Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia.

Essentials The immunogenesis of Heparin-induced thrombocytopenia (HIT) is not well understood. Immunization to platelet factor 4 (PF4)-heparin occurs early in life, before any heparin exposure. PF4 and PF4-heparin complexes induce the proliferation of CD14+ cells. Reduced levels of regulatory cytokines contribute to immune dysregulation in HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4-heparin complexes occurs early in life, even before heparin exposure; however, the immunogenesis of HIT is not well characterized. Objectives To investigate cellular proliferation in response to PF4-heparin complexes in patients with HIT. Patients/Methods Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who had undergone cardiopulmonary bypass (CPB) (n = 17) and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4-heparin complexes. Cellular proliferation was assessed by [3 H]thymidine uptake and 5-ethynyl-2'-deoxyuridine detection. Results and Conclusions PBMCs proliferated in the presence of PF4, and this was enhanced by the addition of heparin in all study groups. CPB and HIT patients showed significantly greater proliferative responses than healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14+ cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines, and both CPB and HIT patients produced significantly lower levels of interleukin-10 and transforming growth factor-ß1 than healthy controls. These findings further demonstrate cellular immune sensitization to PF4-heparin complexes occurs before heparin exposure, and suggests immune dysregulation can contribute to HIT.

Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced thrombocytopenia.

BACKGROUND: Many patients exposed to heparin develop antibodies against platelet factor 4 (PF4) and heparin, yet only those antibodies that activate platelets cause heparin-induced thrombocytopenia (HIT). Patients who produce anti-PF4/heparin antibodies without developing HIT either have antibodies that do not cause platelet activation or produce pathogenic antibodies at levels that are insufficient to cause HIT. Understanding the differences between anti-PF4/heparin antibodies with and without HIT will improve test methods and reduce overdiagnosis. AIMS: To investigate the presence of low levels of platelet-activating antibodies in patients investigated for HIT who had anti-PF4/heparin antibodies but failed to cause platelet activation in the (14) C-serotonin release assay (SRA). MATERIALS/METHODS: We developed a platelet activation assay similar to the SRA using exogenous PF4 without added heparin (PF4-SRA). This assay was able to detect low levels of platelet-activating antibodies. We used this PF4-SRA to test for platelet-activating antibodies in patients investigated for HIT. RESULTS: The PF4-SRA detected platelet-activating antibodies in seven (100%) of seven SRA-positive sera even after the samples were diluted until they were no longer positive in the standard SRA. Platelet-activating antibodies were detected in 14 (36%) of 39 patients who had anti-PF4/heparin antibodies but tested negative in the SRA and did not have clinical HIT. The clinical diagnosis of HIT was confirmed by chart review and concordant with the SRA results. CONCLUSIONS: A subset of heparin-treated patients produce subthreshold levels of platelet-activating anti-PF4/heparin antibodies that do not cause HIT. An increase in the titer of these pathogenic antibodies, along with permissive clinical conditions, could lead to HIT.

Severe bleeding events in adults and children with primary immune thrombocytopenia: a systematic review.

BACKGROUND: The burden of severe bleeding in adults and children with immune thrombocytopenia (ITP) has not been established. OBJECTIVES: To describe the frequency and severity of bleeding events in patients with ITP, and the methods used to measure bleeding in ITP studies. PATIENTS/METHODS: We performed a systematic review of all prospective ITP studies that enrolled 20 or more patients. Two reviewers searched Medline, Embase, CINAHL and the Cochrane registry up to May 2014. Overall weighted proportions were estimated using a random effects model. Measurement properties of bleeding assessment tools were evaluated. RESULTS: We identified 118 studies that reported bleeding (n = 10 908 patients). Weighted proportions for intracerebral hemorrhage (ICH) were 1.4% for adults (95% confidence interval [CI], 0.9-2.1%) and 0.4% for children (95% CI, 0.2-0.7%; P < 0.01), most of whom had chronic ITP. The weighted proportion for severe (non-ICH) bleeding was 9.6% for adults (95% CI, 4.1-17.1%) and 20.2% for children (95% CI, 10.0-32.9%; P < 0.01) with newly-diagnosed or chronic ITP. Methods of reporting and definitions of severe bleeding were highly variable in primary studies. Two bleeding assessment tools (Buchanan 2002 for children; Page 2007 for adults) demonstrated adequate inter-rater reliability and validity in independent assessments. CONCLUSIONS: ICH was more common in adults and tended to occur during chronic ITP; other severe bleeds were more common in children and occurred at all stages of disease. Reporting of non-ICH bleeding was variable across studies. Further attention to ITP-specific bleeding measurement in clinical trials is needed to improve standardization of this important outcome for patients.

