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COMPREHENSIVE

LECTURE
IN
HEMATOLOGY I

Prepared by: GISELLE T. ALBOTRA, RMT


An Overview of Clinical
Laboratory Hematology
 Average human person possesses 5-6 L of blood
 Three categories: red blood cells (RBCs), or erythrocytes; white blood cells (WBCs), or
leukocytes; and platelets (PLTs), or thrombocytes.

Loss of WB
HYPOVOLEMIA Loss of plasma
Loss of body water

HYPERVOLEMIA Blood transfusion


Intravenous injection
Characteristics of Blood

Fluid (in vivo)


Red
Slightly alkaline
Average specific gravity of 1.055
Thick and viscous
Makes up 75 to 85 ml per kg body weight
There are about 200g of solids per 100ml of blood
SAFETY
 Universal Precautions- old term for standard precautions

HANDWASHING STEPS
1. Wet hands and wrist with running water
2. Apply germicidal soap and rub hands for 15 seconds
3. Rinse hands thoroughly under running water in downward flow
4. Dry hands with paper towel, use towel to turn off faucet

 Food and drink, including oral medications and tolerance testing beverages, must
not be kept in the same refrigerator as laboratory specimens or reagents or where
potentially infectious materials are stored or tested
 Biohazard symbol is Red/Orange/Fluorescent Orange
 Gloves should not be worn when clean devices are used
 An appropriate disinfectant solution is household bleach, used in 1:10 (10%)
dilution
 Lab employees should receive the HBV vaccination series at no cost before or
within 10 days after beginning work in the lab.
 Most important practice in preventing the spread of disease is Handwashing
SPECIMEN COLLECTION
 Patient identification (most critical step)
 The tourniquet should be applied 3-4 in. (7.5-10 cm) above the site and left on for no
longer than 1 min.
 For obese patients: BP cuff should not be inflated higher than 40mmHg/60mmHg.
 Adult venipuncture: 21G, 1in.
 Most common skin antiseptic: 70% isopropyl alcohol
 Most common complication: ecchymosis
SKIN PUNCTURE
Depth:
Infants and small children: less than 2.0 mm
Adults: 2.0-2.5 mm
Physiological factors that affect results

FACTORS EFFECTS
Increase:
Stress WBCs
Fibrinogen group
Increase:
Diet Glucose
Lipids
Increase:
Smoking WBCs
Cortisol
Hemoglobin
FACTORS EFFECTS

5- Increase:
Posture Lipids Enzymes Proteins

Afternoon:
Diurnal rhythm Decrease Increase
Cortisol Iron
ACTH Eosinophil

Increase:
Exercise Creatinine Protein CK AST LD
Platelets WBCs
ORDER OF DRAW
VACUTAINER SYSTEM SKIN PUNCTURE

Blood culture (yellow) Tube for blood gas

Citrate (blue) Slides

Serum tube (red) EDTA microcollection tube

Heparin (green) Other microcollection tubes with


anticoagulant
EDTA (purple) Serum microcollection tubes

Oxalate (gray)
ISOLATION TECHNIQUES
PURPOSE PPE
STRICT ISOLATION Cases of contagious diseases Gown, mask, and gloves
that can be transmitted by direct
contact via air.
ENTERIC ISOLATION Used when coming in contact Gown and gloves
with patients who have
dysentery and other disorders
that spread through direct
RESPIRATORY ISOLATION The patient has infections that Mask and gloves
are transmitted via droplets or
by an airborne route.
WOUND AND SKIN Used in cases of skin infection Gown and gloves
ISOLATION that maybe transmitted directly
or indirectly.
PROTECTIVE ISOLATION Protect the patient from Gown, mask , gloves and shoe
infection. coverings
BLOOD FILM PREPARATION
I. two-glass slide method (manual wedge technique)- most frequently used
Correct angle: 30-45 degrees
Important considerations:
a. Size of the drop of blood
b. Speed of the spreader
c. Hematocrit of the patient
II. Coverslip method (rarely used)
a. Beacom’s method
b. Ehrlich’s method
TECHNIQUES IN BLOOD FILM STAINING
a. fixative: methanol
b. Stain: wright’s/ wright-giemsa
c. Buffer: sodium phosphate
Evaluation of Peripheral Blood Smear
I. MACROSCOPIC

UNUSUAL FINDINGS PROBABLE REASON

Blood film is bluer than blue Increased blood proteins

Grainy appearance RBC agglutination

Holes all over the film Increased lipid

Blue specks Markedly increased WBCs and PLT


counts
PROBLEMS ENCOUNTERED IN
BLOOD SMEAR STAINING
PROBLEMS USUAL CAUSES
RBCs: gray or blue Stain or buffer is too basic
WBCs too dark Inadequate rinsing
Eosinophils granules: gray Heparinized blood was used

PROBLEMS USUAL CAUSES


RBCs: too pale or red Stain or buffer is too acidic
WBCs barely visible Under buffering
Over-rinsing
II. MICROSCOPIC

