Heparin-platelet Factor 4 Antibodies Research Articles

Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

BackgroundHeparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin.Materials and MethodsSeven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software.ResultsIn the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT.ConclusionIn these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. [Display omitted] DisclosuresWalenga:Eurofarma: Research Funding.

Delayed-onset heparin-induced skin necrosis: a rare complication of perioperative heparin therapy

An uncommon case of delayed-onset dalteparin-induced skin necrosis in an 83-year-old Caucasian female patient associated with heparin-induced thrombocytopaenia (HIT) presenting on day 30 following dalteparin therapy is reported. Investigations revealed...

Outcome of patients with positive heparin–platelet factor-4 antibodies: A retrospective multi-institutional observational study

Studies show increased mortality with positive heparin–platelet factor-4 (H–PF4) antibodies, especially in hemodialysis patients. We aimed to compare mortality and thrombosis in hospitalized patients with positive, equivocal and negative H–PF4 antibody results. Information was collected on these patients using a multi-institutional retrospective electronic medical record review. Patients tested for H–PF4 antibodies by commercial ELISA during the years 2006 to 2010 were identified. We compared 30-day, 90-day and 1-year mortality in patients with negative, equivocal and positive H–PF4 test and evaluated the relationship between H–PF4 status and rate of thrombosis. Four hundred and seventeen patients had ELISA testing for H–PF4 antibodies. Forty-four patients had equivocal (optical density value 0.4–0.9) and 21 had positive (value 1) H–PF4 antibody test. There were no statistically significant differences in mortality between patients with negative, equivocal and positive results at all three time points (p = 0.22, 0.27 and 0.38, respectively) even after excluding patients with thrombosis (p = 0.22, 0.24 and 0.31, respectively). Age and Charlson score were associated with increased 30-day, 90-day and 1 year mortality. Odds ratio of having thrombosis was 23.1 for positive vs. equivocal results (p < 0.001); however, there was no statistically significant difference between equivocal vs. negative results (p = 0.22). Our results revealed no association between H–PF4 status and mortality, as well as no difference in 1-year survival between the positive and negative groups.

Outcome Of Patients with Positive Heparin-Platelet Factor 4 Antibodies: A Retrospective Observational Study

▪ IntroductionHeparin-induced thrombocytopenia (HIT) is a rare but potentially serious complication of heparin use. Immunoassay (EIA) can detect the presence of antibodies directed toward heparin-platelet factor 4 (H-PF4) complexes, but provide no information about their ability to activate platelets and have only modest specificity. Significance of positive H-PF4 antibodies without clinical HIT is not known. This study evaluates the outcomes of patients with positive H-PF4 antibodies expressed in optical density (OD) units. ObjectivesTo examine the correlation between positive H-PF4 antibodies, mortality and thrombotic events in hospitalized patients with suspected HIT. MethodsA retrospective electronic medical record review was conducted in two institutions. Patients with H-PF4 antibodies detected by commercial ELISA during 2006 - 2010 were identified. We compared 30-day, 90-day and 1-year mortality in patients with negative, equivocal, and positive H-PF4 test and evaluated the relationship between H-PF4 status and rate of thrombosis. One-way ANOVA and Chi square tests were used testing continuous and categorical variables respectively. A multivariate logistic regression model was fitted to estimate relationships between patient characteristics, laboratory findings, and H-PF4 antibody test status. The Kaplan-Meier method and log-rank tests were used to compare survival between groups. ResultsA total of 417 patients were included in the study, of which 44 patients had equivocal (OD value 0.4-0.9) and 21 had positive (OD value ≥1) H-PF4 antibody test result. 30-day, 90-day and 1-year mortalities were 13.6% (48/352), 33.2% (117/352), 46.3% (163/352) respectively for H-PF4 negative patients, 4.5% (2/44), 45.4 % (20/44), 56.8% (25/44) respectively for patients with equivocal H-PF4 status and 14.3% (3/21). 33.3% (7/21), 42.8% (9/21) respectively for H-PF4 positive patients. There were no statistically significant differences in mortality between patients with negative, equivocal, and positive results at all 3 time points (p-value 0.222, 0.273, 0.385, respectively); even after excluding patients with thrombosis (p-value 0.222, 0.248, 0.310 respectively). Age and Charlson score were associated with increased in-hospital, three-months and 1 year mortality; odds ratios were 1.018 and 1.170 (p-value >0.05) for in-hospital mortality, 1.034 and 1.247 (p-value <0.05) for three-months mortality, 1.04 and 1.289 for 1 year mortality (p-value <0.05). Multiple regression analysis showed that age, gender and Charlson score had no statistically significant effect on the occurrence of thrombosis in negative, equivocal, or positive groups. There was a significant correlation between the occurrence of thrombosis and H-PF4 result. Using logistic regression analysis odds ratio of having thrombosis was 2.476 for equivocal vs. negative and positive vs. equivocal results (p <0.0001) (Table 2). ConclusionOur results suggest no association between H-PF4 status positivity and mortality, as well as no difference in survival over one year between the positive and negative groups. H-PF4 positivity however showed positive correlation with thrombosis risk. Disclosures:No relevant conflicts of interest to declare.

