 7 – 14 LEUKOCYTE ALKALINE PHOSPHATASE
days ● Stains ALP present in the neutrophils
● 2. Negative pressure test / Hess test / Suction test ● Helpful in differentiating CML from leukemoid reaction or
o A 2 cm suction cup is used polycythemia vera
o Mid portion of the upper arm is used ● Increased: Polycythemia vera, last trimester of pregnancy,
o The suction cup is applied for 1 minute infections with neutrophilia
o Pressure applied is 200 – 250 torr ● Decreased: CML, PNH, sickle cell anemia, IM, PA
● LAP score: rate 100 neutrophils based on quantity and intensity ● Used to help in the diagnosis of DiGuglielmo’s Syndrome (FAB
of precipitated dye M6)
● Cytoplasm of cells will be colorless to pale blue ● L1 and L2 produce a block pattern
● Mature neutrophils and bands are the only graded cells
● Score ranges from 0-4+ CHLOROACETATE ESTERASE
● N.V.: 30 – 185
● Stains esterases in granulocytes
● Used to differentiate granulocytic cells from monocytic cells
SPECIMEN
● Fresh capillary blood is recommended NONSPECIFIC ESTERASE

● Heparin anticoagulated whole blood may be used alternatively ● Stains esterases present in the monocytic cells, macrophages,
Score No. of cells Score X no. of cells megakaryocytes, and platelets
0 30 0 ● Used to differentiate monocytic leukemias from granulocytic
1 20 20 leukemias
2 25 50
3 30 90 TARTRATE-RESISTANT ACP
4 10 40
● Stains ACP present in the myelogenous cells, lymphocytes,
LAP SCORE = 200
plasma cells, monocytes and platelets
● Using L(+) tartaric acid, the stain is helpful in diagnosing hairy-
MYELOPEROXIDASE STAIN
cell leukemia
● Stains peroxidases present in granulocytes and monocytes
● Used to differentiate acute myelogenous leukemia and monocytic TOLUIDINE BLUE
leukemia from acute lymphocytic leukemia
● Binds with acid mucopolysaccharides in blood cells
● Similar to Sudan Black B
● Useful for the recognition of mast cells and tissue basophils
● Recommended specimen is fresh blood smear from a capillary
puncture
NITROBLUE TETRAZOLIUM NEUTROPHIL REDUCTION
TEST
SUDAN BLACK B
● Screening procedure for the detection of chronic granulomatous
● Stains lipids present in granulocytes and monocytes
disease
● Used to differentiate acute myelogenous leukemia and
● Heparinized whole blood is recommended specimen
myelomonocytic leukemias from acute lymphocytic leukemia
TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE
PERIODIC ACID SCHIFF STAIN ● Stains DNA polymerase
● Present in 90% cases of ALL
● Stains mucoproteins, glycoproteins, and high molecular weight
● Used to differentiate AML from ALL
carbohydrates
● Does not stain carbohydrates on pronormoblasts
HEMATOPOIESIS
● A continuous, regulated process of blood cell production that includes cell renewal, proliferation, differentiation, and maturation
STAGES OF HEMATOPOIESIS_________ MEDULLARY (MYELOID) PHASE

● At 5th month of development, hematopoiesis begins in the bone
MESOBLASTIC PHASE (YOLK SAC PHASE) marrow
● M:E ratio reaches adult levels of 3:1 at 21 weeks of gestation
● Begins during the embryonic development in blood islands of the ● Measurable levels of Hb A1, fetal hemoglobin, and Hb A2
yolk sac at around 19 days of gestation ● After the first 3 weeks postpartum, the bone marrow becomes the
● Primitive erythroblasts arise from mesodermal cells
only normal site of blood cell production and remains so throughout
● Primitive hematopoiesis (does not contribute to definitive life
hematopoiesis)
● Characterized by the development of primitive erythroblasts that
produce hemoglobin (Portland, Gower-1, Gower-2) ADULT HEMATOPOIETIC TISSUE_____
● This phase of hematopoiesis occurs intravascularly
BONE MARROW
HEPATIC PHASE
● Begins at 4th to 5th gestational weeks TWO TYPES
● Characterized by clusters of developing erythroblasts, granulocytes,
and monocytes ● Red Marrow
● Start of definitive hematopoiesis o Hematopoietically active
● Lymphoid cells begin to appear o Predominant type during infancy and childhood
● Liver is the major site of hematopoiesis and retaining activity until o Composed of extravascular cords that contain all developing
1 – 2 weeks after birth cells (stem and progenitor cells, adventitial cells, and
● The spleen, kidney, thymus, and lymph nodes contribute to the macrophages)
hematopoietic process o The hematopoietic cells tend to develop in specific niches
● Detectable levels of hemoglobin (Hb) F, Hb A, and Hb A2 may be within the cords:
present
▪ Normoblasts develop in small clusters adjacent to the STEM CELL CYCLE KINETICS AND
outer surfaces of the vascular sinuses; in addition, some
normoblasts are found surrounding iron-laden CYTOKINES_________________________
macrophages
▪ Megakaryocytes are located close to the vascular walls of TERMINOLOGIES
the sinuses to facilitate release of platelets
▪ Immature myeloid (granulocyte) cells through the ABBREVATION CELL LINE
metamyelocyte stage are located deep within the cords CFU-GEMM Granulocyte, erythrocyte, megakaryocyte, monocyte
o At 18 years of age, the only active hematopoietic sites are: CFU-S Colony Forming Unit Spleen
▪ Sternum, skull, vertebrae, ribs, pelvis, proximal CFU-E Erythrocyte
extremities of long bones
CFU-MEG Megakaryocyte
● Yellow Marrow
o Hematopoietically inactive, composed of adipocytes CFU-M Monocyte
o Between ages of 5 to 7, adipocytes become more abundant CFU-GM Granulocyte, monocyte
o The process of replacing the red marrow by the yellow marrow CFU-BASO Myeloid to basophil
is called retrogression CFU-EO Myeloid to eosinophil
o It is capable or reverting back to active marrow in cases of
CFU-G Myeloid to neutrophil
increased demand
o Approximately, there is equal amount of red and yellow CFU-PRE-T T lymphocyte
marrow in adults CFU-PRE-B B lymphocyte
LIVER STEM CELL THEORY

nd
● Significant role in hematopoiesis in the 2 trimester and the major ● Monophyletic Theory - All blood cells are derived from a single
site during hepatic stage pluripotent stem cell
● Capable of extramedullary hematopoiesis (counter part of hepatic ● Polyphyletic Theory - Suggests that each of the blood cell lineages
phase in adults) in case of bone marrow shut down is derived from its own unique stem cell
● CFU-S-Mice spleens and bone marrows are irradiated, afterwards,
SPLEEN marrow cells are intravenously injected into the mice. Colonies to
HSCs are observed after 7-8 days and were referred to as CFU-s,
● Removes senescent RBCs
which will correspond to CFU-GEMM today
● Sequesters approximately 30% of platelets
● Common Lymphoid Progenitor – T-cell, B-cell and NK-cell lineage
● 3 Types of Tissues
● Common Myeloid Progenitor – Granulocytic, Erythrocytic,
o a. White pulp – consists of scattered follicles with germinal
Monocytic, Megakaryocytic lineage
centers containing lymphocytes, macrophages, and dendritic
cells
KINETICS
o b. Marginal zone – forms a reticular meshwork containing
blood vessels, macrophages, and specialized B cells ● 3 billion RBCs, 2.5 billion platelets, 1.5 billion granulocytes per
o c. Red pulp – comprised on dendritic processes that create a kilogram of body weight are produced by the bone marrow daily
filter the cords of Billroth stagnates and depletes the glucose ● Stem cells exist in a ratio of 1:1000 nucleated blood cells
supply of RBCs that lead to their removal ● Mitotic index: A calculated value used to establish the number of
▪ Culling - cells that are phagocytosed with subsequent cells undergoing mitosis, normally it is 1-2%
degradation of cells and organelles ● Increased mitotic index indicates increased proliferation except in
▪ Pitting - splenic macrophages remove inclusions or megaloblastic anemia wherein mitosis is prolonged
damaged surface membrane forms RBCs
CELL CYCLE
LYMPH NODES
● a. G1 - RNA and proteins synthesis, presynthetic stage
● Formation on new lymphocytes from germinal centers ● b. Synthesis (S) – DNA synthesis
● Processing of specific immunoglobulins ● c. G2 – premitotic stage, postsynthetic stage
● Filter particulate matter, debris, and bacteria entering the lymph ● d. Mitosis – cell division
node o i. Interphase – correlates with G1, S, and G2
o ii. Prophase - chromosomes condense
THYMUS o iii. Prometaphase - centrosomes move to opposite poles
● Densely populated with progenitor lymphoid cells that migrated o iv. Anaphase - sister chromatids segregate
from the bone marrow and will soon give rise to T-cells o v. Telophase - chromatids move to opposite poles, cell
divides
● e. G0 – rest stage, quiescence, limbo, repair
CYTOKINES AND GROWTH FACTORS

● Cytokines – glycoproteins that REGULATE the proliferation,
differentiation and maturation of hematopoietic precursor cells
o POSITIVE INFLUENCE – IL-1, 3, 6, 9, 11, GM-CSF, and kit-
ligand
o NEGATIVE INFLUENCE – Transforming growth factor B,
TNF-a, Interferons
OVERVIEW OF HEMATOPOIESIS_____________________________________________
ERYTHROPOIESIS___________________ RBC DEVELOPMENTAL STAGES
PRONORMOBLAST (RUBRIBLAST)
GENERAL MORPHOLOGIC CHANGES ASSOCIATED
WITH MATURATION ● Nucleus takes up much of the cell (high N:C ratio)
NUCLEUS CYTOPLASM ● Measures 14-20 um and cytoplasm is quite blue
1. Loss of nucleoli 1. Decrease in basophilia ● Globin production begins
2. Decrease in size of 2. Increase in the
nucleus proportion of the BASOPHILIC NORMOBLAST (PRORUBRICYTE)
3. Condensation of cytoplasm
chromatin 3. Appearance of ● N:C ratio decreases to 6:1
4. Possible changes in granules ● Nucleoli usually not visible
shape ● Measures 12-17 um and the cytoplasm stains deep blue
5. Possible loss of nucleus ● Detectable level of hemoglobin synthesis (minute
amount)
MORPHOLOGIC CHANGES ASSOCIATED WITH RBC
MATURATION POLYCHROMATIC NORMOBLAST (RUBRICYTE)
1. Decrease in overall size
● N:C ratio is 4:1
2. Decrease in size of nucleus and in N:C ratio
● Measures 10-15 um and the cytoplasm is pink blue
3. Nuclear chromatin pattern
(murky-gray blue)
4. Nucleoli disappear
● This is the last stage capable of mitosis
5. Decrease in basophilia
● 1st stage where Hb synthesis is visible
ERYTHROCYTIC NOMENCLATURE
ORTHOCHROMIC NORMOBLAST
NORMOBLASTIC RUBRIBLASTIC ERYTHROBLASTIC (METARUBRICYTE)
Pronormoblast Rubriblast Proerythroblast
Basophilic Prorubricyte Basophilic erythroblast ● The nucleus is pyknotic
normoblast ● Pink-orange color of the cytoplasm reflects nearly
Polychromatic Rubricyte Polychromatic complete production of hemoglobin
normoblast erythroblast ● Later in this stage the nucleus is ejected
Orthochromic Metarubricyte Orthochromic
normoblast erythroblast POLYCHROMATOPHILIC ERYTHROCYTE
Polychromatophilic Reticulocyte Polychromatophilic (RETICULOCYTE)
erythrocyte erythrocyte
● No nucleus
Erythrocyte Erythrocyte Erythrocyte
● Cytoplasm is the predominant color of hemoglobin (pink) ● Deficiency in enzymes required for cholesterol exchange between
● Called a reticulocyte when the remnants of the ribosomal RNA the plasma and RBC membrane leads to a decrease in tensile
(reticulum) are stained with supravital stain (E.G., Nmb) strength (acanthocytosis)
● When cholesterol increases, the RBCs gains tensile strength but
ERYTHROCYTE loses elasticity
● No nucleus RBC MEMBRANE PROTEINS

● Biconcave disc measuring 7-8 um in diameter
● Appears salmon pink or red with a pale central pallor ● Skeletal membrane proteins such as spectrin and ankyrin provide
● Has a lifespan of 120 days structural support to the RBC membrane
● Loss of ATP leads to decrease in spectrin phosphorylation and
ERYTHROKINETICS subsequently a loss in membrane deformability
● Erythron: collection of all stages of erythrocyte throughout the body
OSMOTIC BALANCE AND PERMEABILITY
● RBC Mass: cells in the circulation
● Hypoxia is detected by peritubular interstitial cells, which produces ● The RBC membrane is impermeable to Na, K, and Ca
EPO
● It is permeable to water, HCO3, and Cl
● EPO: a glycoprotein hormone which is the major stimulatory ● Damage to the ATPase cation pumps leads to influx of sodium
cytokine for RBC ● As Na enters the cell, water follows causing it to swell and become
o Early release of reticulocytes
spheroid
o Inhibition of apoptosis
o Reduced marrow transit time
ERYTHROCYTE DESTRUCTION
ERYTHROCYTE METABOLISM MACROPHAGE – MEDIATED HEMOLYSIS (NORMAL

EXTRAVASCULAR HEMOLYSIS)
EMBDEN – MEYERHOF PATHWAY
● The spleen generates a stressful environment for the RBCs
● Anaerobic glycolytic pathway ● Glucose is low that leads to reduced glycolysis and reduced ATP
● Results in a net gain of 2 ATP molecules per 1 glucose molecule production
● Generates 90% of RBC’s ATP ● pH is low and leads to oxidation of iron
● Common enzyme being deficient is pyruvate kinase ● ATP dependent cellular processes begin to fail and lead to a loss in
o Leads to hemolytic anemia RBC flexibility
● RBCs become trapped in the splenic sieve and are phagocytosed by
HEXOSE MONOPHOSPHATE PATHWAY macrophages
● Trace urobilinogen in urine, no bilirubinuria
● Aerobic glycolysis ● Urobilinogen and urobilin in urine
● NADP+ is reduced to NADPH
● NADPH reduces glutathione
● Reduced glutathione reduces peroxidases to water
● Common enzyme that is deficient is G6PD
METHEMOGLOBIN REDUCTASE PATHWAY
● NADPH reduces the ferric iron to the ferrous iron in the presence
of methemoglobin reductase
LUEBERING – RAPAPORT SHUNT
● Generates 2, 3 - diphosphoglycerate
● 2,3 – DPG regulates oxygen delivery to tissues by competing with
oxygen for hemoglobin
● When 2-3 DPG binds hemoglobin, oxygen is released which
enhances delivery of oxygen to tissues
RBC MEMBRANE
RBC DEFORMABILITY
● Has an excess surface-to-volume ratio which enables RBCs to

stretch as they pass capillaries and splenic pores
● When hemoglobin (viscosity) increases, RBCs become less MECHANICAL HEMOLYSIS OR INTRAVASCULAR
deformable HEMOLYSIS
RBC ELASTICITY ● Small portion of RBCs rupture in the blood vessels due to turbulence
● Haptoglobin and Hemopexin salvage released hemoglobin so that
● RBC elasticity is attributed to the membrane composition which is iron is not loss in the urine
8% CHO, 52% proteins, and 40% lipids ● Albumin temporarily holds metheme and passes it eventually to
hemopexin
o Occurs in the mitochondria and cytoplasm of bone marrow
RBC precursors
o Begins with condensation of glycine and succinyl coenzyme A,
catalyzed by aminolevulinic acid synthase to form
aminolevulinic acid
o ALA dehydratase (porphobilinogen synthase) in the
presence of ALA catalyzes the formation of porphobilinogen
o Porphobilinogen deaminase (hydroxymethylbilane
synthase) in the presence of porphobilinogen catalyzes
formation of hydroxymethylbilane
o The pathway continues until, in the final step of production of
heme
o Fe2 combines with protoporphyrin IX in the presence of
ferrochelatase / heme synthase to make heme
o Heme leaves the mitochondria and is joined to the globin chains
in the cytoplasm
EXCESSIVE EXTRAVASCULAR PATHWAY
● Pathologic processes that produce changes in the exterior membrane

of the RBCs causes premature removal by the macrophages
● E.g., Heinz bodies, intracellular parasites, immunoglobulins or
complement on RBC membranes
● Increased in plasma unconjugated bilirubin with subsequent
increase of urobilinogen that is excreted by the kidneys due to
increased presentation of unconjugated bilirubin to the liver
EXCESSIVE INTRAVASCULAR PATHWAY
● Traumatic physical lysis of RBCs causes by prosthetic heart valves,

malarial parasites
● Hemoglobinemia, Hemoglobinuria, Hemosiderinuria eventually
increase in urinary urobilinogen
● Decreased haptoglobin and hemopexin
SAMPLE TEST RESULT INTRAVASCULAR EXTRAVASCULAR

