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

SHIMIZU, T., NODA, Y., GOTO, S., MORISHIMA, Y., HASEGAWA, I., FUKUDA, T. and KATO, K. Determination of Low Affinity Platelet Factor 4 in Frozen and Thawed Human Platelets by the Newly Developed Enzyme Immunoassay System. Tohoku J. exp. Med., 1984, 142 (4), 453-460 - 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 β-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 β-thromboglobulin which is immunologically identical with LA-PF4 (γ=0.961, slope=1.056, y-intercept=-3.739ng/ml; n=22). The contents of LA-PF4 per 109 Triton-X 100 lysed platelets in platelet-rich plasma were 23.65±3.14μg (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. - low affinity platelet factor 4; enzyme immunoassay; intraplatelet content; freeze thawing