TRANSCRIBED GEN CAMATO
HEMATOLOGY
LABORATORY
HEMATOLOGY
1
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LABORATORY
HEMATOCRIT
Parallel to hemoglobin
Volume occupy by erythrocytes in the given volume of blood
Useful in determining erythrocyte indices and for calculation of a
blood volume
9 Very useful to establish anemia; & other disorder in blood
ESR
Erythrocyte Sedimentation Rate
Rate of setting of RBC from the plasma after an addition of
anticoagulant
Importance to measure rate of fall
Associated with the net-surface charge of RBC with normal
surface of charge negative
9 MCH & MCV Hemoglobin, Hematocrit & RBC
Useful for total erythrocyte mass determination
Useful in establishing anemia
IMPORTANCE OF ESR
Use as a good index for determination of a hidden but active
disease such as tuberculosis and carcinoma
Measures the suspension stability of the RBC
Measures the abnormal concentration of fibrinogen and protein
globulin
METHODS FOR HEMATOCRIT
1) Macro method Use large volume of blood
a) Winthrobe method
Uses Winthrobe tube
Flat bottom tube graduated at both side
Fill the tube with blood sample using capillary pipet
Uses oxalated blood sample
Alternative anticoagulant: EDTA
Centrifuge the tube with the speed of 3,500rpm for
30minutes
Calculation for % Hematocrit
Volume % of Hematocrit = Hematocrit of Packed RBC
X 100
Hematocrit of White Blood
Reference range: Conventional unit
Male:
47 vol.%
Female:
43 vol.%
ESR METHODS
1. Winthrobe Method
Uses double oxalate; alternative: EDTA
Fill in the capillary pipet, stand for an hour
Westergren Method * For RESEARCH purpose
Most sensitive method of ESR determination use for serial
studies of chronic diseases such as carcinoma and
tuberculosis
Done in 2 readings after an hour and after 2 hours
Anticoagulant: 3.8% Na Citrate
3.
Brays Method
Anticoagulant: 1.3% Na Oxalate
4.
Cutler Method
Anticoagulant: 3.8% Na Citrate
5.
Micro Method
Especially for neonates
a) Landau Smith Method
b) Smith Method
b)
CONDITIONS OF ESR
r FASTER RATE ESR
Pregnant women
NON-PATHOLOGICAL
Menstruating women
Suffering with:
Tuberculosis
PA
TH
Cancer
OL
OG
Rheumatic fever
IC
AL
Malignant lymphoma
!!!!!!0$
2)
Haydens modification
Anticoagulant is 1.1% of NaC2O4 (Na Oxalate)
of 3.8% Na Citrate
c) Van Allens modification method
1.6% or 1.3% NaC2O4 or 3.8% Na Citrate
d) Sanford Magath
1.1% of 1.3% Na oxalate or 3.8% Na citrate
e) Brays Method
Heparin is the anticoagulant
Micro method
a) Micro Adams method Most popular and simple
Uses heparinize capillary tube
Mix to avoid coagulation
Seal with clay sealer then wax
Spin: 10,00012,000 rounds per minute (RPM) for
5minutes
Read with micro hematocrit capillary reader
2.
ESR$Scale$100$!!!!
Note:
The first drop of blood is usually discarded because it is
contaminated with dead epidermal cells and tissue juices.
1
SLOWER RATE ESR
Spherocytosis
Poikilocytosis
Sickle cell anemia
Severe Iron Deficiency Anemia (IDA)
Thalassemia
Jaundice
PA
TH
OL
OG
I
CA
L
TRANSCRIBED GEN CAMATO
HEMATOLOGY
LABORATORY
HEMATOLOGY
1
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LABORATORY
ZETA SEDIMENTATION RATIO
Determines the suspension stability of RBC that is not
affected by anemia
Measures the degree of packing of erythrocyte that occurs
during a 45 degrees cycle of dispension and compaction of
RBC in a special capillary tube and special centrifuge:
zetafuge
Zetafuge four spin cycle of 400 RPM/45 sec. For 4 mins.
Reference Value: 4051%
HEMOCYTOMETRY
Numerical evaluation of the formed elements of blood
Estimation of the number of blood cells in a given
volume of blood
METHODS FOR HEMOCYTOMETRY * OBSOLETE METHODS; NO LONGER USED
1) Turbidimetry
Base on the assumption that the more turbid the solution,
the more cells are present.
2)
Microscopic Method * MANUAL METHOD
Using pipet method/techniques or test tube dilution
method
Principle: blood cells are counted under the microscope
with the use of counting chamber, pipets, and diluting fluid
TYPES OF COUNTING CHAMBER
1. Spencer New Improved Neubauer Counting Chamber
Open type
It has 2 center flat form
1 ruled are divided in 9 primary square (1 sq.mm)
4 large square use for
count
Central primary square
count
Depth: 0.1 mm
2.