SERUM Increased total EXPECTED EXPECTED
bilirubin
Increase indirect EXPECTED EXPECTED
bilirubin ● Globin
Normal direct bilirubin EXPECTED EXPECTED o Produced on specific ribosomes in the cytoplasm of RBCs
DECREASED EXPECTED MINOR o Genetic coding
HAPTOGLOBIN COMPONENT ▪ Chromosome 11 = Gamma, Beta, Delta, Epsilon
DECREASED EXPECTED MINOR
▪ Chromosome 16 = Alpha, Beta
HEMOPEXIN COMPONENT
URINE INCREASED EXPECTED EXPECTED
▪ 2 genes: Beta, Delta, Epsilon, Zeta
UROBILINOGEN ▪ 4 genes: Alpha, Gamma
HEMOGLOBINURIA EXPECTED NONE
WHOLE Decreased Hb, Hct and EXPECTED EXPECTED NORMAL HUMAN HEMOGLOBINS
BLOOD RBC
Decreased Glycated EXPECTED EXPECTED HEMOGLOBIN MOLECULAR STRUCTURE STAGES OF LIFE
hemoglobin Gower I 2 Zeta, 2 epsilon Embryonic
Gower II 2 Alpha, 2 epsilon Embryonic
HEMOGLOBIN METABOLISM Portland 2 Zeta, 2 gamma Embryonic
Fetal (HbF) 2 Alpha, 2 gamma Newborn & adult
STRUCTURE A1 2 Alpha, 2 beta Newborn & adult
A2 2 Alpha, 2 delta Newborn & adult
● Hemoglobin = 4 globin chains + 4 heme groups (4 Fe ++ + 4
protoporphyrin)
● M.W. 64,000 D NORMAL HEMOGLOBIN CONCENTRATION IN ADULTS
● 92 – 95% HbA
SYNTHESIS ● 2 – 3% HbA2
● 1 – 2% HbF
● Heme
FUNCTION NEUTROPHILIC MYELOCYTE
● The primary function of hemoglobin is to carry oxygen for tissue ● Appearance of secondary/specific granules
oxygenation and carry CO2 for excretion in the lungs ● Nucleus is slightly indented (D-shaped)
● 1g of Hb = 1.34 mL of oxygen ● N:C ratio = 1:1
● The affinity of hemoglobin depends on the partial pressure of ● Last stage capable of mitosis
oxygen ● 1st stage that allow granulocyte differentiation
● P50: the amount of oxygen needed to saturate 50% of hemoglobin
(normally around 27mmHg) NEUTROPHILIC METAMYELOCYTE
OXYGEN DISSOCIATION CURVE ● 10 – 15 um

● Kidney – shaped nucleus
● Describes the relationship between pO2 and the oxygen content of ● AKA juvenile cells
hemoglobin
● Hemoglobin has a low affinity for oxygen at low oxygen tension and NEUTROPHILIC BAND
a high affinity for oxygen at high oxygen tension
● It has a normal sigmoid shape ● 9-15 um
● BOHR EFFECT: Shifts of curve to the left or right occur if there are ● Curved nucleus / sausage shaped
changes in the pH of the blood
Shift to the left Shift to the right POLYMORPHONUCLEAR NEUTROPHIL
RESULT Increased affinity for Decreased affinity for
● 9 – 15 um
oxygen leading to a oxygen leading to an
● Pinky to rosy violet granules
decreased oxygen delivery increased oxygen delivery ● Neutrophilic granules contain ACP, acid hydrolase, muramidase,
FACTORS Alkalinity (a decrease in Caused by an increase in: and lactoferrin, which are essential for phagocytosis
H+) CO2 ● Nucleus has 2 – 5 lobes (>6 lobes indicates shift to the right)
Decrease in 2-3 DPG Acidity (an increase in H+) ● Hypersegmentation is seen in vitamin B12 deficiency
Decrease in PCO2 2-3 DPG
NEUTROPHIL POOLS
Decreased temperature Etcetera (Hb variants with
Hb variants with increased decreased affinity for O2)
BONE MARROW PERIPHERAL BLOOD
affinity for O2 Increased temperature
MITOTIC /PROLIFERATING POOL CIRCULATING POOL
CONDITIONS Lowered body temperature High fever
HSC, CMP, CFU-GEMM, GMP,
Blood transfusions with Acidosis
MYELOBLAST, PROMYELOCYTE,
depleted 2-3 DPG Conditions that produce
MYELOCYTE
Alkalosis hypoxia
STORAGE POOL MARGINATING POOL
Methemoglobinemia
Metamyelocyte, Band, Segmented Neutrophil
Increased
carboxyhemoglobin
PRIMARY SECONDARY TERTIARY
Some Hb variants
(AZUROPHILIC) (SPECIFIC)
PROMYELOCYTE MYELOCYTE AND METAMYELOCYTE
LEUKOPOIESIS______________________ STAGE METAMYELOCYTE AND BAND
MPO, cathepsins, b2-Microglobulin, Gelatinase,
defensins, elastase, Collagenase, Gelatinase, Collagenase,
proteinase 3, acid-b- Lactoferrin, lipocalin, Lysozyme,
glycerophosphate Transcobalamin I Acetyltransferase, b2-
Microglobulin
EOSINOPHIL MATURATION
NEUTROPHIL MATURATION ● Secondary granules stain heavily with eosin

● Myelocyte is distinguishable because of the presence of granules
containing major basic protein
MYELOBLAST ● Eosinophilic granules contain peroxidase, ACP, and other
proteolytic enzymes, but do not contain ALP
● 15 – 20 um ● Eosinophils spend less than a week in blood
● Deeply basophilic cytoplasm, round or oval nucleus ● A mature eosinophil has a bilobed nuclei
● N:C ratio of 4:1 ● Charcot-Leyden crystals: remnants of eosinophils
PROMYELOCYTE BASOPHIL MATURATION

● 15 – 21 um ● Characterized by the presence of large heavily staining granules
● Deeply basophilic cytoplasm which are irregularly shaped, unevenly distributed and deep purple
● Appearance of primary granules (procoagulants) to black
● N:C ratio of 3:1 to 2:1 ● Basophilic granules contain histamine, heparin, and chondroitin
sulfate
● <1% of total WBC count
Mast cells: similar to basophil but originates from C.T. mesenchyme
●
PLATELET PRODUCTION,____________
MONOCYTE MATURATION STRUCTURE AND FUNCTION_________
● Reticulated platelets, sometimes known as stress platelets, appear in
compensation for thrombocytopenia
MONOBLAST
o They are markedly larger than ordinary mature circulating
● 12 – 20 um platelets; their diameter in peripheral blood films exceed 6 mm,
● Basophilic cytoplasm and their MPV reaches 12 to 14 fL
● N:C ratio: 4:1 o They also carry free ribosomes and fragments of rough
endoplasmic reticulum, analogous to red blood cell
PROMONOCYTE reticulocytes, which triggers speculation that they arise from
early and rapid proplatelet extension and release
● 14 – 18 um
● Blue-grape cytoplasm GENERAL CHARACTERISTICS OF PLATELETS
● N:C ratio: 3:1 – 2:1 ● Anucleate blood cells
● NV: 150 – 450 x 109/L
MONOCYTE ● Approximately 7 – 21 per OIF
● Life span: 8 – 11 days
● 14 – 20 um ● Average diameter: 2.5 um
● Blue gray cytoplasm ● Mean platelet volume of 8 – 10 fL
● Many fine, azurophilic granules giving the cell a characteristic ● 20 – 30% is sequestrated in the spleen
“ground glass appearance” ● On a Wright – stained wedge – preparation blood film, platelets
● Nucleus is horse-shoe shaped / kidney shaped appear CIRCULAR to irregular, lavender, and granular
MACROPHAGE – TISSUE MONOCYTE MEGAKARYOCYTOPOIESIS

● Platelets arise from the bone marrow cells called megakaryocytes
LYMPHOCYTE MATURATION ● Thrombopoietin: primary hormone influencing platelet maturation
● Endomitosis: No cytokinesis and telophase (no daughter cells)
LYMPHOBLAST STAGES
● 10 – 18 um ● Megakaryoblast
● Moderate to dark blue cytoplasm o 20 – 50 um
● N:C ratio: 4:1 o Blue cytoplasm
o N:C ratio: 10:1
PROLYMPHOCYTE ● Promegakaryocyte
o 20 – 60 um
● Size may be the same or smaller than lymphoblast, moderate to dark o Less basophilic cytoplasm
blue cytoplasm o Nucleus is irregularly shaped
● Granular megakaryocyte
LYMPHOCYTE o Granules become prominent
● Mature megakaryocyte
● Exists as small (8 – 10 um), medium (10 – 12 um), or large (12 – 16 o 2,000 – 4,000 platelets
um)
WBC MATURATION AND LIFESPAN

WBC TYPE MATURATION AND LIFESPAN
Neutrophil MITOTIC POOL: 2 – 3 days
STORAGE POOL: 5 – 7 days
LIFESPAN: 9 – 10 days from myeloblast to death
Eosinophil Maturation: 3.5 days
Half life: 18 hours
Basophil Maturation and storage: 4.3 days or 12 hours
Transit time in the peripheral blood: 3.7 days
Monocyte Maturation: 30 – 48 hours (60 hours)
TISSUE PHASE: macrophage: months, possibly longer
Few hours (inflammatory macrophage)
Lymphocyte MATURATION: B-cell: 30 – 36 hours
LIFESPAN: some live for 3 – 4 days, majority live for
months to years
MATURATION STAGE CYTOPLASMIC GRANULES CYTOPLASMIC TAGS NUCLEAR FEATURES THROMBOCYTES VISIBLE
Megakaryoblast Absent Present Single nucleus, fine No
chromatin, nucleoli
Promegakaryocyte Few Present Double nucleus No
Megakaryocyte Numerous Usually absent Two or more nuclei No
Metamegakaryocyte Aggregated Absent Four or more nuclei Yes
MK-I (MEGAKARYOBLAST) MK-II (PROMEGAKARYOCYTE) MK-III (MEGAKARYOCYTE)

Nucleus Round Indented Multilobed
Nucleoli 2-6 Variable Not visible
Chromatin Homogenous Condensed Deeply but variably condensed
Demarcation System Present Present Present
PLATELET STRUCTURE MEMBRANOUS SYSTEM
● Dense tubular system – arachidonic acid metabolism; activation

center
● Surface connecting system – granule release
o Canaliculi system: opens up then releases the granules
PLATELET FUNCTION
ADHESION
● Platelet roll and cling to nonplatelet surfaces

● Reversible
● Seals endothelial gaps
● Some secretion of growth factors
PERIPHERAL ZONE AGGREGATION
● Glycocalyx - where glycoproteins gbIb and gpIIbIIIa are found ● Platelets adhere to each other
o Unique to platelets ● Irreversible
● Plasma membrane ● Platelet plugs form
● Submembranous area ● Secretion of all platelet contents
● Requires fibrinogen
SOL-GEL ZONE
SECRETION
● Microfilaments (actin, myosin)
● Thrombosthenin / Actomysin - contractile elements ● Irreversible
● Microtubules (Tubulin) - retains platelet shape ● Occurs during aggregation
● Essential to coagulation
ORGANELLE ZONE
● Alpha and dense granules

HEMATOLOGY 2
Notes Compiled by: Renz Louie Galanto
Notes Prepared by: John Alvin O. Reyes, RMT
OUTLINE
• Hemostasis • Special Handling and Processing

o Primary Hemostasis o Effects of Ph
▪ Blood Vessels o Temperature
• Intact Vessels o Centrifugation
• Damaged Vessels • Routine Evaluation of Coagulation
• Substances Released by Endothelial Cells o Test for the Intrinsic and Common Pathway
▪ Platelets ▪ Lee and White Whole Blood Coagulation Time
• Basic Sequence of Events in Primary and • Procedure
Secondary Hemostasis ▪ Plasma Recalcification Time
o Secondary Hemostasis ▪ (Activated) Partial Thromboplastin Time
▪ Coagulation Factors o Test for the Extrinsic and Common Pathway
▪ Pathways ▪ Prothrombin Time
• Mechanisms of Coagulation and Fibrinolysis o Other Coagulation Time
o Primary Hemostasis ▪ Stypven Time
▪ Platelet Adhesion ▪ 5M Urea Solubility Test
▪ Platelet Activation ▪ Thrombin Time
▪ Platelet Secretion ▪ Reptilase Time
▪ Platelet Aggregation ▪ Clause Fl Assay
o Secondary Hemostasis ▪ Substitution Studies
▪ Coagulation Cascade • Tests for Primary Hemostasis
▪ Intrinsic Pathway o Bleeding Time
• Factor XII activation ▪ Detection
• Factor XI activation ▪ Methods
• Factor IX activation • Modified Duke Method
▪ Extrinsic Pathway • Ivy Method
▪ Alternate Pathways Linking the Extrinsic and o Capillary Resistance (Fragility) Test
Intrinsic Pathways ▪ Methods
• Intrinsic Activation of Extrinsic System • Positive Pressure Test / Rumple – Leede /
• Extrinsic Activation of the Intrinsic System Torniquet Test
• Feedback Pathway • Negative Pressure Test
▪ Common Coagulation Pathway o Clot Retraction Time
▪ Thrombin Feedback Mechanism ▪ Procedure
▪ Coagulation Factors ▪ Factors
• Coagulation Factor Groups o Platelet Count
o Fibrinolysis ▪ Direct Methods
▪ Activation of Fibrinolytic System • Tonkantin Method
• Intrinsic Activation • Brecher – Cronkite Method
• Extrinsic Activation • Unopette Method
• Activators in Secretions ▪ Platelet Estimation on Peripheral Blood Smear
• Exogenous Activation • Reporting of Platelet Estimate
▪ Fibrin(ogen) Degradation by Plasmin • Significant Platelet Levels
• Naturally Occurring Inhibitors of Fibrinolysis o Platelet Aggregation
and Coagulation o Platelet Adhesiveness
• Specimen Collection in Hemostasis Testing ▪ Glass Bead Retention Test
o General Considerations ▪ Procedure
▪ Causes of Activation • Disorders of Hemostasis
o Equipment o Basic Terminologies
▪ Needle size ▪ Petechiae
▪ Evacuated Tubes/Syringes ▪ Purpura
▪ Anticoagulants ▪ Ecchymosis
• Sodium Oxalate ▪ Epistaxis
• Trisodium citrate ▪ Hemarthrosis
▪ Hematemesis
• EDTA
▪ Hematoma
• Heparin
▪ Hematuria
OUTLINE
▪ Hemoglobinuria • Malignancy
▪ Hemoptysis • Pregnancy
▪ Melena • Hemorrhagic Disorders
▪ Menorrhagia o Intrinsic Pathway Disorders
o Bleeding Disorders Due to Vascular Defects ▪ Factor XI Deficiency (Hemophilia C)
▪ Hereditary Connective Tissue Defects ▪ Factor VIII:C Deficiency (Hemophilia A)
• Ehlers-Danlos Syndrome ▪ Factor IX Deficiency (Hemophilia B, Christmas
• Pseudoxanthoma Elasticum Disease)
▪ Acquired Connective Tissue Defect ▪ Von Willebrand’s Disease
• Scurvy (Vitamin C deficiency) o Extrinsic and Common Pathway Disorders
• Senile purpura ▪ Factor VII Deficiency
▪ Hereditary Alterations of Vessel Wall Structure ▪ Factor X (Stuart – Prower Factor) Deficiency
• Hereditary Hemorrhagic Telangiectasia ▪ Factor V Deficiency (Owren’s Disease)
• Congenital Hemangiomata (Kasabach – Meritt ▪ Factor II (Prothrombin) Deficiency
Syndrome) ▪ Factor I Deficiency
▪ Acquired Alterations of the Vessel Wall Structure • Afibrinogenemia
• Diabetes Mellitus • Hypofibrinogenemia
• Amyloidosis • Dysfibrinogenemia
▪ Endothelial Damage ▪ Factor XIII Deficiency
▪ Autoimmune Vascular Purpura o Acquired Disorders of Coagulation and Fibrinolysis
o Quantitative Platelet Disorders • Laboratory Evaluation of Fibrinolysis
▪ Thrombocytopenia o Whole Blood Clot Lysis Time
• Decreased production ▪ Principle
• Dilutional Loss o Euglobulin Lysis Time
• Nonimmune Destruction ▪ Principle
• Immune Platelet Destruction ▪ Procedure
• Disseminated Intravascular Coagulation ▪ Reference Range
• Hemolytic Uremic Syndrome and TTP o Protamine Sulfate Gelation Test
▪ Principle
• Increased Platelet Sequestration by Spleen
▪ Reference Range
▪ Thrombocytosis
o Ethanol Gelation Test
• Primary
▪ Principle
• Secondary (reactive)
▪ Reference Range
o Qualitative Platelet Disorders
o Latex D-Dimer Assay
▪ Adhesion Defects
o Anticoagulant Therapy
• Bernard – Soulier Syndrome
• Anemia
• Von Willebrand’s Disease o Causes
▪ Aggregation Defects o Absolute Vs. Relative Anemia
• Glanzmann’s Thrombasthenia o Laboratory Evaluation of Anemias
• Afibrinogenemia ▪ Complete Blood Count
▪ Storage Pool Defects ▪ RBC Indices
• Gray Platelet Syndrome • MCV and MCHC
• Wiskott – Aldrick Syndrome • MCHC
• Hermansky – Pudlak Syndrome ▪ Red Cell Distribution Width
• Chediak – Higashi Syndrome ▪ Peripheral Blood Smear
▪ Acquired Defects ▪ Bone Marrow Examination
• Disorders of Thrombosis ▪ Other Laboratory Tests
o Primary o Types of Anemias
▪ Antithrombin – III Deficiency ▪ Anemia of Impaired or Defective Production
▪ Protein C and S Deficiency • Iron Deficiency Anemia
▪ Fibrinolytic System Disorders • Anemia of Chronic Disease
▪ Dysfibrinogenemia • Sideroblastic Anemia
▪ Homocystinuria • Thalassemia
o Secondary • Lead Poisoning
▪ Lupus Anticoagulant
• Megaloblastic Anemia
▪ Hemostatic Protein Abnormalities
• Pernicious Anemia
• Postoperative States
• Nonmegaloblastic Anemia
•

