BLOOD PLATELETS 



41 



BLOOD PLATELETS 



acid, the diluting fluid should be made 

 fresh every week and only 38 per cent 

 neutral formaldehyde, U.S. P. should 

 be employed. A concentration of 0.1 

 per cent of formaldehyde in the dilut- 

 ing fluid is sufficient. Higher con- 

 centrations may hemolyze the erythro- 

 cytes and form precipitates with the 

 plasma. 



It is generally' conceded that the in- 

 corporation into the fluid of a dye, such 

 as brilliant cresyl blue, methyl violet, 

 methylene blue, toluene red, crystal 

 violet and nile blue, is superfluous, 

 since it does not assist in the differenti- 

 ation of platelets from artefacts and 

 may even itself be the source of arte- 

 facts. Diluting fluids containing no 

 anticoagulant, such as Tyrode's or 

 Ringer's solution, produce many arte- 

 facts and, in addition, cause rapid 

 disintegration of platelets unless a 

 fixative, such as solution of formalde- 

 hyde, is incorporated in the solution. 

 A satisfactory diluting fluid, therefore, 

 is the one proposed by Rees, H. M., 

 and Ecker, E. E. (J. A. M. A., 1923, 

 80, 621) without the brilliant cresyl 

 blue dye. This solution is prepared 

 by adding 0.22 ml. of neutral formalde- 

 hyde (38 per cent U.S. P.) to 100 ml. 

 of 3.8 per cent sodium citrate. The 

 solution should be kept in a well-stop- 

 pered bottle in a refrigerator and should 

 be filtered each time just before use. 

 Tocantins, L. M. (Arch. Path., 1937, 

 23, 850) also recommends sterilizing 

 the solution in order to reduce the num- 

 ber of artefacts caused by bacterial 

 contamination. 



Despite the use of a satisfactory dilut- 

 ing fluid, the adhesion of platelets to 

 each other and to the walls of the 

 pipette is almost impossible to avoid 

 when capillary blood is used. Drawing 

 the diluting fluid to the 0.5 mark on the 

 pipette and subsequently drawing the 

 head of the column of fluid to the 1.0 

 mark while aspirating the blood into 

 the pipette is unsatisfactory, since 

 there is little opportunity for the blood 

 to mi.\ with the diluting fluid within 

 the narrow confines of the capillary 

 stem of the pipette. Furthermore, 

 even the very short time required to 

 draw the exact quantity of blood into 

 the pipette and to mix it with the dilut- 

 ing fluid is sufficient in many instances 

 to allow marked clumping of the plate- 

 lets to occur. The clumping of plate- 

 lets can best be avoided by mixing the 

 blood with the diluting fluid before it 

 is drawn into the capillary pipette. 

 This can be accomplished by the use of 

 venous blood. The blood may be drawn 

 into a silicone-lined syringe and then 



immediately transferred to a tube con- 

 taining the diluting fluid or, preferably, 

 a given amount of blood is drawn into 

 a syringe containing a measured amount 

 of diluting fluid. Needles varying in 

 size from No. 18 to No. 26 have been 

 employed without any apparent effect 

 on the quality of the prep.-iration. A 

 satisfactory method for obtaining ve- 

 nous blood is as follows. Place exactly 

 1 cc. of diluting fluid in a 5 cc. syringe. 

 Attach a sterile dry No. 24 needle and 

 aspirate 1 cc. of blood from the vein, 

 without stasis, by withdrawing the 

 plunger to the 2 cc. mark. Withdraw 

 the needle from the vein and aspirate 

 the blood contained in it into the syr- 

 inge. Remove the needle and thor- 

 oughly mix the diluted blood in the 

 syringe and expell it into a collecting 

 bottle. It is important to use a syr- 

 inge whose plunger and needle fit 

 snugly, in order to avoid the leakage of 

 any air into the syringe while aspirat- 

 ing the blood. A 1 to 200 dilution of 

 the blood can be made by drawing this 

 mixture to the 1.0 mark on the Thoma 

 pipette and subsequently filling it to 

 the 101 mark with the diluent. If this 

 procedure is carried out carefully, 

 it is unnecessary to resort to methods, 

 such as those suggested bv Aynaud, 

 M. (Compt. Rend. Soc. Biol., 1910 

 68, 1062) and Preiss, W. (Zeitsch. Ges. 

 Exp. Med., 1932, 84, 1932) in which no 

 attempt is made to obtain an accurate 

 initial dilution in the syringe, and con- 

 sequently the final dilution of blood in 

 the counting chamber must be deter- 

 mined b}' establishing the ratio of an 

 erythrocyte count done on the platelet 

 preparation with an independent eryth- 

 rocyte count done in the usual manner. 

 There is still another limitation in 

 the accuracy of the direct platelet 

 count, imposed by the relatively small 

 concentration of platelets in the count- 

 ing chamber. The dilution of 1 part 

 blood in 200 parts of fluid is required 

 because with any greater concentration 

 the platelets would be obscured by the 

 erythrocytes. However, since there 

 are only appro.ximately 5 per cent as 

 many platelets as erj^throcytes present, 

 the statistical error is much greater 

 than that of the erythrocyte count, 

 even if all the platelets in the entire 1 

 square mm. central ruled area are 

 counted. Attempts have been made 

 to overcome this difficulty by increas- 

 ing the concentration of platelets in 

 preparations from which the erythro- 

 cytes have been eliminated. In some 

 methods, such as that of Brecher, G. 

 and Cronkite, E. P. (J. Applied Phj's- 

 iol., 1950, 3, 365) the erythrocytes are 



