Professional Documents
Culture Documents
LECTURE
IN
HEMATOLOGY I
Loss of WB
HYPOVOLEMIA Loss of plasma
Loss of body water
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
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
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
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
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
RBC WBC
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
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?
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
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
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
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
(-) (+)
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
MICROCYTIC, HYPOCHROMIC
ASSOCIATED CONDITIONS
Thalassemia
Anemia of chronic disease
Iron deficiency anemia
Lead poisoning
Sideroblastic anemia
MACROCYTIC, NORMOCHROMIC
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
(-) (+)
Two crystals:
I. HEMOGLOBIN SC crystal
• found protruding the RBC membrane
• Characterized by “Washington monument” appearance
ALPHA-THALASSEMIA
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
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
M7 - - - - Localized +
(Megakaryocytic) positivity
LEUKOCYTE ALKALINE PHOSPHATASE TEST
KAPLOW’S METHOD:
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
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)