OBJECTIVE COMMENTS
10X Assess the overall film quality, color and distribution of cells
Locate rare abnormal WBCs
Detect “snowplow” effect
Detect fibrin strands
Recognize rouleaux formation
Assess the area suitable for exam
40X high-dry or 50X oil immersion objective Areas to choose:
Red cells have central pallor and are near each other but not
Multiplication factor: overlapping
40X: 2000 Cells are appropriately stained
50X: 3000 Avoid the feathered edge
Avoid thick part
100 X Normal RBC count: 200-250 per OIF
(Ave: 2000)
Examine nuclear details of WBCs
Significant anemia or erythrocytosis: WBC differential and plt count estimation
Ave. no. of PLT per field x total RBC count Ave. no. of PLTs/OIF X 20,000 = est. plt. Ct. per ul
200 RBCs per field
HEMATOPOIESIS
 CD34: marker of hematopoietic stem cells
 Three phases:
I. MESOBLASTIC/MEGALOBLASTIC (19th day)
chief site: yolk sac
first blood cell: Primitive erythroblast
II. HEPATIC (5th to 7th week)
chief site: fetal liver
with contributions: spleen, thymus, lymph nodes
III. MEDULLARY/MYELOID
chief site: bone marrow
begins in fifth month of gestation and continues throughout life
 Main sites of adult hematopoiesis:

R
S (3)
V
P (2)
 Bone marrow collection sites:
Adults: posterior superior iliac crest & anterior superior iliac crest
Children less than 2 years: anterior medial surface of the tibia
ERYTHROPOIESIS
RUBRIBLAST NORMOBLAST ERYTHROBLAST

Rubriblast Pronormoblast Proerythroblast

Prorubricyte Basophilic normoblast Basophilic erythroblast

Rubricyte Polychromatic normoblast Polychromatic erythroblast

Metarubricyte Orthochromic normoblast Orthochromic erythroblast

Reticulocyte Reticulocyte Reticulocyte

Mature erythrocyte Mature erythrocyte Mature erythrocyte


RUBRIBLAST/PRONORMOBLAST/PROERYTHROBLAST

 Size: 12-20 um
 Contains 1 or 2 nuclei
 Cytoplasm is dark blue
 Globin production begins
 N:C ratio=8:1
 Earliest recognizable erythroid precursor
using a light microscope
PRORUBRICYTE / BASOPHILIC NORMOBLAST /
BASOPHILIC ERYTHROBLAST

 Size: 10-15 um
 0-1 nucleoli
 N:C ratio=6:1
 Last stage with nucleolus
 First stage of hemoglobin synthesis
RUBRICYTE / POLYCHROMATIC NORMOBLAST /
POLYCHROMATIC ERYTHROBLAST
 Size: 10-12 um
 No nucleolus
 First stage in which cytoplasm becomes pink
 Last stage capable of mitosis
 N:C ratio= 4:1
 May be confused with a lymphocyte
 Lymphocyte
Nucleus: Crush velvet
cytoplasm: sky blue/ robin egg blue color
 Rubricyte
Nucleus: checkerboard
Cytoplasm: muddy/gray
METARUBRICYTE / ORTHOCHROMIC NORMOBLAST /
ORTHOCHROMIC ERYTHROBLAST

 Size: 8-10 um
 Last stage with a nucleus
 N:C ratio= 1:2
 Cytoplasm is salmon-pink
 Nucleus is extruded
 Pyrenocyte (enveloped extruded nucleus)
is engulfed by BM macrophages
RETICULOCYTE

 Young RBCs containing residual RNA


 Last stage of hemoglobin production
 Spends 2-3 days in BM and 1 Day in
peripheral blood before developing into a
mature RBC
 Shift cells ( polychromatophilic macrocyte)
Seen in cases of increased RBC
production
 Stress reticulocytes (macroreticulocytes)
Seen in more severe conditions
MATURE ERYTHROCYTE

 Biconcave disk
 Thickness: 1.5-2.5 um
 Ave. life span: 120 days
 Central pallor is 1/3 of the diameter of the
cell
 No protein or hemoglobin synthesis
GRANULOCYTIC SERIES

AS GRANULOCYTES MATURE:
 Nuclear chromatin becomes more condensed
 Nucleoli disappear
 Abundant basophilic cytoplasm with non-specific granulation progresses to more scant
cytoplasm containing specific granules
 The nucleus indents and becomes segmented
 Overall cell size decreases
MYELOBLAST
 EARLIEST recognizable granulocytic precursor using the light microscope
 SIZE: 14-20 um
 THREE TYPES:
I. TYPE I MYELOBLASTS:
• Nucleus occupies most of the cell, with very little cytoplasm
• Slightly basophilic cytoplasm, fine nuclear chromatin, two-four visible nucleoli,
no visible granules
II. TYPE II MYELOBLASTS
• Presence of dispersed primary non-specific granules in the cytoplasm
• No. of granules does not exceed 20 per cell
III. TYPE III MYELOBLASTS
• Have darker chromatin and more purple cytoplasm
• Contain more than 20 granules that do not obscure the nucleus
• Rare in normal BM
• Can be seen in certain types of acute myeloid leukemias
PROMYELOCYTE

 SIZE: 16-25 um
 1-5% of the nucleated cells in BM
 Nucleus round to oval, often eccentric
 Cytoplasm is evenly basophilic and full of primary (azurophilic)
granules
 One to three nucleoli can be seen but may be obscured by the
granules
MYELOCYTE

 SIZE: 15-18 um
 6-17% of the nucleated cells in BM
 LAST STAGE CAPABLE OF MITOSIS
 STAGE OF SYNTHESIS OF SECONDARY GRANULES (SPECIFIC
GRANULES)
 TWO TYPES:
I. EARLY MYELOCYTES
• May look very similar to the promyelocytes
• DAWN OF NEUTROPHILIA
II. LATE MYELOCYTES
• Smaller than promyelocytes
• Nucleoli are difficult to see by light microscopy
METAMYELOCYTE

