Platelets Count

Platelets, also called "thrombocytes", are blood cells whose function (along with the coagulation factors) is to stop bleeding. Platelets have no nucleus: they are fragments of cytoplasm which are derived from the megakaryocytes of the bone marrow, and then enter the circulation. These unactivated platelets are biconvex discoid (lens-shaped) structures, 2–3 µm in greatest diameter. Platelets are found only in mammals, whereas in other animals (e.g. birds, amphibians) thrombocytes circulate as intact mononuclear cells. There are two methods for estimation of erythrocyte count:
•    Manual or microscopic method
•    Automated method

 

 

Principle
Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent at a specific volume in the Unopette reservoir.  The diluents (1% ammonium oxalate) lyses the erythrocytes but preserves leukocytes and platelets.  A 20 µL pipette is used with 1.98 ml of diluents to make a 1:100 dilution. The diluted blood is added to the hemacytometer chamber.  Cells are allowed to settle for 10 minutes before leukocytes and platelets are counted. (Always refer to the manufacturer’s instructions for the procedure.)

 

Equipment
Hemocytometer with cover glass, compound microscope. Unopette capillary pipette, lint-free wipe, alcohol pads,  hand counter, petri dish with moist filter paper.

 

Reagent
Ammonium oxalate: 11.45 gm
Sorensen’s phosphate buffer: 1.0 gm
Thimerosal: 0.1 gm
Distilled water: 1000 ml

 

Specimen
EDTA-anticoagulated blood or capillary blood is preferred.

 

Method
(1) Using the protective shield on the capillary pipette, puncture diaphragm of  Unopette reservoir.    
(2) Remove shield from pipette assembly by twisting. Holding pipette almost horizontally, touch tip of pipette to blood.  Pipette will fill by capillary action. Filling will cease automatically when the blood reaches the end of the capillary bore in the neck of the pipette.

(3) Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor.
(4) Squeeze reservoir slightly to force out some air while simultaneously maintaining pressure on reservoir.
(5) Cover opening of overflow chamber of pipette with index finger and seat pipet securely in reservoir neck.
(6) Release pressure on reservoir. Then remove finger from pipette opening. At this  time negative pressure will draw blood into reservoir.
(7) Squeeze reservoir gently two or three times to rinse capillary bore forcing diluent up int, but not out of, overflow chamber, releasing pressure each time to return mixture to reservoir.
(8) Place index finger over upper opening and gently invert several times to thoroughly mix blood with diuent.
(9) Cover overflow chamber with pipette shield and incubate at room temperature for 10 minutes before charging the hemacytometer.
(10) Meticulously clean the hemacytometer with alcohol or other cleaning solution. This is important because dust particles and other debris can be mistaken for platelets especially on a light microscope. Allow to dry completely before charging with diluted specimen.
(11) To charge the hemacyto-meter, convert to dropper assembly by withdrawing pipette from reservoir and reseating securely in reverse position.
(12) Invert reservoir and discard the first 3 or 4 drops of mixture.
(13) Carefully charge hemacyto-meter with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.
(14) Place hemacytometer in moist Petri dish for 10 minutes to allow platelets to settle.  (Moistened filter paper retains evaporation of diluted specimen while standing.)
(15) Mount the hemacytometer on the microscope and lower its condenser.
(16) Procedure for counting platelets:

• Under 40x magnification, scan to ensure even distribution.  Platelets are counted in all twenty-five small squares within the large center square. Platelets appear greenish, not refractile.
• Count cells starting in the upper left of the large middle square.  Continue counting to the right hand square, drop down to the next row; continue counting in this fashion until the total area in that middle square (all 25 squares) have been counted.
• Count all cells that touch any of the upper and left lines, do not count any  cell that touches a lower or right line.
• Count both sides of the hemocyt-ometer and take the average.

 

 

cells/mm3 =      Tc x Rd     
                    Ns x As x Ds

 

     Where Tc is the number of cells counted, Rd is the reciprocal of dilution, Ns is the number of squares counted, As area of each square and Ds is the depth of the solution.

 

Example:
Total number of cells= 230
Dilution 1:100
Number of squares counted: 1
Area of each square: 1 mm3
Depth of solution: 0.1mm

cells/mm3 =         230 x 100        
                  1 x 1 mm2 x 0.01 mm

               = 230,000/mm3 (µL)
               = 230 x 103/L

 

• 150,000 - 450,000/µL
• 150 - 450 x 109/L

 

REFERENCES
1. Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
2. Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
3. Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
4. Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
5. Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
6. Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.