Particle Gel Immunoassay Research Articles

Limited concordance of heparin/platelet factor 4 antibody assays for the diagnosis of heparin-induced thrombocytopenia: an analysis of the TORADI-HIT study

BackgroundAnecdotal reports suggest that the correlation between heparin/platelet factor 4 (PF4) antibody assays for the diagnosis of heparin-induced thrombocytopenia (HIT) is limited. ObjectivesTo investigate the correlation between widely used assays and examine possible factors contributing to variability. MethodsThis is a large, prospective cohort study with 10 participating tertiary care hospitals including 1393 patients with suspected HIT in clinical practice. HIT was defined by a positive heparin-induced platelet activation (HIPA) assay (washed platelet reference standard test). Three different immunoassays were used to measure heparin/PF4 antibodies: chemiluminescent immunoassay, enzyme-linked immunosorbent assay, and particle gel immunoassay. Various factors that could influence the assays were examined: sex (male or female), age (<65 years or ≥65 years), unfractionated heparin exposure, presence of thrombosis, cardiovascular surgery, and intensive care unit. Spearman’s correlation coefficients were calculated. Z-scores and diagnostic odds ratios were determined in the aforementioned subgroups of patients. ResultsAmong 1393 patients, 119 were classified as HIT-positive (prevalence, 8.5%). The median 4Ts score was 5 (IQR, 4-6) in patients with HIT compared with 3 (IQR, 2-4) in patients without HIT. Correlations (rs) between immunoassays were weak (0.53-0.65). Inconsistencies between immunoassays could not be explained by further analyses of z-scored test results and diagnostic odds ratios in subgroups of patients. ConclusionThe correlation between widely used heparin/PF4 antibody assays was weak, and key factors could not explain this variability. Standardization of immunoassays is requested to improve comparability.

Diagnostic Performance of a Particle Gel Immunoassay in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of adenoviral vector-based COVID-19 vaccines. Similar to heparin-induced thrombocytopenia (HIT), antibodies reacting to platelet factor 4 (PF4) are responsible for platelet activation in VITT. The diagnosis of VITT includes the detection of anti-PF4 antibodies. Particle gel immunoassay (PaGIA) is one of the rapid immunoassays that is commonly used in the diagnosis of HIT to detect anti-PF4 antibodies. The aim of this study was to investigate the diagnostic performance of PaGIA in patients suspected of VITT. In this retrospective, single-center study, the correlation between PaGIA, enzyme immunoassay (EIA), and modified heparin-induced platelet aggregation assay (HIPA) in patients with findings suggestive of VITT was investigated. A commercially available PF4 rapid immunoassay (ID PaGIA H/PF4, Bio-Rad-DiaMed GmbH, Switzerland) and an anti-PF4/heparin EIA (ZYMUTEST HIA IgG, Hyphen Biomed) were used according to manufacturer's instructions. Modified HIPA was accepted as the gold standard test. Between March 8 and November 19, 2021, a total of 34 samples from clinically well-characterized patients (14 males, 20 females, mean age: 48.2 ± 18.2 years) were analyzed with PaGIA, EIA, and modified HIPA. VITT was diagnosed in 15 patients. Sensitivity and specificity of PaGIA were 54 and 67%, respectively. Anti-PF4/heparin optical density values were not significantly different between PaGIA positive and negative samples (p = 0.586). The sensitivity and specificity of EIA, on the other hand, were 87 and 100%, respectively. In conclusion, PaGIA is not reliable in the diagnosis of VITT because of its low sensitivity and specificity.

A machine-learning model for reducing misdiagnosis in heparin-induced thrombocytopenia: A prospective, multicenter, observational study.

