Prospective Evaluation of Laboratory Tests for the Diagnosis of Heparin-Induced Thrombocytopenia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1051-1051 ◽  
Author(s):  
John L. Francis ◽  
Alane Drexler ◽  
Mary Kathryn Duncan ◽  
Hina Desai ◽  
Mildred Amaya ◽  
...  
Keyword(s):  
High Probability ◽  
Laboratory Diagnosis ◽  
Serotonin Release ◽  
Immunological Methods ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Wide Range ◽  
Immunological Assays ◽  
Positive Results ◽  
Test Probability

Abstract The laboratory diagnosis of heparin-induced thrombocytopenia (HIT) relies on the demonstration of antibodies to the heparin-platelet factor 4 (H-PF4) complex. Assays are based on the functional ability of H-PF4 antibodies to activate platelets, or detect the antibody directly by immunological methods. Multiple assays in each category are currently in clinical use and newer, rapid immunological assays are becoming available. The aim of this study was to compare available methods for detecting H-PF4 antibodies in a prospective study of patients with clinically suspected HIT. Functional assessment included serotonin release assay (SRA) and lumi-aggregometry (LA). Immunological assessment included ELISA (GTI), and particle gel immunoassay (PGIA; Diamed and Akers). Circulating platelet microparticles (PMP) were assessed by flow cytometry. Patients were also assessed for the pre-test probability of HIT using the Warkentin 4-T scoring system. 151 patients were enrolled. 54/151 patients (35.8%) had a positive GTI ELISA, while 53/151 (35.1%) and 39/151 (25.8%), respectively, had positive Akers and Diamed PGAI tests. Only 15/149 (10.1%) patients had a positive SRA, while only 5/150 (3.3%) gave a positive result by lumi-aggregometry. There was a strong correlation between the ELISA OD values obtained in serum and plasma using both fresh (r=0.98) and frozen (r=0.99) samples, although slightly more positive results were obtained using serum. Differences were only seen with OD values around the cut-off of 0.4. The majority (77.8%) of H-PF4 antibodies detected by ELISA were neutralized by heparin in the ‘confirmatory’ procedure. Weak antibodies (OD 0.4–0.5) were more likely to be non-neutralizable (5/12; 42%) than strong antibodies (OD>1.0; 4/23; 17%). 47 patients positive by ELISA were retested to determine the predominant immunoglobulin subclass. 15/47 (32%) were positive (OD>0.4) for IgG; 27/47 (57%) for IgM, and 12/47 (25%) for IgA. The Diamed assay more closely correlated with the GTI ELISA than the Akers test (82.1% vs. 56.7%, respectively). The PGIAs were only moderately correlated with each other (64%) with the Akers assay giving more “false positive” results relative to the ELISA. PMP were higher in patients with a positive ELISA (6.2 vs 4.7 × 106/ml) or positive SRA (5.5 vs. 5.1 ×106/ml) but this was not statistically significant due to the wide range of results. Of 119 patients assessed, 87 had a low pre-test probability of HIT (4-T score 0–3), 27 had an intermediate probability (4–5), and 5 had a high probability (6–8). The GTI ELISA was positive in 24, 56 and 80% of low, intermediate and high probability cases. The Akers PGIA was positive in 39, 41 and 40% respectively; the Diamed assay in 21, 33 and 40%, and the SRA in 7, 11 and 40%, respectively. This study was conducted in a patient population biased towards cardiovascular surgery, and confirms previously reported observations that immunoassays are more frequently positive than functional assays. The ELISA correlated better than the PGIA tests with the pre-test probability of HIT, although the Diamed test showed acceptable correlation with the ELISA. In contrast, the Akers assay correlated poorly with the ELISA, often producing positive results when the latter test was negative. We conclude that while the PGIA tests are rapid and convenient, further studies are needed to determine the basis for disparate results relative to the widely used ELISA.

scholarly journals Low Pre-Test Probability Scores Are Not Sufficient to Exclude Heparin-Induced Thrombocytopenia in Critically Ill Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3256-3256
Author(s):  
Noppacharn Uaprasert ◽  
Benjaporn Akkawat ◽  
Rattaporn Vichitratchaneekorn ◽  
Chantiya Chanswangphuwana ◽  
Ponlapat Rojnuckarin
Keyword(s):  
Critically Ill ◽  
Critically Ill Patients ◽  
Diagnostic Performance ◽  
High Probability ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Scoring Model ◽  
Low Probability ◽  
Positive Results ◽  
Test Probability

