期刊
TRANSFUSION
卷 50, 期 1, 页码 32-39出版社
WILEY
DOI: 10.1111/j.1537-2995.2009.02360.x
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资金
- German Federal Ministry for Education and Research [CAN04/006]
- Landesforderungsprogramm Mecklenburg-Vorpommern EFRE
- Alfried Krupp von Bohlen und Halbach-Stiftung
- Forschungsverbund Molekulare Medizin of the Ernst-Moritz-Arndt-University Greifswald [FOMM 2007-06]
- Heart and Stroke Foundation of Ontario [T6157]
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.
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