Platelet Manual Count

8/13/2019 Platelet Manual Count

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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 markedthrombocytopenia.

Physiological variation in the platelet count: there may be a sex difference, thus inwomen 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 collectionor within 24 hours if the blood has been refrigerated.


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**. 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 plateletcount. 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 smallsquares 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.


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**. 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 tothe 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 preventevaporation 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.


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**. Calculation of the Platelet Count:

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

Platelets/ L =

Number ofPlatelets

X Correctionfor Volume

XCorrection for

Dilution

Example:

400X 25 X 20

Correction for Volume:the platelet count is given as the number of platelets per1 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 correctionfactor for dilution is 20.


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**. 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 dilutingfluid 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 50platelets, 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 andstorage.

Understanding of the analytical conditions of the test: dilution, counting chamberfilling, 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.

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Platelet Manual Count