Diagnosing heparin-induced thrombocytopenia (HIT) at the bedside remains challenging, exposing a significant number of patients at risk of delayed diagnosis or overtreatment. We hypothesized that machine-learning algorithms could be utilized to develop a more accurate and user-friendly diagnostic tool that integrates diverse clinical and laboratory information and accounts for complex interactions. We conducted a prospective cohort study including 1393 patients with suspected HIT between 2018 and 2021 from 10 study centers. Detailed clinical information and laboratory data were collected, and various immunoassays were conducted. The washed platelet heparin-induced platelet activation assay (HIPA) served as the reference standard. HIPA diagnosed HIT in 119 patients (prevalence 8.5%). The feature selection process in the training dataset (75% of patients) yielded the following predictor variables: (1) immunoassay test result, (2) platelet nadir, (3) unfractionated heparin use, (4) CRP, (5) timing of thrombocytopenia, and (6) other causes of thrombocytopenia. The best performing models were a support vector machine in case of the chemiluminescent immunoassay (CLIA) and the ELISA, as well as a gradient boosting machine in particle-gel immunoassay (PaGIA). In the validation dataset (25% of patients), the AUROC of all models was 0.99 (95% CI: 0.97, 1.00). Compared to the currently recommended diagnostic algorithm (4Ts score, immunoassay), the numbers of false-negative patients were reduced from 12 to 6 (-50.0%; ELISA), 9 to 3 (-66.7%, PaGIA) and 14 to 5 (-64.3%; CLIA). The numbers of false-positive individuals were reduced from 87 to 61 (-29.8%; ELISA), 200 to 63 (-68.5%; PaGIA) and increased from 50 to 63 (+29.0%) for the CLIA. Our user-friendly machine-learning algorithm for the diagnosis of HIT (https://toradi-hit.org) was substantially more accurate than the currently recommended diagnostic algorithm. It has the potential to reduce delayed diagnosis and overtreatment in clinical practice. Future studies shall validate this model in wider settings. Swiss National Science Foundation (SNSF), and International Society on Thrombosis and Haemostasis (ISTH).

Application of a clinical decision rule and laboratory assays in pediatrics: Adult heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is rare among pediatric patients. The diagnosis of HIT depends upon clinical decision tools to assess its pretest probability, supported by laboratory evidence of anti-platelet factor 4 (anti-PF4)/heparin antibodies. To compare the use of the 4Ts score clinical decision tool, clinical characteristics, and laboratory findings between pediatric and adult patients with suspected HIT. We compiled all pediatric patients in our center for whom HIT testing was performed during the years 2015-2021. These were compared with a cohort of consecutive adult patients. Laboratory diagnosis of HIT was performed with particle gel immunoassay (PaGIA) as screening test and confirmed by an automated latex-enhanced immunoturbidimetric assay (LIA) and/or by functional flow cytometry assay (FCA). The cohort included 34 children (under 18years) and 105 adults. Adults mostly received heparins for thromboembolism prophylaxis and treatment (72.4%, n=76), and were more frequently treated with low-molecular-weight heparin (LMWH). Children were mostly exposed during cardiopulmonary bypass and extracorporeal membrane oxygenation (ECMO, 61.8%, n=21), and were more frequently treated with unfractionated heparin (UFH). Compared with adults, children had significantly higher 4Ts scores. Nevertheless, adults had a slightly higher rate of a positive diagnosis of HIT. Six out of 16 adults with confirmed HIT presented with thrombosis (37.5%), whereas all three pediatric patients with HIT presented with thrombosis (p= .087). 4Ts scores are higher in children compared with adult patients for whom laboratory tests for HIT were obtained. A potentially higher incidence of thrombosis in children with HIT may be attributable to the severity of underlying illness.

Managing argatroban in heparin-induced thrombocytopenia: Aretrospective analysis of 729 treatment days in 32 patients with confirmed heparin-induced thrombocytopenia.

Managing argatroban in heparin-induced thrombocytopenia: Aretrospective analysis of 729 treatment days in 32 patients with confirmed heparin-induced thrombocytopenia.