Thrombocytopenia caused by low-dose heparin supplementation of parenteral nutrition solution

TO THE EDITOR: Heparin-induced thrombocytopenia (HIT) is a significant complication of heparin therapy. Risk factors for HIT include the type of heparin used, duration of heparin exposure, and clinical setting. Here, we report a 76-year-old man who presented with life-threatening pulmonary thromboembolism and deep-vein thrombosis, preceded by a dramatic decrease in platelet count 1 week after radical cystectomy for transitional cell bladder carcinoma. He was diagnosed with type II HIT caused by a small amount of unfractionated heparin mixed into his total parenteral nutrition (TPN) solution. One thousand units heparin per day were added to his TPN for 1 week. A heparin-platelet factor-4 antibody enzyme-linked immunosorbent assay of his serum was strongly positive. After cessation of TPN infusion, argatroban was started for thrombosis. His platelet count gradually increased to a normal level, and the thrombosis was treated successfully. This case suggests HIT should be suspected in patients with typical clinical manifestations and risk factors, even if the infused heparin dose is small. Heparin is widely used in a variety of medical therapies from the treatment of life-threatening acute thromboembolisms to the maintenance of indwelling vascular catheter patency. However, heparin therapy is often complicated by thrombocytopenia. The incidence of heparin-induced thrombocytopenia (HIT) in patients who receive heparin including unfractionated heparin and low-molecular-weight heparin is 0.1-5% [1]. There are 2 types of HIT. Type I HIT is a relatively common non-immune reaction that causes an asymptomatic, transient, and mild decrease in platelet count. In contrast, type II HIT is an immune complex disorder involving heparin bound to the platelet-specific chemokine, platelet factor-4 (PF4). These heparin-PF4 complexes activate both platelets and endothelial cells, resulting in platelet consumption and endothelial injury with thrombosis and disseminated intravascular coagulation [2]. Heparin use could go unnoticed if it is not applied as a conventional anticoagulant but rather as a supportive agent to maintain vasc,ular patency. However, unrecognized HIT can lead to life-threatening complications. Therefore, it is important to suspect HIT in patients who develop acute thrombosis or thrombotic tendency preceded by a decrease in platelet count during or soon after heparin therapy, even if the infused amount is small. Immediate discontinuation of heparin administration and initiating alternative anticoagulant therapy are critical for avoiding further thrombosis, even before laboratory results are available [3]. To the best of our knowledge, this is the first report on a patient who experienced type II HIT after low-dose heparin added to his total parenteral nutrition (TPN) for 1 week.

Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Abstract 2189Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA).Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk.Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA.From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive.Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation.Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay.LFI-HITPaGIAIgG-ELISAOD IgG-ELISAHIPA assayMedian (range)Samples n=121Pos 10Pos 10Pos 82.366 (0.902-3.000)7/7 posNeg 20.199 and 0.1701/2 pos, 1/2 negNeg 0----Neg 111Pos 9Pos 21.318 and 2.0192/2 posNeg 70.110 (0.054-0.139)6/6 negNeg 102Pos 70.436 (0.404-1.463)1/2 pos, 1/2 negNeg 950.082 (0.013-0.376) Disclosures:No relevant conflicts of interest to declare.