FcγRIIa proteolysis as a diagnostic biomarker for heparin-induced thrombocytopenia.

BACKGROUND: A significant challenge in the management of heparin-induced thrombocytopenia (HIT) patients is making a timely and accurate diagnosis. The readily available enzyme immunoassays (EIAs) have low specificities. In contrast, platelet activation assays have higher specificities, but they are technically demanding and not widely available. In addition, ~ 10% of samples referred for HIT testing are initially classified as indeterminate by the serotonin release assay (SRA), which further delays accurate diagnosis. HIT is characterized by platelet activation, which leads to FcγRIIa proteolysis. This raises the possibility that identification of the proteolytic fragment of FcγRIIa could serve as a surrogate marker for HIT. OBJECTIVES: To determine the specificity of platelet FcγRIIa proteolysis induced by sera from patients with HIT, and to correlate the results with those of the SRA. METHODS/PATIENTS: Sera from HIT patients and control patients with other thrombocytopenic/prothrombotic disorders were tested for their ability to proteolyse FcγRIIa. The results were correlated with anti-platelet factor 4 (PF4)/heparin antibodies (EIA), and heparin-dependent platelet activation (SRA). RESULTS: Only HIT patient samples (20/20) caused heparin-dependent FcγRIIa proteolysis, similar to what was shown by the SRA. None of the samples from the other patient groups or hospital controls caused FcγRIIa proteolysis. Among nine additional samples that tested indeterminate in the SRA, FcγRIIa proteolysis resolved five samples that had a positive anti-PF4/heparin EIA result; three had no FcγRIIa proteolysis, and two were shown to have heparin-dependent FcγRIIa proteolysis CONCLUSIONS: This study suggests that heparin-dependent FcγRIIa proteolysis is at least as specific as the SRA for the diagnosis of HIT.

A systematic evaluation of laboratory testing for drug-induced immune thrombocytopenia.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) can be confirmed by the demonstration of drug-dependent platelet antibodies in vitro; however, laboratory testing is not readily accessible and test methods are not standardized. OBJECTIVE: To identify drugs with the strongest evidence for causing DITP based on clinical and laboratory criteria. PATIENTS/METHODS: We developed a grading system to evaluate the quality of DITP laboratory testing. The 'DITP criteria' were: (i) Drug (or metabolite) was required for the reaction in vitro; (ii) Immunoglobulin binding was demonstrated; (iii) Two or more laboratories obtained positive results; and (iv) Platelets were the target of immunoglobulin binding. Laboratory diagnosis of DITP was considered definite when all criteria were met and probable when positive results were reported by only one laboratory. Two authors applied the DITP criteria to published reports of each drug identified by systematic review. Discrepancies were independently adjudicated. RESULTS: Of 153 drugs that were clinically implicated in thrombocytopenic reactions, 72 (47%) were associated with positive laboratory testing. Of those, 16 drugs met criteria for a definite laboratory diagnosis of DITP and thus had the highest probability of causing DITP. Definite drugs were: quinine, quinidine, trimethoprim/sulfamethoxazole, vancomycin, penicillin, rifampin, carbamazepine, ceftriaxone, ibuprofen, mirtazapine, oxaliplatin and suramin; the glycoprotein IIbIIIa inhibitors abciximab, tirofiban and eptifibatide; and heparin. CONCLUSIONS: We identified drugs with the strongest evidence for an association with immune thrombocytopenia. This list may be helpful for ranking potential causes of thrombocytopenia in a given patient.

Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays.