Abstract Background: Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin administration. Diagnosis of HIT is a clinical challenge. The 4Ts scoring model (0-3 low, 4-5 intermediate, 6-8 high probability of HIT) has been validated in several studies including the recent meta-analysis (Blood 2012;120:4160-7), which demonstrated that a low probability 4Ts score conferred a high negative predictive value (0.998; 95% CI, 0.970-1.000) for excluding HIT. Some experts propose a diagnostic approach for HIT based on the 4Ts scoring model and exclude HIT in patients with a low probability score without serologic testing for HIT. Another recently constructed model 'HIT expert probability' (HEP) score (< 2 unlikely, ≥ 2 likely) demonstrated better diagnostic performance in mainly surgical patients. However, in critically ill patients who receive heparin, other concomitant causes of thrombocytopenia are common and may interfere with clinically diagnosis of HIT. In this study, we aimed to determine the diagnostic accuracy of the 4Ts and the HEP score for excluding HIT in a population of critically ill patients. Methods: Consecutive patients admitted in critical care units during 2006-2015 were included in this study. Clinical and laboratory data of individuals were retrospectively reviewed from medical records. The 4Ts and the HEP score were blindly computed by two independent reviewers (NU and RV). The rapid particle gel immunoassay (platelet factor 4/heparin-PaGIA) was used for HIT screening. Subjects yielding positive PaGIA were sent for the confirmatory testing using the in-house platelet aggregometry measuring heparin-induced platelet aggregation (HPA). However, during the shortage of PaGIA, HPA was performed in all cases. HPA using platelet-rich plasma from healthy donors with known reactive platelets was performed as previously described with a few modification. Aggregation values of at least 20% above negative controls in the presence of 0.5 or 1.0 U/ml of heparin, which were subsequently inhibited by the addition of 100 U/ml of heparin, were defined as positive results. Results: There were 92 critically ill patients with suspected for HIT. Among them, 56 (60.9%), 33 (35.9%) and 3 (3.3%) yielded low, intermediate and high probability 4Ts score, respectively, while 33 (35.9%) and 59 (64.1%) yielded unlikely and likely high probability HEP score, respectively. Of 78 with obtainable PaGIA, 25 cases (37.2%; 6/6 HPA+ and 19/72 HPA-) yielded positive results. Eleven patients (12.0%) yielded positive results for HPA were diagnosed as HIT. There were 6 (54.5%) developing thrombosis (4 new proven and 2 progressive). Clinical data of all documented HIT were summarized in the table 1. Documented HIT was diagnosed in 5.4%, 18.2% and 66.7% of low, intermediate and high probability 4Ts score, respectively, whereas HIT was demonstrated in 9.4% and14.3% of unlikely and likely probability HEP score, respectively. The receiver operating characteristic curve analysis demonstrated that the 4Ts score was tended to display better diagnostic performance than the HEP score with the area under curve of 0.740 and 0.587 (P = 0.053), respectively. The HIT cases with low pre-test probability scores were due to concomitant causes of thrombocytopenia resulting in earlier onset, lower nadir of platelet counts and/or more minus scores from alternative etiologies of thrombocytopenia. Conclusions: The diagnostic performance of the 4Ts and the HEP score is limited in complicated and/or critically ill patients due to multiple etiologies affecting onset and severity of thrombocytopenia. Both low probability 4Ts score and unlikely HEP score are unsafe for excluding HIT in this patient group. Table 1. Clinical characteristics of patients documented heparin-induced thrombocytopenia Case Age (years)/ sex Patient type Heparin type Thrombosis PaGIA 4Ts score HEP score 1 61/ M CVT UFH New + 2 -6 2 37/ M CCU UFH, LMWH Progressive NA 3 -3 3 74/ F CVT UFH No NA 3 3 4 83/ M GenS UFH, LMWH New + 4 -6 5 80/ F Med LMWH No + 4 6 6 62/ M CCU UFH No NA 5 4 7 51/ M CCU UFH No NA 5 5 8 80/ M Med UFH No + 5 6 9 76/ M CCU UFH Progressive NA 5 10 10 50/ F Med UFH, LMWH New + 6 5 11 87/ M CCU UFH New + 7 9 M: male, F: female, CVT: cardiovascular thoracic surgery, CCU: coronary care unit, GenS: general surgery, Med: medicine, UFH: unfractionated heparin, LMWH: low molecular weight heparin, NA: not available Disclosures No relevant conflicts of interest to declare.

