Heparin-platelet Factor Research Articles

Successful Use of Bivalirudin for Combined Carotid Endarterectomy and Coronary Revascularization with the Use of Cardiopulmonary Bypass in a Patient with an Elevated Heparin-Platelet Factor 4 Antibody Titer

We report a combined carotid endarterectomy and coronary revascularization surgery with cardiopulmonary bypass using bivalirudin for systemic anticoagulation in a patient with a positive titer for the heparin-platelet factor 4 antibody. The patient experienced procedural success for both the carotid and coronary surgeries. Increased blood and blood product transfusion was required postoperatively.

Prevalence of heparin-induced antibody in African-American hemodialysis patients – comparison to non-dialysis patients

Heparin-induced antibody formation and thrombocytopenia are well-established complications of heparin use and may be associated with thromboembolic events. Our study aimed to establish the prevalence of heparin-induced antibodies (HIA), otherwise known as platelet factor 4 (PF4) antibodies, and their relationship to thrombocytopenia and thrombotic events in a group of predominantly African-American hemodialysis patients over 12 months. For comparison, the prevalence of HIA in thrombocytopenic patients with serum creatinine of less than 2.0 mg/dl tested for the antibody in the same period was evaluated. 36 hemodialysis patients on thrice-weekly, in-center treatments receiving heparin on dialysis were selected to have blood drawn predialysis for PF4 heparin antibodies (HIA). Complete blood counts (CBC) were also checked that day and monthly for the next 3 months. Outpatient records were reviewed for thromboembolic events, including thrombosis of dialysis access lines, grafts and fistulas. The HIA results for the dialysis patients were compared with HIA performed at the same lab for non-dialysis thrombocytopenic patients during the same period. 17 out of 36 (47%) hemodialysis patients had detectable HIA. There were no differences in gender, ethnicity, disease states, or duration of hemodialysis. None of the patients had thrombocytopenia, and the incidence of thromboembolic events was statistically not different from patients without HIA. The non-CKD thrombocytopenic group had a lower incidence, 16/88 (18%) of positive HIA. Heparin-induced antibodies are relatively common in patients receiving hemodialysis. However, their presence does not appear to be associated with clinical consequences. Therefore, testing for HIA should be done only if a clinical indication is present.

Heparin-induced Thrombocytopenia in Two Patients Undergoing Abdominal Aortic Aneurysm Surgery

We have experienced 2 cases of heparin-induced thrombocytopenia during unfractionated heparin treatment for disseminated intravascular coagulation after surgery for an abdominal aortic aneurysm. In the first case, as a symptom of disseminated intravascular coagulation gradually improved with antithrombin concentrates and heparin treatment, mesenteric artery thrombosis suddenly occurred, associated with a >50% decrease in platelet count on the 11th day. Although the platelet counts were increasing due to heparin cessation, clinical symptom and coagulation abnormalities worsened to multiple organ failure. In the second case, the platelet count decreased to <10 x 10(4)/microL on the 13th day after the start of unfractionated heparin anticoagulation along with continuous hemodiafiltration, which was indicated for postoperative renal failure. The extracorporeal circuit clotted frequently under an adequate dose of unfractionated heparin. Serologically, heparin-platelet factor 4 complex antibodies were repeatedly detected by enzyme-linked immunosorbent assay. Argatroban, a direct thrombin inhibitor, was introduced as an alternative to unfractionated heparin, and the platelet count improved with a decrease in titers of the antibodies. Disseminated intravascular coagulation is a common complication in cases of abdominal aortic aneurysm and is usually treated in association with unfractionated heparin. It is important to recognize the onset of heparin-induced thrombocytopenia that acute declines in the platelet count and appearance of thrombosis with positive for heparin-platelet factor 4 complex antibodies would suddenly occur in clinical course of disseminated intravascular coagulation.

