Argatroban anticoagulation in patients with a history of heparin-induced thrombocytopenia

INTRODUCTION: Heparin therapy is not recommended for patients with a history of heparin-induced thrombocytopenia (HIT), except in specialized situations, because this treatment can lead to severe reactions including thrombocytopenia and thrombosis. However, the optimal management of patients with a history of HIT requiring acute anticoagulation has not yet been clarified because of the lack of prospective studies. We evaluated the safety and efficacy of argatroban, a direct thrombin inhibitor, as an anticoagulant in patients with a history of HIT needing acute anticoagulation. METHODS: Thirty-six patients with a history of serologically confirmed HIT were treated prospectively with argatroban [median (5th-95th percentile) dose of 2.0 (1.0-4.3) microg/kg/min for 4.0 (0.7-8.4) days]. Prospectively defined endpoints included successful anticoagulation (therapeutic activated partial thromboplastin time), and bleeding, new thromboembolic events, or other adverse effects during therapy or within 30 days following its cessation. RESULTS: All patients required acute anticoagulation with the most common admission diagnoses being deep venous thrombosis or pulmonary embolism (n=13) and chest pain or acute coronary syndrome (n=12). Eleven patients had previously received argatroban therapy for HIT; one patient underwent two treatment courses of argatroban for a history of HIT. The median (5th-95th percentile) time between the past diagnosis of HIT and initiation of argatroban was 7.5 (0.4-114.6) months. All evaluable patients were successfully anticoagulated. No patient had major bleeding, new thromboembolic events, or other adverse effects. There were no adverse events related to reexposure. CONCLUSIONS: Argatroban can provide safe and effective anticoagulation, on initial or repeat exposure, in patients with a history of HIT.     

Results of a systematic evaluation of treatment outcomes for heparin-induced thrombocytopenia in patients receiving danaparoid, ancrod, and/or coumarin explain the rapid shift in clinical practice during the 1990s

INTRODUCTION: Randomized controlled trials evaluating treatment of acute, transient, but uncommon diseases are difficult to perform. The prothrombotic adverse drug reaction, heparin-induced thrombocytopenia (HIT), is such an example. During the mid-1980s, the defibrinogenating snake venom, ancrod (+/-warfarin, Canada), or coumarin (warfarin, Canada; phenprocoumon, Germany) alone, were often used to treat HIT. During the 1990s, danaparoid+/-coumarin began to replace ancrod (+/-coumarin), or coumarin alone, for treating HIT, despite danaparoid not being approved for treatment of HIT. METHODS: We performed a retrospective evaluation of treatment outcomes from 1986 to 1999, comparing danaparoid+/-coumarin (n=62) versus ancrod+/-coumarin or coumarin alone (controls, n=56). RESULTS: The predefined composite endpoint of adjudicated new, progressive, or recurrent thrombosis (including thrombotic death), or limb amputation, at day 7 (maximum, one event per patient) was significantly lower in danaparoid-treated patients, compared with controls: 8/62=12.9% (95% CI, 4.3-21.5) vs. 22/56=39.3% (95% CI, 26.1-52.5); p=0.0014. We also found a lower frequency of the composite endpoint at end of study (day 35) in danaparoid-treated patients: 12/62=19.4% vs. 24/56=42.9% (p=0.0088). Major bleeding (by day 7) occurred in 7/62 (11.3%) and 16/56 (28.6%) of danaparoid-treated and control patients, respectively (p=0.0211). CONCLUSIONS: The replacement of ancrod+/-coumarin, or coumarin alone, by danaparoid (+/-coumarin) in the mid-1990s for the treatment of HIT was justified by improved efficacy and safety.     