Serologic Results in >1000 Patients With Suspected Heparin-Induced Thrombocytopenia

2007 ◽  
Vol 14 (4) ◽  
pp. 410-414 ◽  
Author(s):  
Suresh G. Shelat ◽  
Anne Tomaski ◽  
Eleanor S. Pollak
Keyword(s):  
Laboratory Diagnosis ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Life Threatening ◽  
Release Assay ◽  
Pathogenic Antibodies

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

A New Enzyme Immuno-Assay That Only Detects IgG Antibodies to Heparin-PF4 Complexes (Zymutest HIA IgG®) Improves the Specificity of the Diagnosis of HIT without Loss of Sensitivity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2096-2096
Author(s):  
Claire Pouplard ◽  
Sandra Regina ◽  
Jean Baptiste Valentin ◽  
Yves Gruel
Keyword(s):  
Platelet Activation ◽  
High Specificity ◽  
Serotonin Release ◽  
Igg Antibodies ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Release Assay ◽  
Immuno Assay ◽  
Enzyme Immuno Assay ◽  
Test Probability

Abstract Heparin-induced thrombocytopenia (HIT) is associated in most patients with the development of antibodies to heparin-modified platelet factor 4 (PF4). Commercial immuno-assays frequently detect these antibodies after cardiac surgery but only few patients develop clinical HIT. Therefore, platelet activation tests such as serotonin release assay (SRA) are necessary to ensure the diagnosis of HIT with high specificity. Another approach to increase diagnosis specificity could be to detect IgG antibodies which are of major clinical relevance since they are the only class able to directly activate platelets in the presence of heparin. Therefore, we evaluated the performances of a new commercial immuno-assay specific to IgG for the diagnosis of HIT Abs (Zymutest HIA IgG®, Hyphen Biomed, Neuville sur Oise, France). Samples from 101 patients with suspected HIT were analysed. 40 cases had developed significant levels of Abs to PF4 measured with PVS/PF4 ELISA (HAT45®,GTI, Brookfiled, WI, USA) and the diagnosis of HIT had been confirmed since SRA was positive. Every sample was then tested with a global assay named Zymutest HIA G/A/M® as followed: each diluted plasma (200 μl) was incubated for 1 hour with 50 μl of platelet lysate providing PF4 into wells previously coated with unfractionated heparin. After washings and incubation (1 hour) with anti IgG/A/M-HRP immunoconjugate, the enzyme activity was developed and absorbance was read at 450nm. In case of positive result (A450 ≥ 0.5), the isotype distribution was analysed with a specific and standardized assay using monospecific anti-IgG-, anti-IgA- and anti IgM-HRP conjugates (Zymutest HIA-IgG® or -IgA® or -IgM®). A450 values ≥ 0.5 were also considered as positive. GTI assay that detected IgG/A/M Abs to PVS/PF4 complexes in the 40 patients with HIT (Ss 100%) was also positive in 30 of the 61 cases with no HIT (Sp 50.8%). Comparatively, Zymutest HIA® global assay was positive in 39 of the 40 patients with HIT (Ss 97.5%) and in 14 of 61 cases without HIT (Sp 77%). On the other hand, significant levels of heparin-dependent IgG antibodies were also measured in these 39 HIT patients using Zymutest HIA IgG® assay (mean A450: 1.81; range A450: 0.5 – 2.76), and only in 6 patients without HIT (Sp: 90%). However, IgG levels measured in patients without HIT were significantly lower (mean A450: 0.60; range A450: 0.08 – 2.20) than in those with HIT (p &lt; 0.0001). In addition, IgA or IgM heparin-dependent antibodies were only present, i.e. without IgG, in samples from patients for whom the diagnosis of HIT had been ruled out (n = 3). In conclusion, this study supports that the detection of significant levels of IgG heparin-dependent antibodies improves the diagnosis specificity in patients with a suspicion of HIT without loss of sensitivity. Nonetheless, whether IgG-specific immunoassays could avoid to perform platelet activation tests in patients with a strong pre-test probability of HIT warrant further study.