Heparin-induced thrombocytopenia with thrombosis syndrome managed with plasmapheresis

Heparin-induced thrombocytopenia with thrombosis syndrome is an antibody-mediated disorder that has a high mortality in cardiac surgical patients in spite of early diagnosis and management with direct thrombin inhibitors. Plasmapheresis, an extracorporeal technique that has been designed for the removal of large molecular weight substances from the plasma, can remove the offending antibodies from these desperately ill patients. We describe a case of a postoperative cardiac surgery patient with heparin-induced thrombocytopenia with thrombosis syndrome and multi-system failure who was dependent upon a left ventricular assist device. He was treated successfully with plasmapheresis with recovery of his platelet count from 25,000/microl to over 200,000/microl, along with multi-organ recovery. This patient survived because of plasmapheresis. Removing the antibodies to the heparin-platelet factor four complex with plasmapheresis is an effective strategy to treat these patients. We believe that the use of plasmapheresis as a bail-out procedure in these often desperately ill post-operative cardiac surgical patients who have heparin-induced thrombocytopenia with thrombosis syndrome could be lifesaving.

A Case of Acute Myocardial Infarction With Repetitive Stent Thrombosis During Emergent Percutaneous Coronary Intervention Transient Decrease in Antithrombin III Activity and Heparin Resistance

A 59-year-old woman was admitted to our hospital for the treatment of an acute anterior myocardial infarction. She had a history of uncontrolled diabetes mellitus, hypertension, hyperlipidemia, obesity, and smoking. Coronary angiography revealed 90% stenosis with spontaneous dissection in the proximal portion of the left anterior descending artery. At this time, heparin was initiated for the first time. Although direct stenting (Be-stent, 3.0-18 mm) was performed for the culprit lesion, coronary dissection occurred at the left main trunk and additional stenting (Multi Link ZETA stent 3.5-15mm) was performed for the left main trunk. Soon after stenting, repetitive stent thrombosis occurred. Aspiration of the thrombus using an aspiration catheter was ineffective and repetitive angioplasty and intraaortic balloon pumping were required. Although we used 17,000 units of unfractionated heparin during the intervention, the activated coagulation time (ACT) was not prolonged (157 seconds). In the coronary care unit, the ACT and activated partial prothrombin time (aPTT) were not prolonged despite the use of large amounts of heparin (69,000 units in 2 days). Protein-S, protein-C, and hepaplastin testing were within normal limits and heparin-platelet factor IV complex antibody was not detected. In the acute phase, a decrease in the antithrombin III activity (65%) was noted and with administration of argatroban, prolongation of the aPTT was achieved. In the chronic phase, the decrease in antithrombin III activity and heparin resistance had improved spontaneously. It is important to recognize the existence of transient decreases in antithrombin III activity in the acute phase of myocardial infarction.

Polymorphisms in TNFA and CTLA-4 Genes and the Risk of Heparin Induced Thrombocytopenia.

Backgroud and objectives: The formation of antibodies (Abs) to heparin platelet factor 4 complexes (H/PF4) associated with heparin-induced thrombocytopenia (HIT) is a T-helper cell dependent event that involves antigen presenting cells (APC) and B-lymphocytes. Polymorphisms of the CTLA-4 (cytotoxic T lymphocyte antigen 4) gene have been described as a risk factor in several autoimmune diseases. In addition, TNFα is a major inflammatory cytokine with potent regulatory functions and polymorphisms in TNFA have also been associated with autoimmune antibody-mediated diseases. We therefore evaluated the possibility that an association between polymorphisms in CTLA-4 (−318 C/T and +49 A/G) or TNFA (−308 G/A) and the development of Abs to H/PF4 and HIT might exist.Methods: Eighty-three patients identified as having developed definite HIT with significant levels of Abs to PVS/PF4 in ELISA (HAT 45, GTI, Brookfield, WI, USA) and positive serotonin release assay were studied (HIT group). Two control groups were studied: the Abneg group consisted of 83 patients who had undergone heart surgery with high doses of unfractionated heparin administered during cardiopulmonary bypass (CPB), and who were tested negative for Abs to PVS/PF4 at the 8th post operative day. The Abpos group consisted of 58 patients who had also undergone CPB but had developed high levels of Abs to PVS/PF4 without significant change in the platelet count post-operatively. Three single nucleotide polymorphisms (SNPs), one in TNFA (−308G/A) and two in CTLA-4 (−318 C/T and +49A/G) were studied by conventional RFLP analysis as described (Astermark et al, Blood 2006 and Astermark et al, Thromb Haemost 2007).Results: The CTLA-4 +49 A/G and −318 C/T genotypes and allele distributions were similar in the 3 groups of patients (Table). In contrast, the frequency of TNFA –308 G/G homozygotes was higher in the HIT group compared to patients without HIT whether they had developed PF4-specific Abs or not (p=0.035). Therefore, the A allele was less frequent in HIT patients (p=0.026, OR 0.49; CI95% 0.26–0.93) but there was no significant difference when comparing patients with and without PF4-dependent antibodies.Genotype Allele frequencyAbneg (n = 82)Abpos (n = 58)HIT (n = 82)CTLA-4(+49)A/A31 (38%)24 (41%)35 (43%)A/G40 (49%)26 (45%)41 (50%)G/G11 (13%)8 (14%)6 (7%)CTLA-4(−318)C/C63 (77%)49 (84%)67 (82%)C/T19 (23%)9 (16%)17 (21%)T/T000 (0%)TNFα(−308)G/G59 (72%)41 (71%)68 (84%)G/A20 (24%)15 (26%)12 (15%)A/A3 (4%)2 (3%)1 (1%)A Allele0.1600.1600.09G Allele0.8400.8400.910Conclusion: The TNFA –308 A allele appears to be protective regarding the risk of heparin-induced thrombocytopenia in patients having developed PF4-specific antibodies. A similar effect has been suggested in immune thrombocytopenic purpura (Foster et al, Brit J Haematol 2001) despite individuals with this allele have been identified as high TNFα producers. Therefore, the mechanisms involved for explaining this apparent protective effect of the TNFA −308A allele in patients at risk for HIT have to be identified.

