TRANSCRIBED GEN CAMATO

HEMATOLOGY LABORATORY

HEMATOLOGY 1 | 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 | 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 | 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
3