Clinical evaluation of acute systemic reaction and detection of IgG antibodies against PF4/heparin complexes in hemodialysis patients

Heparin-induced thrombocytopenia (HIT) is a pathophysiological syndrome caused by platelet-activating antibodies that recognize PF4/heparin complexes. The abrupt onset of HIT following intravenous bolus heparin is known as an acute systemic reaction. Clinical features of this type of HIT may be similar to those of common complications during hemodialysis. The aim of the study was to identify whether the clinical features of the acute systemic reaction are caused by HIT or dialytic complications. Twenty-seven dialytic patients who had thrombocytopenia and clinical features of an acute systemic reaction were enrolled out of 202 HIT-suspected patients. Thirteen patients had HIT confirmed due to the presence of positive functional and immunoassays. Eight of the thirteen patients presented with acute systemic reactions due to HIT. The most common symptom of acute systemic reaction was dyspnea. The other nineteen patients, involving both HIT and non-HIT patients, had dialysis-complicated ASR. The major feature of the acute systemic reaction in hemodialysis was hypotension and its relevant symptoms. An immunoassay for the detection of IgG antibodies against PF4/heparin complexes (HIT-IgG) showed the wide-range linearity of the calibration curve by employing three concentrations of recombinant mouse monoclonal antibodies for PF4/heparin complexes. The results are expressed as micrograms of IgG in one milliliter. Significantly high levels in thirteen HIT patients were compared with levels in fourteen non-HIT patients. The highest median of 1,530 μg/ml (IQR: 3,267-813) was obtained in the presence of HIT associated with an acute systemic reaction. In HIT patients who did not show characteristics of an HIT-derived acute systemic reaction, the median was 339 μg/ml (1,178-834). Despite showing a positive ELISA, nine non-HIT patients without any platelet-activating antibodies showed a value of 97 μg/ml (166-56). The lowest median of 8.3 μg/ml (11-6) was in non-HIT patients with a negative ELISA. In conclusion, measurements of HIT-IgG -specific antibodies can facilitate an appropriate estimation in hemodialysis patients of whether the clinical features of an acute systemic reaction are caused by HIT or dialytic complications.     

Heparin-induced thrombocytopenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. MATERIALS AND METHODS: By photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. RESULTS AND CONCLUSIONS: The main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and--for a given degree of platelet activation (PF4 availability)--as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.     

Diagnosis and treatment of heparin-induced thrombocytopenia in neonates and children

Heparin-induced thrombocytopenia (HIT), a well-known side effect of heparin therapy, occurs with an incidence of 1-2% in certain pediatric patient groups. In affected children, HIT markedly increases the risk of venous and arterial thromboembolism. The use of alternative anticoagulation with danaparoid, lepirudin and argatroban in adults and children has demonstrated to be safe and could reduce morbidity and mortality also in affected pediatric patients. Thus, in children and neonates, an early diagnosis and accurate management is crucial to avoid the deleterious consequences of HIT. This review article will focus on the presentation of HIT in neonates and children. It reviews the pathophysiology of HIT and it summarizes epidemiological data. Finally important diagnostic and therapeutic issues are discussed.     

New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells

Introduction

The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required.

Materials and Methods

We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin.

Results

The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab.

Conclusion

The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.     

Rapid exclusion of the diagnosis of immune HIT by AcuStar HIT and heparin-induced multiple electrode aggregometry

Background

Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential but remains challenging. We have previously demonstrated, in a retrospective study, the usefulness of the combination of the 4Ts score, AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) with optimized thresholds.

Objectives

We aimed at exploring prospectively the performances of our optimized diagnostic algorithm on suspected HIT patients. The secondary objective is to evaluate performances of AcuStar HIT-Ab (PF4-H) in comparison with the clinical outcome.

Methods

116 inpatients with clinically suspected immune HIT were included. Our optimized diagnostic algorithm was applied to each patient. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) of the overall diagnostic strategy as well as AcuStar HIT-Ab (at manufacturer’s thresholds and at our thresholds) were calculated using clinical diagnosis as the reference.

Results

Among 116 patients, 2 patients had clinically-diagnosed HIT. These 2 patients were positive on AcuStar HIT-Ab, AcuStar HIT-IgG and HIMEA. Using our optimized algorithm, all patients were correctly diagnosed. AcuStar HIT-Ab at our cut-off (> 9.41 U/mL) and at manufacturer’s cut-off (> 1.00 U/mL) showed both a sensitivity of 100.0% and a specificity of 99.1% and 90.4%, respectively.

Conclusion

The combination of the 4Ts score, the HemosIL® AcuStar HIT and HIMEA with optimized thresholds may be useful for the rapid and accurate exclusion of the diagnosis of immune HIT.     