High Incidence of True Heparin-Induced Thrombocytopenia and Heparin-Induced Thrombocytopenia Antibody Development in Critically Ill Medical Patients

Background: Thrombocytopenia is very prevalent in critically ill medical patients. Heparin-induced thrombocytopenia (HIT), which is a serious immunologic complication of heparin administration, is one of the major concerns. Diagnosis of HIT in critically ill patients is challenging due to concomitant illnesses. We previously reported the HIT cohort with the high proportion of critically ill medical patients (61.7%) in Thailand (Uaprasert N, etal. Blood Coagul Fibrinolysis 2013;24:261-8). However, data of the true incidence and risk factors for HIT in this particular population in Thailand are still lacking. In addition, the confirmatory assay is not readily available and unlikely to be useful for timely clinical management. Therefore, clinical predictors and screening assays are important for immediate clinical judgment. This study aimed to determine the incidence and define risk factors for HIT and HIT antibody (HIT-Ab) development as well as an appropriate clinical predicting model for HIT in critically ill medical patients.Methods: Consecutive critically ill medical patients who received heparins and were admitted in the intensive care unit (ICU), coronary care unit (CCU) and intensive coronary care unit (ICCU) at King Chulalongkorn Memorial Hospital between October 2013 and May 2017 were included in this study. Patients admitted in general wards who met one of the following criteria: 1) on mechanical ventilator 2) receiving inotropic agents 3) recent undergoing renal replacement therapy due to acute kidney injury and/or 4) APACHE II score > 25, were also included for analysis. Blood samples were collected between 6 and 8 days after heparin exposure. Patients died or discharged before day 6th after heparin exposure were excluded from the study. The rapid particle gel immunoassay (PaGIA) was used as the screening test for detecting antibodies against the heparin/platelet factor 4 complex. Subjects yielding positive PaGIA were sent for the confirmatory test using the in-house platelet aggregometry measuring heparin-induced platelet aggregation (HPA). Details of the medical history and hospital course of individual patient were reviewed to obtain risk factors for HIT as well as to compute two well-defined clinical probability scores (4Ts and HIT expert probability, HEP).Results: There were 217 patients included for analysis. The average age was 64.9 ± 18.7 (mean ± standard deviation) years. The baseline platelet counts were 217 ± 116 x 109/L. A platelet fall ≥ 50% from baseline was found in 135 (62.2%) patients. The most common type of heparin administered was unfractionated heparin (165; 76%). HIT-Ab using PaGIA was detected in 52 (24%) patients, while true HIT was confirmed using HPA in 3 patients (1.38%). Most common underlying illnesses found in patients developing HIT-Ab were hypertension, coronary arterial disease and active infection. Acute stroke, a platelet fall ≥ 50% from baseline, 4Ts score of > 3 and HEP score of ≥ 2 were significantly associated with development of HIT-Ab in univariate analysis, while heparin types and hemodialysis were not statistically associated with HIT-Ab development. Notably, only acute stroke was significantly associated with HIT-Ab development in multivariate analysis (Odds ratio 6.56; 95% confidence interval 1.42-30.31, p=0.016).Conclusion: The incidence of HIT-Ab detected in critically ill medical patients in our cohort was comparable to most studies, while the incidence of true HIT in this cohort was apparently higher than previous reports, which their incidences were ≤ 0.5%. The reasons underlying the high incidence of true HIT in critically ill medical patients in Thailand remains elusive. Acute stroke is a significant risk for HIT-Ab development in critically ill medical patients receiving heparin. DisclosuresNo relevant conflicts of interest to declare.

THE HEPARIN INDUCED THROMBOCYTOPENIA IN CARDIAC SURGERY PATIENTS

Objective: Combination of ‘4Ts’ clinical scoring system and Particle gel immunoassay to determine the frequency of heparin-induced thrombocytopenia during heparin treatment in cardiac surgery patients.&#x0D; Study Design: Prospective observational study.&#x0D; Place and Duration of Study: This study was conducted in the Pathology Department, Army Medical College incollaboration with Armed Forced Institute of Cardiology and Armed Forced Institute of Transfusion, Rawalpindi,from Jan 2019 to Dec 2019.&#x0D; Methodology: A total of 115 suspected cases of heparin-induced thrombocytopenia irrespective of age and gender were included in the study. A clinical scoring system the ‘4Ts’ was used for the classification of patients based on probability into three groups such as low, intermediate, and high probability groups. For the detection of antibodies against heparin/platelet factor 4 complexes, the Particle gel immunoassay was used.&#x0D; Results: There were 39 (33.9%) females and 76 (66.1%) males, the age range of 20 to 86 ± 12.9 years. Among the low probability group, there was no positive result, 2 (2.3%) patients showed positive serological evidence in the intermediate probability group. In the high probability group, 4 (23.5%) patients showed positive results with the chosen assay.&#x0D; Conclusion: Heparin-induced thrombocytopenia was found an overall 5.2% of patients undergoing cardiacsurgery receiving unfractionated heparin. A combination of ‘4Ts’ pretest clinical scoring systems followed byPaGIA constitutes a simple strategy to screen for heparin-induced thrombocytopenia in suspected patients. This will avoid serious complications if the detection is not delayed.

Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated hypercoagulable state featuring high thrombosis risk and distinct pathogenesis involving immunoglobulin G-mediated platelet activation. The target of the immune response is a cationic “self” protein, platelet factor 4 (PF4), rendered antigenic by heparin. A key problem is that only a minority of anti-PF4/polyanion antibodies induced by heparin are pathogenic, i.e., capable of causing platelet activation and thereby clinical HIT. Since thrombocytopenia occurs frequently in hospitalized, heparin-treated patients, testing for “HIT antibodies” is common; thus, the problem of distinguishing between pathogenic and nonpathogenic antibodies is important. The central concept is that those antibodies that have platelet-activating properties demonstrable in vitro correlate well with pathogenicity, as shown by platelet activation tests such as the serotonin-release assay (SRA) and heparin-induced platelet activation assay. However, in most circumstances, immunoassays are used for first-line testing, and so it is important for clinicians to appreciate which immunoassay result profiles—in the appropriate clinical context—predict the presence of platelet-activating antibodies (Bayesian analysis). Clinicians with access to rapid, on-demand HIT immunoassays (e.g., particle gel immunoassay, latex immunoturbidimetric assay, chemiluminescent immunoassay) can look beyond simple dichotomous result interpretation (“negative”/“positive”) and incorporate semiquantitative interpretation, where, for example, a strong-positive immunoassay result (or even combination of two immunoassays) points to a greater probability of detecting platelet-activating antibodies, and hence supporting a diagnosis of HIT. Recent recognition of “SRA-negative HIT” has increased the importance of semiquantitative interpretation of immunoassays, given that strong immunoassay reactivity is a potential clue indicating possible HIT despite a (false) negative platelet activation assay.

Evaluate the diagnostic performance of three methods in testing heparin induced thrombocytopenia antibodies

Objective Evaluate the diagnostic performance of three methods in testingheparin induced thrombocytopenia antibodies the clinical diagnosis performance of the three heparin induced thrombocytopenia(HIT) antibody testing. Methods 143 patients were collected from the Tianjin Medical university general hospital and China-Japan friendship hospital from the 2017 Sep to 2018 Dec, 67 males and 76 females, with a mean age of (56.1±11.4) years, the pretest probability is estimated to be low (1-3 points) with 24 patients, intermediate (4-5 points) with 79 patients and high(6-8 points) with 40 patients. According to therapeutic regimen, the intermediate and high probability patients were divided into two groups: 31 cases in the heparin discontinuing group and 88 cases in the continuing heparin group. The three methods including:the particle immunofiltration assay to detect the plasma whole HIT antibody; the particle gel immunoassayto detect the plasma whole HIT antibody by ACL TOP 700 coagulation analyzer,; the chemiluminescent immunoassay to detect the plasma IgG-specific HIT antibody by ACL AcuStar chemiluminescent analyzer. Results There was no significant difference between the high and intermediate probability patients for particle immunofiltration assay, particle gel immunoassay and chemiluminescent immunoassay(χ2 was 3.15, 2.89 and 1.31 respectively, P>0.05). The positive rate with three assays were higher in the heparin discontinuing group than the continuing heparin group(χ2 was 16.09, 43.37 and 26.94 respectively, P 0.05) between the continuing heparin group and the heparin discontinuing group for positive results with particle immunofiltration assay; all of the patients with there assays negative simultaneous in the continuing heparin group with a significant difference between the two groups(χ2=14.27, P<0.01). Conclusions Whole HIT antibodies had high sensitivity, and was especially suitable for excluding diagnosis, andthe diagnositic performance withtheparticle gel immunoassay precede the particle immunofiltration assay.For the patients with intermediate or high pretest probability and whole antibodies positive, even if they had been treated with alternative anticoagulant therapy, the IgG specific antibodies(chemiluminescent immunoassay) should be detected for definite diagnosis(if the conditions was appropriate), to verify the rationality of alternative anticoagulant therapy and to avoid overtreatment. Key words: Heparin; Thrombocytopenia; Immunoglobulin G; Predictive value of tests; Sensitivity and specificity