OUTLINE
• Aplastic Anemia o Monocytic Disorders
• Myelophthisic Anemia (Marrow Replacement) ▪ Gaucher Disease
Anemia/Myeloid Metaplasia ▪ Niemann – Pick Disease
▪ Blood Loss Anemia o Non-Malignant Lymphocytosis Associated with Viral Infections
▪ Infectious Mononucleosis
• Acute Blood Loss Anemia
▪ Cytomegalovirus
• Chronic Blood Loss Anemia
▪ Infectious Lymphocytosis
▪ Hemolytic Anemias o Malignant Leukocyte Disorders
• Hemolytic Anemias Due to Intrinsic Defects ▪ Hematopoietic Malignancy Classifications
• Hemolytic Anemias Due to Extrinsic Immune ▪ Cytochemical Stains
Defects o Acute Lymphoproliferative Disorders
• Hemolytic Anemias Due to Extrinsic Non- ▪ FAB Classifications of ALL
Immune Defects • FAB L1
• Hemoglobinopathies • FAB L2
o Sickle Cell Disease (Hb SS) • FAB L3
o Sickle Cell Trait (Hb AS) o Acute Myeloproliferative Disorders
o Hb C Disease ▪ FAB Classifications of ALL
o Hb SC Disease • FAB M0 – w/o differentiation
• Poikilocytosis • FAB M1 (AML w/o maturation)
o Echinocytes / Burr Cells • FAB M2 (AML w/ maturation)
o Acanthocytes • FAB M3 (Acute Promyelocytic Leukemia) /
o Target cells / Codocytes / Mexican Hat Cells Hypergranular Promyelocytic Leukemia
o Spherocytes • FAB M4 (Acute Myelomonocytic Leukemia /
o Pyropoikilocytes / Microspherocytes Naegeli Syndrome)
o Teardrops / Dacrocytes • FAB M5 (Acute Monocytic Leukemia / Schilling
Leukemia)
o Sickle cells / Drepanocytes
• FAB M6 (Acute Erythroleukemia / Di Guglielmo
o Helmet cells / Horn cells / Keratocytes
Syndrome)
o Schistocytes / RBC fragments
• FAB M7 (Acute Megakaryocytic Leukemia)
o Stomatocytes / Mouth Cells
o Chronic Leukemias
o Elliptocytes / Ovalocytes
▪ Chronic Myelogenous Leukemia (CML) / Chronic
• RBC Inclusions Granulomatous Leukemia (CGL)
o Nucleated RBCs ▪ Essential Thrombocythemia
o Howell-Jolly Bodies ▪ Polycythemia Vera
o Basophilic Stippling ▪ Chronic Idiopathic Myelofibrosis
o Pappenheimer Bodies ▪ Chronic Lymphocytic Leukemia
o Cabot Rings ▪ Hairy Cell Leukemia
o Hemoglobin C Crystals ▪ Prolymphocytic Leukemia
o Hemoglobin SC Crystals / “Washington Monument” Crystals o Other Lymphoid Malignancies
o Heinz Bodies ▪ Multiple Myeloma
o Malarial Parasites ▪ Waldenstrom’s Macroglobulinemia
o Abnormal RBC distribution ▪ Hodgkin’s Lymphoma
▪ Rouleaux ▪ Non-Hodgkin’s Lymphoma
▪ Mycosis Fungoides
▪ Agglutination
▪ Adult T-Cell Leukemia
• Leukocyte Disorders
o Relationship of Leukemias and Lymphomas
o Non-malignant Granulocytic Disorders
▪ Shift / Physiologic Pseudoneutrophilia • Automation
▪ Pathologic Neutrophilia o Methods
▪ Neutrophilic Leukomoid Reaction ▪ Electrical Impedance
▪ Leukoerythroblastic Reaction ▪ Light Scattering Optical Method
▪ Functional Disorders of Neutrophils o Histograms
▪ RBC Histogram
• Chronic Granulomatous Disease (CGD)
▪ Platelet Histogram
• Chediak – Higashi Syndrome
o Errors in Cell Counting
▪ Nuclear Abnormalities
▪ Instrumental Errors
• Hypersegmentation
▪ Errors Caused by Nature of the Specimen
• Hyposegmentation o Automation in Hemostasis: Detection of Fibrin Clot Formation
▪ Inherited Cytoplasmic Anomalies o Automation Summary
• May – Hegglin Anomaly ▪ Evaluation of Peripheral Blood Smear
• Alder – Reily Anomaly ▪ Percentage Grading for Anisocytosis / Poikilocytosis
● 2. Tubulin – Retains platelet shape
HEMOSTASIS
● The stoppage of blood flow BASIC SEQUENCE OF EVENTS IN PRIMARY AND
● Involves the interaction of blood vessels, platelets, coagulation, SECONDARY HEMOSTASIS
fibrinolysis, and tissue repair
1. VASOCONSTRICTION: Controlled by vessel smooth muscle;
enhanced by serotonin and enhanced by chemicals secreted by platelets
PRIMARY HEMOSTASIS_____________ thromboxane A2
● Platelets
2. PLATELET ADHESION Platelets adhere to exposed subendothelial
● Blood Vessels
connective tissue
3. PLATELET Interaction and aggregation of platelets to one
BLOOD VESSELS
AGGREGATION: another to form initial platelet plug
enhanced by serotonin,
INTACT VESSELS thromboxane A2 and ADP
4. FIBRIN – PLATELET Coagulation factors interact on platelet
● Antithrombotic PLUG FORMATION surface to produce fibrin; fibrin-platelet plug
● Does not activate or promote coagulation 5. FIBRIN Fibrin clot must be stabilized by coagulation
● Facilitates blood flow and reduces turbulence STABILIZATION Factor XIII
DAMAGED VESSELS
SECONDARY HEMOSTASIS___________
● Vasoconstriction (Neurogenic)
● Exposed collaged causes platelets to adhere COAGULATION FACTORS
● Promotes thrombus formation by exposing collagen that initiates FACTOR PREFERRED OTHER NAME
contact phase of coagulation NAME
● Tissue thromboplastin is released which initiates extrinsic pathway I Fibrinogen
● Release of tissue plasminogen activators (TPAs) II Prothrombin Prethrombin
III Tissue Factor Tissue thromboplastin
SUBSTANCES RELEASED BY ENDOTHELIAL CELLS
IV Calcium
- Mineral
coagulation
SUBSTANCES RELEASED BY ENDOTHELIAL CELLS
factor
SUBSTANCE ACTION ROLE
V Proaccelerin Labile factor
PROSTACYCLIN Inhibits platelet activation Anticoagulant Accelerator globulin
(PGI2) – product of Stimulates vasodilation Reduces blood (aCg)
endothelial system; flow rate VII Proconvertin Stable factor
produced by Serum Prothrombin
endothelial cells when Conversion
intact Accelerator (SPCA)
Adenosine Stimulates vasodilation Reduces blood Autoprothrombin I
flow rate VIII:C Antihemophilic Antihemophilic
Thrombomodulin Endothelial surface Anticoagulant - Coagulation factor globulin (AHG)
receptor for thrombin and Fibrinolytic Antihemophilic factor
enhances fibrinolytic A
activity of protein C Platelet cofactor 1
Heparan sulfate Coats endothelial cell Anticoagulant IX Platelet Christmas factor
surface and weakly thromboplastin Antihemophilic factor
enhances activity of component (PTC) A
antithrombin- III Platelet cofactor 2
Tissue plasminogen Converts plasminogen to Fibrinolytic X Stuart-Prower Factor Stuart factor
activators plasmin Prower factor
Von Willebrand factor Secreted by endothelium, Coagulation Autoprothrombin III
(VWF) required for platelet XI Plasma Antihemophilic factor
adhesion thromboplastin C
antecedent
PLATELETS XII Hageman factor Glass factor
● Adhere to injured vessels - Decreased Contact factor
● 1. They aggregate at the site of injury concentration
● 2. Promote coagulation on their phospholipid surface leads to
● 3. They release biochemicals important to hemostasis clotting
● 4. Clot retraction disease
o Consumes Ca++ and ATP - May lead to
o In vitro phenomenon deep vein
o Pulling forces are provided by contractile elements thrombosis
o Participates in vascular constrictive response XIII Fibrin stabilizing Laki-Lorand factor
o Stabilization of fibrin clot factor Fibrinase
o Debulking of clot to help reestablish blood flo Fibrinoligase
● 1. Thrombosthenin – platelet contraction
Serum
Transglutaminase
SECONDARY HEMOSTASIS___________
Prekallikrein Fletcher factor COAGULATION CASCADE
High Molecular Fitzgerald factor
Weight Kininogen Flaujeac factor
(HMWK) Williams factor
Contact activation
factor
PATHWAYS
Intrinsic Extrinsic Common
Factors VIII, IX, XI, Tissue thromboplastin, Factors X, V, II, I,
XII, HMWK, PK, Factor VII, Calcium, PL Calcium, PL
Calcium, PL
MECHANISM OF COAGULATION AND

FIBRINOLYSIS
PRIMARY HEMOSTASIS_____________
PLATELET ADHESION
● Platelets attach to the damaged blood vessel through VWF
● Gp1B found on platelet surface acts a receptor for VWF, allowing
adhesion to blood vessels
o Gp1B is found in the peripheral zone
● DISEASES OF PLATELET ADHESION
o Von Willebrand Disease – no VWF, patient is also deficient of
Factor VIII:C
o Bernard-Soulier Syndrome – lack of Gp1B, giant platelets
PLATELET ACTIVATION
● Platelets undergo viscous metamorphosis/pseudopod formation
● Platelets change shape, and their organelles become centralized
PLATELET SECRETION
● Alpha granules: All other factors
● Dense granules: CAPAS (Ca, ATP, Pyrophosphate, ADP,
Serotonin) + Mg2 INTRINSIC PATHWAY
● Activated in vivo by contact of coagulation proteins with
PLATELET AGGREGATION subendothelial tissue
● Platelets attach to each other through the receptor GpIIb:IIIa and
fibrin FACTOR XII ACTIVATION
● Exposure of phospholipids on the platelet surface, providing a site
for fibrin formation and thrombogenesis ● Begins with absorption of FXII with a negatively charged surface
(collagen)
● Prekallikrein-HMWK complex is adsorbed in vivo to the negatively
charged surface with FXII
● FXI also complexes with HMWK on the surface
● Kallikrein accelerates conversion rate to FXIIa
● Factor XIIa is cleaved into XIIf by plasmin and more importantly
kallikrein, XIIa and XIIf are capable of activating prekallikrein to
kallikrein
● Functions of Factor XII
o Initiates intrinsic pathway, in the presence of HMWK it
converts XI to Xia
o It initiates fibrinolysis, FXIIa and kallikrein activate
plasminogen to plasmin which in turn can initiate the
complement system
o It initiates kinin and complement system, the formation of
kallikrein causes the conversion of HMWK to kinins
(bradykinin)
● Functions of Kallikrein
o It perpetuates FXII production and its own production
o It initiates kinin system
o Initiates the fibrinolytic and complement system together with o Activate V to Va and VIII to VIIIa
FXIIa o Activates XIII to XIIIa
o Directly activates IX to IXa o Platelet aggregation
● Functions of Plasmin ● Thrombin as an inhibitor (high levels of thrombin)
o Begins clot dissolution o Inhibit factor V and VIII activation
o Activates complement system o Initiates fibrinolysis by converting plasminogen to plasmin
o Cleaves factor XIIa to XIIf o Activates protein C (a potent anticoagulant)
▪ And protein S = inactivates Factor V and VIII
FACTOR XI ACTIVATION
COAGULATION FACTORS
● More important in coagulation system, weak in plasminogen
activation ● Circulating proteins waiting to be activated
● Cleaves FIX to IXa (requires Ca++ as a cofactor) ● Produced by the liver except FIII and FVIII (VWF)
● Can be activated directly by contact activation ● 1. Zymogens – substrates which are converted to serine proteases;
● Also activates plasminogen (fibrinolytic and complement system) inactive/resting enzymes
o e.g., Factors II, VII, IX, X, XI, XII and prekallikrein
FACTOR IX ACTIVATION ● 2. Serine protease – Active enzyme
● 3. Cofactors – Factors V, VIII, tissue factor HMWK
● Completes contact activation phase of coagulation ● 4. Transglutaminase – Factor XIII
● Can also be activated by kallikrein
● FIXa combines FVIIIa to and Ca++ platelet PL to activate FX COAGULATION FACTOR GROUPS
EXTRINSIC PATHWAY CONTACT PROTHROMBIN FIBRINOGEN

- Needs to
● Initiated with the release of tissue factor be
● Consists of tissue factor, FVII and Ca++ exposed
● Tissue factor is lipoprotein released from cell membranes into on
plasma when there is vascular injury negatively
● PL portion of tissue factor activates FVII to FVIIa charged
● The FVIIa – Ca++ - Tissue factor complex on platelet PL converts surface
FX to FXa FACTORS XII, XI, II, VII, IX, X, I, V, VIII, XIII
prekallikrein, protein C, protein
ALTERNATE PATHWAYS LINKING THE EXTRINSIC AND HMWK S
INTRINSIC PATHWAYS Vit. K No Yes No
dependent
INTRINSIC ACTIVATION OF EXTRINSIC SYSTEM Adsorbed by Partially Yes No
Ba2SO4
● FXIIa can activate FVII, the FVIIa that is formed is a two-chain Consumed in Partially No (Except II) – Yes
factor that has a greater effect on FX than a single-chain FVIIa coagulation almost depleted
● FIX and kallikrein activation of FVII has been demonstrated in Destroyed by No No Yes
plasma exposed to glass (in vitro) plasmin or
high
EXTRINSIC ACTIVATION OF INTRINSIC SYSTEM concentrations
of thrombin
● FVIIa – Ca++ - Tissue factor complex can slowly activate FIX to Reduced by No Yes No
FIXa with subsequent activation of X to Xa oral
anticoagulants
FEEDBACK PATHWAY Stability Fairly stable Heat labile: VII, Heat labile: I,
IX, X V, VIII
● FXa can hydrolyze FVII to produce a two-chain form that is reported Well preserved in Storage labile:
to have 85 times procoagulant activity than the normal single-chain stored plasma V, VIII
FVIIa
COMMON COAGULATION PATHWAY

FIBRINOLYSIS_______________________
● Body’s defense against occlusion of blood vessels
● Extrinsic activation occurs when VIIa-Ca++ - Tissue factor on the PL ● Dependent on the enzyme plasmin
surface is formed and converts X to Xa ● Plasmin is a serine protease that can digest fibrinogen, fibrin, V, VIII
● Intrinsic activation occurs when the multimolecular complex of IXa o Needs to be regulated; “very powerful”
– Ca++ - VIIIa binds with the PL on the platelet surface which inn
turn activates FX to Xa
● FVIII must be modified by thrombin to become functional (VIIIa)
● On formation of FXa, the prothrombinase complex (Xa-Va-Ca++ +
PL) is formed
● Prothrombinase converts prothrombin to thrombin
● The reactions are completed once fibrinogen is converted to fibrin
and is stabilized into clot
THROMBIN FEEDBACK MECHANISM