Whole blood impedance aggregometry detects heparin-induced thrombocytopenia antibodies

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin use. IgG antibodies to complexes of platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. Only a subset of these antibodies will activate platelets and these can only be identified with platelet aggregation (functional) assays. Heparin-induced platelet aggregation (HIPA) and ¹⁴C-serotonin release (SRA) assays for HIT are time-consuming and complex to perform. We have developed a whole blood impedance (WBI) test using the new Multiplate^® analyser.All samples referred to our laboratory over a 10 month period were screened for heparin-PF4 antibodies by an ELISA method (Zymutest HIA IgG). The 4T's score was used to assess HIT pretest probability.Twenty antibody positive samples were further tested by all three functional assays: light transmission aggregometry (LTA), SRA and WBI. Thirteen out of twenty samples were positive by LTA (10 patients) and 15 by WBI (11 patients). SRA, considered to be the gold standard, was used as a confirmatory test and 11 were found to be positive (10 patients); four discrepant samples were weakly positive by WBI. The prevalence of a positive functional test was strongly correlated with the 4T's clinical risk score, but a small number of low-risk patients had positive functional assays.In this study, the WBI assay detected all SRA positive patients and was positive for two others suggesting greater sensitivity. The rapid and easy to perform assay may be a useful tool for haematology laboratories to detect platelet-activating HIT antibodies.     

A clinical-laboratory approach contributing to a rapid and reliable diagnosis of heparin-induced thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) remains a very challenging diagnosis. The first objective of this study was to compare the performance of the ID-H/PF4 PaGIA^® with the Asserachrom^® HPIA ELISA. The main purpose was to evaluate the diagnostic utility of the combination of the H/PF4 PaGIA^® with the clinical “4T's” score as a screening strategy.

Materials and Methods

102 patients with clinical suspicion of HIT were classified into risk groups using the 4T's score. The presence of HIT antibodies was assessed by two immunoassays and confirmed by a functional flow cytometric assay.

Results

Comparison of the ID-H/PF4 PaGIA^® with the Asserachrom^® HPIA ELISA demonstrated a comparable technical performance, being an excellent screening test to rule out HIT (negative predictive value or NPV = 100%). According to the 4T's score, HIT was excluded in all low risk patients (NPV = 100%). ELISA optical density levels were significantly different between all risk groups (P-values < 0.01). In contrast, due to the low positive predictive value (22%) and weak positive likelihood ratio (2.6), a positive ID-H/PF4 PaGIA^® result did not considerably increase the probability of HIT.

Conclusion

Our study confirms the combination of the 4T's score with the ID-H/PF4 PaGIA^® as a reliable strategy to rule out HIT. Yet, confirming positive ID-H/PF4 PaGIA^® results by flow cytometry within 1-2 h after blood sampling remains necessary. This novel clinical-laboratory approach can contribute in a rapid and reliable way to the definite diagnosis of HIT.     

Frequent off-label use of fondaparinux in patients with suspected acute heparin-induced thrombocytopenia (HIT) – findings from the GerHIT multi-centre registry study

Introduction

In life-threatening immune heparin-induced thrombocytopenia (HIT), treatment with an approved non-heparin anticoagulant is essential. However, off-label use with fondaparinux has been reported in the literature. The study aim was to collect data on “real-life” management of patients with suspected acute HIT regarding diagnostic and therapeutic strategies.

Patients and Methods

In a national multi-centre registry study, patients with a 4 T’s HIT-probability score of ≥ 4 points and treatment with at least one dose of (A)rgatroban, (L)epirudin, (D)anaparoid, or (F)ondaparinux were retrospectively evaluated.

Results

Of 195 patients, the 4 T’s scores were 4/5/6/7/8 points in 46 (23.6%)/50 (25.6%)/74 (38.0%)/13 (6.7%)/7 (3.6%) patients, respectively. During heparin therapy, 47 (24.1%) thromboembolic events, 5 (2.6%) skin lesions, 1 (0.5%) amputation, 24 (12.3%) Hb-relevant bleedings, and 2 (1.0%) fatalities occurred. A functional heparin-induced platelet activation assay was performed in 96.9%, a platelet factor 4/heparin-dependent enzyme immunoassay in 89.2%, a particle gel immunoassay in 12.3%, and a serotonin-release assay in none of the patients. Argatroban was used in 16.4%, lepirudin in 2.1%, danaparoid in 23.6%, fondaparinux in 40.0% of the patients; the sequential therapy strata were: AF (5.6%), DA (5.6%), DF (2.6%), DL (2.1%), ADF (1.5%), and DFL (0.5%).