• SIZE: 14-16 um
• 3-20% of the nucleated cells in the BM
• Nucleoli are absent
• Synthesis of tertiary granules may begin at this stage
• JUVENILE CELL
• FIRST STAGE OF NUCLEAR IDENTATION
BAND/STAB/STAFF
CELLS
• SIZE: 9-15 um
• 9-32% of the nucleated cells in the BM
• YOUNGEST GRANULOCYTIC PRECURSOR TO
NORMALLY APPEAR ON PBS
• PRODUCTION OF SECRETORY GRANULES
• Nucleus: elongated, curved, or sausage-shaped with rounded
ends
• Included within the neutrophil count
SEGMENTED NEUTROPHIL
SIZE: 9-15 UM
• Most common WBC
• Commonly the first phagocytes to reach the infected areas
Nucleus: 3-4 segments connected by threadlike filaments
Cytoplasm: pink to tan with violet or lilac granules
• Three pools:
Stem cell pool: HSCs
Proliferation/ mitotic pool: CMP, GMP, MYELOBLAST,
PROMYELOCYTE, MYELOCYTE
Maturation/storage pool: metamyelocytes, bands, and mature
neutrophils
Half-life: 7 hrs
EOSINOPHIL
Size: 9-15 um
Half-life: 18 hrs
Survival time in human tissues ranges from 2-5 days
An elevated eosinophil count is called eosinophilia and often
signals a response to allergy or parasitic infection.
Nucleus:
Dark purple, usually has two lobes
Cytoplasm:
Filled with large, spherical granules of uniform size that stain
bright orange
BASOPHIL
• Size: 10-16 um
• Least common WBC
• Are cells with dark purple, irregular cytoplasmic granules that
obscure the nucleus. The basophil granules contain
histamines and various other proteins.
• Life span: 60 hrs
• Nucleus: difficult to observe because of overlying granules
• Cytoplasm: densely stained, dark to violet to purple-black
granules
MONOCYTE
Size: 15-20 um
Nucleus:
Round, horse-shoe shaped or lobulated, usually with some
degree of folding
Cytoplasm:
Abundant, blue-gray, containing fine, indistinct granules called
azure dust (ground-glass appearance)
Small pseudopods or blebs may be observed
• Best identified by its strong positive reaction with
NONSPECIFIC ESTERASE stain
 An immature macrophage passing through the blood from its point of origin,
usually the bone marrow, to a targeted tissue location.
 Macrophages - most abundant cell type in the body
 On a Wright-stained blood film, monocytes have a slightly larger diameter than
other WBCs, blue-gray cytoplasm with fine azure granules, and a nucleus that is
usually indented or folded.

LIVER
LUNGS
BRAIN
SPLEEN
SKIN
BONE
SYNOVIAL
KIDNEYS
LYMPH NODES
PLACENTA
LYMPHOCYTE
Sizes: 7-10 um, 10-12 um, 11-25 um
Nucleus: deep purple, round, oval, or indented
Cytoplasm: sky-blue or “robin-egg” blue
VARIANT LYMPHOCYTE:
TYPE I (Turk’s irritation cell/ Plasmacytoid lymphocyte)
Seen in: GERMAN MEASLES
TYPE II (Infectious mononucleosis cell)
“flared skirt” or “fried egg” appearance
TYPE III
Nucleus: finely reticulated nuclear chromatin pattern
RBC COUNT/WBC
COUNT

 3 mm by 3mm (divided into 9 sq. mm) Improved Neubauer Counting


Chamber
 The 4 corner (large) squares – used for manual
WBC count
 The middle (central) square – used for manual
RBC count
0.1 mm – space between the thick coverglass
and the ruled platform

RBC WBC

Markings 0.5, 1, 101 0.5, 1, 11

Color of bead Red White

Volume 100 10
RBC DILUTING FLUIDS WBC DILUTING FLUID
1. Hayem’s 1. 2% glacial acetic acid
2. Gower’s 2. 1% hydrochloric acid
3. Toisson’s 3. Turk’s solution
4. Bethel’s
5. Formol-citrate (Dacie’s fluid)- BEST
6. NSS
7. 3.8% sodium citrate
STEPS IN MICROSCOPIC HEMOCYTOMETRY
1. Suck blood to 0.5 mark of the pipet
2. Suck diluting fluid to 101 and 11
3. Shake pipet to mix
4. Discard few drops
5. Charge
6. Count RBCs in 5 intermediate squares in the central large square
7. Computation

RBC count = RBC counted x 10 x 200 x 5


Reference ranges:
Children and adult female: 4.00-5.40 x 10 12/L
Adult male: 4.60-6.00 x 10 12/L

WBC count = WBC counted x 10 x 20


4
Reference ranges:
Adult: 4.5-11.0 x 10 9 /L or 4,500-11,000/mm3
Newborns: 13.5-38.0 x 10 9 /L or 13,500-38,000/mm3
SAMPLE PROBLEMS
1. A manual leukocyte count was performed on an EDTA-anticoagulated specimen.
The specimen was diluted 1:20, and a total of 150 leukocytes were counted in the
four corner squares of the hemocytometer. What is the leukocyte count?

2. When a 1:20 dilution is used, the four large squares on one side of the chamber yield
counts of 23, 26, 25, and 27. The four large squares on the other side of the chamber
yield counts of 28, 24, 29 and 27. What is the leukocyte count?

3. When a 1:20 dilution is used, the four large squares on one side of the chamber yield
counts of 35, 14, 28, and 27. The four large squares on the other side of the chamber
yield counts of 18, 24, 19, and 21. What is the leukocyte count?