Adherence to Guidelines for Monitoring for Heparin-Induced Thrombocytopenia in Patients Treated with Low Molecular Weight Heparin

Laboratory monitoring for early detection of adverse drug reactions is recommended for many drugs. For patients treated with low molecular weight heparin (LMWH), the Summary of Product Characteristics (SPC) and clinical guidelines recommend to monitor the platelet count for heparin-induced thrombocytopenia (HIT), a potentially life-threatening adverse event, characterised by a typical drop in platelet count. When the platelet count drops without obvious explanation in these patients, testing for heparin-platelet factor 4 antibodies (HPF4-Ab) and initiating alternative anticoagulation are advised. In the current study adherence to recommended platelet count monitoring in clinical patients without thrombocytopenia-associated diseases treated with LMWH for at least five days at our institution, and adherence to recommended testing for HPF4-Ab and initiation of alternative anticoagulation in patients with potential HIT (defined as a drop of at least 50% in platelet count between days 5 and 14 following the start of LMWH treatment, or stopdate, whichever occurred first, compared to the highest platelet count within days 1–4) were investigated. Data from the Utrecht Patient Oriented Database (UPOD) were used for this retrospective cohort study. Inpatients exposed to the LMWHs dalteparin or nadroparin for at least five days during the period 2004–2005 were included. Patients with thrombocytopenia-related diseases were excluded. Firstly, adherence to recommended platelet count monitoring, based on recommendations from SPCs and clinical guidelines, was investigated. Secondly, the association between patient- and treatment characteristics and obtaining at least 2 platelets counts during treatment was investigated. Thirdly, adherence to recommended testing for HPF4-Ab and initiating treatment with danaparoid was investigated in patients with potential HIT. 6,804 patients with 7,770 episodes of LMWH treatment of at least five days were included. Adherence to the recommendations for platelet count monitoring from the SPC of nadroparin and dalteparin was 36.5% and 26.3% respectively. Adherence to the different platelet count monitoring recommendations from the 2002 clinical guideline on HIT was 23.0% and 41.5%. Obtaining at least 2 platelet counts during treatment was found to be strongly associated with ICU admission, previous UFH exposure, and a treatment duration of at least 10 days. There were 98 patients with potential HIT. Adherence to testing for HPF4-Ab in patients with potential HIT was 6.1%. Adherence to starting alternative anticoagulation in patients with potential HIT treatment was 0%. The results of this study suggest that adherence to recommendations for monitoring for HIT with LMWH is low at our institution. The results of this study justify to say that there is a need to think of appropriate actions for improving the awareness of HIT as an adverse reaction to LMWH, and to secure the safe use of LMWH.

Heparin-induced thrombocytopenia--therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4-heparin complexes.

