背景:疫苗诱导的免疫性血栓性血小板减少症(VITT)是基于腺病毒的针对SARS-COV-2的疫苗的并发症,这是由于针对血小板因子4(PF4)的血栓前IgG抗体,在接受肝素治疗的患者中,可能难以与肝素诱导的血小板减少症(HIT)区分。
目的:在这种情况下,我们评估了竞争性抗PF4酶免疫测定(EIA)的有用性。
方法:1E12,1C12和2E1的F(ab\')2片段,3单克隆抗PF4抗体的能力,使用EIA评估抑制人VITT或HIT抗体与PF4的结合。进行丙氨酸扫描诱变以确定参与单克隆抗体和PF4之间相互作用的氨基酸(AA)。
结果:用1E12测量VITTIgG与PF4结合的强抑制作用(中位抑制作用93%,n=8),而它对HIT抗体的结合没有影响(中位数:6%,n=8)。相比之下,1C12和2E1抑制VITT(中位数:74%和76%,分别)和HIT抗体(中位数:68%和53%,分别)与PF4结合。当用1E12对19个额外的VITT样品进行竞争性抗PF4EIA时,它强烈抑制IgG与PF4的结合,除了一个病人,根据临床病史,谁实际上发展了HIT。表位图谱显示,1E12与PF4上的5个关键AA相互作用,其中4个也是人类VITT抗体结合所必需的,从而解释了竞争性抑制。
结论:使用1E12的简单竞争性抗PF4EIA可以帮助确认VITT诊断,并在两种诊断都可能的情况下将其与患者的HIT区分开。
BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin.
OBJECTIVE: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context.
METHODS: The ability of F(ab\')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4.
RESULTS: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition.
CONCLUSIONS: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.