A modified ELISA procedure for platelet factor 4 with decreased heparin sensitivity

Abstract number: P0776

Grimaux M., Caron N., Quignon V., Woodhams B.

Serbio SA (Diagnostica Stago), France

Platelet factor 4 (PF4) ELISA procedures were criticized because of significant heparin sensitivity and nonconformity to the WHO PF4 standard by Merryman et al. (Throm Res 1997: 86: 525–6). They noted that the two different kits tested displayed different heparin sensitivity and that neither conformed to the WHO standard. This nonconformity may be related to the tetrameric structure of PF4 which is sensitive to ionic strength. We report here on the introduction of changes in the Asserachrom^® PF4 assay (Diagnostica Stago, France) to decrease heparin sensitivity and to ensure conformity to the WHO standard. The introduction of an anti-heparin into the plasma dilution buffer reduces heparin sensitivity with no effect on assay performance. The assay can now be performed in the presence of up to 2 im mL^-1 of either standard or fractionated heparin. Studies showed that the reported divergence of assay values from the WHO standard related to a nonparallel relation to the WHO standard. This was only apparent with higher PF4 levels and not in the normal range. This divergence was due to ionic strength and once adjustment were made so that the ionic strength used in the assay was equivalent to that used in the WHO standard the previously reported divergence disappeared. The alteration in ionic strength of the buffers and the use of higher plasma dilutions (change from 1:5 to 1:10) altered the assay characteristics so as to be fully compliant with the WHO standard over the whole assay range (y = 1.00 + 0.048x; r = 0.998). These modifications result in changes in assay performance so that the assay can now be used with plasma samples containing up to 2 IU mL^-1 of heparin and gives good correlation with the WHO standard over the whole measuring range.