Treatment of heparin-induced thrombocytopenia (HIT), a disorder in which anti-platelet factor 4 (PF4)-heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non-heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin-mimicking pentasaccharide). PF4-heparin complexes form at optimal stoichiometric ratios. To compare the effects of these various non-heparin anticoagulants in disrupting the formation of PF4-heparin complexes, and PF4-containing immune complexes. Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4-heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4-heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4-transfected HEK-293 EBNA cell line expressing the PF4 receptor CXCR3-B (P = 0.0408), reduced PF4-heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4-heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4-heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4-containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.

Heparin-Induced Thrombocytopenia: Diagnosis and Management

Heparin-induced thrombocytopenia (HIT) is an immune reaction in response to platelet factor 4-heparin complexes, which results in increased platelet activation and thrombocytopenia beginning on the 4th-5th day after heparin exposure induced by IgG antibody production. Platelet activation can lead to arterial thrombosis, but more commonly platelet microparticle formation contributes to venous thrombosis. Accurate diagnosis of HIT is based on the presence of clinical features, including a 50% fall in platelet count, appropriate timing of thrombocytopenia, development of new thrombosis despite thrombocytopenia and heparin therapy, and the absence of a more likely cause of thrombocytopenia. Documentation of an anti-PF4-heparin antibody is necessary, but is not sufficient to make the diagnosis since antibody formation occurs in a variety of clinical settings without the development of thrombocytopenia or thrombosis. Once HIT is suspected or confirmed, all forms of heparin should be discontinued and an alternative form of anticoagulation should be administered until the platelet count recovers. Treatment options include intravenous administration of argatroban, lepirudin, and bivalirudin; subcutaneous administration of fondaparinux has also been described. Warfarin therapy, if indicated, should be avoided until platelet recovery. Re-exposure to heparin can be avoided by use of alternative anticoagulants for most circumstances. Heparin-induced thrombocytopenia (HIT) has been the focus of increasing attention over the past 15-20 years. As interventions for HIT are developed, there is a need to accurately diagnose the condition, which can be challenging especially in severely ill patients.

A prospective analysis of heparin–platelet factor 4 antibodies in pregnant women treated with the low-molecular-weight heparin, dalteparin

Indications for heparin during pregnancy are expanding. Heparin-induced thrombocytopenia caused by heparin-platelet factor 4 antibodies (HPF4-As), however, remains a serious concern. While up to 50% of cardiovascular surgical patients develop HPF4-As while receiving heparin, the rate of seroconversion is lower in medical patients, suggesting an impact of the patient population on the underlying immune response. We therefore prospectively analyzed HPF4-As development in 31 pregnant women (32 +/- 5 years) receiving thromboprophylaxis with dalteparin for more than 4 weeks. According to their individual risk, study individuals were stratified to receive subcutaneous dosages of 2500-10 000 IU/day. The median treatment duration was 33 weeks (6-45 weeks). HPF4-As isotypes (IgG, IgM, IgA) were measured at baseline, on days 6-9, days 19-21 and subsequently every month until the end of therapy. Platelet counts and clinical examinations were carried out routinely or earlier if indicated. Throughout the study no thromboembolic event occurred, and in none of the patients was HPF4-As seroconversion noted. A prolonged drop in platelets to less than 50% from baseline was observed in one case (3%) after 35 weeks of treatment, which spontaneously resolved after child delivery. These findings suggest that long-term thromboprophylaxis with low-molecular-weight heparin is associated with a low rate of HPF4-As seroconversion in pregnancy.

Heparin-induced thrombocytopenia complicated by warfarin-induced skin necrosis

A case of heparin-induced thrombocytopenia (HIT) complicated by warfarin-induced skin necrosis (WISN) is reported. A patient with a history of hypertension, heart failure, and myocardial infarction was admitted to the hospital after complaining of a two-day history of shortness of breath, diaphoresis, and chest pain. The patient underwent a cardiac catheterization and received several medications, including heparin. Suspicions of HIT occurred when her platelets began to decrease severely and she developed a left groin hematoma and a pseudoaneurysm. Lepirudin was initiated and a heparin platelet factor 4 (PF4) antibody test was performed. The results were negative and lepirudin was discontinued. She was rechallenged with unfractionated heparin (UFH) after surgery of the pseudoaneurysm, but her platelets began to decrease again. A second PF4 test was performed, the results of which were positive. The UFH treatment was discontinued. Warfarin was also initiated after surgery and the patient's platelets rapidly increased after heparin was discontinued. She was discharged one week later. Three days after discharge, she was readmitted after complaining of severe pain and swelling of the fatty tissue of her right flank that began the day after she was discharged. Some blistering and necrosis were noted on the lesion. Histological sections showed focal thrombosis of vessels in the deep reticular dermis consistent with WISN. Local wound care was given to manage the WISN, lepirudin was initiated, and warfarin was discontinued and reinstated one week later at a low dosage. A patient with HIT developed severe skin necrosis after initiation of warfarin therapy.