● Thrombin as an activator (low levels of thrombin)
ACTIVATION OF FIBRINOLYTIC SYSTEM Inhibits plasmin
Protein C Complexed with protein S, inhibits Va, VIIIa
INTRINSIC ACTIVATION Inactivates inhibitors of plasminogen activators
(enhances fibrinolysis)
● Factor XIIa, kallikrein, HMWK, and a specific plasma protein Protein S Complexed with protein C, inhibits Va, VIIIa
(proactivator) can activate plasminogen to plasmin Inactivates inhibitors of plasminogen activators
(enhances fibrinolysis)
EXTRINSIC ACTIVATION A2-antiplasmin Principal inhibitor of plasmin
● Plasminogen activators are present in organ tissues and endothelial

cells (TPAs)
SPECIMEN COLLECTION IN
HEMOSTASIS TESTING
ACTIVATORS IN SECRETION
● Urokinase, tears, saliva, semen, milk GENERAL CONSIDERATIONS_________

● Primary concern is to prevent premature activation (falsely
EXOGENOUS ACTIVATION shortened tests) of the clotting process
● Therapeutic destruction of thrombin (urokinase, streptokinase, CAUSES OF ACTIVATION

manufactured TPAs)
● 1. Contamination with tissue thromboplastin
o Tissue factor is released form injured cells and activates the
FIBRIN (OGEN) DEGRADATION BY PLASMIN
extrinsic pathway
● a. The process of fibrin degradation by plasmin produces fragments ● 2. Inappropriate container
called fibrin(ogen) degradation products or fibrin(ogen) split o Glass provides a negatively charged surface activating the
products intrinsic pathway and attracts platelets
● b. The four principal fragments are X, Y, D, E ▪ Never use glass
● c. Fragment X is the first and largest fragment, it results from the o Use polystyrene tubes or glass tubes coated with silicone
cleavage if the alpha chains by plasmin ● 3. Improper temperature
● d. When fragment X is cleaved by plasmin, two fragments called Y o Labile factors at room temperature (Factors V and VIII)
and an intermediate complex DXD, is formed o Prematurely activated at ref temperature (Factors VII and XI)
● e. The complex is further cleaved into DED complex and DY/YD ● Hemolysis
complex, until finally fragments E and D are formed o a. Prolonged torniquet application (<1 minute)
● f. D-dimer is an indicator of in vivo fibrinolysis, it indicates the ▪ Stasis elevates the concentration of vWF and Factor VIII;
presence of fibrin (not fibrinogen) split products falsely decreases fibrinolytic parameters; and falsely
o D-dimer (+): increased clotting = increased lysis shortens clot-based test results
● g. Fragments X and Y, along with intermediate FDPs, appear to be ▪ Hemolysis -> TF-like substance released -> extrinsic
the most important in exerting anticoagulant effects, fragments Y pathway gets activated -> falsely shortened coagulation
and D inhibit fibrin polymerization, fragment E is a powerful results
inhibitor of thrombin o b. Moisture or contamination
o c. Using needles with too small a bore
o d. Frothing of sample due to air entry
o e. Expelling of blood from the syringe through the needle
o f. Excessive and vigorous mixing
EQUIPMENT_________________________
NEEDLE SIZE
● a. Needles with small bores are more likely to cause hemolysis
● b. 20-gauge needle – most commonly used for hemostasis sample
collection
● c. 19-gauge needle – for collection of 20 mL of blood or more
● d. 21-gauge needle – narrow or small veins
NATURALLY OCCURING INHIBITORS OF FIBRINOLYSIS AND EVACUATED TUBES/SYRINGES

COAGULATION
● Syringes or evacuated tubes should be silicone coated
INHIBITOR FUNCTION
Antithrombin III Inhibits Thrombin, XIIa, XIa, Xa, IXa, and ANTICOAGULANTS
kallikrein
Principal inhibitor of coagulation SODIUM OXALATE
Inhibits plasmin and kallikrein
A2-Macroglobulin Inhibits thrombin and kallikrein ● Forms insoluble complexes or precipitates once recalcified and
Inhibits plasmin and kallikrein produces an interference in OD testing
A1-Antitrypsin Potent inhibitor of Xia, weak inhibitor of
thrombin TRISODIUM CITRATE
Inhibits plasmin
C1 Inactivator Inhibits XIIa, XIIf, Xia, and Kallikrein ● Binds Ca++
● Preserves factor V and VIII most satisfactory for platelet ● N.V.: 7 – 15 minutes
aggregation studies, anticoagulant of choice today
● Ratio: Nine parts of blood to one part of anticoagulant (9:1) PLASMA RECALCIFICATION TIME
● This ratio is ideal for patients with normal hematocrit
● a. A modification of L-W clotting time
● A specimen with a hematocrit over .50L/L, or an incompletely filled
● b. This test is based on the fact that except for calcium (as it is bound
tube has excess unbound citrate which causes the coagulation tests
to sodium citrate), PRP contains all the components necessary for
to be prolonged
creating a fibrin clot
● Concentration: Coagulation tests are more sensitive to excess citrate
● c. The time required for blood to clot after Ca++ is added is a general
than calcium
measure of the intrinsic and common pathway
o 3.2% Na Citrate (Preferred)
● d. Reference ranges:
o 3.8% Na Citrate – Overanticoagulation for patients with high
o PRP: 100 – 150 seconds
hematocrit
o PPP: 130 – 240 seconds
o PRP should clot at least 20 seconds faster than PPP
EDTA
● NEVER USE FOR COAGULATION TESTS (ACTIVATED) PARTIAL THROMBOPLASTIN TIME

● Unsuitable for coagulation ● a. Thromboplastins - are lipoproteins which can be fractioned into
● Inhibits fibrinogen – thrombin reaction lipids and proteins
● Factor is unstable V in its presence ● b. Partial thromboplastin – only the phospholipid component is
present
HEPARIN ● c. It screens for factor deficiencies in the intrinsic and common
pathways (measures all factors except VII and XIII)
● Acts with antithrombin III and inhibits all stages of coagulation ● d. Reagent consists of two components
● Can be used for platelet retention test o Platelet substitute (phospholipid)
o Activators: Kaolin, Celite, Ellagic acid, Micronized silica
● e. Procedure
SPECIMEN HANDLING AND o PPP + APTT reagent (phospholipid + activator + CaCl2)
PROCESSING o N.V.: 20 – 45 seconds
● f. Used for monitoring heparin therapy
● Preferred sample: PPP
EFFECTS OF PH______________________ o We want phospholipid to be provided by the reagent
● 1. Changes in pH can prolong clotting times o PRP results into shortened coagulation results
o “Do not remove the cap when not ready to process/test” o Temperature requirement for PT and APTT testing: 37 degrees
● 2. Mediated by loss of CO2 Celsius
o pH becomes more alkaline
● 3. Samples should be unopened if immediate testing is not possible TESTS FOR THE EXTRINSIC AND
TEMPERATURE_____________________ COMMON PATHWAY_________________
● 1. Labile: Factors V and VIII
PROTHROMBIN TIME
● 2. Prematurely activated at 4oC: VII and XI
● a. Tests for factor I, II, V, VII and X deficiency (Extrinsic and
CENTRIFUGATION__________________ ●
Common)
b. Test of choice for monitoring anticoagulant therapy with Vit. K
● 1. 2000 g for 10 minutes to obtain platelet poor plasma
antagonist (warfarin)
● 2. Only upper ¾ of plasma layer should be used for testing
● c. Procedure: PPP + PT reagent (Thromboplastin + CaCl 2)
● d. N.V.: 10 – 12 seconds
ROUTINE EVALUATION OF ● e. International Normalized Ratio
o Provides a means of standardizing PT reporting worldwide; not
COAGULATION dependent on Thromboplastin reagent
o Used to monitor coumarin/warfarin therapy
TESTS FOR THE INTRINSIC AND o Therapeutic range is dependent on condition being treated, but
is generally considered to be between 2.0 – 3.0
COMMON PATHWAY_________________ ▪ Applicable if taking vitamin K antagonist (warfarin /
coumarin)
LEE AND WHITE WHOLE BLOOD COAGULATION TIME Formula = (Patient PT/control PT)ISI
● a. Based on the fact that when venous blood is put into a glass tube, o International Sensitivity Index (ISI) for the thromboplastin
it will form a solid clot reagent is provided by the manufacturer and is based on the
● b. The time required for this response is a measure of the overall WHO standards
intrinsic and common pathways o The closer the ISI is to 1, the more sensitive the reagent
● c. Insensitive to factor deficiencies ▪ MANCHESTER REAGENT: most sensitive PT reagent
● Increased INR = increased patient PT; prolonged clotting time
PROCEDURE leading to bleeding
● Whole blood is dispensed into three 13 x 100 mm or 12 x 75 mm OTHER COAGULATION TEST_________

tubes (tube 3, tube 2, tube 1)
● Incubate in a water bath at 37oC for 5 minutes STYPVEN TIME
● Checking is done every 30 seconds until a solid clot has formed (tube
1, tube 2, tube 3) ● a. Utilizes the powerful coagulant properties of Russel’s viper
venom obtained from the snake Viperarusselli
o Russel’s viper venom: can directly activate Factor X -> Xa V A A N C C NC
● b. PRINCIPLE: The venom is capable of bypassing the action of VII A N N C NC C
factor VII and directly activates Factor X to Xa VIII N A N C C NC
o Abnormal: Factor X deficiency IX N A N C NC C
o Normal: Factor VII X A A N C NC C
● c. It was used to help distinguish between factor X and VII deficiencies XI N A N C C C
● d. N.V.: 6 – 10 seconds XII N A N C C C
● Factor XI and XII aged serum are to be differentiated, always read
5M UREA SOLUBILITY TEST the patient’s history
● a. AKA Duckhert’s Test
● b. Clots formed in normal plasma are insoluble in 5M urea because
of the action factor XIII TESTS FOR PRIMARY HEMOSTASIS
o No factor XIII: the clot will dissolve
BLEEDING TIME_____________________
THROMBIN TIME ● 1. The actual time it takes for a standard would to stop bleeding
● a. Used to screen for fibrinogen deficiency and the presence of fibrin
degradation products DETECTION
● b. Addition of thrombin to plasma bypasses all coagulation reactions
● It detects:
except polymerization of fibrinogen
o Abnormalities of platelet number and function
● c. Dysfibrinogenemia, hypofibrinogenemia, FDPs, immunologic
o Factor VIII:VWF deficiency
antithrombins, and abnormal globulins will prolong thrombin time
o Abnormalities of vessel wall structure
(=heparin therapy)
● d. PROCEDURE: PPP + Thrombin reagent METHODS
● e. N.V.: 10 – 14 seconds
REPTILASE TIME MODIFIED DUKE METHOD

● a. Used to assess fibrinogen deficiency in patients undergoing ● 15 – 20mm rounded fatty portion of the earlobe
heparin therapy ● No. 11 sterile Bard-Parker Surgical Blade
● b. Reagent: Bothropsatrox snake venom ● Blade passes through ear and hits glass slide with a clicking sound
● c. Principle: The venom has thrombin like activity and is unaffected ● N.V.: <8 minutes
by heparin
● d. N.V.: 10 – 15 seconds IVY METHOD
Thrombin Time Reptilase Time ● Volar surface of the forearm
Heparin Therapy Prolonged Normal ● 40 mmHg
Fibrin Split Products Prolonged Prolonged ● Puncture twice
Hypofibrinogenemia Greatly Prolonged Prolonged ● Collect blood using filter paper strips after 2 minutes and again after
Dysfibrinogenemia Prolonged Greatly Prolonged every 30 seconds
Immunologic Prolonged Normal
antithrombins Platelet Count Bleeding Time Probable disease or cause
Normal Prolonged Qualitative platelet defect
CLAUS FL ASSAY VWD
● a. Modification of the TCT, recommended procedure for estimating Vessel wall structure
the functional fibrinogen level abnormality
● b. SAMPLE: PPP REAGENT: bovine thrombin Low Normal Autoimmune thrombocytopenia
● c. Thrombin reagent concentration = 50 NIH units/mL Low Very prolonged Qualitative and quantitative
● d. PPP to be tested is diluted 1:10 with Owren buffer platelet deficiency
SUBSTITUTION STUDIES CAPILLARY RESISTANCE____________

● a. Used to distinguish factor deficiencies (done after obtaining an (FRAGILITY) TEST___________________
abnormal APTT or PT) ● 1. Detects abnormality of the capillaries due to a structural weakness
● b. Reagents in the capillary walls or thrombocytopenia
o Normal plasma – All factors are present except Ca2+ ● 2. Abnormal hereditary telangiectasia, may also be abnormal in
o Adsorbed plasma – Factor I, V, VIII, XI, XII are present hemophilia and Vit. K deficiency
▪ Utilized Barium Sulfate – no prothrombin group since
it is absorbed METHODS
o Aged serum – Factors VII, IX, X, XI, XII are present;
fibrinogen group is absent
o Thrombin time – tests for Fl (fibrinogen) deficiency POSITIVE PRESSURE TEST / RUMPLE – LEEDE /
o APTT – screens intrinsic and common pathway TORNIQUET TEST
o PT – screens extrinsic and common pathway
● Blood pressure cuff is applied to the upper arm for 5 minutes
Deficiency PT APTT TT Normal Adsorbed Aged ● The blood pressure cuff is set at 100 mmHg (80 mmHg for females)
Plasma Plasma Serum ● Observe for petechiae formation after 5 minutes of removal of cuff
I A A A C C NC
II A A N C NC NC
NEGATIVE PRESSURE TEST ● There are approximately 10 – 40 red cells per platelet in normal
peripheral blood
● A suction cup (2 cm in diameter) is placed on the midportion of the ● Ex. An OIO field with 100 RBCs has approximately 3 to 10
upper arm platelets; an OIO field with 200 RBCs has approximately 5 to 20
● 200 – 250 torr pressure is applied for 1 minute platelets
● Petechiae is counted after 5 minutes ● RODAKS: 1 platelet = 25 RBCs
o Example: 500 RBCs = 20 platelets
CLOT RETRACTION TIME___________ o 500 / 25 = 20 platelets
● McFarlane Clot Retraction Technique
REPORTING OF PLATELET ESTIMATE
ESTIMATE REPORTING
0 – 49,000/uL Markedly decrease
50,000 – 99,000/uL Moderately decreased
100,000 – 149,000/uL Slightly decreased
150,000 – 199,000/uL Low normal
200,000 – 400,000/uL Normal
401,000 – 599,000/uL Slightly increased
600,000 – 800,000/uL Moderately increased
Greater than 800,000/uL Markedly increased
PROCEDURE SIGNIFICANT PLATELET LEVELS
● 5 mL of whole blood is incubated at 37oC for 1 hour
● Clot will begin to shrink and retract from the walls of the tube Less than 100,000/uL Abnormally low; abnormal BT
● N.V.: 44 – 67% 30,000 – 50,000/uL Bleeding possible with trauma
● FORMULA = % CRT = (Vol. of serum/Total vol. of whole blood) Less than 30,000/uL Spontaneous bleeding possible
x 100% Less than 5,000/uL Severe spontaneous bleeding
FACTORS PLATELET AGGREGATION___________

● a. Number of contractile platelets ● Principle
● b. Presence of calcium and ATP o Aggregating agents are added to PRP to induce shape change
● c. Normal concentration of fibrinogen and aggregation of platelets
● Abnormal with thrombocytopenia, low or abnormal fibrinogen, o Due to platelet aggregation, the sample becomes clearer and
paraproteinemias (multiple myeloma; Waldenstrom syndrome) and transmits more light which is measured by an aggregometer
Glanzmann’s thrombasthenia ● CONCEPT SUMMARY
o The LIGHT TRANSMITTANCE is DIRECTLY
PROPORTIONAL to the PLATELET AGGREGATION
PLATELET COUNT___________________
● N.V.: 150 – 450 x 109/L (150,000 – 450,000/uL)
DIRECT METHODS
TONKANTIN METHOD
● Uses Rees – Ecker as diluent

● Uses light microscope
● Rees – Ecker Diluting Fluid
● Sodium Citrate – Anticoagulant
● Formalin – Preservative
● Brilliant Cresyl Blue – Stain
BRECHER – CRONKITE METHOD AGGREGATION DISORDERS ADHESION DEFECTS

