Effect of paraformaldehyde on platelet size and on measurement of surface IgG

In many platelet assays, as in measurement of platelet-adherent IgG (PAIgG), platelets are fixed in paraformaldehyde (PFA). To better clarify the effect of PFA on platelet size and on PAIgG measurement we compared PAIgG levels in a series of 40 samples, with or without PFA fixing. We used an ELISA which was set up on unfixed platelets and gave excellent results in terms of linearity (r = 0.923), precision (mean CV = 5%) and correlation with a platelet suspension immunofluorescence test. We found PAIgG values in unfixed platelets were about 10-fold higher than in PFA-fixed (0.411 +/- 0.172 fg/platelet vs. 0.035 +/- 0.019 fg/platelet). This discrepancy could be a consequence of the smaller mean platelet volume (MPV) of washed platelets when fixed in PFA (8.0 +/- 0.8 fl as compared to 10.1 +/- 1.07). This effect of PFA could decrease the amount of binding sites for IgG exposed on the platelet membrane and hence explain the significantly lower PAIgG values observed in fixed platelets. The PAIgG measurements on unfixed platelets from 200 healthy subjects displayed a Gaussian distribution with a mean +/- SD of 0.32 +/- 0.13 fg/platelet, i.e., 1200 +/- 500 molecules/platelet.