Heparin-platelet factor 4 antibodies are frequent after vascular surgery but are not a frequent cause of graft thrombosis or thrombocytopenia

Approximately 10% of infrainguinal bypass surgeries are complicated by early conduit failure. The cause is unclear in most cases. A prospective study was conducted to monitor the development and function of platelet factor 4 (PF4)/heparin antibodies after infrainguinal bypass procedures and to evaluate their clinical significance in early graft occlusion. Blood samples were obtained before surgery and at the 7-, 14-, and 28-day postsurgical evaluation. Relevant demographic and laboratory data were collected, and plasma samples were assayed for the presence and function of PF4/heparin-antibody by enzyme-linked immunosorbent assay (ELISA) and a two-point platelet aggregation assay. All tests were performed in duplicate or triplicate. Of the 79 patients who were enrolled, 67 reported previous heparin exposure. Six patients (7.6%) tested positive for the presence of PF4/heparin antibodies before surgery with ELISA, and four of these (67%) also had a positive result on the aggregation assay. During the 28-day follow-up, 22 subjects (32%) converted to positive according to the ELISA results; and five (22.7%) of these also tested positive for platelet-activating antibodies. No participants presented with thrombocytopenia or a >/=50% decrease in platelet count during the study period. Early graft occlusion was detected in three patients, all with negative ELISA and functional assay results throughout the study. Patients undergoing vascular surgery frequently develop PF4/heparin antibodies, with platelet-activating antibodies detected in up to 11% of these individuals. However, thrombocytopenia and vascular graft thrombosis both appear to be an uncommon consequence.

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.

Usefulness of Optical Density Values From Heparin-Platelet Factor 4 Antibody Testing and Probability Scoring Models to Diagnose Heparin-Induced Thrombocytopenia

Usefulness of Optical Density Values From Heparin-Platelet Factor 4 Antibody Testing and Probability Scoring Models to Diagnose Heparin-Induced Thrombocytopenia

Anti-PF4/heparin antibodies associated with repeated hemofiltration-filter clotting: a retrospective study

IntroductionHeparin-induced thrombocytopenia is an immune-mediated adverse drug reaction that is associated with a procoagulant state and both arterial and venous thrombosis. After observing two cases of repeated hemofiltration-filter clotting associated with high anti-PF4/heparin antibody concentrations, we systematically measured the anti-PF4/heparin antibody concentration in all cases of unexpected and repeated hemofiltration-filter clotting during continuous veno-venous hemofiltration (CVVH). The aim of this study was to identify factors associated with positive anti-PF4/heparin antibody in the case of repeated hemofiltration-filter clotting.MethodsWe reviewed the charts of all patients who had an anti-PF4/heparin antibody assay performed for repeated hemofiltration-filter clotting between November 2004 and May 2006 in our surgical intensive care unit. We used an enzyme-linked immunoabsorbent assay (heparin-platelet factor 4-induced antibody) with an optical density (OD) of greater than 1 IU considered positive.ResultsDuring the study period, anti-PF4/heparin antibody assay was performed in 28 out of 87 patients receiving CVVH. Seven patients were positive for anti-PF4/heparin antibodies (OD 2.00 [1.36 to 2.22] IU) and 21 were antibody-negative (OD 0.20 [0.10 to 0.32] IU). Baseline characteristics, platelet counts, and activated partial thromboplastin time ratios were not different between the two groups. CVVH duration was significantly decreased in antibody-positive patients (5.0 [2.5 to 7.5] versus 12.0 [7.5 to 24.0] hours; P = 0.007) as was CVVH efficiency (urea reduction ratio 17% [10% to 37%] versus 44% [30% to 52%]; P = 0.04) on heparin infusion. Anti-PF4/heparin antibody concentration was inversely correlated with CVVH duration. The receiver operating characteristic curve showed that a 6-hour cutoff was the best CVVH session duration to predict a positive antibody test (sensitivity 71%, specificity 85%, and area under the curve 0.83). CVVH duration (32 [22 to 37] hours; P < 0.05) and urea reduction (55% [36% to 68%]; P < 0.03) were restored by danaparoid sodium infusion.ConclusionRepeated hemofiltration-filter clotting in less than 6 hours was often associated with the presence of anti-PF4/heparin antibodies, regardless of the platelet count. In antibody-positive patients, replacement of heparin by danaparoid sodium allowed the restoration of CVVH duration and efficiency.