Evaluating Heparin-Induced Thrombocytopenia: The Old and the New

Heparin-induced thrombocytopenia (HIT) is a rare but potentially serious complication of heparin use. Prompt diagnosis is crucial and requires the integration of clinical assessment and laboratory testing. Pretest clinical scoring systems (i.e., 4 Ts) have been established. Immunoassays can detect the presence of antibodies directed toward heparin-platelet factor 4 (H-PF4) complexes, but provide no information about their ability to activate platelets. A low clinical score, when combined with a negative immunoassay result obviates the need for further testing. However, immunoassays and 4 Ts scores have only modest specificity. Functional testing (serotonin release assay or heparin-induced platelet activation) remain important in confirming the presence of pathogenic H-PF4 antibodies, but are technically demanding to perform and limited in guiding clinical decisions in the acute setting. This review evaluates current immuno- and functional assays available in the laboratory diagnosis of HIT, and describes recent attempts to improve the specificity of enzyme immunoassays, including adopting an immunoglobulin G-specific assay and raising the optical density value cutoff for a positive result. The importance of donor selection and newer functional assays, including flow cytometry-based assays, are also discussed. A current approach to integrating clinical scoring, immunoassays, and functional testing for HIT is also outlined.

Heparin Platelet Factor 4 Antibody Positivity in Pseudothrombocytopenia

Pseudothrombocytopenia (PTCP) is a laboratory event of platelet clustering related to drugs used for anticoagulation. This condition is engendered by autoantibodies against platelets in usually EDTA-anticoagulated blood. Pseudothrombocytopenia has no clinical significance but when evaluated as true thrombocytopenia, this misconception may lead to unnecessary diagnostic procedures. Heparin-induced thrombocytopenia with thrombosis (HITT) is a complication of heparin treatment caused by heparin platelet factor 4 (HPF-4) antibodies, leading to platelet activation and hypercoagulability. In our study, 48 patients with PTCP and 36 healthy volunteers were included. Heparin platelet factor 4 antibody positivity was detected in 12 patients from PTCP group; nobody from control group had. Citrated serum samples and peripheral blood smears showed normal platelet count. Of the 4 patients using heparin derivative, 1 (2.1%) had antibody positivity but without any bleeding symptoms. In conclusion, HPF-4 antibody positivity might be a risk factor for PTCP. Clinicians should be aware of this kind of condition.

Assessment of the 4Ts Pretest Clinical Scoring System as a Predictor of Heparin‐Induced Thrombocytopenia

To evaluate the utility of the 4Ts clinical scoring system as a pretest probability method for the detection of heparin-induced thrombocytopenia (HIT). Prospective observational study. Medical and surgical inpatients at a tertiary care medical center. Eighty consecutive patients with suspicion of HIT who had a polyspecific enzyme-linked immunosorbent assay (ELISA) performed between December 1, 2008, and April 1, 2009, for detection of platelet factor 4 (PF4)-heparin antibodies. The predictive value of the 4Ts scoring system as determined by using a standard laboratory marker of HIT--the ELISA--and the interrater reliability of the scoring system were assessed. Sixty-seven (84%) of the 80 patients had low clinical probability of HIT based on the calculated 4Ts score. The ELISA result was negative for PF4-heparin antibodies in 74 patients (93%). Based on the results of the ELISA, the negative predictive value of the 4Ts score was 91%. Each 4Ts score was calculated by two independent investigators and adjudicated by a third investigator when necessary. The interrater reliability of the scoring system was fair (Cohen κ coefficient 0.362, 95% confidence interval [CI] 0.222-0.502; weighted κ coefficient 0.554 (95% CI 0.441-0.667). Determination of the timing of HIT was associated with the largest number of discrepancies (16) between evaluators, followed by other causes of thrombocytopenia (15), degree of decline in platelet count (14), and the presence of thrombosis or other sequelae (2). A low 4Ts score supports a low probability of HIT based on the results of the polyspecific ELISA. Overall, the interrater reliability of the scoring system was fair. Components of the 4Ts scoring system need to be further clarified or modified in order to improve interrater reliability and thereby increase the clinical utility of this pretest probability model.

