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A Platelet Factor 4-Dependent Platelet Activation Assay Facilitates Early Detection of Pathogenic Heparin-Induced Thrombocytopenia Antibodies

2017; Elsevier BV; Volume: 152; Issue: 4 Linguagem: Inglês

10.1016/j.chest.2017.06.001

1931-3543

Curtis G. Jones, Shannon M. Pechauer, Brian R. Curtis, Daniel W. Bougie, Mehraboon S. Irani, Binod Dhakal, Brenda M. Pierce, Richard H. Aster, Anand Padmanabhan,

Platelet Disorders and Treatments

Heparin-induced thrombocytopenia (HIT) is a dangerous complication of heparin therapy. HIT diagnosis is established by recognizing thrombocytopenia and/or thrombosis in an affected patient and from the results of serological tests such as the platelet factor 4 (PF4)/heparin immunoassay (PF4 ELISA) and serotonin release assay (SRA). Recent studies suggest that HIT antibodies activate platelets by recognizing PF4 in a complex with platelet glycosaminoglycans (and/or polyphosphates) and that an assay based on this principle, the PF4-dependent P-selectin expression assay (PEA), may be even more accurate than the SRA for HIT diagnosis. Here, we demonstrate that the PEA detected pathogenic antibodies before the SRA became positive in two patients with HIT studied serially, in one case even before seropositivity in the PF4 ELISA. In one of the patients treated with plasma exchange, persistent dissociation between the PEA and SRA test results was observed. These results support a role for the PEA in early HIT diagnosis. Heparin-induced thrombocytopenia (HIT) is a dangerous complication of heparin therapy. HIT diagnosis is established by recognizing thrombocytopenia and/or thrombosis in an affected patient and from the results of serological tests such as the platelet factor 4 (PF4)/heparin immunoassay (PF4 ELISA) and serotonin release assay (SRA). Recent studies suggest that HIT antibodies activate platelets by recognizing PF4 in a complex with platelet glycosaminoglycans (and/or polyphosphates) and that an assay based on this principle, the PF4-dependent P-selectin expression assay (PEA), may be even more accurate than the SRA for HIT diagnosis. Here, we demonstrate that the PEA detected pathogenic antibodies before the SRA became positive in two patients with HIT studied serially, in one case even before seropositivity in the PF4 ELISA. In one of the patients treated with plasma exchange, persistent dissociation between the PEA and SRA test results was observed. These results support a role for the PEA in early HIT diagnosis. HIT is characterized by thrombocytopenia, usually beginning 5 to 10 days after starting heparin therapy and poses a high risk of thromboembolic complications.1Cuker A. Management of the multiple phases of heparin-induced thrombocytopenia.Thromb Haemost. 2016; 116: 835-842Crossref PubMed Scopus (31) Google Scholar, 2McKenzie S.E. Sachais B.S. Advances in the pathophysiology and treatment of heparin-induced thrombocytopenia.Curr Opin Hematol. 2014; 21: 380-387Crossref PubMed Scopus (29) Google Scholar Patients with HIT produce antibodies that recognize the chemokine platelet factor 4 (PF4) in complex with heparin, or a similar negatively charged polyanion, and can be rapidly detected in immunoassays that use PF4: heparin (or polyvinylsulfonate) complexes as targets (PF4 enzyme-linked immunosorbent assay [ELISA]).3Visentin G.P. Moghaddam M. Beery S.E. McFarland J.G. Aster R.H. Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/thrombosis.J Lab Clin Med. 2001; 138: 22-31Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Amiral J. Bridey F. Dreyfus M. et al.Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia.Thromb Haemost. 1992; 68: 95-96Crossref PubMed Google Scholar, 5Kelton J.G. Smith J.W. Warkentin T.E. Hayward C.P. Denomme G.A. Horsewood P. Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4.Blood. 1994; 83: 3232-3239Crossref PubMed Google Scholar, 6Greinacher A. Potzsch B. Amiral J. Dummel V. Eichner A. Mueller-Eckhardt C. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen.Thromb Haemost. 1994; 71: 247-251PubMed Google Scholar Recent studies demonstrate that "pathogenic" (platelet-activating) HIT antibodies preferentially recognize PF4 in a complex with endogenous platelet surface glycosaminoglycans7Padmanabhan A. Jones C.G. Bougie D.W. et al.Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.Blood. 2015; 125: 155-161Crossref PubMed Scopus (63) Google Scholar and/or polyphosphates.8Cines D.B. Yarovoi S.V. Zaitsev S.V. et al.Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia.Blood Advances. 2016; 1: 62-74Crossref PubMed Google Scholar On the basis of these findings, we described a novel diagnostic test, the PF4-dependent P-selectin expression assay (PEA).9Padmanabhan A. Jones C.G. Bougie D.W. et al.A modified PF4-dependent, CD62p expression assay selectively detects serotonin-releasing antibodies in patients suspected of HIT.Thromb Haemost. 