BACKGROUND: Many laboratories test for heparin-induced thrombocytopenia (HIT) using a PF4-dependent enzyme-immunoassay (EIA). An advantage of the EIA is its simplicity; a disadvantage is that it only indirectly detects heparin-dependent, platelet-activating antibodies ('HIT antibodies'). OBJECTIVES: To determine whether the magnitude of a positive EIA result, expressed in optical density (OD) units, predicts risk of HIT antibodies, defined as a strong-positive platelet serotonin-release assay (SRA) result (>or=50% serotonin release). PATIENTS/METHODS: We determined the risk of a strong-positive SRA result for five categories of OD reactivity (<0.40, 0.40-<1.00, 1.00-<1.40, 1.40-<2.00, and >or=2.00 OD units) using two EIAs (commercial anti-PF4/polyanion IgG/A/M and in-house anti-PF4/heparin-IgG). RESULTS: For patient sera investigated for HIT antibodies, a weak-positive result (0.40-<1.00 OD units) in either EIA indicated a low probability (or= 2.00 units. Quantifying the EIA-SRA relationship for 1553 referred patient sera, we found that for every increase of 0.50 OD units in the EIA-IgG, the risk of a strong-positive SRA result increased by OR = 6.39 [95% confidence interval (CI), 5.13, 7.95; P < 0.0001]. For every increase of 1.00 OD units in the EIA-IgG, the risk increased by OR = 40.81 (95% CI, 26.35, 63.20; P < 0.0001). CONCLUSIONS: The probability of HIT antibodies (strong-positive SRA result) inferred by a positive PF4-dependent EIA varies considerably in relation to the magnitude of the EIA result, expressed as OD values. In our laboratory, the probability of HIT antibodies being present reached >or=50% only when the OD level was >or=1.40 units.

Increased mortality in hemodialysis patients having specific antibodies to the platelet factor 4-heparin complex.

Heparin-induced thrombocytopenia is a serious complication of heparin therapy that can lead to thromboembolism, cardiovascular events, and death. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing antibodies to the platelet factor 4-heparin (PF4-H) complex. We sought to determine the association between PF4-H antibodies and mortality in a prospective cohort of 419 asymptomatic hemodialysis patients. Pre-dialysis blood samples were screened for nonspecific PF4-H antibodies, and all positive and indeterminate samples were subsequently tested using immunoglobulin (Ig)G-specific PF4-H and platelet serotonin-release assays. During a median follow-up of 2.5 years there were 129 all-cause deaths. After controlling for potential confounding variables, the relative risk of death was significantly increased for patients with IgG-specific PF4-H antibodies and further elevated with an indeterminate serotonin-release assay. Our study suggests that IgG-specific PF4-H antibody formation is associated with increased mortality in hemodialysis patients.

Intravenous immunoglobulin as an adjunct to plasma exchange for the treatment of chronic thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease. Therapeutic plasma exchange (TPE) is the most effective therapy; however, despite TPE, about one-third of TTP patients will relapse. A subset of patients with TTP has antibodies to ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) and may become resistant to conventional treatments. We describe a patient with TTP and high-titre anti-ADAMTS13 antibodies who developed a chronic, relapsing course of TTP despite frequent TPE. Once adjuvant treatment with intravenous immunoglobulin (IVIG) was added, remission was achieved. Even during remission, anti-ADAMTS13 antibodies remained elevated. We conclude that IVIG may sustain remission in some patients with chronic, relapsing TTP.

Assessing the effectiveness of whole blood-derived platelets stored as a pool: a randomized block noninferiority trial.

BACKGROUND: Prestorage pooling of whole blood-derived platelets (PLTs) would simplify bacterial detection. This study evaluated the in vivo effect of the prestorage pooling of PLTs stored for up to 5 days, by assessing the corrected count increment (CCI) 18 to 24 hours after transfusion of the product. STUDY DESIGN AND METHODS: A randomized block noninferiority design was used. Eligible patients had chemotherapy-induced thrombocytopenia and were considered likely to need at least six PLT transfusions. For every block of two transfusion events, one consisted of PLTs stored individually and then pooled before transfusion, and the other was a product pooled before storage. The primary outcome was categorized as a successful (>4.5) or unsuccessful (

In vitro evaluation of prestorage pooled leukoreduced whole blood-derived platelets stored for up to 7 days.

