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. 2023 Dec 28;142(26):2305-2314.
doi: 10.1182/blood.2023022136.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure

Linda Schönborn  1 , Olga Esteban  2 , Jan Wesche  1 , Paulina Dobosz  2 , Marta Broto  2 , Sara Rovira Puig  2 , Jessica Fuhrmann  1 , Raquel Torres  2 , Josep Serra  2 , Roser Llevadot  2 , Marta Palicio  2 , Jing Jing Wang  3 , Tom Paul Gordon  3 , Edelgard Lindhoff-Last  4 , Till Hoffmann  5 , Lorenzo Alberio  6 , Florian Langer  7 , Christian Boehme  8 , Eugenia Biguzzi  9 , Leonie Grosse  10 , Matthias Endres  11   12   13   14 , Thomas Liman  12   13   15 , Thomas Thiele  16 , Theodore E Warkentin  17 , Andreas Greinacher  1
Affiliations

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure

Linda Schönborn et al. Blood. .

Abstract

Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

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Conflict of interest statement

Conflict-of-interest disclosure: A.G. reports grants and nonfinancial support from Aspen, Boehringer Ingelheim, Merck Sharp & Dohme (MSD), Bristol Myers Squibb (BMS), Paringenix, Bayer HealthCare, Gore Inc, Rovi, Sagent, and Biomarin/Prosensa; personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, and Werfen; and nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V. outside the submitted work. O.E., P.D., M.B., S.R.P., R.T., J.S., R.L., and M.P. are employees of Werfen. O.E., P.D., M.B., R.L., and A.G. have filed a patent application (PCT/EP2023/062615) covering the design and use of the new rapid anti-PF4 assay. E.L.-L. has received lecture honoraria and advisory fees from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb/Pfizer, Daiichi Sankyo, Portola, CSL Behring, Viatris, Werfen, Norgine, and Aspen; and institutional research support from Bayer AG, Bristol-Myers Squibb/Pfizer, Daiichi-Sankyo, and CSL Behring for a different research project. F.L. has received personal fees for lectures or consultancy from Alexion, AstraZeneca, Bayer, BioMarin, BioNTech, Bristol Myers Squibb, Chugai, CSL Behring, Daiichi Sankyo, Grifols, Janssen-Cilag, LEO Pharma, Mitsubishi Tanabe Pharma, Novo Nordisk, Pfizer, Roche, Swedish Orphan Biovitrum, Takeda, Viatris, and Werfen; and institutional research support from Bayer, Chugai, CSL Behring, Intersero, Novo Nordisk, Pfizer, and Swedish Orphan Biovitrum. L.A. reports institutional research support from Bayer, CSL Behring, Novartis, Novo Nordisk, Octapharma, Roche, Sobi, Takeda/Shire, and Werfen; and fees for lectures or consultancy from Bayer, Biotest, Boehringer-Ingelheim, CSL Behring, Daiichi Sankyo, Novo Nordisk, OrPha Swiss, Roche, Sanofi-Aventis, Sanofi-Genzyme, Siemens, Sobi, Takeda/Shire, Viatris, and Werfen. M.E. reports grants from Bayer; and fees paid to the Charité from Abbot, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, Sanofi, Novartis, and Pfizer, all are outside the submitted work. T.T. reports personal fees and/or other from Bristol Myers Squibb, Pfizer, Bayer, Chugai Pharma, Novo Nordisk, Novartis, Daichii Sankyo, and LFB Pharma, all of which are outside the submitted work. L.S. receives a young investigator grant of the medical faculty of the Universitätsmedizin Greifswald and a Global Research Award of the American Society of Hematology. T.H. has received lecture honoraria and advisory fees from CSL Behring, Werfen, and Takeda. C.B. has received lecture honoraria from AbbVie; and travel grants from Pfizer and Sanofi outside the submitted work. T.E.W. has received lecture honoraria from Werfen (Instrumentation Laboratory), and royalties from Informa (Taylor & Francis); consulting service fees from Ergomed, Paradigm Pharmaceuticals, Octapharma, and Veralox Therapeutics; research funding from Werfen (Instrumentation Laboratory); and has provided expert witness testimony relating to heparin-induced thrombocytopenia (HIT) and non-HIT thrombocytopenic and coagulopathic disorders. The remaining authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Results of the 9 patients with VITT-like features (Table 1., Table 2.) with thrombocytopenia and thrombosis without proximate exposure to heparin or vaccination. Results of the rapid anti-PF4/heparin assay are given on the x-axis (cutoff: dashed vertical line). Results of new rapid anti-PF4 assay are given on the y-axis (cutoff: dashed horizontal line). Patient samples shown in green (patients 4, 7, and 8) were positive both in the PF4-dependent functional assay and a heparin-dependent functional assay, whereas patient samples in orange (patients 1, 2, 3, 5, and 6∗) exclusively tested positive in the PF4-dependent functional assay. Detail courses of patients 1, 2, and 8 were previously described. ∗Some samples of patient 6 from previous time points were weakly positive in the heparin-dependent functional assay and strongly positive in the PF4-dependent functional assay. Treatment of this patient after the time covered by this report is summarized in Lindhoff-Last et al.
Figure 2.
Figure 2.
Rapid assay results of patient sera with HIT, VITT, and negative controls. Results of the rapid anti-PF4/heparin assay are given on the x-axis (cutoff: dashed vertical line). Results of the new rapid anti-PF4 assay are given on the y-axis (cutoff: dashed horizontal line). Sera of patients with heparin-induced thrombocytopenia are shown in red (n = 131), VITT sera in blue (n = 103), and negative controls in gray symbols (n = 155). None of the 16 VITT sera reacting positive with the rapid anti-PF4/heparin assay (blue dots upper right quadrant) tested positive with 0.2 aFXaU heparin in the heparin-dependent platelet activation assay (12 undiluted; 4 at an informative serum dilution [giving negative buffer reactivity]).
Figure 3.
Figure 3.
Exploratory cohort of patients with strong reactivity in the in-house anti-PF4/heparin IgG EIA but negative heparin-dependent functional assay (n = 188). Results of the rapid anti-PF4/heparin assay are given on the x-axis (cutoff: dashed vertical line). Results of the new rapid anti-PF4 assay are given on the y-axis (cutoff: dashed horizontal line). Of these 188 patients, 40 (21.3%) tested positive by the new rapid anti-PF4 assay (upper left and right quadrant). Of those with sufficient available serum (n = 33, dark blue), 13 (39.4%) tested positive by PF4-dependent platelet activation assay (dark blue with red outline; 4 in the upper left quadrant and 9 in the upper right quadrant). ASA, acetylsalicyl acid; DVT, deep vein thrombosis; LMWH, low molecular weight heparin; PE, pulmonary embolism; VKA, vitamin K antagonists.
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