2015; 114PubMed Google Scholar, 10Padmanabhan A. Jones C.G. Curtis B.R. et al.A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis.Chest. 2016; 150: 506-515Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In the PEA, platelets pretreated with PF4 are incubated with samples from patients suspected of having HIT and platelet surface p-selectin expression is measured as a marker of platelet activation. Preliminary findings suggest that the PEA may be even more accurate than the SRA for diagnosis of HIT.10Padmanabhan A. Jones C.G. Curtis B.R. et al.A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis.Chest. 2016; 150: 506-515Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In the aforementioned study, stringent criteria were used to define a patient as having HIT (high 4Ts score (thrombocytopenia, timing, thrombosis, other) and PF4 ELISA optical density [OD] ≥ 1.0, or intermediate 4Ts score and PF4 ELISA OD ≥ 2.0), leaving little doubt that classification of patients as "HIT-positive" was accurate. Notably, HIT-positive patients who were PEA-positive but SRA-negative in that study tended to have lower levels of P-selectin expression than those who were positive in both assays. Mean (1 SD) P-selectin expression was 70% (33%) and 97% (10%) in SRA-/PEA+ and SRA+/PEA+ HIT-positive patients, respectively (P = .05 by the Mann-Whitney test; data not shown), and patient antibodies studied produced positive results in the PEA at higher dilutions than in the SRA,10Padmanabhan A. Jones C.G. Curtis B.R. et al.A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis.Chest. 2016; 150: 506-515Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar suggesting that the former assay may be inherently more sensitive for the detection of pathogenic platelet-activating antibodies. This led us to question whether the PEA may be particularly valuable for identifying patients with HIT early in their clinical course, before the SRA becomes positive. To investigate this possibility, we studied serial blood samples collected from two local patients who developed thrombotic HIT. Findings show that, in both patients, the PEA detected pathogenic HIT antibodies before the SRA, and in one case, earlier than the highly sensitive PF4 ELISA. Patient 1, a 67-year-old woman, was admitted for treatment of a Bochdalek hernia and was started on unfractionated heparin (UFH) for thrombosis prophylaxis. Her course was complicated by a declining platelet count suggestive of HIT. IgG-specific PF4 ELISA and SRA tests were strongly positive ∼180 h (7.5 days) after starting heparin (Fig 1A) and imaging demonstrated venous thrombosis in both upper and lower extremities. The patient was treated with argatroban, but her condition deteriorated further because of sepsis-associated multisystem organ failure and therapeutic plasma exchange (TPE) was instituted. Four TPE treatments led to a gradual improvement in platelet counts from 18,000/μL to 47,000/μL (Fig 1B). Unfortunately, the patient expired after the fourth TPE treatment from complications of multisystem organ failure. Patient 2, a 57-year-old man, was admitted for creation of a thigh flap in preparation for knee replacement. Postoperatively (day 0), the flap became pale with poor pulses and UFH was started. On day 5 after heparin exposure, platelets dropped 54% from the postoperative peak platelet ("baseline") count.11Warkentin T.E. Roberts R.S. Hirsh J. Kelton J.G. An improved definition of immune heparin-induced thrombocytopenia in postoperative orthopedic patients.Arch Intern Med. 2003; 163: 2518-2524Crossref PubMed Scopus (271) Google Scholar HIT was suspected and PF4 ELISA and SRA testing were performed, both of which were negative (Fig 2). A strong suspicion of HIT prompted the physician caring for the patient to repeat testing the next day and positive results were obtained in both the SRA and PF4 ELISA. An additional sample obtained ∼1 week later was also strongly positive in both assays (Fig 2). Thrombocytopenia persisted and extensive venous thrombosis of the right upper extremity was noted on day 16. At this time, a disseminated intravascular coagulation-like picture was present with fibrinogen of 190 mg/dL, D-dimer of 1.6 mg/L fibrinogen-equivalent units and prothrombin time of 23.5 s (international normalized ratio, 2.5). Partial thromboplastin time (on bivalirudin treatment) was 81.3 s (∼2.8× upper limit of normal). This picture may suggest "partial thromboplastin time confounding"12Warkentin T.E. Anticoagulant failure in coagulopathic patients: PTT confounding and other pitfalls.Expert Opin Drug Saf. 2014; 13: 25-43Crossref PubMed Scopus (65) Google Scholar and undertreatment with bivalirudin that possibly led to the thrombotic event. The patient was subsequently discharged on warfarin upon full recovery. IV immunoglobulin G treatment may have been beneficial in this patient with persistent HIT, as shown by our group recently in a separate study.13Padmanabhan A. Jones C.G. Pechauer S.M. et al.IVIg treatment of severe refractory heparin-induced thrombocytopenia.Chest. 