Background: Heparin-induced thrombocytopenia (HIT) is an iatrogenic immune-mediated prothrombotic disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Immunoglobulin G (IgG) antibodies bind to PF4/heparin complexes and cause Fc-receptor-mediated activation of platelets and monocytes. PF4 binds endogenous heparin-like polyanions to reveal cross-reactive epitopes that can also bind anti-PF4/heparin antibodies. Based on this observation, researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we utilized two assays: (1) a 3H-thymidine uptake assay to measure peripheral blood mononuclear cell (PBMC) proliferation in response to in vitro stimulation with PF4 and (2) a PBMC ELISPOT assay to measure the preoperative frequency of PF4-specific antibody-secreting cells. Proliferation was quantified as a stimulation index (SI). We then utilized a PF4/heparin-dependent enzyme immunoassay to measure the in vivo levels of anti-PF4/heparin antibodies produced by these patients in the postoperative period. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative polyspecific anti-PF4/heparin antibody production [Spearman’s ρ (95% CI) = –0.02 (–0.32, 0.28), P = 0.91]. PF4-dependent proliferation had a weak negative association with postoperative anti-PF4/heparin IgG antibody production [Spearman’s ρ (95% CI) = –0.31 (–0.56, –0.02), P = 0.04], but was not associated with postoperative IgM or IgA anti-PF4/heparin antibody production [IgM: Spearman’s ρ (95% CI) = –0.04 (–0.33, 0.26), P = 0.78; IgA: Spearman’s ρ (95% CI) = –0.05 (–0.34, 0.25), P = 0.73]. Qualitative analysis demonstrated that two patients who had the strongest preoperative PF4-dependent proliferation responses produced the highest postoperative levels of anti-PF4/heparin IgM antibodies, but this relationship was not observed with postoperative anti-PF4/heparin IgG antibodies. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin IgM or IgG antibodies [IgM: Spearman’s ρ (95% CI) = 0.30 (–0.79, 0.93), P = 0.683; IgG: Spearman’s ρ (95% CI) = –0.21 (–0.92, 0.83), P = 0.600]; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that PF4-dependent proliferation increases 5–6 days following cardiopulmonary bypass surgery [geometric mean (GM) postoperative PF4 alone proliferation (in SI) vs. GM preoperative PF4 alone proliferation (in SI) ± SEM: 23.7 ± 1.3 vs. 6.9 ± 1.5, P = 0.009]. Conclusions: Based on our findings, we conclude that preoperative PF4-dependent proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of cardiopulmonary bypass patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative PF4-specific ASC frequency and postoperative anti-PF4/heparin antibody production, but our findings would suggest that an association does not exist between these two variables in this patient cohort. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the postoperative HIT immune response. / Thesis / Master of Science (MSc) / Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Antibodies bind to PF4/heparin complexes and cause activation of platelets and monocytes. Researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we measured cellular proliferation in response to PF4 stimulation and the preoperative frequency of PF4-specific antibody-secreting cells. We also measured the level of anti-PF4/heparin antibodies following surgery. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative anti-PF4/heparin antibody production. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin antibodies; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that proliferation increases 5–6 days following cardiopulmonary bypass surgery. Conclusions: Based on our findings, we conclude that preoperative proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative ASC frequency and postoperative anti-PF4/heparin antibody production. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the HIT immune response.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21081 |
| Date | January 2017 |
| Creators | Staibano, Phillip |
| Contributors | Nazy, Ishac, Arnold, Donald, Bowdish, Dawn, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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