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A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest-sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 ± 0.60 μm3 (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest-sized platelets, designated Fraction 7, was 8.19 ± 0.64 μm3. The MPV for the original platelets prior to fractionation was 6.57 ± 0.61 μm3. The mean density of Fraction 1 platelets was 1.067 ± 0.002 g/cm3, while Fraction 7 had a mean density of 1.072 ± 0.001 g/cm3. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 ± 0.9 dense bodies per platelet, and Fraction 7 had 12.6 ± 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 ± 0.92 iu per 1010 platelets; Fraction 7 platelets had 14.88 ± 1.23 iu per 1010 platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 ± 1.45 iu per 1010 platelets.