scholarly journals Clinical Significance of the Serotonin Release Assay and Platelet Count Monitoring After Cardiac Surgery

2017 ◽  
Vol 24 (6) ◽  
pp. 944-949 ◽  
Author(s):  
Shinya Motohashi ◽  
Takefumi Matsuo ◽  
Hidenori Inoue ◽  
Makoto Kaneko ◽  
Shunya Shindo
Keyword(s):  
Cardiac Surgery ◽  
Platelet Count ◽  
Clinical Course ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Release Assay ◽  
Immunological Assays ◽  
Platelet Count Monitoring ◽  
The Relationship

Heparin-induced thrombocytopenia (HIT) is one of the serious complications in patients who undergo cardiac surgery. However, there remains a major problem in diagnosing HIT because the current immunological assays for detection of HIT antibody have limitations. Furthermore, the clinical course of thrombocytopenia in this surgery makes it increasingly difficult to diagnose HIT. We investigated the relationship between platelet count and HIT antibody in 59 patients who underwent cardiac surgery using cardiopulmonary bypass (CPB). The number of postoperative HIT antibody-positive patients evaluated using enzyme-linked immunosorbent assay kit (polyanion IgG/IgA/IgM complex antibodies/antiplatelet factor 4 enhanced) was 37 (62.7%). In contrast, platelet activation by HIT antibody was evaluated using the serotonin release assay (SRA). More than 20% and 50% release of serotonin was obtained from 12 patients (20.3%) and 8 patients (13.6%), respectively. The levels of d-dimer were significantly different on postoperative day 14 between SRA-positive and SRA-negative groups; however, postoperative thrombus complication was not detected using sonography in the patients with positive serotonin release at all. After being decreased by the operation, their platelet count recovered within 2 weeks in both groups equally. In our study, although the patients were positive in the platelet activating HIT antibody assay, they remained free from thrombosis and their platelet count recovered after early postoperative platelet decrease. Therefore, in addition to the SRA, monitoring of platelet count might be still considered an indispensable factor to facilitate the prediction of HIT thrombosis prior to manifestation in the patients undergoing cardiac surgery using CPB.

Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT)

2011 ◽  
Vol 106 (12) ◽  
pp. 1197-1202 ◽  
Author(s):  
Jakob Hesberg ◽  
Sentot Santoso ◽  
Gregor Bein ◽  
Tamam Bakchoul ◽  
Ulrich Sachs
Keyword(s):  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Medical Patients ◽  
High Potency ◽  
Platelet Factor ◽  
Predictive Values ◽  
Heparin Induced Thrombocytopenia ◽  
Clinical Probability ◽  
Short Period ◽  
Test Probability

SummaryHeparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid singlesample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

Diagnosis and treatment of heparin-induced thrombocytopenia

Perfusion ◽  
2003 ◽  
Vol 18 (1) ◽  
pp. 47-53 ◽  
Author(s):  
William J DeBois ◽  
Junli Liu ◽  
Leonard Y Lee ◽  
Leonard N Girardi ◽  
Charles Mack ◽  
...  
Keyword(s):  
New York ◽  
Platelet Inhibition ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Antibody Detection ◽  
Thrombin Inhibitor ◽  
Heparin Infusion ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Life Threatening

Heparin-induced thrombocytopenia (HIT) is a major side effect secondary to the administration of heparin. This syndrome is serious and potentially life threatening. This response is the result of antibodies formed against the platelet factor 4 (PF4)/heparin complex. The incidence of this immune-mediated syndrome has been estimated to be 1-3% of all patients receiving heparin therapy. The occurrence of HIT in patients requiring full anticoagulation for cardiopulmonary bypass (CPB), therefore, presents a serious challenge to the cardiac surgery team. The diagnosis of HIT should be based on both clinical and laboratory evidence. While functional assays, platelet aggregation tests, and the serotonin release assay can be used to support the diagnosis, the negative predictive value of these tests is generally less than 50%. In contrast, although non-functional antibody detection assays are more sensitive, they have a low specificity. HIT can be treated in several ways, including cessation of all heparin and giving an alternative thrombin inhibitor, platelet inhibition followed by heparin infusion, and the use of low molecular weight heparins. In this presentation, the pathology and current diagnostic tests, as well as the successful management of patients with HIT undergoing CPB at New York Presbyterian Hospital, are reviewed.