Patient mit heparininduzierter Thrombozytopenie Typ II und implantiertem linksventrikulärem Assistdevice

The administration of heparin can lead to life-threatening heparin-induced thrombocytopenia (HIT) type II, which is caused by antibodies against heparin-platelet factor 4-complexes. The multimorbid patient presented here suffered from HIT II. Preoperative and postoperative prevention of thrombosis was successfully conducted with argatroban and the management of anticoagulation is presented. During therapy with argatroban, discrepancies in the partial thromboplastin time (PTT) and the international normalized ratio (INR) appeared sporadically. The clinical causes for these differences remain unclear.

Comparison of Antigen and Activation Assays with a Probability Scoring Model for the Diagnosis of Heparin Induced Thrombocytopenia.

Background: Heparin induced thrombocytopenia (HIT) is a syndrome characterized by thrombocytopenia and an elevated risk of thrombosis. In the evaluation of patients with suspected HIT, laboratory testing is used to help differentiate between those patients who have HIT and those who do not and therefore do not require non-heparin anticoagulation. Laboratory tests include activation (serotonin release, heparin-induced platelet aggregation, ATP luminescence) and antigen (enzyme-linked immunoabsorbant assay (ELISA)) assays directed against the platelet factor 4-heparin or PF4/PVS complex. A pre-test scoring system (Warkentin 4T’s) has been derived to assess the probability of HIT which can be used in conjunction with laboratory testing in the evaluation for HIT. Objectives: To compare the activation assay used at The Ottawa Hospital (ATP luminescence) with the PF4 enhanced® ELISA and correlate results with clinical data. Methods: Patients undergoing HIT testing were identified. ELISA and activation assays were performed. Charts were reviewed in order to derive a 4T’s score and to assess response to therapeutic non-heparin anticoagulation (when administered). Correlation between ELISA, activation assay, 4T’s score and platelet response to alternative anticoagulation was determined. Sensitivity, specificity, PPV and NPV of laboratory tests or 4T’s scores were calculated depending on the measure chosen as the reference standard to diagnose HIT. Results: 111 patients undergoing HIT testing were evaluated. 41 and 43 were positive by ELISA (OD &gt; 0.4) or activation assay respectively. 12 were positive only by ELISA (mean optical density (OD): 1.27) and 10 were positive by both activation assay and ELISA (mean OD: 2.28). Clinical information was available for 70 patients. 26 of these received therapeutic non-heparin anticoagulation as treatment for suspected HIT. Reference standard= activation assay: ELISA: Sens 95% (95% CI 73–99%), Spec 80% (66–89%), PPV 66% (46–81%), NPV 98% (86–100%). 4T’s (low vs. high score): Sens 85% (54–97%), Spec 95% (83–99%), PPV 85% (54–97%), NPV 95% (83–99%). ELISA OD &gt; 1.5 + high 4T’s score: Sens 61% (36–82%), Spec 100% (56–100%), PPV 100% (68–100%), NPV 50% (24–76%). Reference standard= 4T’s score (high and low score only, excludes intermediate category): ELISA: Sens 85% (53–97%), Spec 77% (61–88%), PPV 52% (30–74%), NPV 94% (79–99%). Activation: Sens 85% (54–97%), Spec 95% (83–99%), PPV 85% (54–97%), NPV 95% (83–99%). Reference standard= “clear response to non-heparin anticoagulation” defined as significant platelet increase within 48 hours of start of therapy and no other explanation for platelet recovery: ELISA: Sens 100% (72–100%), Spec 54% (26–80%), PPV 68% (43–86%), NPV 100% (56–100%). Activation: Sens 85% (54–97%), Spec 69% (39–90%), PPV 73% (45–91%), NPV 82% (48–97%). 4T’s (low vs. high score): Sens 90% (54–99%), Spec 100% (60–100%), PPV 100% (63–100%), NPV 89% (51–99%). ELISA OD &gt; 1.5 + 4T’s (low vs. high score): Sens 89% (51–99%), Spec 100% (56–100%), PPV 100% (60–100%), NPV 88% (47–99%). Conclusions: A high 4T’s score best predicts a clinical response to non-heparin therapeutic anticoagulation when HIT is suspected. ELISA OD &gt; 1.5 does not add additional information in this respect. A negative ELISA and a low 4T’s score have comparable NPVs when an activation assay is the reference standard. Future trials should employ a clinical reference standard such as “clear response to non-heparin anticoagulation” when evaluating the operator characteristics of HIT assays.

