Antibodies to Macromolecular Platelet Factor 4-Heparin Complexes in Heparin-induced Thrombocytopenia: a Study of 44 Cases

Abstract

SummaryAs heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 <0.3) and 35 patients with other causes of thrombocytopenia (A492 <0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 ± 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped ≤ 100 × 109/l only at days 11–12. Surprisingly, among 41 patients under heparin for >7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.

Platelet factor 4/heparin antibody (IgG/M/A) in healthy subjects: a literature analysis of commercial immunoassay results

“…Positive results were typically, but not always, of low OD and close to the cut-off by ELISA or of the lowest titer (titer of 1) by PGIA, that is, they were "weak" positives. However, three of the 14 seropositive subjects by PF4/heparin ELISA with available data had OD 492 values exceeding 0.8 (including one value > 1.0), higher positive values that are often seen in patients with HIT [13,16,17,20]. An OD 405 > 1.0 in the PF4/PVS ELISA has been associated with thrombotic predisposition [30], although the specific relationship between OD values from the different ELISAs is unclear.…”

“…Seven articles were excluded because healthy volunteers were tested using a noncommercial ("inhouse") assay for PF4/heparin antibodies. In three of these seven articles, the described assay ultimately served as basis of a commercial assay [12,13,19]. The literature set comprised the remaining 11 articles [10,16,17,[20][21][22][23][24][25][26][27], each of which reported the prevalence of PF4/heparin antibodies in healthy subjects, as assessed by a commercial immunoassay according to manufacturer's directions (Table 1).…”

“…At least four commercial immunoassays have been developed for detecting human antibodies (IgG, IgA or IgM) to PF4/heparin. Two enzyme-linked immunosorbent assays (ELISAs), initially described, respectively, by Amiral and colleagues [12,13] and by Visentin and colleagues [14,15], differ most notably in the antigen coated on the microtiter plate, i.e., PF4 with heparin (Asserachrom® HPIA, Diagnostica Stago, Asnieres, France) or PF4 with polyvinyl sulfonate (PVS) (PF4 Enhanced®, GTI, Waukesha, WI, USA). The upper cut-off value for normality, as established by the manufacturer, is an optical density (OD) at 492 nm of ≥0.5 in the PF4/heparin ELISA and an OD at 405 nm of ≥0.4 in the PF4/ PVS ELISA.…”

Heparin‐induced thrombocytopenia and thrombosis

“…However, available commercial kits are designed to detect antibodies of all Ig classes, in part because of early reports describing patients with HIT (some with thrombosis) whose antibodies were exclusively IgM [70,71]. Another argument for detecting IgM antibodies is that they are produced earlier in the course of the immune response to heparin and could presage later production of strong and ''clinically significant'' antibodies of the IgG class [44].…”

Antibodies to Platelet Factor 4–Heparin After Cardiopulmonary Bypass in Patients Anticoagulated With Unfractionated Heparin or a Low-Molecular-Weight Heparin

“…4,10 For the overall assay measuring IgG, IgA, and IgM isotypes, the result was defined as negative if absorbance at 492 nm (A 492 ) was Ͻ0.5 or as positive if A 492 was Ն0.5.…”