EPINEPHRINE, COLLAGEN, EPINEPHRINE, COLLAGEN,
● Uses 1% ammonium oxalate as diluting fluid ADP = ABNORMAL ADP = NORMAL
● REFERENCE METHOD for manual platelet count RISTOCENTIN = NOTMAL RISTOCENTIN = ABNORMAL
DISEASES: Glanzmann DISEASES: VWD, Bernard –
UNOPETTE METHOD thrombasthenia, Soulier Syndrome
Afibrinogenemia
● Uses tripotassium EDTA and ammonium oxalate
PRINCIPLE: Hemolysis of red cells by hypotonicity and complete
●
block of platelet activity by chelating Mg and Ca with EDTA and PLATELET ADHESIVENESS___________
ammonium oxalate
GLASS BEAD RETENTION TEST
PLATELET ESTIMATION ON PERIPHERAL BLOOD SMEAR ● When blood is passed through a glass bead column, normal platelets
● 8 – 20 platelets / OIF = 9 - 21 that have vWF will adhere and aggregate to the beads
● Count 10 oil immersion fields
● Factor used is 20,000
PROCEDURE
BLEEDING DISORDERS DUE TO______
First sample is collected using routine EDTA tube
●
● Second sample is collected using a glass bead collecting system
VASCULAR DEFECTS________________
● Platelet count is performed on both specimens and platelet
HEREDITARY CONNECTIVE TISSUE DEFECTS
adhesiveness is calculated
● PA = (PC1-PC2/PC1) x 100%
o PC1 – EDTA tube count EHLERS-DANLOS SYNDROME
o PC2 – Glass bead tube count
● N.V.: 70% or greater ● The individual has hyperextensible joints and hyperplastic skin
● Skin, vasculature, and bones of affected individuals lack structural
support
DISORDERS OF HEMOSTASIS ● The defect may lie in a peptidase enzyme deficiency that converts
procollagen to collagen
BASIC TERMINOLOGIES_____________ PSEUDOXANTHOMA ELASTICUM
PETECHIAE
● Connective tissue elastic fibers in small arteries are calcified and
● Purplish, red, pinpoint hemorrhagic spots in the skin caused by loss structurally abnormal
of capillary ability to withstand normal blood pressure and trauma ● Subarachnoid and gastrointestinal bleeding are the most common
causes of death
PURPURA
ACQUIRED CONNECTIVE TISSUE DEFECTS
● Hemorrhage of blood into small areas of skin, mucous membranes,
and other tissues
● Appears first as red but later on turns purple and finally brownish SCURVY (VITAMIN C DEFICIENCY)
yellow color
● Acquired disorder of dietary deficiency of vitamin C
ECCHYMOSIS ● Ascorbic acid is required for the formation of intact structure of the
vascular basement membrane
● A form of purpura in which blood escapes into large areas of skin or ● Without Vitamin C, hydroxylation of the amino acids proline and
mucous membranes, but not into deep tissue lysine cannot take place and collagen will not be formed
o No formation of collagen = basement membrane / becomes
EPISTAXIS fragile
● Nosebleed
SENILE PURPURA
HEMARTHROSIS
● The aging process brings about a degeneration of collagen, elastin,
● Leakage of blood into a joint cavity and subcutaneous fat
HEMATEMESIS HEREDITARY ALTERATIONS OF VESSEL WALL STRUCTURE

● Vomiting of blood
HEREDITARY HEMORRHAGIC TELANGIECTASIA
HEMATOMA
● A swelling or tumor in the tissues or a body cavity that contains ● Characterized by vascular malformations and surface skin lesions
blood called telangiectasias
o “Dilation”
HEMATURIA ● The small blood vessels are focally disorganized and dilated
throughout the body, their wall support is poor, and their ability to
● Presence of intact RBCs in urine contract is diminished
● Smoky, cloudy red urine
CONGENITAL HEMANGIOTOMA (KASABACH – MERITT
HEMOGLOBINURIA SYNDROME)
● Presence of hemoglobin in the urine
● A disorder associated with tumors composed of vessels that
HEMOPTYSIS commonly swell and bleed at the surface
o Usually starts to diminish slowly after/starting 2 years of age
● Expectoration of blood secondary to hemorrhage in the larynx, (takes 7 years)
trachea, bronchi, or lungs
ACQUIRED ALTERATIONS OF THE VESSEL WALL
MELENA STRUCTURE
● A stool containing dark red or black blood
● Signs of upper GI bleeding DIABETES MELLITUS
MENORRHAGIA ● The large vessels may become atherosclerotic, and the capillary
basement membrane may thicken, thus blocking the normal flow of
● Excessive menstrual bleeding
blood
● Most often affected are the glomerulus and retina
AMYLOIDOSIS HEMOLYTIC UREMIC SYNDROME AND TTP
● It can involve and obstruct the function of may organs, including the ● Platelets aggregate and lodge in the endothelium and cause damage
vascular system, in which there is deposition of the fibrillar protein
called amyloid INCREASED PLATELET SEQUESTRATION BY SPLEEN
ENDOTHELIAL DAMAGE ● Normal is 30 – 45%

● Damaged caused by autoimmune of infectious agents to the THROMBOCYTOSIS
endothelial lining of the blood vessels may lead to hypercoagulation
or hypocoagulation
PRIMARY
AUTOIMMUNE VASCULAR PURPURA
● Uncontrolled proliferation of platelets, a characteristic of
● Drug – induced purpura – quinine, procaine, penicillin, aspirin, myeloproliferative disorders, polycythemia vera, essential
sulfonamides, sedatives, coumarins thrombocythemia, Chronic Granulomatous Leukemia\
● Allergic purpura
o Drug - induced- purpura associated with abdominal pain SECONDARY (REACTIVE)
secondary to GIT hemorrhaging
o Schonlein purpura - associated with joint pain especially in the ● A broad spectrum of acute and chronic illnesses illicit an increase in
knees and ankles platelet production
● Infectious purpura – Purpura may form for several reasons
o a. Result on an inflammatory response to infection
o b. An autoimmune response QUALITATIVE PLATELET DISORDERS
o c. Bacterial products
o d. Toxins or direct injury by infectious agent ADHESION DEFECTS
QUANTITATIVE PLATELET__________ BERNARD – SOULIER SYNDROME

DISORDERS_________________________ ● Platelets lack GpIIb which is necessary for bibding vWF
● Characterized by abnormal bleeding time and giant platelets
THROMBOCYTOPENIA
VON WILLEBRAND’S DISEASE
DECREASED PRODUCTION
● Lack of vWF
● Aplastic anemia: Generalized bone marrow suppression leading to a
decrease on all cell types AGGREGATION DEFECTS
o “Pancytopenia”
● Selective suppression of the megakaryocyte by chrolothiazide GLANZMANN’S THROMBASTHENIA
(diurectic)
o May inhibit megakaryocyte ● Lack of GpIIbIIIa
● Thrombocytopenia with absent radius (TAR): absent or decreased
and abnormal bone-marrow megakaryocytes, congenital deformities AFIBRINOGEMIA
of the arm
● Myelophthisic process: a space occupying lesion in the bone marrow ● Complete absence of fibrinogen
such as metastatic tumor, fibrosis or leukemia
STORAGE POOL DEFECTS
DILUTIONAL LOSS
● Platelets lack certain granules
● Extensive blood transfusion often is accompanied by
thrombocytopenia (since PRBC is stored in ref temperature, platelets GRAY PLATELET SYNDROME
become inactivated (non-viable)), the degree of which is directly
proportional to the number of units transfused ● Alpha granules are missing, platelets are larger than normal and
appear gray or blue-gray in Wright stain
NONIMMUNE DESTRUCTION o Also characterized by “GIANT PLATELETS”
● Artificial surfaces: induced platelet adherence as well as formation WISKOTT – ALDRICH SYNDROME
of platelet microaggregates
● Ex. Cardiovascular prosthetic devices, vascular grafts, dialysis ● Characterized by a triad of thrombocytopenia, recurrent infections,
membranes and eczema
o Triad: Thrombocytopenia, Eczema, Recurrent infections (TER)
IMMUNE PLATELET DESTRUCTION ● Platelets lack dense & alpha granules
● Platelets are small
● Platelet destruction by immune mechanisms, associated with o ToRCH
increased levels of IgG or complement on the platelet surfaces
HERMANSKY – PUDLAK SYNDROME
DISSEMINATED INTRAVASCULAR COAGULATION
● Characterized by a triad of tyrosinase – positive oculocutaneous
● Platelets are consumed and destroyed albinism, accumulation of ceroid – like pigment in macrophages and
a bleeding tendency
o Triad: Tyrosinase (+) oculocutaneous albinism, Accumulation HEMOSTATIC PROTEIN ABNORMALITIES
of ceroid – like pigment in macrophages, bleeding tendency
(TAB)
POSTOPERATIVE STATES
● Associated with a lack of beta (dense) granules
● Due to the release of tissue thromboplastin which initiates
CHEDIAK – HIGASHI SYNDROME
coagulation
● Characterized by albinism, recurrent infections, and giant lysosomes
MALIGNANCY
o Triad: Recurrent infections, albinism, giant lysosomes (RAG)
● Beta-granule deficiency
● Release of coagulating factors by neoplastic cells
ACQUIRED DEFECTS________________ PREGNANCY

● a. Drugs – aspirin, carbanicillin
o Decreased plasma aggregation, PROLONGED bleeding ● Placenta is rich in tissue thromboplastin
time
● b. Diet
● c. Diseases (acquired platelet defects) HEMORRHAGIC DISORDERS
o Myeloproliferative disorders
o Uremia
▪ Acquired platelet defect
INTRINSIC PATHWAY DISORDERS____
o DIC
FACTOR XI DEFICIENCY (HEMOPHILIA C)
o Paraproteneimias
▪ Decreased plasma retention, abnormal aggregation of ● Rosenthal Syndrome
platelets ● Mild bleeding
● Autosomal
● More than half of the cases have been described in Ashkenazi jews
DISORDERS OF THROMBOSIS
● Refers to an increased tendency to develop thrombi and emboli, FACTOR VIII:C DEFICIENCY (HEMOPHILIA A)
sometimes called the hypercoagulable states
● Sex-linked
● Factor VIII:C is complexed with Factor VIII:Vwf
PRIMARY___________________________ ● VIII:C is attached (carrier) to VWF
ANTITHROMBIN-III DEFICIENCY FACTOR IX DEFICIENCY (HEMOPHILIA B, CHRISTMAS

DISEASE)
● Principal inhibitor of thrombin is deficient
● Sex-linked
PROTEIN C AND S DEFICIENCY ● Milder bleeding compared to Hemophilia A
● Protein C and S are Vit. K dependent glycoproteins that inactivate VON WILLEBRAND’S DISEASE
factors Va and VIIIa
o Factor V Leiden: Factor V that is resistant to Protein C and S ● Defects of both Factor VIII:C and vWF
o vWF: carrier of FVIII:C; no carrier = no FVIII:C = symptoms
FIBRINOLYTIC SYSTEM DISORDERS similar to Hemophilia A
● PROLONGED APTT, PROLONGED BT, NORMAL PT
● Quantitative and functional abnormalities of plasminogen ● MOST FREQUENTY ENCOUNTERED HEREDITARY
● Deficiency in Factor XII COAGULOPATHY
o Thrombotic Disease o 2nd Hemophilia A
● CBC, APTT, PT ARE RECOMMENDED FOR INITIAL
DYSFIBRINOGENEMIA WORKUP
● An abnormal functional fibrinogen molecule is produced and is
resistant to fibrinolysis EXTRINSIC AND COMMON PATHWAY
HOMOCYSTINURIA DISORDERS_________________________
● Abnormal platelet-vessel wall interaction
FACTOR VII DEFICIENCY
Abnormal PT, Normal APTT and thrombin time
SECONDARY________________________ ●
FACTOR X (STUART – PROWER FACTOR) DEFICIENCY

LUPUS ANTICOAGULANT
● Abnormal APTT and PT, normal TCT
● Immunoglobulin directed against the phospholipid
● Associated with thrombosis due to prostacyclin inhibition (in vitro) FACTOR V DEFICIENCY (OWREN’S DISEASE)
● Prolonged APTT that does not correct with normal plasma
substitution ● Abnormal APTT and PT, normal TCT
FACTOR II (PROTHROMBIN) DEFICIENCY

● Abnormal APTT and PT, normal TCT
FACTOR I DEFICIENCY ANTITHROMBIN Normal Decreased
III ASSAY
● Abnormal APTT, PT and TCT
D-DIMER Negative Positive
AFIBRINOGENEMIA
WHOLE BLOOD CLOT LYSIS TIME___
● Complete absence
HYPOFIBRINOGENEMIA PRINCIPLE
● Low levels/decreased (<100 mg/dL) ● Whole blood will clot spontaneously when collected in a glass tube
without anticoagulant
DYSFIBRINOGENEMIA ● The clot should remain intact for 48 hours at 37oC, dissolution of the
clot prior to 48 hours is indicative of excessive systemic fibrinolysis
● Dysfunctional EUGLOBULIN LYSIS TIME___________
● Avoids the problems that arise from plasmin(ogen) inhibitors, a
FACTOR XIII DEFICIENCY more rapid and sensitive assay of lytic activity
● Clot dissolves in 5M urea
PRINCIPLE
ACQUIRED DISORDERS OF___________ ● Euglobulins are proteins that precipitate when plasma is diluted with
water and acidified
COAGULATION AND FIBRINOLYSIS__ ● They include plasminogen, plasmin, fibrinogen, and plasminogen
● 1. Hepatic disease – decreased coagulation factors (PT is a sensitive activators
test for liver function)
o Liver synthetic function test: why PT and not APTT? Factor PROCEDURE
VII is the first one to be depleted
● 2. Vitamin K Deficiency – Vit. K is needed for gamma carboxylation ● Diluted PPP + acid
of PT group ● Euglobulin (precipitate) + Thrombin = clot
● 3. Disseminated Intravascular Coagulation – coagulation factors and ● Clot is incubated at 37oC
platelets are destroyed leading to decreased coagulation proteins
o Hard to reverse, usually leading to death REFERENCE RANGE
● 4. Primary firbinogenolysis – increased activation of plasmin, ● Lysis in less than 2 hours is indicative of increased fibrinolytic
fibrinogen is broken down (pathologic fibrinogenolysis) activity
● 5. Secondary fibrinolysis – increased consumption of fibrinogen
(DIC)
o Consumptive coagulopathy PROTAMINE SULFATE GELATION TEST
o Increased clotting = increased amount of fibrinolysis; ● A test for secondary (smaller) fibrin degradation products
decreased fibrinogen because it is converted into O clot
PRINCIPLE
Test Reference Interval Value in DIC ● PARACOAGULATION - Protamine sulfate replaces secondary
Platelet count 150,000 – <150,000/uL degradation products from fibrin monomers and primary FDPs,
450,000/uL resulting to a gel formation
Prothrombin time 11 – 14 sec >14 sec o Gel formation: indicates increased fibrinolytic activity
Partial thromboplastin 25 – 35 sec >35 sec
time REFERENCE RANGE
D-dimer 0 – 240 ng/mL >240 ng/mL, often
10,000 to 20,000 ● Normally, no gel formation is seen
ng/mL
Fibrinogen 220 – 498 mg/dL <220 mg/dL, often ETHANOL GELATION TEST__________
higher, because ● Less sensitive but more specific than protamine sulfate test in
fibrinogen is an acute detecting soluble fibrin monomers and polymers in plasma
phase reactant
(Decreased or Normal) PRINCIPLE
PRIMARY SECONDARY ● 50% ethanol causes soluble fibrin monomer to dissociate, resulting
Fibrinogen Decreased Decreased in polymerization of the monomers and subsequent gel formation
Fibrin monomer None Present
REFERENCE RANGE
Fibrin polymer None Present
Stable clot None Present ● There should be no gel formation under normal conditions
LABORATORY EVALUATION OF LATEX D-DIMER ASSAY______________

● Measures a specific fragment arising from degradation of fibrin (D-
FIBRINOLYSIS dimer)
● Measures fibrinolysis and not fibrinogenolysis (secondary
PRIMARY SECONDARY fibrinolysis)
EUGLOBULIN Shortened Normal or slightly ● Already positive after 4 hours of DIC onset
CLOT LYSIS shortened
PLATELET Greater than 100 x Less than 100 x
COUNT 109/L 109/L
RBC INDICES
ANTICOAGULANT THERAPY_________
HEPARIN THERAPY WARFARIN THERAPY
Action: enhances antithrombin Action: antagonize vitamin K, MCV AND MCHC
III, inhibits thrombin induces formation of PIVKAs
Treatment: Protamine sulfate Treatment: Vitamin K ● Are most commonly used
administration ● MCV = (Hct x 10 / RBC Ct.)
Monitoring: APTT Monitoring: PT ● Indicator of average volume of RBC
● Heparin: used during coronary artery bypass graft surgery, during ● N.V.: 80 – 100 fL
cardiac-catheterization, and in several medical conditions; prolongs ● Microcytic: <80 fL (ATIS)
APTT and TCT ● Normocytic: 80 – 100 fL (AHA)
● RAT POISON ACTIVE INGREDIENT: Warfarin/Coumarin; ● Macrocytic: Megaloblastic, Nonmegaloblastic
utilizes PT monitoring
MCHC
ANEMIA ● Measure of the average concentration of hemoglobin