The utility of pre-test clinical scoring for clinical diagnosis of heparin-induced thrombocytopenia in cardiac surgery patients of a tertiary care centre in North India

Heparin-induced thrombocytopenia (HIT) should be diagnosed clinically as well as by laboratory assays for timely recognition, prevention and management of complications. To evaluate the clinical utility of pre-test clinical scoring system in combination with two immunoassays for the diagnosis of HIT in cardiac surgery patients. A total of 100 consecutive patients undergoing cardiac surgery were studied. Pre-test clinical scoring was carried out in patients with thrombocytopenia and further tested by two immunoassays, i.e., Heparin platelet factor 4 (H-PF4) enzyme-linked immunosorbent assay (ELISA) and particle gel immunoassay (PaGIA). Of the 100 patients studied, 42 patients developed thrombocytopenia post-operatively. On pre-test clinical scoring, low T-score was observed in 6 patients, intermediate in 28 and high score in 8 patients, whereas 19 patients (45.2%) were positive by H-PF4 ELISA and 10 (23.8%) by PaGIA for H-PF4 antibody. The difference in the incidence of clinically significant HIT antibodies in the three categories was statistically significant. A good correlation was also observed with ELISA optical density, T-scoring and PaGIA. Pre-test clinical scoring correlates well with the development of H-PF4 antibodies which are incriminated in the causation of thrombotic complications in patients with HIT. We also propose a protocol for diagnosing patients with clinical suspicion of HIT using pre-test clinical scoring and immunoassay.

Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands

While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.

Mesenteric and coeliac occlusions following heparininduced thrombocytopenia after aortobiprofunda bypass surgery

A 56-year-old man developed mid-gut bowel ischaemia following an elective aortobiprofunda bypass for short-distance claudication. The bowel was resected and he was commenced on lifelong total parenteral nutrition. He was found to have developed heparin-induced thrombocytopenia and thrombosis, confirmed by high levels of heparin-platelet factor 4-antibody on enzyme-linked immunosorbent assay (ELISA). He subsequently had foregut ischaemia with a second bout of thrombocytopenia despite not being on heparin. The case describes the first report of bowel ischaemia as a consequence of heparin-induced thrombocytopenia causing sequential superior mesenteric and coeliac arterial thrombosis in this scenario and highlights the importance of the awareness of the association of these pathological entities and subsequent management.

Higher Prevalence of Heparin-Induced Thrombocytopenia Antibodies in Asian Indian Population: Is This Due to Contaminated Heparin?

Abstract 4182Heparin treatment may result in an adverse effect because of the formation of antibodies against heparin-platelet factor 4 (H-PF4) complexes, which in turn leads to platelet/endothelial cell activation followed by heparin-induced thrombocytopenia (HIT). Several recent publications have reported a higher incidence of H-PF4 antibodies in patients treated with contaminated heparin. The main contaminant in recalled heparin was identified to be oversulfated chondroitin sulfate (OSCS), the same compound known to have caused several deaths due to generation of bradykinin, a potent vasoactive mediator, and generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. It is known that the quality-control measures in the European community and the United States are stringent; such control measures are not enforced in other countries. In our experience, that was performed prior to the global recall of contaminated heparins and the implementation of new analytical procedures, 11 of 47 cardiovascular surgery patients who have undergone heparin therapy were H-PF4 antibody positive by serotonin release assay. Of the 11 patients who showed positive H-PF4 antibody, only 5 of whom had thrombocytopenia and the remaining 6 patients did not show thrombocytopenia or any adverse effects. Even though a greater prevalence of H-PF4 antibody has also been reported with the bovine heparin preparations, the increase in prevalence of these antibodies may also be due to the quality of heparin used. Our subsequent study in another fifty cardiovascular surgery patients, who underwent heparin therapy, did not show any positive H-PF4 antibody. This demonstrates that the quality of heparin used may play a role in the generation of H-PF4 antibodies. Published reports suggest that the hypersulfated glycosaminoglycans such as the pentosan polysulfate also produces a much higher incidence of H-PF4 antibodies. Several other contaminants, such as the oversulfated derivatives of dermatan sulfate, heparin sulfate and heparin, may also be present in substandard heparin preparations. Thus, the higher generation of H-PF4 antibodies in these patients observed may be due to the heparin used; and heparin needs to be re-evaluated and subject to quality-control measures when a batch of heparin shows higher prevalence of H-PF4 antibodies. Also, to confirm true HIT in heparin treated patients, it is important to identify the functional forms of these H-PF4 antibodies. Disclosures:No relevant conflicts of interest to declare.

