SRA Does Not Detect Potentially Relevant HIT Antibodies.
Authors: Prechel Margaret , McDonald Meredith K. , Jeske Walter P. , Drenth Amanda F. , Walenga Jeanine M. 
Published: April 2020

Abstract
Even before the nature of the heparin:platelet factor 4 (H:PF4) antigenic target was identified, it was known that pathogenic activity in sera/plasma from Heparin-Induced Thrombocytopenia (HIT) patients could be recovered in the immunoglobulin G (IgG) fraction. In in vitro diagnostic tests, blockade of platelet FcγIIa receptors completely prevents platelet activation by HIT sera. For these reasons, the IgG isotype is considered the predominant HIT pathogenic agent. H:PF4 ELISA techniques have made it possible to distinguish between antibodies (Abs) of the IgG, IgA and IgM isotypes, and have shown that IgA and IgM Abs are more prevalent than previously thought. In a group of 247 H:PF4 ELISA-positive specimens studied at Loyola University Medical Center, 65% contained IgG, 53% had IgA, and 56% had an IgM component. The biological activity and pathogenic relevance of non-IgG H:PF4 Abs is as yet unknown. These studies were undertaken to specifically test whether H:PF4 IgA or IgM isotypes, in the absence of IgG, could cause platelet activation in the Serotonin Release Assay (SRA), the reference standard activation assay for diagnosis of HIT. Four SRA positive specimens with multiple isotypes were fractionated using Protein G (Amersham Biosciences) chromatography to separate IgGs. Isotyping of the fractions demonstrated that the H:PF4 IgA and IgM were present in the flow through peak; IgGs were in fractions eluted from the Protein G by low pH. Only fractions that bound to Protein G, ie IgG isotype, caused the characteristic SRA positive response of platelet activation in the presence of low heparin (0.1 U/ml) and not in the presence of excess heparin (100 U/ml). Study of an SRA positive specimen that tested negative for the IgG isotype, showed that the SRA activity was in fractions that bound to Protein G even though the antibody was not detected in the ELISA. Another four SRA positive specimen with multiple isotypes were fractionated using immobilized jacalin chromatography (Pierce, Rockford IL) to separate IgA Abs. Isotyping demonstrated that IgG Abs did not bind to the column; IgA Abs were in fractions eluted from the jacalin column in the presence of melibiose. Only the flow through H:PF4 IgG, and not the eluted IgA Abs, tested positive in the SRA. However, three of the isolated H:PF4 IgAs elicited some degree of platelet serotonin release in the absence of heparin only, without the characteristic 2-point response to heparin. In conclusion, the platelet activation by SRA positive specimens with multiple isotypes is attributable to the IgG component only. Neither IgA nor IgM H:PF4 Abs are detected in the 2-point SRA. While the SRA does not detect IgA or IgM Abs, these isotypes may still have pathogenic activity. Preliminary studies with H:PF4 IgA, indicate that these Abs can bind to platelets and cause serotonin release in the absence of heparin. Until there is better understanding of in vivo biological activity of non-IgG HIT isotypes, it should be noted that the SRA, the reference standard activation assay for HIT, may not detect all physiologically relevant H:PF4 Abs.