Pathophysiology of Heparin-Induced Thrombocytopenia

2000 ◽  
Vol 124 (11) ◽  
pp. 1657-1666 ◽  
Author(s):  
Fabrizio Fabris ◽  
Sarfraz Ahmad ◽  
Giuseppe Cella ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
New Information ◽  
Study Selection ◽  
Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

Implementation of a Fully Automated Latex Immunoturbidimetric Assay for the Diagnosis of Heparin-Induced Thrombocytopenia in a High-Volume University Hospital Laboratory

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S30-S30
Author(s):  
Christina Pierre ◽  
Mary Acker ◽  
Surabhi Palkimas ◽  
Lindsay Bazydlo
Keyword(s):  
High Volume ◽  
Turnaround Time ◽  
University Hospital ◽  
Reference Laboratory ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Hospital Laboratory ◽  
Assay Precision ◽  
Positive Results ◽  
Interassay Precision

Abstract Background Heparin-induced thrombocytopenia (HIT) is an immune-mediated, adverse reaction to heparin in which heparin binds platelet factor 4 (PF4), triggering the development of heparin-PF4 antibodies (HITAb). HITAbs bind and activate platelets, causing thrombosis, platelet consumption, and thrombocytopenia. Heparin is replaced with relatively costly nonheparin anticoagulants until HIT can be ruled out. HIT diagnosis consists of a HITAb immunoassay with reflex to a serotonin release assay (SRA) for confirmation of positive results. Recently, a fully automated latex immunoturbidimetric assay (LIA) for detection of HITAbs received FDA clearance. We sought to verify the performance characteristics of the LIA with the aim of implementing the test in a high-volume university hospital laboratory. Methods The in-house HITAb LIA was performed on the Instrumentation Laboratory TOP700 analyzer using HemosIL HIT-Ab(PF4-H) reagent. The comparator method, a HITAb ELISA, was performed at a reference laboratory with positive results reflexed to SRA. All samples (36 total) sent to the reference laboratory for HIT testing from December 2017 to November 2018 were aliquoted and run in parallel by LIA. Intra-assay precision was assessed by running manufacturer-provided low and high control samples 10 times in succession, while interassay precision was assessed by running low and high samples every day for 10 days. Turnaround time to HITAb result was retrieved from the electronic medical records for HIT testing performed 60 days before and after in-house test implementation. Results The agreement between the LIA and ELISA was 92% (33/36). One discordant sample tested negative by ELISA and was not assessed by SRA. Another tested positive by ELISA and negative by LIA and was confirmed negative by SRA. The final discordant sample tested negative by LIA but positive by ELISA and was confirmed positive by SRA. Thirty-three percent (12/36) of samples tested positive for HITAb by ELISA and were reflexed to SRA. Both the ELISA and LIA showed 83% agreement (10/12) with the SRA. The coefficient of variance (CV) for the intra-assay precision studies was 18% and 5% for the low and high controls, respectively. The CV for the interassay precision studies was 28% and 5% for the low and high controls, respectively. Postimplementation quality control data revealed 61% and 20% imprecision on the low and high level controls, respectively, which declined significantly when reagents were removed from the instrument and refrigerated within 2 hours. The turnaround time for HITAb results was reduced by 74% (10.5 vs 41 hours) after in-house test implementation, significantly reducing the need for administration of nonheparin anticoagulants. Conclusion The LIA and ELISA methods compared favorably, allowing for clinical implementation of the LIA. The shortened turnaround time of the LIA significantly reduced the time to rule out HIT, enhancing patient care and reducing drug costs. The assay imprecision warrants further investigation regarding reagent stability.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3414-3414
Author(s):  
Dorothee Leroux ◽  
Claire Pouplard ◽  
Benoit Guillet ◽  
Beatrice Cosne ◽  
Marc Antoine May ◽  
...  
Keyword(s):  
Heart Surgery ◽  
Cytotoxic T Lymphocyte ◽  
Rflp Analysis ◽  
Serotonin Release ◽  
Nucleotide Polymorphisms ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Significant Difference ◽  
Patients At Risk ◽  
High Doses

