title: 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.
abstract: 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.
  
  
  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.
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt Universität, Greifswald, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Selleng
authors.forename: Antje
authors.initials: A
authors.lastname: Schütt
authors.forename: Sixten
authors.initials: S
authors.lastname: Selleng
authors.forename: Theodore E
authors.initials: TE
authors.lastname: Warkentin
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants / Q000009:adverse effects / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000302:isolation & purification; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D005260:Female; D006493:Heparin / Q000009:adverse effects* / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010978:Platelet Factor 4 / Q000276:immunology*; D013921:Thrombocytopenia / Q000139:chemically induced / Q000175:diagnosis / Q000276:immunology
chemical_list: D000925:Anticoagulants; D001323:Autoantibodies; D000939:Epitopes; D010978:Platelet Factor 4; D006493:Heparin