4. WBC count: 15,000/mm3


12 NRBCs were seen (in 100 WBCs)
 CORRECTION OF WBC COUNT
• Performed if there are > 5 NRBCs seen in 100 WBCs
FORMULA:

Corrected WBC count = Uncorrected WBC x 100


(100+NRBCs)
WBC DIFFERENTIAL COUNT:
A. 100-cell differential
routinely performed
B. 200- cell differential
WBC count is > 40𝑥 10 9/𝐿
C. 300 or 400-cell differential
WBC count is > 100 𝑥 10 9/𝐿
D. 50-cell differential
WBC count is < 1.0 𝑥 10 9/𝐿
RELATIVE COUNT
FORMULA:
No. of specific WBC x 100 = Relative count (%)
100 WBCs counted

REFERENCE RANGES:
Neutrophil= 51-67%
Lymphocyte= 25-33%
Monocyte= 3-6%
Eosinophil= 1-4%
Basophil= 0-1%
ABSOLUTE COUNT
FORMULA:
Absolute count= Relative count (%) x WBC count

REFERENCE RANGES:
Neutrophil= 1,600-7260/mm3
Lymphocyte= 960-4,400/mm3
Monocyte= 180-880/mm3
Eosinophil= 45-440/mm3
Basophil= 45-110/mm3
RETICULOCYTE COUNT
 Assessment of RBC production by the BM
 adults: 0.5-1.5%
 Newborns: 1.8-5.8%
 Reticulocytosis/polychromatophilia – considered as first sign of accelerated erythropoiesis
 Reticulocytopenia- observed in aplastic anemia

MATERIALS: MILLER DISK


1. New Methylene Blue (preferred stain)
Components: 1. sodium oxalate A
2. sodium chloride
2. Brilliant Cresyl Blue B
Components: 1. sodium citrate
2. sodium chloride
METHODS OF COUNTING:
A. ROUTINE LIGHT MICROSCOPE
1. Combine equal amounts of blood and supravital stain and allow to incubate at
room temperature for ≥10 minutes.
2. Remix the preparation
3. Prepare 2 blood smears
4. Count 1000 RBCs under OIO
5. calculate:
Retic (%) = No. of retics observed x 100
1,000 RBCs observed
B. CALIBRATED MILLER DISK
1. Count a minimum of 112 RBCs in small square (B)
2. Retic (%) = total retics in square A x 100
(total RBCs in Sq. B x 9)
 ABSOLUTE RETICULOCYTE COUNT
ARC= reticulocytes (%) x RBC count (x10 12/L) x 1000
100
 CORRECTED RETICULOCYTE COUNT
CRC= Retics (%) x Hct in L/L
0.45 L/L
 RETICULOCYTE PRODUCTION INDEX
RPI= Corrected Reticulocyte Count
Maturation Time in peripheral blood
HEMATOCRIT (L/L) MATURATION TIME (DAYS)
40-45 1.0
35-39 1.5
25-34 2.0
15-24 2.5
<15 3
RED BLOOD CELL ANOMALIES
Anisocytosis Variation in size of RBC
Poikilocytes Variation in shapes of RBCs
Anisochromia Variation in the normal coloration
Spherocyte Small, round dense RBC with no central pallor
Elliptocyte/Ovalocyte Cigar or egg shaped RBC
Stomatocyte RBC with slit-like area of central pallor; found
in SHARE
Sickle cell/ Drepanocyte Thin, dense, elongated RBC pointed at each
end
Hb C crystal Hexagonal crystal of dense Hgb
Hb SC crystal Fingerlike or quartz-like crystal
Target cells RBC with hemoglobin concentrated in
(codocytes) center and periphery
(leptocytes)
Schistocyte (Schizocyte) Fragmented RBC due to rupture
Folded cell Membrane folded over
Acanthocyte (spur cell) RBC with irregularly spaced projections
Found in severe live disease,
abetalipoproteinemia and Mcleod syndrome
Burr cell (Echinocyte) (crenated RBCs) RBC with blunted short projections that are
evenly spaced
Found in uremia and PK deficiency
Teardrop cell (dacryocyte) Myelofibrosis, myelophthisic anemia,
pernicious anemia
Bite cell (degmacyte) Demonstrate a semicircular defect in their
edge
G-6-PD deficiency
Semilunar body Pale-pink staining ghost of the red cell
Seen in malaria
INCLUSIONS
Lead poisoning
Thalassemias
Basophilic stippling Precipitated RNA Hemoglobinopathies
Megaloblastic anemia
Howell-jolly bodies DNA Post splenectomy
Heinz bodies Denaturated Hgb G6PD deficiency
Unstable hemoglobins
Pappenheimer bodies Iron Sideroblastic anemia
Cabot ring Mitotic spindle Pernicious anemia
Hb H B-globin chains Hb H disease
GRADING OF HYPOCHROMIA
1+ ½
2+ 2/3
3+ ¾
4+ Thin rim of hemoglobin

GRADING OF POLYCHROMASIA
GRADE PERCENTAGE OF POLYCHROMATOPHILIC RBCs
SLIGHT 1%
1+ 3%
2+ 5%
3+ 10%
4+ >11%
RED CELL MORPHOLOGY GRADING
CHART
“SSAD PH”
1+ = aggregates
Polychromatophilia 1+ = 1-5 per field
of 3 to 4 RBCs
Helmet cells 2+ = 6-10 per field
2+ = aggregates
Dacryocytes 3+ = greater than 10
ROULEAUX of 5 to 10 RBCs
Acanthocytes per field
3+ = many
Schistocytes
aggregates with
Spherocytes
only a few free
RBCs
“P-BEBOTS”
Poikilocytes 1+= 3 to 10 per field “ HOW SICK BA SI
Ovalocytes 2+= 11 to 20 per field PAPS”
Elliptocytes 3+= greater than 20 Sickle cells POSITIVE ONLY
Burr cells per field Basophilic stippling
Bizarre-shaped RBC Pappenheimer bodies
Target cells Howell-jolly bodies
Stomatocytes
WBC ANOMALIES
ABNORMAL WBCs MORPHOLOGIC DEFECT REMARKS
Smudge cells Nuclear remnants of lymphocytes May be associated with CLL
Similar to thumbprint
Basket cells Nuclear remnants of granulocytic May be seen in leukemias
cells
Hand-mirror lymphocytes Lymphocyte with hand-mirror May be seen in certain types of
appearance ALL and AML
May also be associated with
infectious mononucleosis