Conclusions

The current diagnostic laboratory strategy for suspected HIT is mostly (> 96%) based on the recommended 2-step strategy (immunoassay plus functional assay). However, there is a wide fondaparinux off-label use (up to 50.3%) for suspected HIT, even in those patients with a high clinical pretest probability. Efficacy and safety of fondaparinux for HIT-treatment require further evaluation.     

Platelet microparticle generation assay: A valuable test for immune heparin-induced thrombocytopenia diagnosis

Background

Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance.

Objectives

The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of ¹⁴C-serotonin release assay (SRA) on the same series of patients.

Methods

Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1 IU and 500 IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient’s medical records. Performances of PMPGA and SRA (n = 53) were evaluated using ROC curve analysis with clinical outcome as reference.

Results

In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of ¹⁴C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2).

Conclusions

PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with ¹⁴C-SRA performances and predominately with clinical outcome.     

Results of a consensus meeting on the use of argatroban in patients with heparin-induced thrombocytopenia requiring antithrombotic therapy - a European Perspective

Argatroban has been introduced as an alternative parenteral anticoagulant for HIT-patients in several European countries in 2005. In 2009 a panel of experts discussed their clinical experience with argatroban balancing risks and benefits of argatroban treatment in managing the highly procoagulant status of HIT-patients. This article summarizes the main conclusions of this round table discussion. An ongoing issue is the appropriate dosing of argatroban in special patient groups. Therefore, dosing recommendations for different HIT-patient groups (ICU patients; non-ICU patients, paediatric patients, and for patients undergoing renal replacement therapies) are summarized in this consensus statement. Because of the strong correlation between argatroban dosing requirements and scores used to characterize the severity of illness (APACHE; SAPS, SOFA) suitable dosing nomograms are given.This consensus statement contributes to clinically relevant information on the appropriate use and monitoring of argatroban based on the current literature, and provides additional information from clinical experience. As the two other approved drugs for HIT, danaparoid and lepirudin are either currently not available due to manufacturing problems (danaparoid) or will be withdrawn from the market in 2012 (lepirudin), this report should guide physicians who have limited experience with argatroban how to use this drug safely in patients with HIT.     

Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay

Background

IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain.

Objective

To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined.

Methods

We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test.

Results

Platelet-activating antibodies were rarely detected (< 0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83 = 17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD ≤ 1.0). However, decreased sensitivity was observed with strong-positive sera (OD > 1.0): 11/69 such sera (16%) that did not show > 40% inhibition by high heparin nevertheless contained platelet-activating antibodies.

Conclusion

Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5- ≤ 1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (> 1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies.     

Physiological changes in membrane-expressed platelet factor 4: Implications in heparin-induced thrombocytopenia

Background

Many heparin-induced thrombocytopenia (HIT) antibodies cause platelet activation in the serotonin release assay (SRA) in the absence of heparin. This in vitro observation may help unravel the mechanism of delayed-onset HIT, where seropositive patients develop thrombocytopenia and associated thrombosis after cessation of heparin.

Objective

Studies were conducted to examine the relationship between platelet environment, surface PF4 expression, and the extent of heparin-independent platelet activation in the SRA.

Methods

Ex vivo platelets were washed and labeled for SRA, then used either before or after 45 minutes of recovery at 37 °C. HIT antibody-mediated serotonin release in the absence of heparin was compared to the extent of surface staining of the platelets with fluorescent anti-human PF4 antibodies.

Results

Handling of platelets for in vitro studies resulted in transient expression of surface PF4, and it was during this interval that platelets were most sensitive to activation by HIT antibodies in the absence of heparin. Heparin-independent platelet activation was attenuated when SRA-positive specimens were retested after platelets were incubated 45 minutes at 37 °C. Surface PF4 expression was diminished on the rested platelets, compared to the same platelets labeled immediately after handling. Thus compared to rested platelets, mildly activated platelets had elevated surface PF4 expression and a higher level of HIT antibody-mediated, heparin-independent platelet activation.

Conclusion

Surface expression of PF4 reflects HIT antigen presentation, and varies with the physiological state of platelets. Thus there can be differences in HIT antibody target availability among patients which may explain the variability in consequences of HIT antibody seropositivity.     

