Characterization of patient-derived anti-PF4 antibodies in chemiluminescence assays

Introduction: Heparin-induced thrombocytopenia (HIT) and Vaccine-induced immune thrombotic thrombocytopenia (VITT) are misdirected immunothrombotic diseases against platelet factor4 (PF4). PF4 complexed with heparin or PF4 act as antigens in HIT and VITT respectively. Recently developed chemiluminescence assay, namely rapid anti-PF4 assay combined with an available chemiluminescence rapid anti-PF4/polyanion assay differentiate between typical HIT (anti-PF4/heparin antibodies) and VITT antibodies (anti-PF4 antibodies). We recently identified patients with anti-PF4 disorders, who tested positive in both assays. However, it is unclear, if the patients harbor both typical HIT and VITT antibodies or merely one type of antibodies that are cross-reactive to PF4/heparin and PF4 antigens.

Fig. 1 Characterization of affinity-purified antibodies in rapid chemiluminescence assays. A)Patient sera represented in red (n=4) tested in rapid anti-PF4/polyanion assay(x-axis) and rapid anti-PF4 assay(y-axis).Each symbol represents a patient.B)Affinity purified antibodies with PF4 and PF4/polyanion complexes are shown in blue (n=4) and black (n=4) respectively.C)Antibodies purified with PF4/polyanion complexes from patient1 in rapid anti-PF4/polyanion assay and D)in rapid anti-PF4 assay. E)Antibodies purified with PF4 from patient1 in rapid anti-PF4/polyanion assay and F)in rapid anti-PF4 assay.Dashed lines represent threshold values in respective rapid chemiluminescence assay.

Method: Patient sera testing positive in both assays were used to affinity purify either anti-PF4/heparin antibodies or anti-PF4 antibodies by beads coated with PF4/polyanion complexes or PF4. The affinity-purified antibodies were tested in the assays.

Results: The reactivity of the serum (Panel A) is given as red symbols and of the respective eluates from PF4 beads as blue or PF4/heparin beads as black symbols (Panel B). Eluates of patient 2 (squares) and 3 (triangles) give the same results, regardless, whether purified by PF4 or PF4/heparin beads. This is typical for cross-reactivity. Eluates from patient 1 (circles) and 4 (rhomboid), however, give different results depending on the solid phase used for purification, indicating 2 types of antibodies. Concentration dependent reactivity testing of eluates of patient 1 show that antibodies purified by anti-PF4/heparin beads bind exclusively to PF4/heparin beads but not to PF4 alone. Antibodies purified by PF4 beads bind primarily to PF4. At very high concentrations they also bind to PF4/heparin beads, most likely by competitive displacement of heparin from the heparin binding site on PF4 ([Fig. 1]).

Conclusion: Patients with anti-PF4 disorders reacting in in both chemiluminescence assays should not be treated with heparin as some of them have a mixture of anti-PF4 and of anti-PF4/heparin antibodies (Patient 1 and 4).

 

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Conflict of Interest:

M. Broto and M. Palicio are Werfen employees. The rest of authors declare no conflict of interests.

Publication History

Article published online:
13 February 2025