Abstract 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 frequency Ab neg (n = 82) Ab pos (n = 58) HIT (n = 82) CTLA-4(+49) A/A 31 (38%) 24 (41%) 35 (43%) A/G 40 (49%) 26 (45%) 41 (50%) G/G 11 (13%) 8 (14%) 6 (7%) CTLA-4(−318) C/C 63 (77%) 49 (84%) 67 (82%) C/T 19 (23%) 9 (16%) 17 (21%) T/T 0 0 0 (0%) TNFα(−308) G/G 59 (72%) 41 (71%) 68 (84%) G/A 20 (24%) 15 (26%) 12 (15%) A/A 3 (4%) 2 (3%) 1 (1%) A Allele 0.160 0.160 0.09 G Allele 0.840 0.840 0.910 Conclusion: 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.

Heparin-Induced Thrombocytopenia: A Rapid, Reliable and Practical, Functional Flow-Cytometric Assay

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1130-1130
Author(s):  
Varda Deutsch ◽  
Michal Cipok ◽  
Sigi Kay ◽  
Yvette Levy ◽  
Shoshana Bar On ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
High Sensitivity ◽  
Relative Sensitivity ◽  
Serotonin Release ◽  
Specific Test ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Laboratory Confirmation ◽  
Relative Specificity ◽  
Biological Probe

Abstract Background Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin treatment, associated with morbidity and mortality. HIT is characterized by thrombocytopenia and thrombotic complications secondary to the formation of antibodies (Abs) against heparin-platelet-factor 4 (PF4) complexes. The pathologic mechanism involves the binding of the heparin-immune-complex to the platelet-Fc-receptor, resulting in platelet activation, aggregation, and rapid elimination. The diagnosis of HIT requires laboratory confirmation. Common laboratory testing is based on immune detection of antibodies directed against the PF4/heparin complex (ID-H/PF4-PaGIA or ELISA). However, these assays suffer from methodological limitations, especially low specificity, as compared to the platelet functional assays. The “gold standard” functional test for detecting of platelet-activating antibodies is the radioactive [14C] serotonin-release assay (14C-SRA) (Sheridan D, et al, Blood. 1986;67:27-30, Kelton JG, et al.,Blood.1988;72:925-30). However, the assay includes the use of a radiolabeled biological probe and requires considerable expertise to obtain reliable results. Consequently, its use is limited to research laboratories. Aim To overcome the methodological limitations associated with current assays, we modified a functional flow-cytometry assay (FCA), which exhibits high sensitivity and specificity (Tomer, A. Br J Haematol, 1997;98: 648-656 , Tomer, A., et al, Am J Hematol, 1999;61: 53-61). This assay, similar in concept to the 14C-SRA, determines the capacity of the patient's serum to activate platelets in the presence of heparin, using a fluorescent probe. Methods Consecutive samples from 254 patients clinically suspected for HIT were tested. The FCA assay was compared with the standard ID-H/PF4-PaGIA antigenic assay (DiaMed, Switzerland) with two dilutions to assess specificity (Nellen, V., et al.,Haematologica, 2012;97: 89-97). Results Of the total 254 samples tested, 48 (19%) were positive by PaGIA, compared to 13 (5.1%) positive by the functional FCA (Table 1). The number of PaGIA positive samples was reduced to 24 (9.4%) by 1:16 dilution, and to 14 (5.5%) by 1:32 dilution. All FCA positive samples were positive at all PaGIA dilutions (relative sensitivity 93%). Thirty PaGIA negative samples were all negative by the FCA (relative specificity 100%). Conclusion The results suggest that the functional FCA is a practical, sensitive, and highly specific test for the reliable diagnosis of HIT. Disclosures: No relevant conflicts of interest to declare.

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