Interleukin-10 promoter microsatellite polymorphisms influence the immune response to heparin and the risk of heparin-induced thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) results from an atypical immune response with synthesis of IgG antibodies (Abs) to platelet factor 4/heparin complexes (PF4/H), and probably involves both B and T cells. We investigated whether 3 single nucleotide polymorphisms (SNPs), rs1800896 (− 1082G/A), rs1800871 (− 819C/T) and rs1800872 (− 592C/A) and the polymorphic CA repeat microsatellites IL10R [5325CA(11_15)] and IL10G [8134CA(14_29)] are associated with the synthesis of Abs to PF4/heparin and HIT.

Materials and methods

Eighty-two patients with definite HIT and two control groups were studied. The first control group (Ab^neg) consisted of 85 patients without Abs to PF4/heparin after cardiopulmonary bypass (CPB). The second control group (Ab^pos) consisted of 84 patients who had developed significant levels of PF4-specific antibodies after CPB, but without HIT.

Results

Allele frequencies of the 3 SNPs were similar in HIT patients and controls. Fourteen alleles in IL10G (G16 to G29) and 3 alleles in IL10R (R13 to R15) were defined. The short G20 allele of IL10G was more frequent in Ab^neg patients (8.2%) than in Ab^pos (2.9%) and HIT patients (3%). It thereby appeared to protect against developing Abs to PF4/heparin (OR 0.29; 95% CI [0.12-0.70], p = 0.006). Combined haplotypes cH1/cH8 comprising the short G20 + R13 alleles were less frequent in HIT (OR 0.33; 95% CI [0.11-0.97], p = 0.036), and levels of Abs to PF4 in Ab^pos patients were lower in cH1/cH8 subjects (p = 0.019).

Conclusion

These results suggest that IL10 promoter microsatellite polymorphisms might influence the immune response against PF4/heparin and the risk of HIT.     

Antiplatelet Factor 4—Heparin Antibodies in Patients with Antiphospholipid Antibodies

Antibodies directed against platelet factor 4-heparin are present in patients with heparin-induced thrombocytopenia (HIT). Additionally, it has been suggested that heparin can be an antigenic target of antiphospholipid antibodies (aPL). We investigated the presence of heparin-platelet factor 4-induced antibodies (HPIA) in 33 patients with aPL. There were 30 patients with lupus anticoagulant, 25 with anticardiolipin antibodies, 21 with anti-β₂ glycoprotein I, and 18 with antiprothrombin antibodies. 20 patients had a history of thrombosis and 19 had received heparin during the last 60 months. We found 7 (21.2%) who had HPIA; 5 of them also had anti-β₂ glycoprotein I antibodies. Four patients had severe thrombocytopenia and suspicion of HIT. Among them, two presented high positive HPIA results, one of them with positive platelet aggregation test. The third patient showed grey zone HPIA and borderline aggregation test and the fourth one had negative results. Among patients without a history of HIT, 2 who had never received heparin presented high positive, one a moderate positive, and one a grey zone HPIA result; all of them with negative aggregation tests. Five positive sera samples were incubated with cardiolipin liposomes in the presence of β₂ glycoprotein I, and whereas an inhibition greater than 50% was achieved in anticardiolipin and anti-β₂ glycoprotein I activities, HPIA results did not change. We demonstrate that HPIA could be frequently found in patients with aPL. They are responsible for HIT in some cases but can also be found in patients who have not received heparin. Whether they predispose patients with aPL to HIT is not known; nevertheless, a close follow-up of heparin treatment in these patients seems to be mandatory.     

Platelets of patients with peripheral arterial disease are hypersensitive to heparin