● Symptoms of a disease ● MCHC = (Hb x 100 / Hct)
● Decreased RBC, hemoglobin and hematocrit ● N.V.: 32 – 37 g/dL
● Results in reduction in the oxygen-carrying capacity of the blood ● ANISOCHROMIA – variation in Hb content
and subsequent hypoxia ● Hypochromic: <32 g/dL
● Normochromic: 32 – 37 g/dL
● Hyperchromic: >37 g/dL
CAUSES_____________________________ ● Increased in the presence of spherocytes or error in RBC or
● 1. Blood loss hemoglobin measurements
● 2. Impaired red cell production ● Decreased in IDA and thalassemia
● 3. Accelerated hemolysis
RED CELL DISTRIBUTION WIDTH
ABSOLUTE VS. RELATIVE ANEMIA___
● Absolute – true decrease in red cell mass ● Marker of anisocytosis
● Relative – cause by shift of extravascular fluid to the intravascular ● Derived from RBC histogram; coefficient of variation of the MCV
compartment ● Directly proportional to the degree of anisocytosis
o RBC becomes more diluted ● High: post-transfusion, post-treatment (iron, vitamin B12 or folate),
● Absolute reticulocyte count is used to determine the mechanism of idiopathic sideroblastic anemia and in the presence of two
anemia concurrent deficiencies (iron and folic acid deficiency)
o Increase RBC loss – Due to hemolysis or acute blood loss, high ● N.V.: 11.5 – 14.5%
reticulocyte
o Decreased production – low reticulocyte count
▪ Microcytic: IDA, thalassemia
▪ Normocytic: Aplastic anemia
▪ Macrocytic: Vitamin B12 deficiency (megaloblastic),
liver disease
LABORATORY EVALUATION OF______

ANEMIAS___________________________
HYPOCHROMIA GRADING
1+ Area of central pallor is one-half of cell diameter
2+ Area of pallor is two-thirds of cell diameter
3+ Area of pallor is three quarters
4+ Thin rim of hemoglobin
POLYCHROMASIA GRADING
Slight 1%
1+ 3%
2+ 5%
3+ 10%
4+ > 11% PERIPHERAL BLOOD SMEAR
● a. Anisocytosis: variation in cell size
ROULEAUX GRADING ● b. Poikilocytosis: variation in cell shape
1+/Slight Aggregates of 3-4 RBCs ● c. Anisochromia: variation in cell hemoglobin content
2+/ Moderate Aggregates of 5-10 RBCs
3+/ Marked Numerous aggregates with only few free RBCs BONE MARROW EXAMINATION
COMPLETE BLOOD COUNT ● Collection sites

o Posterior superior iliac crest – most preferred
● Decreased Hb, Hct, RBC count o Anterior iliac crest
o Posterior iliac crest o Schistosoma mansoni, haematobium
o Sternum ● Laboratory findings
o Ribs and vertebrae o Microcytic hypochromic RBC
▪ For bone marrow differential count at least 500 cells but o Decreased serum iron, serum ferritin, and reticulocyte count
preferably 1000 cells must be counted o Increased RDW, TIBC
▪ Normal M:E ratio is 2:1 – 4:1 or 3:1 o Smear shows ovalocytes/pencil forms
▪ Target cells, possible
OTHER LABORATORY TESTS
ANEMIA OF CHRONIC DISEASE
● Iron studies
Serum Ferritin ● Reflect body’s tissue iron stores ● Due to an inability to use iron for hemoglobin production
● 1st test to become abnormal when iron ● Impaired release of storage iron associated with increased hepcidin
stores begin to decrease levels
Serum iron ● Not necessary for differential diagnosis ● Laboratory findings
● Tested when the disease is not obvious o Normocytic normochromic, but can become microcytic
Transferrin Iron Binding ● Measures the ability of transferrin to hypochromic in some patients
bind iron o Increased ESR
● Indirect measure of transferrin o Increased or normal ferritin levels (opposite of IDA)
Transferrin saturation = Serum Fe x 100 o Decreased TIBC, serum iron (opposite of IDA)
TIBC
Free Erythrocyte ● Increases when Fe is deficient SIDEROBLASTIC ANEMIA
Protoporphyrin/ZEP ● Protoporphyrin is the substance to
which Fe binds to form heme ● Caused by blocks in the protoporphyrin pathway resulting to
● Urinalysis: check for hematuria (glomerular disease) ineffective hemoglobin synthesis and iron overload
● Fecal analysis: occult blood test (+ = chronic GIT bleeding) = may ● Ringed sideroblasts – excess iron accumulates in the mitochondrial
lead to IDA region of the immature erythrocyte in the bone marrow and encircles
● Renal and hepatic function tests the nucleus
● Siderocytes – excess iron accumulates in the mitochondrial region
of the mature erythrocytes in the circulation
● Siderotic granules – inclusions in the siderocytes using Prussian blue
stain
● Pappenheimer Bodies – inclusions demonstrated using Wright’s
stain
● Laboratory findings
o Microcytic, hypochromic RBCs
o Increased ferritin and serum iron
o Decreased TIBC
THALASSEMIA
● Genetic disorder characterized by a primary, quantitative reduction

of in globin chain synthesis
● Characterized by microcytic, hypochromic RBCs and target cells
● Types
o Beta-thalassemia
▪ Major/homozygous (Cooley anemia)
● Markedly decrease rate of synthesis or absence of
both beta chains resulting in an excess of alpha
chains
● No HbA produced; compensated with up to 90%
HbF
o Severe microcytic, hypochromic anemia
o Target cells, teardrop cells, many nRBCs,
TYPES OF ANEMIAS_________________ basophilic stippling, Howell-Jolly bodies,
Pappenheimer bodies, Heinz bodies
o Increased serum iron, bilirubin
ANEMIAS OF IMPAIRED OR DEFECTIVE PRODUCTION
▪ Minor heterozygous
● One beta chain is normal
IRON DEFICIENCY ANEMIA ● HbA is slightly decreased, but HbA2 is slightly
increased to compensate
● A microcytic hypochromic anemia caused by inadequate supplies of ● Laboratory findings
iron needed to synthesize hemoglobin o Mild microcytic, hypochromic anemia
● Can be caused by blood loss, hookworm infection, pregnancy o Target cells, basophilic stippling
o Hookworms: A. duodenale, N. americanus o Alpha-thalassemia
o T. trichuria ▪ Major
● All four alpha chains are deleted; no normal Hb is o Pancytopenia
produced o Macrocytic, normochromic anemia with oval macrocytes and
● Disease is known as hydrops fetalis teardrop cells
● 80% Bart’s Hb (y4) is produced; cannot carry o Howell-Jolly bodies, nucleated RBCs, basophilic stippling,
oxygen; incompatible with life Pappenheimer bodies and Cabot rings
o Fetus dies in utero or shortly after delivery; o Hypersegmented neutrophils
Hydrops fetalis: generalized edema of o Increased LDH, bilirubin, iron levels
fetus/newborn
▪ Hb H disease (gulf butt cell)
● Three alpha genes are deleted; decrease in alpha
chains lead to beta chain excess
● Hb H (B4) is produced, it is unstable and leads to
the formation of Heinz bodies and causes the RBC
to become rigid and destroyed by the spleen
o Heinz bodies
▪ Bite cells becomes hemolyzed
▪ Aka HbH inclusion
o Moderate microcytic, hypochromic anemia
o Up to 30% Hb H, the rest is Hb A
▪ Minor/Trait
● Two alpha genes are deleted
● Patients are usually asymptomatic and discovered
accidentally
● Up to 6% Hb Bart’s, in newborns may be helpful in
diagnosis
● Mild microcytic, hypochromic anemia often with a
high RBC count and target cells
LEAD POISONING
● Multiple blocks in protoporphyrin pathway ● Vegan persons are at risk of vitamin B12 deficiency
o Blocks conversion of ALA by porphobilinogen ● Folate deficiency: persons who do not eat vegetables
o Blocks coupling of iron to PIC by ferrochelatase
● Laboratory findings PERNICIOUS ANEMIA
o Normocytic normochromic, may progress to microcytic,
hypochromic ● An autoimmune disease wherein no intrinsic factor is produced by
parietal cells in the stomach due to antibody destruction
● Schilling’s test: Detects pernicious anemia
NONMEGALOBLASTIC ANEMIA
● Include alcoholism and conditions that cause accelerated

erythropoiesis
● Erythrocytes are round, not oval as seen in megaloblastic anemia
o Coarse basophilic stippling (RNA remnants)
● Iron Studies in Microcytic, Hypochromic Anemias APLASTIC ANEMIA
Iron Thalassemia Anemia of Sideroblastic Lead ● Bone marrow failure causes pancytopenia
Deficiency Minor Chronic anemia poisoning ● Laboratory findings
Anemia Inflammation o Decreased hemoglobin, hematocrit, reticulocytes
Serum Ferritin D I or N I or N I N o No response to erythropoietin
● Can be genetic or acquired
Serum Iron D or N I or N D I V CLINICAL/LAB DIAMOND FANCONI
TIBC I N D D or N N FEATURES BLACKFAN ANEMIA ANEMIA
Transferrin D I or N D I I
saturation
Hematologic Congenital pure red cell Congenital aplastic
Classification aplasia anemia
FEP/ZPP I I or N I M I
Brown skin Uncommon Common
Legend: D – decreased, I – increased, N – normal, M – mixed, V –
pigmentation
variable
Thumb Uncommon Common
abnormalities
MEGALOBLASTIC ANEMIA
Renal Uncommon Common
● Defective DNA synthesis causes abnormal nuclear maturation abnormalities
o DNA strands cut short due to deficient thymidine Onset of < 1 year of age 5 – 10 years of age
● Asynchronism: Abnormal nuclear maturation, normal cytoplasm hematologic
maturation abnormalities
● Caused by either a vitamin B12 or folic acid deficiency Bone marrow Cellular Hypoplastic to
● Laboratory findings biopsy aplastic
Bone marrow Marked decreased in Pancytopenia o Membrane defects caused by polarization of cholesterol at the
aspirate erythroid precursors only ends of the cell rather than around pallor area
Peripheral Blood Decrease in RBC; Pancytopenia ● Hereditary Stomatocytosis
normal WBC and o Membrane defects due to abnormal permeability both to
platelets sodium and potassium; causes erythrocyte swelling
Cytogenetics No associated Multiple ▪ Where sodium goes, water follows
abnormalities chromosomal ● Hereditary Acanthocytosis (Abetalipoproteinemia)
abnormalities in o Increased cholesterol: lecithin ratio in the membrane due to
many tissues abnormal lipid concentrations; absence of serum beta-
lipoprotein needed for transport
MYELOPHTHISIC ANEMIA (MARROW REPLACEMENT) ● Glucose – 6 – Phosphate Dehydrogenase Deficiency
ANEMIA / MYELOID METAPLASIA o Most common enzyme deficiency in HMP
o Reduced glutathione levels are not maintained because of
● Substitution/replacement decreased NADPH generation -> oxidation of hemoglobin to
● Hypoproliferative anemia caused by replacement of bone marrow methemoglobin (Fe3+) -> Denaturation of Hb to Heinz bodies
hematopoietic cells by malignant cells or fibrotic tissue ● Pyruvate Kinase Deficiency
● Associated with cancers o Most common enzyme deficiency in Embden – Meyerhof
● Laboratory findings Pathway
o Normocytic, normochromic anemia o Lack of ATP causes impairment of the cation pump that
o Leukoerythroblastic blood picture controls intracellular sodium and potassium level
▪ Poikilocyte: dacrocytes o Decreased RBC deformability reduces their lifespan
▪ Young RBCs and WBCS in smear ● Paroxysmal Nocturnal Hemoglobinuria
o Acquired defect in which RBC membrane has an increased
BLOOD LOSS ANEMIA sensitivity for complement binding as compared to normal
RBCs
o All cells are abnormally sensitive to complement lysis
ACUTE BLOOD LOSS ANEMIA o Diagnosis
▪ Ham’s acidified serum test
● Characterized by a sudden loss of blood resulting from trauma or ▪ Sugar water test
other severe forms of injury ▪ Flow cytometry to detect deficiencies of surface
● Clinical symptoms expression of glycosyl phosphatidyl inositol (GPI)-linked
o Hypovolemia: leading to shock proteins such as CD 55 and CD 59
o Rapid pulse
o Low blood pressure HEMOLYTIC ANEMIAS DUE TO EXTRINSIC IMMUNE DEFECTS
o Pallor
▪ Body mechanism ● Paroxysmal Cold Hemoglobinuria
● Shifting of EVF to intracellular o An IgG biphasic Donath-Landsteiner antibody with P
o Dilution -> IDA specificity fixes complement to RBCs in the cold (<20 oC);
● Laboratory findings complement-coated RBC lyse when warmed at 37oC
o Normocytic, normochromic anemia ● Warm Autoimmune Hemolytic Anemia
o Initially normal reticulocyte count then reticulocytosis in 3 – 5 o RBCs are coated with IgG or complement
days o Macrophages phagocytize these RBCs, or they may remove the
o Initially normal hemoglobin and hematocrit; in a few hours, antibody complement from the RBC surface, causing
increase in platelet and leukocytosis with a shift to the left, drop membrane loss and spherocytes
in hemoglobin, hematocrit and RBC ● Cold Autoimmune Hemolytic Anemia
o RBCs are coated with IgM and complement at temperatures
CHRONIC BLOOD LOSS ANEMIA <37oC
o RBCs are lysed by complement or phagocytized by
● Characterized by gradual long term loss of blood; often caused by macrophages
gastrointestinal bleeding o Antibody is usually anti-I but can be anti-i
● Laboratory findings ▪ Anti – I: mycoplasma pneumoniae
o Initially normocytic, normochromic anemia that over time ▪ Anti – i: EBV
causes a decrease in hemoglobin and hematocrit, gradual loss ● Hemolytic Transfusion Reaction
of iron causes microcytic, hypochromic anemia o Recipient has antibodies to antigens on donor RBCs; donor
cells are destroyed
HEMOLYTIC ANEMIAS ● Hemolytic Disease of the Newborn (HDN)
o Due to different blood group antigens on the parent of a
HEMOLYTIC ANEMIAS DUE TO INTRINSIC DEFECTS newborn
o E.g., ABO, Rh
● Hereditary Spherocytosis
o Defect in membrane skeletal proteins (spectrin, ankyrin) HEMOLYTIC ANEMIAS DUE TO EXTRINSIC NON-IMMUNE
o Membrane skeletal protein abnormalities cause RBCs to lose DEFECTS
unsupported lipid membrane
o Laboratory findings ● Microangiopathic Hemolytic Anemias (MAHA)
▪ Spherocytes o Disseminated Intravascular Coagulation
▪ MCHC > 37 g/dL ▪ Systemic clotting is initiated by the activation of the
▪ Increased Osmotic Fragility – true elevation of OFT coagulation cascade due to toxins or conditions that
▪ Increased Serum Bilirubin trigger release of procoagulants
● Hereditary Elliptocytosis (Ovalocytosis) ▪ Multiple organ failure occurs due to clotting
▪ Fibrin is deposited in small vessels, causing RBCs to
fragment POIKILOCYTOSIS
o Hemolytic Uremic Syndrome
▪ Occurs most often in children following gastrointestinal
infection (e.g., Escherichia coli) (O157:47)
▪ Clots form causing renal damage
o Thrombotic Thrombocytopenic Purpura
▪ Likely due to a deficiency of enzyme ADAMTS13 that is
responsible for the breakdown of large von Willebrand
factor multimers
▪ When multimers are not broken down, clots form,
causing RBC fragmentation and CNS impairment
● March Hemoglobinuria
o Transient hemolytic anemia that occurs after forceful contact of
the body with hard surfaces
● Other causes
o Infectious agents – malarial parasites & C. perfringes
o Mechanical trauma – Prosthetic heart valves
o Thermal burns
HEMOGLOBINOPATHIES
HEMOGLOBINOPATHIES
Hemoglobin S 6th amino acid of B chain; glutamate is replaced by
valine
Hemoglobin C 6th amino acid of B chain; glutamate is replaced by
lysine
Hemoglobin D 121st amino acid of B chain; glutamate is replaced by
glycine
Hemoglobin E 26th amino acid of B chain; glutamate is replaced by
lysine
Hemoglobin O 121st amino acid of B chain; glutamate is replaced by
lysine
SICKLE CELL DISEASE (Hb SS)_______

● No Hb is produced
● Approximately 80% Hb S and 20% Hb F
● Hb A2 is variable
● Hb insolubility results when deoxyhemoglobin is formed
● Hb crystallizes, forming the classic sickle shape of the cell
SICKLE CELL TRAIT (Hb AS)_________

● Defect is inherited from one parent only
● Approximately 60% Hb A and 40% Hb S
● Normal amounts of Hb A2 and Hb F
Hb C DISEASE_______________________
● No Hb A is produced
● Approximately 90% Hb C, 2% Hb A2 and 7% Hb F
● Folded cell – RBC with membrane folded over
Hb SC DISEASE______________________ ECHINOCYTES / BURR CELLS________