2017; 152: 478-485Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Serial samples obtained from both patients were used to perform the PEA, SRA, and IgG-specific PF4 ELISA. Studies were approved by the institutional review board of the Medical College of Wisconsin (protocol PRO00023318). In patient 1 (Fig 1A), the PEA became positive at 152 h (6.4 days) after heparin exposure (69% P-selectin expression) when the SRA was negative (0% serotonin release). At this time, there was a significant drop in platelet count to > 50% of baseline (platelet count immediately before UFH treatment) and the PF4 ELISA was weakly positive at OD 0.67, a value generally not predictive of HIT.14Warkentin T.E. Sheppard J.I. Moore J.C. Sigouin C.S. Kelton J.G. Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays.J Thromb Haemost. 2008; 6: 1304-1312Crossref PubMed Scopus (277) Google Scholar The SRA was negative in two subsequent samples tested, both of which gave strongly positive PEA results (Fig 1A). The SRA turned positive (100% release) ∼30 h after the first PEA-positive test result (Fig 1A). Similarly, in patient 2, the SRA failed to detect a clinically significant antibody when HIT was first suspected because of a > 50% decline in platelet count 5 days after heparin exposure (Fig 2). The same sample was also negative in the PF4 ELISA (0.31 OD) but positive in the PEA (74%; Fig 2). A second sample collected the following day yielded positive results in the SRA (79%) and PF4 ELISA (2.8 OD) at a time when the PEA test result had increased to 105%. Because of the seemingly discrepant test results obtained with the first sample from patient 2, the sample was retested in the SRA and PF4 ELISA, but results (2% serotonin release and 0.532 OD) were comparable to those obtained initially. Findings made in these cases suggest that the PEA can identify patients with HIT early in the course of their disease at a time when the SRA and PF4 ELISA are negative/weakly positive. The significant drop in platelet count seen in both patients when the PEA first became positive suggests that truly pathogenic antibodies were being detected. Platelet counts decreased further in both patients after SRA and PF4 ELISA seroconversion occurred, likely reflecting increased HIT antibody titers. Of note, the same platelet preparation was used for PEA and SRA testing in patient 1, arguing against platelet donor variability as an explanation for different results obtained with the two assays. As in a previously reported case,15Warkentin T.E. Sheppard J.A. Chu F.V. Kapoor A. Crowther M.A. Gangji A. Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.Blood. 2015; 125: 195-198Crossref PubMed Scopus (58) Google Scholar the strength of SRA test results declined much more rapidly than those obtained with the PF4 ELISA over the course of TPE (Fig 1B). Interestingly, PEA test results tended to parallel those obtained with the PF4 ELISA in the TPE setting (Fig 1B). It is plausible that PEA-positive/SRA-negative antibodies detected during the course of TPE were pathogenic because such antibodies were also detected early in the patient's course when thrombocytopenia was developing. In summary, findings made in these cases suggest that the PEA can aid in early diagnosis of HIT. Repeat SRA/PF4 ELISA testing may be indicated in patients with initially negative serology if sampling occurred early in the course of the disease. Further studies are warranted to define the extent to which use of the PEA facilitates early HIT diagnosis and management. Author contributions: C. G. J., S. M. P., B. R. C., D. W. B., M. S. I., R. H. A., and A. P. were involved in study design. C. G. J. and S. M. P. performed the experiments described. B. D. and B. P. provided helpful suggestions and patient correlates. A. P. analyzed the data. R. H. A. provided advice on all aspects of the study. C. G. J. wrote the first draft of the manuscript, and all authors edited and approved the final version. A. P. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Financial/nonfinancial disclosures: The authors have reported to CHEST the following: A. P., C. G. J., D. W. B., and R. H. A. disclose that a patent application has been filed related to the PF4-dependent P-selectin expression assay (Method of Detecting Platelet-Activating Antibodies That Cause Heparin-Induced Thrombocytopenia/Thrombosis; PCT/US14/62591). None declared (S. M. P., B. R. C., M. S. I., B. D., B .P.). Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.