Methodologic Variations in the Determinations of Anti-Platelet Factor 4-Heparin Antibodies in Patients Suspected of Having Heparin Induced Thrombocytopenia: Diagnostic and Prognostic Implications.

It is now widely believed that the pathogenesis of heparin induced thrombocytopenia (HIT) is largely mediated via the generation of anti-platelet factor 4-heparin antibodies (APFA) in patients who are treated with heparin and related drugs. In addition antibodies to other heparin binding proteins such as neutrophil activating peptide-2 and interleukins also contribute to this syndrome and which are not detectable by the methods based on heparin platelet factor 4 capture probes. Currently, several immunologic methods mostly utilizing sandwich ELISA techniques to measure APFA in sera of patients with HIT syndrome are available. These methods utilize different capture probes including heparin platelet factor 4 complex (ASSERACHROM® HPIA; Diagnostica Stago, France), platelet factor 4 complexed to polyvinyl sulfonate (PF4 Enhanced; GTI, USA) and heparin protamine suflate along with platelet lysates as a source of PF4 (ZYMUTEST HIA IgGAM; Hyphen Biomedical, France). These different capture probes for the APFA have different affinity for the APFAs. Moreover, these capture probes can also bind to certain other heparin binding proteins. To compare these three methods, samples (n = 100) were selected from banked sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were initially positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). For the head to head comparison all of these samples were assayed using the three different methods. In the GTI method the reassayed samples showed 93 out of 100 positive (93%), in the HPIA test 79 out of 100 (79%) and in the ZYMUTEST 56 out of 100 (56%) were positive. Interestingly, the correlation coefficients also showed marked variation (GTI vs ZYMUTEST r2 = 0.38; GTI vs Stago r2 = 0.49; ZYMUTEST vs Stago r2 = 0.67). The prevalence of positive samples was not consistent in the three tests used. This data clearly shows that each of the different ELISA methods exhibit different performance characteristics in binding to not only the APFA but other proteins, which may contribute to the higher false positive prevalence. Moreover, the positive/negative cutoff limits in these assays are arbitrarily set without due consideration of the antibody titer, which can also account for the observed differences. More interestingly, all of the samples positive in the SRA (n = 14) were positive in each of the three methods used with the exception of two samples, which were positive in the GTI, ASSERACHROM® HPIA and negative in the ZYMUTEST. These results clearly indicate the differences in the diagnostic efficacy of various commercially available tests and warrant clinical field trials to validate their reliability in the monitoring of APFA. Furthermore, the diagnostic reliability of these tests can be further improved by immunoglobulin subtyping and utilizing other capture probes with platelet factor 4 and related proteins, which complex with heparins.

Is Heparin-Induced Thrombocytopenia (HIT) Overdiagnosed by the Conventional EIA Detecting IgG/M/A Antibodies Against Heparin-Platelet Factor 4-Complex? First Experience with the New EIA Detecting Only IgG-Class Antibodies.

