Studies of the anti–platelet factor 4/heparin immune response: adapting the enzyme‐linked immunosorbent spot assay for detection of memory B cells against complex antigens
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BACKGROUND: The anti–platelet factor 4 (PF4)/heparin immune response, which underlies heparin‐induced thrombocytopenia (HIT), has several atypical features: relatively rapid onset even without previous heparin exposure, lack of immune anamnesis, and transience of antibody production.STUDY DESIGN AND METHODS: We modified the enzyme‐linked immunosorbent spot (ELISPOT) assay to investigate for PF4/heparin‐specific memory B cells in cardiac surgery patients, in whom the high anti‐PF4/heparin immunization rate made a prospective study feasible. The PF4‐containing antigen complexes were attached to microtiter plates via a spacer, rather than using nitrocellulose, and the final reaction enzyme substrate was added in melted agarose which, after rapid hardening, localized color development of enzyme‐tagged anti‐immunoglobulin G (IgG) probes to single PF4/heparin‐specific B cells. This modified ELISPOT assay was applied to 58 consecutive patients (testing blood from preoperative baseline and Postoperative Days 6 and 10), in which we compared detectability of PF4/heparin‐specific B cells to tetanus toxin‐specific B cells (comparator group with presumed vaccination).RESULTS: No patient had detectable PF4/heparin‐specific memory B cells at baseline. In 2 of 30 patients (6.7%) who formed anti‐PF4/heparin IgG, PF4/heparin‐specific memory B cells (three to four spots/well) were detected by Postoperative Day 10, whereas tetanus toxin–specific memory B cells were found in 12 of 24 (50.0%) patients tested (3‐25 spots/well; p < 0.001).CONCLUSIONS: HIT lacks a strong memory B‐cell response, perhaps explaining transience and lack of anamnesis of the anti‐PF4/heparin immune response. The technical modifications we describe for the ELISPOT assay, which permit detection of B‐cell reactions to complex antigens, could be useful for studying other immunohematologic disorders, for example, drug‐dependent thrombocytopenia and acquired hemophilia.