We sought to verify earlier reports of increased platelet reactivity in patients with peripheral arterial disease (PAD) during perioperative heparin administration, and to test the hypothesis of platelet hypersensitivity to heparin in these patients. Before and after incubation of platelet rich plasma with unfractionated (UH), low molecular weight heparin (LMWH), and a low molecular weight heparinoid, real-time quantitative assessment of platelet function was performed by stagnation point flow adhesio-aggregometry (SPAA) in 21 patients with PAD and 14 healthy volunteers. With SPAA the occurrence of spontaneous aggregation is pathological. In the 15 patients requiring operation, platelet function and count were measured at regular intervals. To detect heparin dependent antibodies, the heparin induced platelet activation assay (HIPA) was performed preoperatively and after 10 days of heparin therapy. Mean baseline platelet adhesion in patients was double that observed in controls (p < 0.001). Spontaneous aggregation was seen in 9 (43%) patients and no controls (p < 0.001). In controls heparinoid reduced, whereas UH and LMWH slightly increased adhesion. Spontaneous aggregation was observed once with UH. Platelets from patients showed significantly enhanced adhesiveness and aggregability (p < 0.05) with UH and LMWH when compared to controls. Effects with the heparinoid were less pronounced and non-significant. In patients requiring operation, postoperative increases in platelet function and reductions in count were significant (p < 0.001). Ten (67%) experienced a fall in platelet count of > 50%. Preoperatively the HIPA assay showed no evidence of antibodies, whereas after heparin administration antibodies were verified in 4 (32%) patients and could not be ruled out in 6 (40%). Three developed postoperative thrombosis, in one case fatal. A hypersensitive in vitro and in vivo platelet response to heparin was verified in patients with PAD and a large number developed the immunological type of heparin-associated thrombocytopenia. Our findings suggest that a thrombin antagonist which does not interact with platelets may give the best perioperative protection in these patients.     

A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin

Introduction

Lower low-molecular-weight heparins are being developed to improve on the safety and efficacy of antithrombotic therapy. Semuloparin and bemiparin are two depolymerized heparins produced by distinct manufacturing processes. The objective of this investigation was to determine whether a common standard could be used to define their potency.

Materials and Methods

Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment.

Results

The activity of semuloparin and bemiparin was comparable in FXa-based assays (anti-FXa, Heptest). However, bemiparin produced a stronger effect in the aPTT, ACT and anti-thrombin assays. Assessment of the parallelism of the concentration-response curves indicated that bemiparin and semuloparin are not equivalent in terms of anti-FIIa activity. Bemiparin had a stronger inhibitory effect on thrombin induced platelet aggregation, and a stronger interaction with HIT antibodies.

Conclusions

These data demonstrate that depolymerized heparins can exhibit a range of biologic activities making them unique agents. Pharmacopoeial parameters such as anti-IIa and anti-Xa potency and molecular weight are insufficient to characterize such agents.     

Assessment of the performances of AcuStar HIT and the combination with heparin-induced multiple electrode aggregometry: A retrospective study

Background

Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is challenging. HemosIL® AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) were recently proposed as rapid diagnostic methods.

Objectives

We conducted a study to assess performances of AcuStar HIT-IgG (PF4-H) and AcuStar HIT-Ab (PF4-H). The secondary objective was to compare the performances of the combination of Acustar HIT and HIMEA with standardised clinical diagnosis.

Methods

Sera of 104 suspected HIT patients were retrospectively tested with AcuStar HIT. HIMEA was performed on available sera (n = 81). The clinical diagnosis was established by analysing in a standardized manner the patient’s medical records. These tests were also compared with PF4-Enhanced®, LTA, and SRA in subsets of patients. Thresholds were determined using ROC curve analysis with clinical outcome as reference.

Results

Using the recommended thresholds (1.00 AU), the negative predictive value (NPV) of HIT-IgG and HIT-Ab were 100.0% (95% CI: 95.9%-100.0% and 95.7%-100.0%). The positive predictive value (PPV) were 64.3% (95% CI: 35.1%-87.2.2%) and 45.0% (95% CI: 23.2%-68.6%), respectively. Using our thresholds (HIT-IgG: 2.89 AU, HIT-Ab: 9.41 AU), NPV of HIT-IgG and HIT-Ab were 100.0% (95% CI: 96.0%-100.0% and 96.1%-100.0%). PPV were 75.0% (95% CI: 42.7%-94.5%) and 81.8% (95% CI: 48.3%-97.7%), respectively. Of the 79 patients with a medium-high pretest probability score, 67 were negative using HIT-IgG (PF4-H) test at our thresholds. HIMEA was performed on HIT-IgG positive patients. Using this combination, only one patient on 79 was incorrectly diagnosed.

Conclusion

Acustar HIT showed good performances to exclude the diagnosis of HIT. Combination with HIMEA improves PPV.