Introduction: If HIT is considered, it should be confirmed or ruled out immediately to guarantee optimal (and low cost) anticoagulation or thrombosis prophylaxis for the patient. When diagnosis is not made, it may cause fatal thromboembolic disease in the patient due to enhanced platelet activation. The pathomechanism is well established: Antibody (ab) binding to heparin/PF4-complex (antigen) forms an immunocomplex. The complex binds to the Fc-Receptor of the platelet membrane resulting in platelet activation and further thrombin generation. Persistent exposure to heparinleads to continuous complex formation. To increase sensitivity and specificity, combination of an antigen test with a functional test is recommended: the antigen-test (EIA) detects ab binding (IgG, IgA und IgM) to heparin/PF4-complex or polyvinylsulfat-complex. Functional tests (HIPA-Test, Serotonin-release test or platelet aggregation studies) detect HIT- ab against different antigens - however, only IgG class. Therefore, the positive predictive value of a functional test is higher than for an antigen test to diagnose HIT. Ab of the IgA and IgM class have no platelet activating capacity and therefore, they are thought to be of minor clinical relevance.Aim of the study: With HIT-IgG-EIA tests approaching the market, the question is, which EIA-test should be performed and will the result change our diagnosis made before by the conventional EIA together with the functional assay and complete clinical data.Material and Methods: We compared the results of 28 out of 85 samples send over a one month period to our lab for HIT-testing. For this study positive or borderline positive samples were selected and only 5 of the negative's. All samples we run the same day with the conventional EIA (GTI) and later with the ID-PaGIA gel-particle-card (IgG/IgM/IgA) (DIAMED Comp., Suisse) and two different EIA (only IgG) by GTI Diagnostics (Waukesha, WI) and Haemochrom Diagnostica (Essen, Germany).Results: Out of the 28 samples, initially 5 were tested negative, 21 were tested positive (OD 0.5– 3.1, mean OD 1.41; positive if OD >0.4) and 2 being at the cut off. All 5 samples were tested negative in all assays, the 2 borderline samples were tested negative in the other assays. Of the 21 positive. samples 5 were negative by the ID-PaGIA gel-particle-card. Of the 21 positive. samples 13 were positive in the GTI-EIA (IgG only) and 7 in the Haemochrom EIA (IgG only) and 4 out of those had positive functional studies (platelet aggregation assay) - confirming the presence of IgG ab. However, the ID-PaGIA gel-particle-card missed 3 positive samples, when compared to the IgG-only EIAs.Conclusion: As expected there were fewer positive samples in the EIA detecting only IgG class ab, however the comparability of those two EIAs was pure, surprisingly. Further investigation will be done to confirm this observation. Sensitivity and specificity of the ID-PaGIA gel-particle-card is lower then of the EIA. In our small series we could not find an OD-cut-off-point for positive EIA-results (IgG/M/A) in comparison with a positive or negative result for the IgG-EIA. In the future we might add an IgG-only EIA to the established GTI-EIA (IgG/M/A) to our work up and use the clinical scoring system (4T's) vigorously before the diagnosis HIT will be made, to avoid overdiagnosing patients having positive results in the GTI-EIA (IgG/M/A) due to other underlying clinical conditions.

The Incidence of Recognized Heparin-Induced Thrombocytopenia in a Large, Tertiary Care Teaching Hospital

Heparin-induced thrombocytopenia (HIT) is estimated to occur in up to 5% of patients receiving unfractionated heparin. The goal was to determine the incidence of HIT within our 1,061-bed tertiary care institution. A retrospective review of three hospital database systems (ie, admission, pharmacy, and laboratory) was undertaken for a 1-year period ending in March 2004. The pharmacy database was queried to identify patients who received heparin and those who received a direct thrombin inhibitor (DTI). The medical records of patients receiving a DTI were reviewed to categorize the indication for DTI therapy. The laboratory system database was queried to retrieve heparin platelet factor 4 immunoassay results. A total of 58,814 patient admissions occurred with an estimated 24,068 patients being exposed to unfractionated heparin. DTI therapy was administered to 133 patients. Of these, 49 new HIT cases and 15 cases of suspected HIT (unconfirmed) were identified. The overall incidence of recognized new HIT was 0.2%. New HIT occurred in 0.76% of patients receiving therapeutic-dose IV heparin and in < 0.1% of patients receiving antithrombotic prophylaxis (subcutaneous heparin). Forty-nine percent of all new HIT cases were in coronary artery bypass and/or valve replacement surgery patients, while no cases were identified in hip/knee arthroplasty patients. The incidence of recognized HIT in a large teaching institution was 0.2%, with a 0.76% incidence in those patients receiving therapeutic-dose IV heparin. The low incidence likely reflects a brief duration of heparin exposure for many patients. Approximately half of all new HIT cases were recognized in the cardiovascular surgery population.