Implementation of a rapid HIT immunoassay at a university hospital – Retrospective analysis of HIT laboratory orders in patients with thrombocytopenia

BackgroundHeparin-induced thrombocytopenia (HIT) is a rare cause of thrombocytopenia and a potentially life-threatening adverse drug reaction. Clinical overdiagnosis of HIT results in costly laboratory tests and anticoagulation. Criteria and algorithms for diagnosis are established, but their translation into clinical practice is still challenging. Study design and methodsIn a retrospective approach we studied all HIT related laboratory test requests within four years and evaluated data before (1st period, 24month) and after (2nd period, 24month) replacing particle gel immunoassay (PaGIA) and enzyme-linked immunosorbent assay (ELISA) by a chemiluminescent immunoassay (CLIA). HIT was confirmed by heparin-induced platelet activation (HIPA) test. Clinical pretest probability for HIT using an implemented simplified 4Ts score and platelet count were evaluated. Costs for laboratory tests and alternative anticoagulation were calculated. ResultsIn 1850 patients with suspected HIT, 2327 laboratory orders were performed. In 87.2% of these orders an intermediate/high simplified 4Ts score was found. Thrombocytopenia was present in 87.1%. After replacing PaGIA and ELISA by CLIA the number of immunological and functional laboratory tests was reduced by 38.2%. The number of positive HIT immunoassays declined from 22.6% to 6.0%, while the number of positive HIPA tests among positive immunological tests increased by 19%. Altogether, acute HIT was confirmed in 59 patients. A decline in the use of alternative anticoagulants was observed in the 2nd period. ConclusionOur study shows that in a university hospital setting HIT is well-known, but diagnosis requires a precise laboratory confirmation. Replacing PaGIA and ELISA by CLIA did not influence laboratory order behavior but results in reduced overall costs for laboratory diagnostics and alternative anticoagulation.

Rapid immunoassays for diagnosis of heparin-induced thrombocytopenia: Comparison of diagnostic accuracy, reproducibility, and costs in clinical practice.

BackgroundImmunoassays are crucial in the work-up of patients with suspected heparin-induced thrombocytopenia (HIT) and rapid tests have been recently developed. However, comparative data on diagnostic accuracy, reproducibility, and analytical costs of different immunoassays in clinical practice are limited.MethodsSamples of 179 consecutive patients evaluated for suspected HIT in clinical practice using a polyspecific enzyme-linked immunoabsorbent assay (GTI diagnostics; ELISA) and a rapid particle gel immunoassay (PaGIA), were additionally analysed with a IgG-specific chemiluminescent immunoassay (AcuStar HIT-IgG). Presence of HIT was defined as a positive functional heparin-induced platelet aggregation test. Diagnostic accuracy was determined for low, intermediate and high thresholds as previously established (ELISA: optical density 0.4, 1.3, and 2.0 respectively; PaGIA: positive/negative, titre of 4, titre of 32; AcuStar HIT-IgG: 1.0 U/ml, 2.8, 9.4) and reproducibility was assessed by repeated measurements. Costs of test determination were calculated taking reagents, controls, and working time of technicians according to Swiss health care system into account.ResultsData on PaGIA results were available for 171 patients (95.5%), ELISA for 144 patients (80.4%), and AcuStar HIT-IgG for 179 patients (100%). Sensitivity was above 95% for all assays at low and intermediate thresholds. Specificity increased with higher thresholds and was above 90% for all assays with intermediate and high thresholds. Specificity of AcuStar HIT-IgG (92.8%; 95% CI 87.7, 96.2) was significantly higher than PaGIA (83.0%; 95% CI 76.3, 88.5) and higher than ELISA (81.8%, 95% CI 74.2, 88.0) at low threshold (p<0.05). Reproducibility was adequate for all assays. Total costs per test were CHF 51.02 for ELISA, 117.70 for AcuStar HIT-IgG, and 83.13 for PaGIA.ConclusionsWe observed favourable diagnostic accuracy measures and a high reproducibility for PaGIA and AcuStar HIT-IgG. Implementation into 24-hours-service might improve patient care but the results must be confirmed in other settings and larger populations as well.

Diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis

AbstractImmunoassays are essential in the workup of patients with suspected heparin-induced thrombocytopenia. However, the diagnostic accuracy is uncertain with regard to different classes of assays, antibody specificities, thresholds, test variations, and manufacturers. We aimed to assess diagnostic accuracy measures of available immunoassays and to explore sources of heterogeneity. We performed comprehensive literature searches and applied strict inclusion criteria. Finally, 49 publications comprising 128 test evaluations in 15 199 patients were included in the analysis. Methodological quality according to the revised tool for quality assessment of diagnostic accuracy studies was moderate. Diagnostic accuracy measures were calculated with the unified model (comprising a bivariate random-effects model and a hierarchical summary receiver operating characteristics model). Important differences were observed between classes of immunoassays, type of antibody specificity, thresholds, application of confirmation step, and manufacturers. Combination of high sensitivity (>95%) and high specificity (>90%) was found in 5 tests only: polyspecific enzyme-linked immunosorbent assay (ELISA) with intermediate threshold (Genetic Testing Institute, Asserachrom), particle gel immunoassay, lateral flow immunoassay, polyspecific chemiluminescent immunoassay (CLIA) with a high threshold, and immunoglobulin G (IgG)-specific CLIA with low threshold. Borderline results (sensitivity, 99.6%; specificity, 89.9%) were observed for IgG-specific Genetic Testing Institute-ELISA with low threshold. Diagnostic accuracy appears to be inadequate in tests with high thresholds (ELISA; IgG-specific CLIA), combination of IgG specificity and intermediate thresholds (ELISA, CLIA), high-dose heparin confirmation step (ELISA), and particle immunofiltration assay. When making treatment decisions, clinicians should be a aware of diagnostic characteristics of the tests used and it is recommended they estimate posttest probabilities according to likelihood ratios as well as pretest probabilities using clinical scoring tools.

Diagnostic accuracy of rapid immunoassays for heparin-induced thrombocytopenia

The platelet factor 4/heparin ELISA has limited specificity for heparin-induced thrombocytopenia (HIT) and frequently does not provide same-day results. Rapid immunoassays (RIs) have been developed which provide results in 30 minutes or less. We conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of RIs for HIT. We searched the literature for studies in which samples from patients with suspected HIT were tested using a RI and a functional assay against which the performance of the RI could be measured. We performed sensitivity analyses of studies that directly compared different RIs with each other and with ELISAs. Estimates of sensitivity and specificity for each RI were calculated. Twenty-three articles, collectively involving six different RIs, met eligibility criteria. All RIs exhibited high sensitivity (0.96 to 1.00); there was wider variability in specificity (0.68 to 0.94). Specificity of the IgG-specific chemiluminescent assay (IgG-CA) was greater than the polyspecific chemiluminescent assay [0.94 (95 %CI 0.89-0.99) vs 0.82 (0.77-0.87)]. The particle gel immunoassay demonstrated greater specificity than the polyspecific ELISA [0.96 (0.95-0.97) vs 0.91 (0.89-0.92)]. The IgG-CA and lateral flow immunoassay [0.94 (0.91-0.97)] exhibited greater specificity than the IgG-specific ELISA [0.86 (0.82-0.90)]. Given their high sensitivity and rapid turnaround time, RIs are a reliable means of excluding HIT at the point-of-care in patients with low or intermediate clinical probability. Additionally, some RIs have greater specificity than HIT ELISAs. In summary, IgG-specific RIs appear to have improved diagnostic accuracy compared with ELISAs in patients with suspected HIT and may reduce misdiagnosis and overtreatment.