In Vitro Characteristics of Fondaparinux On HIT/HITT Samples.

Abstract 1319Poster Board I-335Heparin-induced thrombocytopenia (HIT) and HIT-associated thrombosis (HITT) are secondary to immune-mediated IgG antibodies that develop against the molecular complex of platelet factor 4 (PF4)–heparin, occurring with exposure to unfractionated heparin (UFH) and low molecular weight heparins (LMWH). Fondaparinux, a synthetic pentasaccharide that binds antithrombin and selectively inhibits Factor Xa, is indicated for prophylaxis and treatment of thrombosis. Fondaparinux has occasionally been reported to induce production of PF4–heparin antibodies; whether these antibodies activate platelets, in vitro or in vivo, remains unclear. Thus, fondaparinux has not been approved for patients with HIT/HITT. This study examined the in vitro cross-reactivity of fondaparinux in 2 HIT assays: a serotonin release assay (SRA) and an anti-PF4 enzyme-linked immunosorbent assay (ELISA) with neutralization. Serum samples (n=10) from patients with confirmed HIT/HITT were tested by SRA with UFH, LMWH (enoxaparin), and fondaparinux; and for anti-PF4 using a polyspecific ELISA (GTI, Waukesha, WI). In addition, 20 previously characterized samples strongly positive on the anti-PF4 ELISA (>1.000 OD) were used for confirmatory quenching studies with UFH, LMWH, and fondaparinux. LMWH-SRA and UFH-SRA results were concordant (positive) in all but one sample, which was positive with UFH but indeterminate with LMWH (Table). The fondaparinux-SRA was negative in 6 of 10 cases and indeterminate in 4. The indeterminate fondaparinux-SRA results were reproducible with a second platelet donor and when diluted 1:4 with negative anti-PF4 serum. Of note, platelet activation was observed in the buffer well (ie, without UFH, LMWH, or fondaparinux) in 3 of the 4 indeterminate cases but not in the 6 negative fondaparinux-SRA samples. All 10 samples showed positive results in the anti-PF4 ELISA (range=0.423-3.408 OD; median OD=2.588). In the second group of samples tested by anti-PF4 ELISA (range=1.395-3.204 OD; median=2.417 OD), quenching studies with excess UFH or LMWH yielded ≥50% inhibition, confirming anti-PF4 positivity, in 17 of 20 samples. Quenching with fondaparinux was not confirmatory, yielding only equivocal inhibition (range=0 to 46.7%; median=9.1%). In conclusion, fondaparinux appears to be generally not cross-reactive in SRAs performed on HIT/HITT-positive samples. The four fondaparinux-SRA cases were indeterminate despite heat inactivation of complement and the addition of hirudin to neutralize any thrombin contamination; this suggests that there may be heterogeneous pathogenic subpopulations that bind PF4 even in the absence of heparin or heparin-like molecules. The considerable variability in anti-PF4 confirmation studies with fondaparinux, relative to UFH and LMWH, warrants further study.TableSRA* and Anti-PF4 ELISA** Results from Patients with Clinical HIT/HITTUFH-SRALMWH-SRAFondaparinux-SRAAnti-PF4 ELISASample(% release)(% release)(% release)(OD units)110096indeterminate0.798298100<202.573334indeterminateindeterminate0.42347290<203.0125100107indeterminate3.27569592<202.90378099<202.60287470<202.34995586indeterminate1.75610100108<203.408*Positive result defined as ≥20% serotonin release with standard drug concentration and ≥50% inhibition with excess drug. Negative result defined as <20% serotonin release. Indeterminate result defined as ≥20% serotonin release with standard drug concentration and <50% inhibition with excess drug.**Positive result defined as OD ≥0.400. DisclosuresNo relevant conflicts of interest to declare.