Fuchs Rosenthal
Design for CSF analysis
Depth: 0.2 mm
3.
Speirs Levy
Has 4 center flatforms
PIPETS (Automatic and Non-Automatic)
1) Unopette * COMMONLY USED; PREFERRED FOR PLATELET COUNTING
[ Microblast capillary pipet that automatically suck just
the right amount of a sample, connected into a plastic
container, containing just the right amount of diluting
fluid
2)
Thoma pipet *MANUAL
CLEANING OF THE PIPETS
1. Wash with water, alcohol and then ether. Air dry
2. Wash with water, acetone. Air dry
RBC Pipette
WBC Pipette
Larger
Smaller
Color of Bead
Size of Bulb
Calibration Mark
101
11
Size of Lumen
Smaller
Larger
TRANSCRIBED GEN CAMATO
HEMATOLOGY
LABORATORY
HEMATOLOGY
1
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LABORATORY
(Continuation RBC dilution)
! Formol citrate (Dacies fluid)
STEPS IN HEMOCYTOMETER
A. Pipet Method
1. Suck blood up to 0.5 mark of the pipet
2. Suck diluting fluid up to 101 (for RBC) and 11 (for WBC)
3. Shake pipet to mix
4. Discard first few drops
5. Charge counting chamber
6. Count RBC in 5 intermediate square and WBC in 4 corner
larger square
B.
Best RBC diluting fluid
With preservative action so NO MOLD formation
Cell morphology not altered
! NSS
Used in cases of emergency
Ideal to use in case of excessive rouleaux formation or
agglutination.
Test tube dilution Method
1. Add 4mL of dilution fluid
2. Shake tube
3. Obtain sample by a capillary tube
4. Charger counting chamber
5. Count in counting chamber
WBC DILUTING FLUID * Function: to LYSE RBC
! 1-3% Acetic acid with Gentian violet
! 1% HCl
! Tuerks
Acetic acid
Methyl violet
Distilled water
Note:
to convert mL to L
ex. 0.02 mL x 1000 L = 20 L
1mL
2 TYPES OF AUTOMATED HEMOCYTOMETRY
1) OPTICAL AUTOMATED
Principle: blood cells are counted as deflections of light
beams passes to the dark field area of the machine.
Example: Fisher autocytometer
COMPUTATION FOR
3
RBC in Millions/mm = RBC count x 10 x 200 x 5
I Wherein: 10
200
5
depth correction factor (constant)
dilution factor (variable)
area correction factor (constant)
2)
COMPUTATION FOR
3
WBC in Thousand/mm = WBC counted x 10 x 20
4
I Wherein: 10
20
4
depth correction factor (constant)
dilution factor (variable)
area correction factor (constant)
ELECTRICAL AUTOCYTOMETER
Principle: blood cells are counted as changes in voltage
pulse
Example: Coulter counter (diluting fluid: Isoton)
Inclusion
Composed of
Stain
CHARACTERISTIC OF IDEAL DILUTING FLUID
Isotonic (RBC)
Hypotonic (WBC)
Howell-jolly
DNA
Wright
9 0.85 -0.9% NaCl
9 to Lyse RBC
* To prevent RBC from Shrinking & Swelling
Indications
Chead and economical
Easy to secure and prepare
With preservative action
With high specific gravity
Stable
With buffer action
Non-allergenic
Non-corrosive
Basophilic
stippling
Pappenherimer
bodies
Siderotic
granules
RBC DILUTING FLUID
! Hayems
Initiates mold and rouleaux formations
Can stand for a long period of time and no corrosive
effect
Disturbed
erythropoietin
Hemolytic
Anemia
Megaloblastic
anemia
Postsplenectomy
RNA
Wright
New Methylene
Blue
Thalassemia
Lead poisoning
Denatured
precipitated
hemoglobin
Supravital Stain
G6PD deficiency
Thalassemia
Unstable
hemoglobins
Cabots ring
Remnants of
mitotic spindle
Wright
Megaloblastic
anemia
Parasites
Malaria
Babesia
Trypanosomes
Wright
Parasitic
infection
! Gowers
Prevents rouleaux formation and precipitation of protein
! Toissons
Initiates mold formation so it should be filtered
With high specific gravity
With stain so used by beginners since RBC are easily
identified form WBCs whose nuclei stain are blue.
A wise man will hear, and will increase learning; and a man of understanding shall
attain unto wise counsels:
- Proverbs 1:5
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