Leukocyte Adhesion Deficiency Lack of cell surface adhesion Serious condition with recurrent
(LAD) proteins infections
Myeloperoxidase Deficiency Low or absent myeloperoxidase Commonly benign
(Alius-Grignashi anomaly) enzyme
Chronic Granulomatous Disease Cells are unable to kill Frequent infections esp. in children
microorganisms
ABNORMAL WBCs MORPHOLOGIC DEFECT REMARKS
Niemann-Pick Disease Deficiency of sphingomyelinase More commonly seen in Ashkenazi
Presence of foam cells/ pick cells jews
Spleen and liver are greatly enlarged

Gaucher Disease Defect in the catabolic enzyme β- “crumpled tissue” or “onion-skin” like
glucocerebrosidase appearance

Hypersegmented neutrophils Nucleus has 5-10 lobes Pernicious anemia, folic acid def.,
Undritz anomaly, and myelokathexis

Pelger-Huet Anomaly Failure of the neutrophil nucleus to Most common genetic disorder of
segment WBCs
“Pince-nez” or “spectacle” form of
nucleus

Lupus Erythematosus (LE) Cell Neutrophil that has ingested another Found in SLE
neutrophil

Reider Cell Similar to normal lymphocyte but Found in CLL


nucleus is notched, lobulated, and Can be artificially formed through
cloverleaf-like blood film prep.
ABNORMAL WBCs MORPHOLOGIC DEFECT REMARKS
Flame Cell Abnormal plasma cell with intensely Found in IgA Myeloma
eosinophilic or “flaming” cytoplasm

Grape cell Abnormal plasma cell with cytoplasm Found in Multiple Myeloma
that is completely filled with Russel
bodies
Alder-Reilly Anomaly Characterized by dense azurophilic Found in mucopolysaccharidoses
granulation in all types of leukocytes

Chediak-Higashi Syndrome Presence of large, abnormal Partial albinism


cytoplasmic granules in phagocytes
and occasionally in lymphocytes

Hairy Cells Small lymphocytes with little Found in Hairy Cell Leukemia
cytoplasmic projections
TRAP (+)
Tart Cells A monocyte that has ingested a Seen in drug sensitivity
lymphocyte
Toxic granulations Altered primary granules Seen in severe infections and
chemical poisoning
ABNORMAL WBCs MORPHOLOGIC DEFECT REMARKS
Auer Rods Linear projections of primary granules Seen in certain types of Acute
Myelogenous Leukemia (AML)
Reed-Sternberg Cells Large lymphoid cell which may Definitive histologic characteristic of
demonstrate two nuclei and an Hodgkin’s Disease
abundant cytoplasm
“Owl’s eye” appearance
Lazy Leukocyte Syndrome Poor neutrophil response to Characterized by neutropenia
chemotactic agents
Job Syndrome Neutrophils have poor directional Recurrent severe bacterial infections,
motility skeletal abnormalities, and elevated
levels of IgE
Jordan’s Anomaly Presence of fat-containing vacuoles in Seen in muscular dystrophy and
granulocytes and monocytes ichthyosis

Dohle Bodies Round or oval blue-staining Found in severe burns, infections and
cytoplasmic inclusions in neutrophils pregnancy

May-Hegglin Anomaly Gray-blue spindle-shaped inclusions Leukopenia, variable


in the cytoplasm of granulocytes and thrombocytopenia and giant platelets
monocytes
INCLUSIONS SIZE SHAPE PAS rxn CONTENT

Dohle Smaller Round + Ribosomal


Bodies RNA

MHA Larger Spindle- - Messenger


shape RNA
Hemoglobin, Hematocrit, and Red
Blood Cell Indices
 Hemoglobin measurement relies on a weak solution of potassium cyanide
and potassium ferricyanide, called Drabkin reagent.
 Aliquot of WB + Drabkin reagent= stable cyanmethemoglobin
(hemiglobincyanide)
 Absorbance or color intensity of the solution is measured in a
spectrophotometer at 540 nm wavelength.
 Hematocrit is the ratio of the volume of packed RBCs to the volume of
whole blood also called packed cell volume (PCV), the packed cells
referring to RBCs.
 Buffy coat contains WBCs and platelets and is excluded from hematocrit
exam.
 RBC count, HGB, and HCT—to compute the RBC indices mean cell
volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin
concentration (MCHC)
 The MCV, although a volume measurement recorded in femtoliters (fL),
reflects RBC diameter on a Wright-stained blood film.
 The MCHC, expressed in g/dL, reflects RBC staining intensity and amount
of central pallor.
 The MCH in picograms (pg) expresses the mass of hemoglobin and
parallels the MCHC.
 A fourth RBC index, RBC distribution width (RDW), expresses the degree
of variation in RBC volume.
HEMOGLOBIN
SOURCES OF ERROR (FALSELY ELEVATED) CORRECTION
High WBC count Centrifuge reagent-sample solution, then
High platelet count supernatant is measured