Compliance with Platelet Count Monitoring Recommendations and Management of Possible Heparin-Induced Thrombocytopenia in Hospitalized Patients Receiving Low-Molecular-Weight Heparin

Summaries of product characteristics (SPCs) and clinical guideline recommendations are available for monitoring the platelet count for heparin-induced thrombocytopenia (HIT) in patients receiving low-molecular-weight heparin (LMWH). Testing for the presence of heparin-platelet factor 4 antibodies (HPF4-Ab) and starting alternative anticoagulation is recommended when HIT is suspected. To investigate the frequency of compliance with recommendations for platelet count monitoring and management of possible HIT in hospitalized patients receiving prophylaxis and treatment dosing of LMWH for at least 5 consecutive days. A retrospective cohort study within the Utrecht Patient Oriented Database (UPOD) was conducted. For all inpatients, all episodes of exposure to dalteparin or nadroparin for at least 5 consecutive days in 2004-2005 were selected. In 4 different nonexclusive groups of patients (all pts. receiving dalteparin, all pts. receiving nadroparin, surgical pts. with a prophylactic dose of either dalteparin or nadroparin, and pts. exposed to unfractionated heparin [UFH] within 100 days before receiving either dalteparin or nadroparin), compliance with recommendations for platelet count monitoring from SPCs and a clinical guideline was studied. The frequency of compliance with these recommendations was determined. In addition, it was determined whether patient and treatment characteristics were associated with regular platelet count monitoring. Finally, the frequency of testing for HPF4-Ab and the initiation of danaparoid treatment in patients with a drop of at least 50% in platelet count were investigated. A total of 6804 patients, with 7770 episodes of LMWH treatment, were included in the analysis. The frequency of compliance with platelet count monitoring recommendations was 26.3% for all patients receiving dalteparin, 35.6% for all patients receiving nadroparin, 23.0% for surgical patients receiving prophylactic dosing of either dalteparin or nadroparin, and 41.5% for patients exposed to UFH within 100 days before the start of either dalteparin or nadroparin treatment. Regular platelet count monitoring was strongly positively associated with medical patients (relative risk [RR] 2.33), surgical patients (RR 2.03), critically ill patients (RR 2.80), and those with recent exposure to UFH (RR 2.19). The frequency of testing for HPF4-Ab was 5.4% and the initiation of alternative anticoagulation with danaparoid in patients with a 50% drop in platelet count was 0%. The results suggest that compliance with recommendations for platelet count monitoring and management of possible HIT is low at our institution. Policies and tools to improve compliance with recommended laboratory monitoring should be developed to secure the safe use of LMWH and other medications.

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.

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.

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.

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.

Positive Heparin-Platelet Factor 4 Antibody Complex and Cardiac Surgical Outcomes

Given the large number of patients undergoing cardiac operations annually, it is important to identify populations at high risk for adverse outcomes. This observational study was conducted to determine the incidence of preoperative heparin-platelet factor 4 (HPF4) antibodies and to assess the associated risk of postoperative adverse outcomes in a nonselected cardiac surgery patient population. Between March 2002 and December 2004, 1114 (92%) of 1209 patients undergoing cardiac surgery with heparin were tested in an unselected manner for HPF4 antibodies. Main outcome measures were HPF4 antibody seropositivity and fatal and nonfatal adverse clinical outcomes after cardiac surgery. Of those screened, 60 (5.4%) of 1114 had positive HPF4 antibodies preoperatively. These patients had longer mean postoperative length of stay (14.0 days versus 9.8 days, p = 0.05), a higher incidence of prolonged (> or = 96 hours) mechanical ventilation (20.3% versus 9.2%, p = 0.02), acute limb ischemia (5.1% versus 0.9%, p = 0.03), renal complications including dialysis (20.3% versus 10.5%, p = 0.03), and gastrointestinal complications (15.3% versus 5.9%, p = 0.01). Stepwise logistic regression analysis showed positive HPF4 antibody status to be an independent predictor for adverse outcome and was associated with a higher risk for renal complications, including dialysis (adjusted odds ratio 2.2; 95% confidence interval, 1.1 to 4.3), than was diabetes. In this large patient series, the presence of HPF4 antibodies before surgical heparin administration was an independent and clinically significant risk factor for postoperative adverse events after cardiac surgery. An optimal preoperative cardiac surgery risk profile should include HPF4 antibody status.