● Have evenly spaced round projections; central pallor area present
● Washington monument crystal ● Caused by changes in osmotic pressure
● Double heterozygous condition where an abnormal sickle gene from ● Seen in liver disease, uremia, heparin therapy, pyruvate kinase
one parent and abnormal C gene form the other parent is inherited deficiency or as an artifact
● No Hb A is produced ● Reversible
● Approximately 50% Hb S and 50% Hb C are produced
● Compensatory Hb F may be elevated up to 7%
ACANTHOCYTES____________________
● Have unevenly space pointed projections; lack a central pallor are
● Caused by excessive cholesterol in the membrane
● Associated with alcoholic liver disease, post-splenectomy and
abetalipoproteinemia
● Non-reversible
LEACH Phenotype: RBCs lack Gerbich antigens; there are
TARGET CELLS / CODOCYTES /______ ●
elliptocytes on the PBS
MEXICAN HAT CELLS_______________
● Show central areas of hemoglobin surrounded by a colorless ring
and a peripheral ring of hemoglobin; cells have an increased surface- RBC INCLUSIONS
to-volume ratio
● Caused by excessive cholesterol in the membrane or a hemoglobin
distribution imbalance
NUCLEATED RBCS___________________
● Usually orthochromic normoblasts (metarubricytes) but can appear
● Seen in liver disease, hemoglobinopathies, thalassemia, iron
in any erythropoietic stage of maturation
deficiency anemia
● Indicate bone marrow stimulation or increased erythropoiesis
● Associated with thalassemia major, sickle cell anemia, other
SPHEROCYTES______________________ hemolytic anemias, erythroleukemia and myeloproliferative
● Disk-shaped cells with a smaller volume than a normal erythrocyte; disorders
cells have a decreased surface to volume ratio; lack of a central ● Normal newborns can have a few
pallor area ● Healthy individuals should have none on a peripheral blood smear
● Associated with defects of the red cell membrane proteins
●
●
MCHC may be >37%; increased osmotic fragility
Damaged RBC; seen in hereditary spherocytosis, G6PD deficiency
HOWELL – JOLLY BODIES___________
● Small round DNA fragments usually one per cell, but can be
and immune hemolytic anemia
multiple
● Stain dark purple to black with Wright’s stain
PYROPOIKILOCYTES /_______________ ● Not seen in normal erythrocytes; normally pitted by splenic
macrophages
MICROSPHEROCYTES_______________ ● Seen in sickle cell anemia, beta thalassemia major and other severe
● Fragments at 41 – 45 degrees Celsius hemolytic anemias, megaloblastic anemia, alcoholism, post-
● <4 um splenectomy
● Frequently seen in severe injury (burns) o Splenic atrophy, splenic infarction
TEARDROPS / DACRYOCYTES________ BASOPHILIC STIPPLING_____________

● Pear-shaped cells with one blunt projection ● Multiple, tiny, fine and coarse inclusions (ribosomal RNA remnants)
● Seen in megaloblastic anemia, thalassemias, and extramedullary evenly dispersed throughout the cell, “blueberry bagel” appearance
hematopoiesis (myelofibrosis, myelophthisic anemia) ● Stain dark blue with Wright’s stain
● Seen in thalassemia, megaloblastic anemias, sideroblastic anemia,
SICKLE CELL / DREPANOCYTES______ lead poisoning and alcoholism
● Shapes vary but show thin, elongated, pointed ends and will appear
crescent – shaped; usually lack a central pallor PAPPENHEIMER BODIES_____________
● Contain polymers of abnormal Hb S ● Small, irregular, dark staining, iron granules usually clumped
● Seen in hemoglobinopathies SS, SC, SD and S/B-thalassemia together at periphery of the cell
● Cell shape is caused by cell membrane alterations due to an amino ● Stain with Perl’s Prussian blue stain; appear dark violet with
acid substitution Wright’s stain
● Caused by an accumulation of ribosomes, mitochondria, and iron
HELMET CELLS / HORN CELLS /_____ fragments
● Seen in sideroblastic anemia, hemoglobinopathies, thalassemia,
KERATOCYTES______________________ megaloblastic anemia, myelodysplastic syndrome (RARS)
● Interior portion of cell is hollow, resembling a horn or a helmet cell
● Seen in microangiopathic hemolytic anemias (MAHA)
CABOT RINGS_______________________
● Thin- red-violet, single to multiple ring-like structures that may
SCHISTOCYTES / RBC FRAGMENTS__ appear in loop of figure-eight shapes
● Damaged RBC; fragments of various sizes and shapes are present, ● Composed of fragments of nuclear material
often with pointed projections ● Seen in megaloblastic anemia, myelodysplastic syndromes, lead
● Seen in microangiopathic hemolytic anemia (MAHA), thermal poisoning
injury, renal transplant rejection and G6PD deficiency
HEMOGLOBIN C CRYSTALS__________
STOMATOCYTES / MOUTH CELLS____ ● Condensed, intracellular, rod-shaped crystals
● Characterized by an elongated or slit-like area of central pallor ● Seen in hemoglobin C or SC disease, but not in trait
● Seen in liver disease, hereditary stomatocytosis or as artifact
● Caused by osmotic changes due to cation imbalance (Na +/K+)
● Other correlations: HEMOGLOBIN SC CRYSTALS /_______
o Alcoholism “WASHINGTON MONUMENT”________
o Liver cirrhosis
o Rh null disease CRYSTALS__________________________
● 1 – 2 blunt, finger-like projections extending from the cell
membrane
ELLIPTOCYTES / OVALOCYTES______ ● Seen in hemoglobin SC disease, but not in trait
● Abnormal cholesterol distribution
● Cigar to egg-shaped erythrocytes
● Associated with defects of the red cell membrane proteins HEINZ BODIES______________________
● Degraded hemoglobin
● Multiple inclusions ranging in size, supravital stain must be used to ● Toxic changes are present
be able to visualize ● Shift to the left
● Seen in G6PD deficiency, beta-thalassemia major, Hb H disease,
unstable hemoglobinopathies, drug induced anemias NEUTROPHILIC LEUKEMOID REACTION
● Blood picture mimics chronic myelogenous leukemia (CML)
MALARIAL PARASITES______________ ● Benign, specific response to specific agent or stimulus
● P. vivax, P. falciparum, P. malariae and P. ovale ● WBC count can increase to 50 – 100 x 109/L
● Increased LAP score
There is a shift to the left with toxic changes to neutrophils
ABNORMAL RBC DISTRIBUTION_____ ●
LEUKOERYTHROBLASTIC REACTION
ROULEAUX
● Presence of immature leukocytes and immature erythrocytes in the
● Stacking or “coining” pattern of RBCs due to abnormal or increased blood
plasma protein
● Occurs in marrow replacement disorders such as myelofibrosis
o Removes zeta potential
● May see excessively blue color macroscopically and FUNCTIONAL DISORDERS OF NEUTROPHILS
microscopically
● Seen in hyperproteinemia, multiple myeloma, Waldenstrom’s
macroglobulinemia and conditions that produce increased CHRONIC GRANULOMATOUS DISEASE (CGD)
fibrinogen
● May be artifactual; considered normal in thicker are of the smear ● Morphologically normal, but functionally abnormal because of
enzyme deficiency that results in an inability to degranulate which
AGGLUTINATION causes inhibited bactericidal function
● Characterized by clumping of erythrocytes with no pattern CHEDIAK – HIGASHI SYNDROME
● Occurs when erythrocytes are coated with IgM antibodies and
complement ● Both morphologically and functionally abnormal leukocytes
● Seen in cold autoimmune hemolytic anemia ● WBCs are unable to degranulate and kill invading bacteria
● Warm blood to 37oC to correct falsely low RBC and hematocrit, and ● Abnormal fusion of primary and secondary neutrophilic granules
falsely high MCHC (>37 g/dL) when using an automated cell ● Leukocytes have gigantic granules that are peroxidase positive
counter
NUCLEAR ABNORMALITIES
HYPERSEGMENTATION
● Shift to the right

● 5 or more lobes in the neutrophil
● Associated with megaloblastic anemia
HYPOSEGMENTATION
● Pelger – Huet Anomaly

● Machine would mistakenly count this as 1 big RBC leading to o Nucleus is hyperclumped and does not mature past the two-fold
decreased RBC count, decreased hematocrit, increased MCV and stage
MCHC o Nucleus has a dumbbell or peanut – shaped; “pince-nez”
● The effect of cold agglutinins to: RBCs (decreased) and hemoglobin appearance
(no effect) o Morphologically abnormal but functionally normal
HETEROZYGOUS HOMOZYGOUS
LEUKOCYTE DISORDERS ● Granulocytes whose nucleus ● Characterized by granulocytes
is segmented only once, with round or oval nuclei (no
taking the form of “two very segmentation)
NON-MALIGNANT GRANULOCYTIC distinct segments connected ● May be accompanied by
by a very thin filament” impaired cognitive
DISORDERS_________________________ ● Does not affect function of development, skeletal
neutrophils abnormalities, and heart defects
SHIFT/PHYSIOLOGIC PSEUDONEUTROPHILIA ● Pseudo Pelger – Huet Anomaly
● Redistribution of the blood pools causes a short-term increase in the o Acquired abnormality associated with myeloproliferative
total WBC count and in the absolute number of neutrophils in the disorders and myelodysplastic syndromes; can also be drug
circulating granulocyte pool induced
● Caused by exercise, stress, pain and pregnancy o Nucleus is usually round instead of dumbbell shape
● No toxic changes o Accompanied by hypogranulation
● No shift to left
INHERITED CYTOPLASMIC ANOMALIES
PATHOLOGIC NEUTROPHILIA
● Neutrophils leave the circulating pool, enter the migrating pool and MAY – HEGGLIN ANOMALY
then move to the tissues in response to tissue damage
● WBC count can increase up to 50 x 109/L ● Large crystalline, Dohle-like inclusions in the cytoplasm of
neutrophils on Wright’s stain; gray blue spindle (cigar) shaped
● Morphologically abnormal but functionally normal o Cytochemical stain o Immunologic probes of
● Giant platelets, thrombocytopenia, and clinical bleeding are also results cell markers
associated with this anomaly ● Defines acute leukemia as o Cytogenetics
>30% bone marrow blasts o Molecular
ALDER – REILY ANOMALY ● Easier to use and is still abnormalities
widely taught o Clinical Syndromes
● Large azurophilic granules appear in the cytoplasm of all or only one ● Defines acute leukemia as
cell line >20% bone marrow blasts
● Granules contain degraded mucopolysaccharides due to an enzyme ● Standard for diagnosis
defect
o Metachromatic granules that contains mucopolysaccharides CYTOCHEMICAL STAINS
STAIN PURPOSE COMMENT
MONOCYTIC DISORDERS____________ Perl’s Prussian Blue ● Perl’s reagent ● Stains siderotic
(potassium granules,
GAUCHER DISEASE ferricyanide – Pappenheimer
● Most common lipid storage disorder HCl) bodies and
● Deficiency in glucocerebrosidase causes glucocerebroside to ● Principle: hemosiderin
accumulate in the macrophages of the bone marrow, spleen and liver Prussian blue
● Crumpled tissue paper appearance of macrophage reagent stains
Fe++ a vivid blue
NIEMANN – PICK DISEASE or green color
Leukocyte Alkaline ● Stains ALP High (>100) NAP
● Deficiency in sphingomyelinase causes sphingomyelin to Phosphatase present in the consistent with
accumulate in macrophages in multiple organs and bone marrow neutrophil leukemoid reaction,
● Foamy appearance of macrophages (foam cells) ● Increased: in which
Polycythemia granulocytes have
LIPID STORAGE DISEASE ENZYME DEFICIENCY vera, last increased enzyme
Fabry’s Disease Alpha galactosidase trimester of activity (3+ to 4+)
Gaucher Disease Glucocerebrosidase pregnancy, (KAPLOW
Krabbe Cerebroside beta galactosidase infections with CLOUNT)
Neimann – Pick Sphingomyelinase neutrophilia SPECIMEN: Fresh
Metachromatic leukodystrophy Arylsulfatase A (leukemoid capillary blood is
Sandhoff Hexosaminidase A and B reaction) recommended
Tay Sach Hexosaminidase A ● Decreased:
CML, PNH,
sickle cell
NON-MALIGNANT LYMPHOCYTOSIS__ anemia, IM, PA
ASSOCIATED WITH VIRAL INFECTIONS Myeloperoxidase ● Stains Recommended
stain peroxidases specimen is fresh
INFECTIOUS MONONUCLEOSIS present in blood smear from a
granulocytes and capillary puncture
● Caused by Epstein – Barr Virus monocytes
o CD21: C3d receptor, but also a receptor for EBV ● Used to
● Targets B-cells differentiate
● Reactive T-lymphocytes are present attacking infected B-cells acute
o Downey Cell – atypical or reactive T-lymphocytes myelogenous
● Positive heterophile antibody test leukemia and
monocytic
CYTOMEGALOVIRUS leukemia from
● Symptoms similar to IM acute
● Most common transmitted infection from mother to fetus lymphocytic
● Negative heterophile antibody test leukemia
● No reactive lymphocytes Sudan Black B ● Stains lipids
present in
INFECTIOUS LYMPHOCYTOSIS granulocytes and
monocytes
● Associated with adenovirus and coxsackie A virus ● Used to
● Contagious disease mostly affecting young children differentiate
● Lymphocytosis with no reactive lymphocytes acute
myelogenous
MALIGNANT LEUKOCYTE DISORDERS leukemia and
myelomonocytic
HEMATOPOIETIC MALIGNANCY CLASSIFICATIONS leukemias from
acute
French – American – British World Health Organization lymphocytic
(FAB) Classification (WHO) Classification leukemia
● Based on: ● Based on:
o Cellular morphology o Cellular morphology
o Cytochemical stain
Periodic Acid Schiff ● Stains ● L1 and L2 (TdT) – stains the
Stain mucoproteins, produce a lymphoblasts
glycoproteins, block pattern ● AUER RODS – lysosomes; only seen in AML
and high
molecular weight
carbohydrates
ACUTE LYMPHOPROLIFERATIVE____
● Used to help in DISORDERS_________________________
the diagnosis of
DiGuglielmo’s FAB CLASSIFICATION OF ALL
syndrome (FAB
M6)
Naphthol AS-D ● Stains esterases ● Used to FAB L1
Choroacetate in granulocytes differentiate
Esterase and mast cell granulocytic ● Small lymphoblast, homogenous
granules cells from ● Most T – cell ALLs are FAB L1
monocytic ● Most common ALL (childhood)
cells
● Specific FAB L2
esterase
● Large lymphoblast; heterogenous
a-Naphthyl Acetate ● Stains esterases Non-specific
● Most common ALL in adults
Esterase present in the esterase
o Most common leukemia in adults: Acute Myeloid Leukemia
monocytic cells,
(AML)
macrophages,
megakaryocytes,
FAB L3
and platelets
● Monocytes stain
● Leukemic phase of Burkitt’s lymphoma
red brown
● Large lymphoblasts, homogenous and vacuolated
a-Naphthyl Butyrate ● Identifying Non-specific
● All FAB L3 are B cell
Esterase monocytes, esterase
promonocytes
and monoblasts ACUTE MYELOPROLIFERATIVE_____
Tartrate-resistant
ACP (TRAP)
● Marker for hairy
cell leukemia
In the presence of L
(+) tartaric acid, the
DISORDERS_________________________
activity of acid
FAB M0 – W/O DIFFERENTIATION
phosphatase is
inhibited except for ● Blast exhibit myeloid markers
ACP isoenzyme 5 ● Does not stain
which is produced ● Detected through CD markers: CD13, CD33, CD34. CD117 (flow
by hairy cells cytometry)
Toluidine Blue ● Binds with acid o CD13 and 33: Pan myeloid
mucopolysaccha o CD34: hematopoietic stem cell marker
rides in blood
cells FAB M1 (AML W/O MATURATION)
● Useful for the
● > 90% myeloblasts, may have Auer Rods
recognition of
● Positive staining by: Sudan black, MPO, specific esterase
mast cells and
tissue basophils
FAB M2 (AML W/ MATURATION)
Nitroblue ● Screening ● Heparinized
Tetrazolium procedure for the whole blood is ● < 90% marrow myeloblasts
Neutrophil detection of the o Myeloblasts: very first granular stage (granulocyte)
Reduction Test chronic recommended ● Positive staining by: SBB, MPO, specific esterase
granulomatous specimen
disease FAB M3 (ACUTE PROMYELOCYTIC LEUKEMIA) /
● Sample from HYPERGRANULAR PROMYELOCYTIC LEUKEMIA
patients with
● > 30% marrow promyelocytes with bundles of Auer rods
CGD lack the
● Associated with: DIC
enzyme activity
● SBB, MPO, Sp. Esterase (+)
from neutrophils
● Non sp. Esterase negative (-)
leading to the
inability to kill
FAB M4 (ACUTE MYELOMONOCYTIC LEUKEMIA / NAEGELI
organisms and
SYNDROME)
reduce NBT to
blue – black ● > 30% marrow myeloblasts of monocytic origin
formazan ● Positive for SBB, MPO, Sp. Esterase, and non-specific esterase
deposits
Terminal ● Present in 90% ● Used to FAB M5 (ACUTE MONOCYTIC LEUKEMIA / SCHILLING
Deoxyribonucleotid of cases of ALL differentiate LEUKEMIA)
yl Transferase ALL from
AML ● MPO, SBB, SPE (-)
● Non SPE (+) NON-HODGKIN’S LYMPHOMA
FAB M6 (ACUTE ERYTHROLEUKEMIA / DI GUGLIELMO

