BLOOD PLATELETS 



40 



BLOOD PLATELETS 



direct methods of platelet counting 

 consists in the accurate dilution of a 

 measured quantity of blood with a 

 suitable fluid. The diluted blood is 

 placed in a counting chamber and the 

 number of platelets in a circumscribed 

 volume is counted. These methods of 

 counting platelets involve the use of 

 the same apparatus as is employed in 

 the erythrocyte count. The Thoma 

 diluting pipette is generally used. It 

 consists of a capillary tube graduated 

 in tenths, which opens into a bulb con- 

 taining a glass bead. The bulb, when 

 filled to the mark 101 above it, will 

 hold 100 times the quantity of fluid 

 contained in the 10 divisions of the 

 capillary tube. The counting chamber 

 is a heavy glass slide, with two central 

 ruled platforms. They are separated 

 from each other by one moat, and from 

 elevated shoulders on each side by 

 transverse moats. These lateral shoul- 

 ders are so ground that a cover-slip 

 resting on them lies exactly 0.1 mm. 

 above each platform. A ruled area of 

 3 mm. by 3 mm. (9 sq. mm.) is engraved 

 on each side. This area is divided into 

 9 large 1 mm. squares. The central 

 square is divided into intermediate- 

 sized squares of 1/25 sq.rnm., each of 

 which is further divided into smaller 

 squares of 1/400 sq. mm. 



In making a count, blood is drawn by 

 suction into the Thoma pipette to the 

 0.5 mark. The blood adhering to the 

 outside of the pipette is wiped off and 

 the diluent is drawn in until it fills the 

 bulb and reaches the mark 101. While 

 drawing in the diluent, the pipette is 

 revolved between the finger and the 

 thumb, in order to mix the blood thor- 

 oughly with the diluent. After the 

 desired quantity of solution has been 

 drawn into the pipette, it is held hori- 

 zontally and shaken for one-half niin- 

 ute, or it may be placed in a mechanical 

 shaking device, in order to secure 

 thorough mixing. The coverglass is 

 next placed on the chamber. Several 

 large drops of fluid are expelled from 

 the pipette and discarded, and a small 

 quantity of diluted blood is allowed to 

 run under the coverglass. The plat- 

 form should be completely covered with 

 fluid, and none should run over into 

 the moats. 



The preparation is allowed to stand 

 10 minutes to allow settling of the cells, 

 then the number of platelets seen in 

 the entire central 1 mm. ruled area is 

 counted. This number is multiplied 

 by 2000 [1/10 (depth) X 1/200 (dilu- 

 tion)]. 



There may be errors due to uncleanli- 

 uess of the glassware, inaccurate 



calibration of pipettes or chambers, 

 imperfect dilution, failure to mix the 

 blood and diluting fluid thoroughly, 

 imperfect filling of the chamber and 

 improper sampling, such as might occur 

 if a free flow of blood is not obtained 

 from the finger or ear when capillary 

 blood is employed, or from improper 

 sampling if venous blood is used. The 

 above sources of error can be avoided 

 by an experienced technician, so that 

 their contribution to the inaccuracy of 

 the platelet count is minimal. How- 

 ever, there are additional sources of 

 error when this method, which was 

 originally devised for erythrocyte 

 counting, is used for the enumeration 

 of platelets. Olef, I. (J. Lab. & Clin. 

 Med., 1935, 20, 416) has listed the fol- 

 lowing: 1) clumping of the platelets 

 during drawing of the blood into the 

 pipette, 2) the impossibility of visualiz- 

 ing the smaller platelets with the high 

 dry lens, 3) the failure to visualize 

 some of the platelets in the 100 micron 

 space that exists between the bottom 

 of the counting chamber and the over- 

 lying coverglass, 4) the sticking of the 

 platelets to the side of the pipette and 

 to the parts of the hemacytometer, 

 and 5) the confusion of foreign particles 

 or precipitates with platelets. 



Platelet clumping, the adhesion of 

 platelets to glassware and the occur- 

 rence of morphological changes in the 

 platelets can be prevented only by the 

 immediate mixing of the blood with a 

 diluting fluid which contains both an 

 anticoagulant and a fixative. It is 

 not generally appreciated that de- 

 generative changes occur quite rapidly 

 in platelets, even in the presence of 

 an anticoagulant, unless a fixative is 

 also incorporated in the diluting fluid. 

 The anticoagulants which have been 

 employed are sodium, potassium or 

 ammonium oxalate, sodium citrate, 

 sodium metaphosphate, sodium sulfate, 

 magnesium sulfate, heparin, peptone 

 and gelatin. Of these, 3.8 per cent 

 sodium citrate appears to be the most 

 satisfactory, since it is isotonic with 

 the blood plasma, does not form crystals 

 or precipitates with the plasma, and is 

 not highly conducive to bacterial 

 growth. The fixatives most commonly 

 employed are mercury bichloride, osmic 

 acid, solution of formaldehyde, and 

 methyl or ethyl alcohol. Of these, 

 the solution of formaldehj^de is prefer- 

 able because it does not produce clump- 

 ing of the erythrocytes, does not form 

 protein precipitates with the plasma, 

 and does not produce secondary changes 

 in the platelets. In order to avoid 

 hemolysis from the formation of formic 