Lipemia Add 0.01 ml of the patient’s plasma to 5 ml of the


cyanmethemoglobin reagent and using this solution
as the reagent blank
Cells containing HB S and HB C Make a 1:2 dilution with distilled water (1 part diluted
sample plus 1 part water) and multiplying the results
from the std. curve by 2.
Abnormal globulins Add 0.1 gram of potassium carbonate to the
cyanmethemoglobin reagent.
HEMOGLOBIN ELECTROPHORESIS
 CELLULOSE ACETATE ( pH 8.4-8.6) CITRATE AGAR (pH 6.0-6.2)
 Fastest: Hb H -differentiates S from D and G, and
 Slowest: A2, C, E, D arab, C harlem hemoglobin C from hemoglobins
E, O arab, C harlem
A2 S F A
C D
E G
O arab F A S C
C harlem A2
(-) (+)

(-) (+)
FORMS OF HEMOGLOBIN
HEMOGLOBIN MOLECULAR PROPORTION IN PROPORTION IN
STRUCTURE NEWBORNS ADULTS
PORTLAND ζ2γ2 0 0
GOWER I ζ2ε2 0 0
GOWER II α2ε2 0 0
F a2γ2 80 <1
A1 a2β2 20 97
A2 a2δ2 <0.5 2.5

HEMOGLOBIN DERIVATIVES
TYPE COLOR
OXYHEMOGLOBIN BRIGHT RED
DEOXYGENATED HEMOGLOBIN DARK RED
CARBOXYHEMOGLOBIN CHERRY RED
METHEMOGLOBIN CHOCOLATE BROWN
SULFHEMOGLOBIN MAUVE LAVENDER
HEMATOCRIT DETERMINATION
MICRO-HEMATOCRIT TUBE: TWO TYPES:
APPROXIMATELY: 75 mm With red band (with anticoagulant)
Inner bore: 1.2 mm With blue band (no anticoagulant)
Can hold 0.05 mL of blood Reference ranges:
Plug: 4-6 mm
Conventional S.I.
Adult male 40-54% 0.40 to 0.54 L/L
Procedure:
Adult females 35-49% 0.35 to 0.49 L/L
1. Perform skin puncture
Newborn 53-65% 0.53 to 0.65 L/L
2. Wipe off the first drop of blood
3. Fill two heparinized capillary tubes two-thirds with blood
4. Seal the dry end with the sealing clay
5. Place the tubes in the radial grooves of the microcentrifuge with their heads opposite from each other. Sealed end
should be away from the center of the centrifuge
6. Spin for 5 mins. at 10,000 RPM
7. Read the hematocrit
FALSELY INCREASED FALSELY DECREASED
Dehydration Hemolysis
Insufficient centrifugation Increased anticoagulant concentration
Hemoconcentration Improper sealing of the tube
Buffy coat inclusion Introduction of excess tissue fluid

RULE OF THREE
 3 X RBC = Hb
 3 X Hb = Hct (+ 3%)

ERYTHROCYTE INDICES:

Mean Cell Volume (MCV) Mean Cell Hemoglobin (MCH) Mean Cell Hemoglobin Concentration
(MCHC)
MCV = Hct/RBC ct. x 10 MCH = Hb/RBC ct. x 10 MCHC = Hb/Hct x 100

R.R. = 80 to 100 fL R. R. = 26-32 pg 31-37 g/dL or %


ERYTHROCYTE SEDIMENTATION RATE
METHODS TIME ANTICOAGULANT
WESTERGREN After 1 Hr. and after 2 hrs. 3.8% sodium citrate
MODIFIED WESTERGREN After 1 Hr. 0.85% NaCl or 3.8% sodium
citrate

WINTROBE After 1 Hr. Double oxalate/ EDTA

MODIFIED WESTERGREN WINTROBE


ADULT MALES 0 To 10 mm/hr 0 to 9 mm/hr
ADULT FEMALES 0 to 15 mm/hr 0 to 20 mm/hr

First 10 minutes Next 40 minutes Last 10 mins


LAG PHASE DACANTATION PHASE FINAL SETTLING PHASE
Factors Affecting RBC Sedimentation
FACTORS INCREASED DECREASED
PROTEINS AND LIPIDS ↑ cholesterol ↑ albumin
↑ fibrinogen ↑ glucose
↑ gamma globulins ↑ bile salts
↑ phospholipids
↓ albumin ↓ fibrinogen
↓ gamma globulins
RED BLOOD CELLS Anemia Polycythemia
Macrocytosis Anisocytosis
Poikilocytosis
thalassemia

WHITE BLOOD CELLS Leukemia Marked leukocytosis


TECHNIQUE Refrigerated sample not returned Clotted blood sample
to room temperature Delay in testing
High temperature Bubbles in ESR column
Tilted ESR tube Low temperature
Vibration Narrow ESR column diameter
OSMOTIC FRAGILITY TEST
PRINCIPLE: RBCs are diluted in 0 to 0.85% saline solutions and the amount of hemolysis at
each concentration is determined by measuring the absorbance of supernatant at 540nm.
Anticoagulant: HEPARIN
Normal values: initial hemolysis: 0.45%
complete hemolysis: 0.30%
INCREASED OFT: Hereditary Spherocytosis
DECREASED OFT: anemia with target cells

