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GHOR ALSAFI LAB.

Platelet Manual Count


A.KAREEM AL-AJOURI 1

Platelet Manual Count



**. Definition:

The platelet count denotes the number of platelets in 1L of whole blood.

**. Normal Reference Ranges:

The platelet count falls between:150,000 to 400,000 /L.

**. Clinical Significance:

The platelet count may be elevated (thrombocytosis) in:
Polycythemia vera.
Idiopathic thrombocythemia.
Chronic myelogenous leukemia.
Following splenectomy.
May drop below normal values (thrombocytopenia) in:
Thrombocytopenic purpura.
Aplastic anemia.
Acute leukemia.
Gauchers disease.
Pernicious anemia.
Sometimes following chemotherapy and radiation therapy.
Prolonged bleeding time and poor clot retraction are found when there is marked
thrombocytopenia.
Physiological variation in the platelet count: there may be a sex difference, thus in
women the count has been reported to be about 20% higher than in men. A fall in
the platelet count may occur in women at about the time of menstruation and there
is some evidence of a cycle with a 21-35 day rhythm.

**. Preanalytical Conditions:

1. Preparation of Patient:

No special procedure to follow.

2. Collection of Specimen:

Collection of EDTA whole blood.

3. Storage Considerations:

The blood should be diluted and smears made within 5 hours of blood collection
or within 24 hours if the blood has been refrigerated.
GHOR ALSAFI LAB. Platelet Manual Count
A.KAREEM AL-AJOURI 2





**. Reagents and Equipment:

1. Hemocytometer:

Neubauer ruling consists of two raised platforms.
There is a raised ridge on both sides of two platforms on which a cover glass is
placed.
The space between the top of the platform and the cover glass over it is 0.1 mm.
Each of the platforms contains a large middle square containing 25 smaller
squares of equal size.
The 10 small squares labeled P are the areas to be counted for the platelet
count. The large center square has a volume of 0.1 L. Therefore, the volume of
each of the 25 smaller squares is 0.004 L, or a total volume for the 10 small
squares of 0.04 L.

2. Platelet Diluting Fluid:

Platelet diluting fluid used is Ammonium Oxalate, 1% W/V.
Weigh 1 gram of Ammonium Oxalate and dissolve in 100 mL distilled water in a
volumetric flask.
Store Ammonium Oxalate 1% in refrigerator and filter before use.



















GHOR ALSAFI LAB. Platelet Manual Count
A.KAREEM AL-AJOURI 3
**. Procedure:
Procedure for Platelet Manual Count
Step Procedure
1. Dilution of the blood:
-Mix the specimen of blood for approximately 1 minute.
-In a small 5 mL plastic tube, using the automatic pipette, dilute blood 1:20 (950 L
platelet diluting fluid + 50 L whole blood).
- Close the tube with parafilm.
-Mix the diluted blood for approximately 2 minutes to ensure adequate mixing. This
dilution is stable for 8 hours.
2. Cleaning of the counting chamber:
-Clean the hemocytometer thoroughly and make certain it is completely free of all dirt
and lint. The use of 95% (V/V) Ethyl Alcohol and lint-free cloth is recommended for
this process.
3. Preparation of the moist chamber:
-Obtain a petri dish and a filter paper of approximately the same diameter as the petri
dish.
-Thoroughly moisten and place the filter paper inside the petri dish so that it adheres to
the dish.
-Place two small wooden sticks on top of the filter paper.
4. Filling the counting chamber:
-With a capillary tube draw some of the diluted preparation, place the tip of the
capillary on the edge of the ruled area of the counting chamber.
-Allow the mixture to seep under the cover glass gradually and fill the area (if the
capillary is removed just before the area looks filled, the area will fill without becoming
flooded). Care should be taken not to move the cover glass.
-Place the hemocytometer on top of the two wooden sticks in the moist chamber and
cover the moist chamber (petri dish lid).
-Allow to stand for 15 to 30 minutes (this allows the platelets to settle and prevent
evaporation of the fluid in the counting chamber).
5. Counting the platelets:
-Carefully, keeping the counting chamber horizontal at all times, place the
hemocytometer on the stage of the microscope.
-Using low power only (10x objective), place the large center square in the middle of
the field of vision. This square is further subdivided into 16 even smaller squares. This
facilitates cell counting.
-Turn to the 40x objective (the platelets appear as round or oval bodies).
-Count all the platelets in 10 of the 25 small P squares cells in this square, (See
Figure One), remembering to count the platelets on two of the outer margins but
excluding those lying on the other two outside edges.
-Count the platelets in the same area on both sides of the counting chamber. The total
number of platelets counted on each side should be in agreement (10 platelets) when
the platelet count is in the normal range. Add the two counts together and divide by two
to determine the average number of platelet count.
6. Perform a platelet estimate on a Wright-stained smear
-Make a blood smear.
-Perform a platelet estimate on a Wright-stained smear by counting platelet in 10 oil
fields and taking the average then multiplying by a factor of 15.
-For low platelet counts (< 100,000) multiply by a factor of 20.
-For high platelet counts (> 400,000) multiply by a factor of 10.


GHOR ALSAFI LAB. Platelet Manual Count
A.KAREEM AL-AJOURI 4





**. Calculation of the Platelet Count:

Platelets/ L: calculate the number of platelets per L as shown below:


Platelets/ L =
Number of
Platelets
X Correction
for Volume
X
Correction for
Dilution

Example:
400

X

25

X

20



Correction for Volume: the platelet count is given as the number of platelets per
1 L of blood. Therefore, if the platelets are counted in 10 small squares the total
volume counted is 0.04 L (10 X 0.04). To obtain a volume of 1.0 L, 0.04 is
multiplied by 25 (1/0.04).

Correction for Dilution: since the blood was initially diluted 1:20 the correction
factor for dilution is 20.




















GHOR ALSAFI LAB. Platelet Manual Count
A.KAREEM AL-AJOURI 5


**. Technical Notes:

Technical Note on Platelet Manual Count
Problem What to do
If the platelet count does not reasonably
agree with the platelet estimate.
The count should be repeated.
If clumps of platelets are noted in the
platelet count, due to inadequate mixing
or poor technique in obtaining the blood
sample.
The procedure should be repeated.
If there is an uneven distribution of
platelets in the counting chamber.
The procedure should be repeated.
Inaccurate counting due to interfering
artifacts.
The hemocytometer must be
scrupulously clean and the diluting
fluid must be freshly filtered.
Fewer than 80 platelets are counted in
the 10 small squares.
All the platelets in the large central
square (25 small squares) on both sides
of the hemocytometer should be
counted. Then if fewer than 50 platelets
are counted per slide, the platelet count
should be repeated diluting the original
sample 1:20 to confirm if it is a
dilution problem or not. If after that the
platelet count is still fewer than 50
platelets, a 1:10 dilution should be
made and the correction for dilution
will be 10.
The platelet count is extremely high. A dilution of 1:200 or greater should be
made.
Error Rate 8% - 10 %


***. Critical Elements of Competence for Evaluation

Understanding of the pre-analytical conditions of the test: specimen collection and
storage.
Understanding of the analytical conditions of the test: dilution, counting chamber
filling, technique of counting platelets and calculations.
Ability to perform the proper technique for platelet count.
Ability to act when special situations like high, low and difficulties in counting
arise.