MYCOSIS FUNGOIDES
SYNDROME)
● Sezary cell syndrome
● Erythrocyte lineage involved
● The most common cutaneous lymphoma
● PAS (+)
● Presence of Sezary cells = malignant T lymphocytes; has convulated
nucleus
FAB M7 (ACUTE MEGAKARYOCYTIC LEUKEMIA)
● Malignancy of T cells = nucleus, looks like brain = cerebriform cells
● No stain
o Use flow cytometry ADULT T-CELL LEUKEMIA
● Use anti-vWF abs
● Caused by HTLV – 1 (adult T cell leukemia)
● Flower cells
CHRONIC LEUKEMIAS______________
CHRONIC MYELOGENOUS LEUKEMIA (CML) / CHRONIC
RELATIONSHIP OF LEUKEMIAS AND_
GRANULOMATOUS LEUKEMIA (CGL) LYMPHOMAS________________________
● Absence of Philadelphia chromosome correlates to rapid LEUKEMIA TYPE SOLID TUMOR COUNTERPART
progression of disease = poor prognosis Stem cell leukemia Lymphoma, undifferentiated
● Philadelphia chromosome is a result of T (9, 22) or BCR/ABL gene Acute lymphocytic leukemia Lymphoma, poorly differentiated,
o T stands for translocation lymphocytic
● LAP score is decreased Chronic lymphocytic leukemia Lymphoma, well differentiated,
● The only CHROMIC MYELOPROLIFERATIVE DISEASE lymphocytic
NEGATIVE FOR JAK2 V617F gene Monocytic leukemia Reticulum cell sarcoma
o JAK2 V617F = Janus kinase Acute myelogenous leukemia Chloroma
Plasma cell leukemia Myeloma
ESSENTIAL THROMBOCYTHEMIA
● Platelets can reach up to >1000 x 109/L AUTOMATION
● JAK2 V617F gene positive
POLYCYTHEMIA VERA METHODS___________________________

● JAK2 V617F gene positive
ELECTRICAL IMPEDANCE
● PCV = pancytosis and low EPO (increased viscosity)
● Principle:
CHRONIC IDIOPATHIC MYELOFIBROSIS o Cells pass through an aperture with an electrical current flowing
through simultaneously
o Cells do not conduct current but rather they change electrical
CHRONIC LYMPHOCYTIC LEUKEMIA
resistance, which is then counted as voltage pulses
● Increased presence of smudge cells in the PBS ▪ Number of pulses generated is proportional to cell count
● JAK2 V617F gene positive ▪ Amplitude of the pulse generated is proportional to cell
size
HAIRY CELL LEUKEMIA
LIGHT SCATTERING OPTICAL METHOD
● ACP Isoenzyme S
● TRAP positive ● Uses flow cytometer with laser to measure light scattering properties
● B – cell malignancy of cells
o Forward angle light scatter measures (0 angle): cell size
PROLYMPHOCYTIC LEUKEMIA o Orthogonal / slide angle light scatter: internal complexity or
cellcell granularity
o Differential scatter: A combination of forward low-angle light
OTHER LYMPHOID MALIGNANCIES__ scatter (2-3 degrees) and forward high-angle scatter (5 – 15
degrees) which correlates to cell volume and refractive index or
MULTIPLE MYELOMA with high internal complexity
o Number of pulses generated is proportional to the cell count
● Cancerous plasma cell
● Most common abnormally increased Ig is IgG
● Presence of BJP in the urine and precipitates at 40 – 60 degrees HISTOGRAMS_______________________
Celsius and dissolves at 100 degrees Celsius ● A histogram utilizes impedance technology and it is a representation
of cell number versus one measured property, usually cell size
WALDENSTROM’S MACROGLOBULINEMIA ● WBC Histogram
o 35 – 450 fL is the reference range for WBCs
● The increased Ig is IgM ▪ 1st peak – Lymphocytes / smallest, measures 35 – 90 fL
● Lymphoplasmacytic infiltration of the B.M. ▪ 2nd peak – Monocytes / medium, measures 90 – 160 fL
▪ 3rd peak – Granulocytes / largest, measures 160 – 450 fL
HODGKIN’S LYMPHOMA
o Abnormal WBC histograms
● The pathognomonic cell is Reed – Sternberg cells ▪ Population before 35fL may indicate nucleated RBCs,
giant or clumped platelets
▪ Peak overlap at 90 fL may indicate reactive lymphocytes o Two peaks indicate a dimorphic erythrocyte population
or blast cells o Increased curve width will correlate with an increased RDW
▪ Peak overlap at 160 fL may indicate increase in bands, (anisocytosis)
immature neutrophils, eosinophils, or basophils o Shift to the right indicates an increased MCV (macrocytic)
▪ Population after 450 fL may indicate a high granulocyte o Shift to the left indicates a decreased MCV (microcytic)
count
PLATELET HISTOGRAM
RBC HISTOGRAM
● 2 – 20 fL is the reference range for platelets
● 36 fL and above - is the reference size range for RBCs ● Lower region interference (<2 fL) indicates electrical impedance;
● A normal RBC histogram will show a single peak between 70 and upper region indicates lower microcytic RBCs or schistocytes, giant
110 fL that will correlate with MCV or clumped platelets
● Abnormal RBC histogram
CONDITION PARAMETERS RATIONALE INDICATORS CORRECTIVE ACTION
AFFECTED
Cold Agglutinins Decreased RBC and HCT, RBC agglutination DUAL RBC Warm sample at 37oC
- Counted as 1 big RBC Increased MCV and POPULATION OR
MCHC, GRAINY SHIFT TO RIGHT ON
APPEARANCE RBC HISTOGRAM
Lipemia, icterus, chylomicrons Increased Hb, MCH and Increased turbidity affects HB X 3 IS NOT EQUAL Plasma replacement
MCHC spectrophotometric reading = TO HCT
increased absorbance
Hemolysis Decreased RBC and HCT RBCs LYSED NOT HB X 3 IS NOT EQUAL New sample
COUNTED TO HCT
Lysis – resistant RBC with Increased WBC and Hb RBCs with Hb S, C or F may fail INTERFERENCE AT Manual dilutions, allow
abnormal Hb to lyse an be counted as WBC NOISE – WBC incubation for lysis time
INTERFACE ON
HISTOGRAM
Microcytosis or schistocytes Decreased RBC, Size of RBC or RBC fragments LEFT SHIFT ON RBC Smear review
Increased Platelets lower than RBC threshold HISTOGRAM, MCV
FLAGGED IF BELOW
LIMITS
NUCLEATED RBCs, Increased WBC NUCLEATED RBCS OR NUCLEATED RBC COUNT NUCLEATED RBCS
MEGAKARYOCTE MICROMEGAKARYOBLASTS FLAG OR
FRAGMENTS, OR COUNTED AS WBC MICROMEGAKARYOBLASTS
MICROMEGAKARYOBLASTS PER 100 WBCs AND CORRECT
Platelet clumps Decreased Platelets, LARGE CLUMPS COUNTED PLATELET CLUMPS / N Redraw specimen in sodium
- Platelet satellitosis Increased WBC AS WBCs FLAG citrate, multiply by 1.1
WBC > 100,000/uL Increased Hb and RBC, INCREASED TURBIDITY ON HB X 3 IS NOT EQUAL Spun microhematocrit, manual Hb
If WBC is greater than 100 x incorrect Hct, Abnormal HB, WBCS COUNTED AS TO HCT (read supernatant), correct RBC
109/L, the Hb will be increased indices RBC COUNT count, recalculate indices
and inaccurate
Leukemia with chemotherapy Decreased WBC, FRAGILE WBCs COUNTED INCONSISTENT Smear review, phase platelet count
increased Platelets AS PLATELETS PLATELET COUNT
WITH PREVIOUS
RESULTS
Old specimen Increased MCV and MPV, RBCs swell and as sample ages, ABNORMAL Establish stability and sample
Decreased Platelet, platelets swell and degenerate, CLUSTERING ON WBC rejection criteria
Automated Differential WBCs affected by prolonged HISTOGRAM
may be incorrect exposure to EDTA
ERRORS FROM CELL COUNTING_____ ● 4. Agglutination will cause a false negative result (RBC, WBC, and
platelet)
● 5. Agglutinated RBCs and platelets may cause a falsely positive
INSTRUMENTAL ERRORS
WBC count
● 1. Aperture plugs (+) ● 6. Platelet satellitism will result in falsely low platelet counts
● 2. Extraneous electrical pulses (+) ● 7. Some abnormal RBCs tend to resist lysis, which may result in
● 3. Improper setting of aperture current (+/-) high WBC counts
● 4. Bubbles (+) o Example sickle cells, extremely hypochromic cells and target
● 5. Excessive lysing of RBCs (-) cells
ERRORS CAUSED BY NATURE OF THE SPECIMEN

● 1. Giant platelets may be counted as RBCs or WBCs
● 2. Fragments of WBC cytoplasm may be counted as platelet or
RBCs (chemotherapy)
● 3. Increased number of schistocytes may make accurate RBC and
platelet count impossible
AUTOMATION IN HEMOSTASIS: DETECTION OF FIBRIN CLOT FORMATION_____
VISUAL Tilt tube method
ELECTROMECHANICAL FIBROMETER – fibrin strand formation is detected using a wire loop or hook that is incorporated to a semi-
automated instrument
PHOTO-OPTICAL Fibrin clot detection depends in the light scattering associated with formation of insoluble fibrin polymers
SEMIAUTOMATED: Electra 750 and 750A, Fibrintimer series, FP910 Coagulation Analyzer
AUTOMATED: Ortho Koagulab 16S and 40A, Coag – A – Mate X2 and XC, MLA ELECTRA 700 and 800
PERCENTAGE GRADING FOR ANISOCYTOSIS /

AUTOMATION SUMMARY____________ POIKILOCYTOSIS
● 1. Computed values: MCH, MCHC, HCT
● 2. Derived values: MCV, RDW, MPV, PDW Normal 5%
● 3. Directly measured by electrical impedance: RBC ct., WBC ct., Slight 5 – 10%
PLT ct. 1+ 20 – 25%
o Electrical impedance 2+ 25 – 50%
▪ Hemoglobin: spectrophotmetric determination 3+ 50 – 75%
● 4. Flow cytometer: Laser (light source), Fluidics, Computer, 4+ ➢ 75%
Detection system
● 5. 3 part differential: Granulocytes, Lymphocytes, Monocytes
● 6. 5 part differential: Neutro, Lympho, Mono, Eo, Baso Reference:
EVALUATION OF PERIPHERAL BLOOD SMEAR Notes from Legend Review Center and John Alvin O. Reyes, RMT
Morphological WNL 1+ 2+ 3+ 4+ Disclaimer: All notes in this material are from the following reference
Characteristics above. No additional notes were included for the creation of this material.
Macrocytes 0-5 5-10 10-20 20-50 >50
Microcytes 0-5 5-10 10-20 20-50 >50
Hypochromia 0-2 3-10 10-50 50-75 >75
Poikilocytosis 0-5 5-10 10-20 20-50 >50
Burr cells 0-5 5-10 10-20 20-50 >50
Acanthocytes <1 2-5 5-10 10-20 >20
Schistocytes <1 2-5 5-10 10-20 >20
Dacryocytes 0-2 2-5 5-10 10-20 >20
Codocytes 0-2 2-10 10-20 20-50 >50
Spherocytes 0-2 2-10 10-20 20-50 >50
Ovalocytes 0-2 2-10 10-20 20-50 >50
Stomatocytes 0-2 2-10 10-20 20-50 >50
Sickle cells Abs Report as 1+ do not quantitate
Polychromatophilia
Adult <1 2-5 5-10 10-20 >20
Newborn 1-6 7-15 15-20 20-50 >50
Basophilic Stippling 0-1 1-5 5-10 10-20 >20
Howell – Jolly Abs 1-2 3-5 5-10 >10
Bodies
Siderocytes Abs 1-2 3-5 5-10 >10
(Pappenheimer
bodies)

Review Notes On Hematology

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HEMATOLOGY

Notes Compiled by: Renz Louie Galanto

SAFETY IN THE HEMATOLOGY

ANTISEPTICS ● 1. Blood gases

VEINS FOR ROUTINE VENIPUNCTURE

DIURNAL RHYTHM_________________ DIET_______________________________

ROUTINE HEMATOLOGY PROCEDURES

● Blood Oxygen Capacity (Gasometric/Van Slyke Method)

METHODS ● Whole blood is diluted with isotonic diluting fluid to facilitate

CORRECTED WBC COUNT

● Performed when 5 or more nucleated RBCs are present in the PBS

MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)

● An expression of the average concentration of hemoglobin in red

● “Bretcher – Cronkite Method”

SPECIAL HEMATOLOGY PROCEDURES

RETICULOCYTE COUNT____________ ● ARC = 2 x (2.20 x 1012) = 44 x 109/L

VARIATIONS SUGAR WATER SCREENING TEST

SODIUM METABISULFITE METHOD OSMOTIC FRAGILITY TEST____________

METHODS PRUSSIAN BLUE REACTION

● Fresh capillary blood is recommended NONSPECIFIC ESTERASE

STAGES OF HEMATOPOIESIS_________ MEDULLARY (MYELOID) PHASE

LIVER STEM CELL THEORY

CYTOKINES AND GROWTH FACTORS

ERYTHROPOIESIS___________________ RBC DEVELOPMENTAL STAGES

● No nucleus RBC MEMBRANE PROTEINS

ERYTHROCYTE METABOLISM MACROPHAGE – MEDIATED HEMOLYSIS (NORMAL

METHEMOGLOBIN REDUCTASE PATHWAY

LUEBERING – RAPAPORT SHUNT

● Has an excess surface-to-volume ratio which enables RBCs to

EXCESSIVE EXTRAVASCULAR PATHWAY

● Pathologic processes that produce changes in the exterior membrane

EXCESSIVE INTRAVASCULAR PATHWAY

● Traumatic physical lysis of RBCs causes by prosthetic heart valves,

SAMPLE TEST RESULT INTRAVASCULAR EXTRAVASCULAR

OXYGEN DISSOCIATION CURVE ● 10 – 15 um

NEUTROPHIL MATURATION ● Secondary granules stain heavily with eosin

PROMYELOCYTE BASOPHIL MATURATION

MACROPHAGE – TISSUE MONOCYTE MEGAKARYOCYTOPOIESIS

WBC MATURATION AND LIFESPAN

MK-I (MEGAKARYOBLAST) MK-II (PROMEGAKARYOCYTE) MK-III (MEGAKARYOCYTE)

PLATELET STRUCTURE MEMBRANOUS SYSTEM

● Dense tubular system – arachidonic acid metabolism; activation

● Platelet roll and cling to nonplatelet surfaces

PERIPHERAL ZONE AGGREGATION

● Alpha and dense granules

• Hemostasis • Special Handling and Processing

REVIEW NOTES ON HEMATOLOGY 2

MECHANISM OF COAGULATION AND

EXTRINSIC PATHWAY CONTACT PROTHROMBIN FIBRINOGEN

COMMON COAGULATION PATHWAY

THROMBIN FEEDBACK MECHANISM

● Plasminogen activators are present in organ tissues and endothelial

● Urokinase, tears, saliva, semen, milk GENERAL CONSIDERATIONS_________

● Therapeutic destruction of thrombin (urokinase, streptokinase, CAUSES OF ACTIVATION

NATURALLY OCCURING INHIBITORS OF FIBRINOLYSIS AND EVACUATED TUBES/SYRINGES

● NEVER USE FOR COAGULATION TESTS (ACTIVATED) PARTIAL THROMBOPLASTIN TIME

● Whole blood is dispensed into three 13 x 100 mm or 12 x 75 mm OTHER COAGULATION TEST_________

REPTILASE TIME MODIFIED DUKE METHOD

SUBSTITUTION STUDIES CAPILLARY RESISTANCE____________

DIURNAL RHYTHM_ DIET_______________

Hb SC DISEASE______________ ECHINOCYTES / BURR CELLS

TEARDROPS / DACRYOCYTES BASOPHILIC STIPPLING_____