CONDITIONS INITIAL HEMOLYSIS COMPLETE HEMOLYSIS


NORMAL 0.45 0.30
HEREDITARY SPHEROCYTOSIS 0.65 0.45
THALASSEMIA 0.35 0.20
SICKLE CELL ANEMIA 0.35 0.20
ANEMIA
Defined as the decrease below normal of:
 RBC count
 Hemoglobin
 PCV
MECHANISMS OF ANEMIA
A. HEMORRHAGE
Gastrointestinal tract- common location for clinically significant bleeding
B. HEMOLYSIS
Shortened RBC survival time not explained by bleeding
C. DECREASED RBC PRODUCTION
CLASSIFICATION OF ANEMIAS
MORPHOLOGIC CLASSIFICATION
NORMOCYTIC, NORMOCHROMIC

NORMAL OR DECREASED RETIC. COUNT INCREASED RETICULOCYTE COUNT


APLASTIC ANEMIA PNH
ANEMIA OF RENAL DISEASE PCH
SICKLE CELL DISEASE
PORPHYRIAS
ENZYME DEFICIENCIES

MICROCYTIC, HYPOCHROMIC

ASSOCIATED CONDITIONS
Thalassemia
Anemia of chronic disease
Iron deficiency anemia
Lead poisoning
Sideroblastic anemia
MACROCYTIC, NORMOCHROMIC

MEGALOBLASTIC ANEMIA NONMEGALOBLASTIC


ANEMIA
CAUSES Acute Erythroleukemia Liver disease
Vit. B12 deficiency BM failure
Folic acid deficiency alcoholism
Pernicious anemia
HYPERSEGMENTED PRESENT ABSENT
NEUTROPHILS
SHAPE OF MACROCYTES OVAL ROUND
MEGALOBLASTS IN BM PRESENT ABSENT
PORPHYRIAS
 Greek word: “porphyria” = purple
 Impaired Heme production due to specific enzyme deficiency in biosynthetic pathway

I. ACQUIRED
 LEAD POISONING
inhibit some enzymes like pyrimidine 5’- nucleotidase and ferrochelatase
FINDINGS:
hypochromic RBCs with basophilic stippling
toxic granulation in neutrophils
elevated ZEP test
II. HEREDITARY

CONGENITAL ERYTHROPOIETIC X-LINKED


ERYTHROPOIETIC PROTOPORPHYRIA ERYTHROPOIETIC
PORPHYRIA PROTOPORPHYRIA
ENZYME AFFECTED Uroporphyrinogen III Ferrochelatase ALA-synthase 2
synthase deficiency deficiency
INHERITANCE Autosomal recessive Autosomal dominant X-linked dominant

CLINICAL FEATURES Photosensitivity, Photosensitivity, mild Photosensitivity, mild


hemolytic anemia anemia microcytic, hypochromic
anemia with reticulocyte
response
HEMOGLOBINOPATHIES
 QUALITATIVE globin synthesis defects
 Major groups:
ALPHA, BETA (most common form),GAMMA, DELTA

HEMOGLOBIN S (most common and most severe)


o Glutamic acid on the sixth position of the β chain is replaced by valine
o Degree of sickling depends on the concentration of hemoglobin S in the RBC:
Concentration= 80-100%
sickling of RBCs occurs readily at slightly decreased oxygen concentration
Concentration = 20-40%
oxygen conc. Must be much lower before sickling occurs
TESTS FOR HEMOGLOBIN S
SCREENING TESTS PRINCIPLE POSSIBLE RESULTS
SODIUM METABISULFITE METHOD o Mix whole blood with sodium Positive result- “holly-leaf” form of
metabisulfite, RBC
o Hb S causes formation of sickle Negative result- normal looking or
shaped RBCs slightly crenated RBCs
SODIUM DITHIONITE TUBE TEST o When RBCs are added to the Positive result- turbid solution
(SOLUBILITY TEST) working solution containing Negative result- clear solution
sodium dithionite and saponin,
the red cells lyse immediately
o Hb S in the reduced state,
forms liquid crystals and
produces turbid appearance
HEMOGLOBIN ELECTROPHORESIS o In alkaline buffer solution (pH
(CELLULOSE ACETATE) 8.4-8.6) hemoglobin is
negatively charged
• During electrophoresis, the Hb
molecules travel toward the
anode (+) because of their net
neg charge
CONFIRMATORY PRINCIPLE POSSIBLE RESULT
o Migration distances of the different
hemoglobins are based on the
HEMOGLOBIN electrophoretic charge of the molecules F A S C
ELECTROPHORESIS and their absorption to the agar A2
(CITRATE AGAR) compound

(-) (+)

HEMOGLOBIN C (2ND MOST COMMON)


 Glutamic acid on the 6th position of the beta chain is replaced by lysine

Two crystals:
I. HEMOGLOBIN SC crystal
• found protruding the RBC membrane
• Characterized by “Washington monument” appearance

II. HEMOGLOBIN CC crystal


• Found within RBC membrane
• “Bar of gold” appearance
THALASSEMIAS
 Reduction or total absence of synthesis of one or more of the globin

ALPHA-THALASSEMIA

CLINICAL SYNDROME NO. OF DELETED GENES REMARKS


SILENT CARRIER STATE One of four genes Asymptomatic
(α-thalassemia)
a-THALASSEMIA TRAIT/ a- Two of four genes Mild microcytic
THALASSEMIA MINOR hypochromic anemia
Hb H DISEASE Three of four genes Microcytic hypochromic
anemia
Hb BART HYDROPS FETALIS Four of four genes Hb Bart’s disease
SYNDROME Most severe form may
cause stillbirth/hydrops
fetalis
β-THALASSEMIA

