Determination of low affinity platelet factor 4 in frozen and thawed human platelets by the newly developed enzyme...

By the use of a newly developed sandwich enzyme immunoassay method for low affinity platelet factor 4 (LA-PF4), the effects of repeated freeze-thawing on the contents of this protein in platelets were determined and compared with those of Triton-X 100 lysed platelets. The assay system consisted of polystyrene balls covered with immobilized antibody fragments F(ab')2 and the same antibody Fab' labeled with beta-D-galactosidase from E. coli. The assay was specific to LA-PF4 with no significant cross-reactivity with platelet factor 4. Coefficients of variation within-run and between-run for the assay of LA-PF4 were less than 12%. The results obtained with this enzyme immunoassay correlated well with those of a radioimmunoassay of beta-thromboglobulin which is immunologically identical with LA-PF4 (r = 0.961, slope = 1.056, y-intercept = -3.739 ng/ml; n = 22). The contents of LA-PF4 per 10(9) Triton-X 100 lysed platelets in platelet-rich plasma were 23.65 +/- 3.14 micrograms (mean +/- S.D.). The contents of LA-PF4 in platelets were increased from 46% to 95% of Triton-X 100 values by repeated freezing and thawing 1 to 7 times. The present data indicate that the freeze-thawing technique should be done carefully to obtain the reliable determination of LA-PF4 in platelets.