BLOOD PLATELETS 



42 



BLOOD PLATELETS 



hemolyzed. However, the use of hemo- 

 lytic diluting fluids, such as potassium 

 cyanide, urea, or 1 per cent ammonium 

 oxalate is to be discouraged since 

 hemolyzed erythrocytes may be the 

 source of artefacts. Furthermore, if 

 such diluting fluids contain no fixative, 

 dissolution of some of the platelets 

 may also occur. Another means of 

 attaining a higher concentration of 

 platelets in the counting chamber is 

 to do the platelet count on plasma. 

 In Reimann's modification of Thorn- 

 sen's method (J. Exper. Med., 1924, 

 40, 553) 0.9 CO. of blood is drawn into 

 a tuberculin syringe containing 0.1 

 cc. of 10 per cent sodium citrate. The 

 needle is removed and the open end 

 of the syringe is closed with a piece of 

 rubber tubing fastened to the barrel 

 with rubber bands. The plunger is 

 removed and the syringe is placed in 

 a vertical position. After sedimenta- 

 tion has occurred, a 1 to 20 dilution of 

 the supernatant plasma is made with 

 physiologic sodium chloride solution. 

 The platelets in the mixture are then 

 counted in the counting chamber in 

 the usual manner. If the platelets 

 seen in five intermediate sized squares 

 (80 small squares) are counted, the 

 number is multiplied by 1000 [1/5 (area) 

 X 1/20 (dilution) X 1/10 (depth)], in 

 order to find the number of platelets 

 per cubic millimeter of plasma. A 

 further procedure has been designed to 

 determine the number of platelets per 

 cubic millimeter of blood. The syringe 

 containing the blood is centrifugalized 

 for 20 minutes at 2000 r.p.m. The 

 relative amount of plasma and packed 

 cells is recorded and the number of 

 platelets per cubic millmeter of blood 

 is determined thus: 



Number of 

 platelets per 

 cubic milli- 

 meter of 

 blood 



Number of 

 platelets per 

 cubic milli- 

 meter of 

 plasma 



Amount of plasma 



Total amount 

 mixture 



of 



To the result, 10 per cent of the total 

 number is added to offset the original 

 dilution in the syringe. The determi- 

 nation of the platelet count in plasrna 

 by this method has the advantage of 

 increasing the concentration of plate- 

 lets per unit volume in the counting 

 chamber approximately ten times. 

 However, there is some question as to 

 the stability and uniformity of the sus- 

 pension of platelets in plasma during 

 the time required for sedimentation 

 of the erythrocytes. Furthermore, the 

 determination of the relative plasma 

 and packed cell volumes by the method 

 given is inadequate, since even at a 

 centrifugalization speed of 3000 r.p.m. 



at least 15 per cent of the plasma is 

 retained in the packed cell mass. 



In order to avoid the errors inherent 

 in the Thomsen method and still in- 

 crease the concentration of platelets 

 in the counting chamber, methods have 

 been devised for eluting the platelets 

 from the whole blood. In the method 

 of Villarino and Pimentel (Am. J. 

 Clin. Path., 1942, 12, 362) a 1 to 2 dilu- 

 tion of venous blood with Aynaud's 

 fluid is made in the syringe. Four cc. 

 of pooled eluate from 0.2 cc. of this 

 mixture are obtained by four separate 

 centrifugations for 1 minute at 1500 

 r.p.m. Only an insignificant number of 

 platelets can be recovered by further 

 washing. A similar method has been 

 devised by Scheff, G. I. and Ralph, 

 P. H. (Am. J. Clin. Path., 1949, 19, 

 1113) using an angle centrifuge at a 

 speed of 700 r.p.m., and dark field 

 illumination. Although it would ap- 

 pear that practically all of the plate- 

 lets are recovered by these methods 

 since only an insignificant number can 

 be recovered by further washing and 

 only a very few can be found in stained 

 smears of the washed sediment, there 

 is some question as to whether some of 

 them may not have been destroyed 

 during the process of repeated centri- 

 fugalization. 



Regardless of the source of the speci- 

 men, type of diluting fluid or concentra- 

 tion of platelets per unit volume of 

 fluid in the chamber, there are certain 

 difficulties in differentiating platelets 

 from artefacts when high dry magnifica- 

 tion and bright field illumination are 

 used. Under these circumstances, a 

 platelet is defined simply as "a small 

 refractile body". According to Tocan- 

 tins, L. M. (Arch. Path., 1927, 23, 850) 

 "Only forms from 1 to 3 microns or 

 larger in size, rod or comma-like if seen 

 sidewaj^s, and thin, translucent and 

 disclike if flat on the floor of the count- 

 ing chamber, should be counted. Gran- 

 ules 0.8 micron in diameter or smaller, 

 jerkily moving about more or less ac- 

 tively, globules of oil, irregularly 

 shaped debris floating on the upper 

 layers of the fluid, strings of cocci, 

 and other minute objects may be dis- 

 tinguished from platelets after a little 

 practice. The error of counting too 

 few platelets may be equaled only by 

 the error of counting every particle in 

 the field as a platelet." That experi- 

 enced observers differ significantly, 

 even when using the same method, is 

 shown by the following results of cu- 

 taneous platelet counts on normal 

 adult subjects, using the Rees and Ecker 

 method. 