BACKGROUND: Advantages to storing whole blood-derived platelets (PLTs) as a pool for 7 days would include operational efficiencies and facilitation of bacterial testing and pathogen inactivation. The in vitro quality of pre-storage pooled PLTs stored for up to 7 days was assessed. STUDY DESIGN AND METHODS: Leukoreduced PLTs were pooled before storage (5 units/pool) and stored for either 5 or 7 days. Samples were collected at the time of pooling and either on Day 5 (n=16-29) or on Day 7 (n=4-30) and tested for biochemical and activation markers and morphology and/or shape change. Control PLTs were stored individually for 5 or 7 days and then tested as indicated above. RESULTS: The mean PLT counts (x10(9)/L) were similar: control PLTs, 1344 (464 SD); and prestorage pooled PLTs, 1327 (220 SD; p=0.93). On Day 5, the pH value was significantly lower (p

Heparin-induced thrombocytopenia: an overview.

Heparin-induced thrombocytopenia (HIT) is the most important immunological drug reaction that patients face today. HIT typically develops in patients 5 days after starting heparin therapy, but can occur sooner with recent heparin exposure or rarely have a delayed onset. The platelet count typically drops below 150 x 10(9)/L (average 60 x 10(9)/L), and patients may experience a thrombotic episode simultaneously or shortly after the onset of thrombocytopenia. The thrombocytopenia and the associated thrombotic episodes are now considered to be overlapping outcomes of the same syndrome. The pathophysiology of HIT has been characterized: immune complexes of IgG and heparin in association with a small platelet peptide, platelet factor 4 (PF4), activate platelets by binding to the Fc receptors (FcR) and releasing procoagulant-active, platelet-derived microparticles. The recognition that HIT is characterized by intense thrombin generation dictates the use of antithrombin agents in HIT therapy. Therapeutic approaches that are currently prevalent in the management of HIT will be discussed.

Delayed-onset heparin-induced thrombocytopenia and thrombosis.

BACKGROUND: Heparin-induced thrombocytopenia is a prothrombotic drug reaction caused by platelet-activating antibodies that recognize complexes of platelet factor 4 and heparin. OBJECTIVE: To describe a syndrome termed delayed-onset heparin-induced thrombocytopenia, in which thrombocytopenia and thrombotic events begin 5 or more days after withdrawal of heparin. DESIGN: Case series. SETTING: Secondary and tertiary care hospitals. PATIENTS: 12 patients who presented with serologically confirmed, delayed-onset heparin-induced thrombocytopenia, including 6 outpatients presenting after hospital discharge. MEASUREMENTS: The platelet serotonin-release assay was used to measure IgG-induced heparin-dependent and heparin-independent platelet activation; an enzyme immunoassay that detects IgG against platelet factor 4-heparin complexes was also used. RESULTS: Patients with delayed-onset heparin-induced thrombocytopenia presented with thrombocytopenia and associated thrombosis a mean of 9.2 days (range, 5 to 19 days) after stopping heparin therapy. Nine patients received additional heparin, with further decrease in platelet counts. Compared with controls, patients with delayed-onset heparin-induced thrombocytopenia had higher titers of IgG antibodies to platelet factor 4-heparin and greater IgG-induced heparin-dependent and heparin-independent platelet activation. CONCLUSIONS: Delayed-onset heparin-induced thrombocytopenia should be suspected when patients present with thrombocytopenia and thrombosis up to 3 weeks after exposure to heparin. This syndrome could be caused by high titers of platelet-activating IgG induced by heparin.

Decreased von Willebrand factor protease activity associated with thrombocytopenic disorders.