CLINICAL SYNDROMES REMARKS


β-THALASSEMIA Asymptomatic; normal hematologic
(SILENT CARRIER STATE) parameters
β-THALASSEMIA TRAIT/ β-THALASSEMIA Asymtomatic; mild hemolytic anemia;
MINOR microcytic, hypochromic RBCs
Β-THALASSEMIA MAJOR Severe hemolytic anemia; microcytic,
hypochromic RBCs;
TRANSFUSION-DEPENDENT
Β-THALASSEMIA INTERMEDIA Moderate clinical symtoms, mild to
moderate hemolytic anemia, microcytic,
hypochromic RBCs
TRANSFUSION-INDEPENDENT
LEUKEMIA

Overproduction of various types of immature or mature


cells in BM and/or peripheral blood

Involves WBCs of the myelogenous or lymphocytic cell


types
Malignant cells easily trespass BBB

LEUKEMIA
 Malignant neoplasm of blood forming tissues of the BM, spleen, and lymph system
 More blasts: shorter, more fatal course of disease
 ↑ WBC count with shift to the left
 M:E ratio of 10:1
 Type of anemia: normocytic, normochromic
LYMPHOCYTIC LEUKEMIAS
o Myeloperoxidase : NEGATIVE
o Sudan Black B: NEGATIVE

ACUTE LYMPHOCYTIC LEUKEMIA (ALL)


 Most common form of childhood leukemia

FACTORS L1 L2 L3
Patients 70% of childhood ALL 70% of adult ALL Rare in children and adults
Cell size Homogenous, small Heterogenous, large Homogenous, large
Nucleus Uniformly round, small Irregular Round to oval
Cytoplasm Scant, blue Moderate, pale Moderate, blue, prominently
vacuolated
Cytochemistry
Periodic acid-Schiff + + -
Methyl Green Pyronin - - +
Oil Red O + + +
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
• Most common type of leukemia in elderly
Characterized by persistent lymphocytosis
Increased number of Reider cells and smudge cells in PBS

NON-LYMPHOCYTIC LEUKEMIAS/ MYELOGENOUS LEUKEMIAS


• Myeloperoxidase: POSITIVE
• Sudan Black B: POSITIVE

ACUTE MYELOGENOUS LEUKEMIA (AML)

SUBGROUP ORIGIN REMARKS


M0 Myelocytic AML, minimally differentiated
M1 Myelocytic AML, without maturation
M2 Myelocytic AML, with maturation
M3 Myelocytic Acute Promyelocytic Leukemia
(APL)
SUBGROUP ORIGIN REMARKS
M4 Myelocytic Acute Myelomonocytic leukemia
Monocytic (AMML)
M5 Monocytic Acute Monocytic Leukemia (AMoL)
M6 Erythrocytic Acute erythroleukemia
Myelocytic
M7 Megakaryocytic Acute Megakaryocytic Leukemia

CHRONIC MYELOGENOUS LEUKEMIA (CML)


 characterized by presence of Philadelphia Chromosome (Ph1)
 Due to reciprocal translocation of the long arms of chromosome 9 and 22

LEUKEMOID REACTION (LR)


 Excessive leukocytic response in PB
 WBC ct.= greater than 50 x 10 9/L
MPO SBB a- a-NAPHTHYL
NAPHTHOL AS-D NAPHTHYL ACETATE
CHLOROACETATE BUTYRATE ESTERASE FACTOR
(SE) ESTERASE (NSE) VIII
(NSE)
M1, M2, M3 + + + - - -
M4 (AMML) + + + + + -
M5 (AMoL) - +/- - + + -
M6 +/- +/- +/- - - -
(Erythroleukemia)

M7 - - - - Localized +
(Megakaryocytic) positivity
LEUKOCYTE ALKALINE PHOSPHATASE TEST

 Distinguish LR from CML

KAPLOW’S METHOD:

Principle: Hydrolysis of sodium alpha naphthyl phosphate by alkaline phosphatase produces a


colored precipitate with a diazotized amine

Reagents:
Fixative: methanol and formalin
Buffer: propanediole
Substrate: sodium alpha naphthyl phosphate
Initial stain: brentamine fast garnet salt
Counterstain: aqueous Mayer’s hematoxylin
PROCEDURE:
1. Immerse dry blood smear in fixative for 30 seconds
2. Pour unto smear the working substrate and allow to stand at least 10 minutes
3. Rinse with distilled water and dry
4. Counterstain for 10-15 minutes
5. Rinse with distilled water and mount in mounting solution
6. Examine immediately under the microscope and look for the presence of reddish-brown to
black precipitate in the cytoplasm of neutrophils
7. Count 100 segmented neutrophils and bands and score each cell

SCORE DESCRIPTION
0 No reddish brown to black precipitate
1+ Slightly diffused reddish brown to black precipitate
2+ Moderately diffused reddish brown to black precipitate
3+ Heavily diffused reddish brown to black precipitate
4+ Very heavily diffused reddish brown to black precipitate
 Normal Kaplow’s Score: 20-100

Increased LAP score: Leukemoid Reaction


Decreased LAP score: CML

INCREASED KAPLOW’S (LAP) SCORE DECREASED KAPLOW’S (LAP) SCORE


3rd trimester of pregnancy Myelodysplastic syndrome
Polycythemia vera Sideroblastic anemia
Intoxications Paroxysmal Nocturnal Hemoglobinuria
Infection CML
Sample problem:

SCORE NO. OF LAP SCORE


NEUTROPHILS
0 32 0
1+ 24 24
2+ 21 42
3+ 15 45
4+ 8 32
TOTAL 143
CELL CYCLE

G1- cell growth and synthesis of components necessary for replication. (10
hours)
S stage - DNA replication. (8 hours.)
G2 - tetraploid DNA is checked for proper replication and damage. (4 hours)
Mitosis or M stage - division of chromosomes and cytoplasm into 2 daughter
cells. (1 hour)

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