Recent studies investigating thrombotic thrombocytopenic purpura (TTP) have implicated abnormal plasma von Willebrand factor (vWF)-cleaving metalloprotease activity in this disorder. It has been proposed that a metalloprotease cleaves unusually large (UL) multimers of vWF, which enter the circulation from the endothelium. Abnormal metalloprotease activity could result in ULvWF, which could participate in TTP. However, the diagnostic specificity of abnormalities in the plasma metalloprotease activity has not been established. A prospective study of vWF protease activity was performed using samples from 20 healthy controls, 20 patients with acute TTP, 20 patients with immune idiopathic thrombocytopenic purpura (ITP), 10 patients with disseminated intravascular thrombocytopenia (DIC), 10 patients with systemic lupus erythematosus (SLE,) and 5 thrombocytopenic patients with leukemia. Studies were performed blinded to the diagnosis. Samples from hospitalized patients with normal platelet counts were also tested. The vWF digests and multimer analysis were done using previously described methods. Six laboratory personnel independently scored each of the multimer gels. Reduced protease activity was observed in 9 of 20 patients with TTP. Reduced activity was also observed in 6 of 20 patients with ITP, 6 of 10 patients with DIC, 5 of 10 patients with SLE, 1 of 5 patients with leukemia, 2 of 20 healthy controls, and 3 of 25 hospitalized patients. This study indicates that abnormalities of vWF protease activity are not restricted to patients with the diagnosis of TTP.

Argatroban anticoagulant therapy in patients with heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome caused by heparin. Complications range from thrombocytopenia to thrombocytopenia with thrombosis. We report a prospective, historical- controlled study evaluating the efficacy and safety of argatroban, a direct thrombin inhibitor, as anticoagulant therapy in patients with HIT or HIT with thrombosis syndrome (HITTS). METHODS AND RESULTS: Patients with HIT (isolated thrombocytopenia, n=160) or HITTS (n=144) received 2 microgram. kg(-1). min(-1) IV argatroban, adjusted to maintain the activated partial thromboplastin time 1.5 to 3.0 times baseline value. Treatment was maintained for 6 days, on average. Clinical outcomes over 37 days were compared with those of 193 historical control subjects with HIT (n=147) or HITTS (n=46). The incidence of the primary efficacy end point, a composite of all-cause death, all-cause amputation, or new thrombosis, was reduced significantly in argatroban-treated patients versus control subjects with HIT (25.6% versus 38.8%, P=0.014). In HITTS, the composite incidence in argatroban-treated patients was 43.8% versus 56.5% in control subjects (P=0.13). Significant between-group differences by time-to-event analysis of the composite end point favored argatroban treatment in HIT (P=0.010) and HITTS (P=0.014). Argatroban therapy, relative to control subjects, also significantly reduced new thrombosis and death caused by thrombosis (P<0.05). Argatroban-treated patients achieved therapeutic activated partial thromboplastin times generally within 4 to 5 hours of starting therapy and, compared with control subjects, had a significantly more rapid rise in platelet counts (P=0.0001). Bleeding events were similar between groups. CONCLUSIONS: Argatroban anticoagulation, compared with historical control subjects, improves clinical outcomes in patients who have heparin-induced thrombocytopenia, without increasing bleeding risk.

Temporal aspects of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia is a relatively common antibody-mediated drug reaction. We studied the temporal relation between previous or current heparin therapy and the onset of heparin-induced thrombocytopenia. METHODS: We examined the time between the start of heparin therapy and the onset of thrombocytopenia in 243 patients with serologically confirmed heparin-induced thrombocytopenia. We also investigated the persistence of circulating heparin-dependent antibodies by performing a platelet serotonin-release assay and an assay for antibodies against platelet factor 4. The outcome in seven patients who had previously had an episode of heparin-induced thrombocytopenia and were later treated again with heparin was also examined. RESULTS: A fall in the platelet count beginning four or more days after the start of heparin therapy occurred in 170 of the 243 patients (70 percent); in these patients, a history of previous heparin treatment did not influence the timing of the onset of thrombocytopenia. In the remaining 73 patients (30 percent), the onset of thrombocytopenia was rapid (median time of onset, 10.5 hours after the start of heparin administration); all these patients had been treated with heparin within the previous 100 days. During recovery from thrombocytopenia, heparin-dependent antibodies in the serum fell to undetectable levels at a median of 50 to 85 days, depending on the assay performed. In the seven patients who were given heparin again after the disappearance of heparin-dependent antibodies, a new episode of heparin-induced thrombocytopenia did not occur. CONCLUSIONS: Heparin-induced thrombocytopenia can begin rapidly in patients who have received heparin within the previous 100 days. Heparin-dependent antibodies do not invariably reappear with subsequent heparin use.