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

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 1Clinical characteristics of patients documented heparin-induced thrombocytopeniaCaseAge (years)/ sexPatient typeHeparin typeThrombosisPaGIA4Ts scoreHEP score161/ MCVTUFHNew+2-6237/ MCCUUFH, LMWHProgressiveNA3-3374/ FCVTUFHNoNA33483/ MGenSUFH, LMWHNew+4-6580/ FMedLMWHNo+46662/ MCCUUFHNoNA54751/ MCCUUFHNoNA55880/ MMedUFHNo+56976/ MCCUUFHProgressiveNA5101050/ FMedUFH, LMWHNew+651187/ MCCUUFHNew+79M: 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 DisclosuresNo relevant conflicts of interest to declare.

Combination of 4Ts score and PF4/H-PaGIA for diagnosis and management of heparin-induced thrombocytopenia: prospective cohort study

AbstractRapid exclusion of heparin-induced thrombocytopenia (HIT) is needed to determine which patients can continue to receive heparin. In this prospective management study, 526 participants had a 4Ts score, rapid particle gel immunoassay (platelet factor 4/heparin [PF4/H]-PaGIA), and serotonin-release assay (SRA) performed. While awaiting SRA results, participants with a low 4Ts score (irrespective of PF4/H-PaGIA result) or intermediate 4Ts score plus a negative PF4/H-PaGIA result received prophylactic doses of danaparoid or fondaparinux; all others received therapeutic doses of nonheparin anticoagulants. The primary outcome was the frequency of management failures defined as HIT-positive participants with a low 4Ts score (irrespective of PF4/H-PaGIA result) or intermediate 4Ts score plus negative PF4/H-PaGIA result. Six participants (1.1–; 95– confidence interval [CI], 0.2-2.1–) were management failures. A negative PF4/H-PaGIA result reduced the pretest probability of HIT from 1.9– to 0– (95– CI, 0-1.3–), 6.7– to 0– (95– CI, 0-2.7–), and 36.6– to 0– (95– CI, 0-14.3–) in the low, intermediate, and high score groups, respectively. A positive PF4/H-PaGIA result increased the probability of HIT in the low score group to 15.4– (95– CI, 5.9-30.5). Thus, a low or intermediate 4Ts score plus negative PaGIA result excluded HIT, whereas any other combination of results justified the use of alternative anticoagulants until HIT could be excluded. This trial was registered at www.clinicaltrials.gov as #NCT00489437.

Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia.

A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the 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

IntroductionIn 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 MethodsIn a national multi-centre registry study, patients with a 4T’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. ResultsOf 195 patients, the 4T’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%). ConclusionsThe 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.

Detection of anti-IgA antibodies using the particle gel immunoassay: a rapid test for increased patient safety.

Patient safety is a major issue in transfusion medicine and commands continuous efforts to develop valid control methods aiming to avoid serious transfusion-related complications. Anti-IgA antibodies can cause anaphylactic transfusion reactions in IgA-deficient individuals. Since standard quantitative methods for anti-IgA measurement require considerable time to be performed, in an emergency situation it can be a challenge to prevent or to quickly interpret and manage acute transfusion reactions suspected to be a consequence of anti-IgA. The purpose of this study was to test and validate at our transfusion centre a rapid assay for the identification of patients with anti-IgA antibodies. Forty-six samples (6 from healthy controls and 40 from IgA-deficient patients) were collected. Sera were analysed blindly by three different clinical laboratory technologists using two DiaMed particle gel immunoassays (ID-PaGIA) for IgA deficiency and for antibodies to IgA. The results were subsequently checked with the results of a fluorescence enzyme immunoassay conducted in the reference immunology laboratory. The ID-PaGIA had a sensitivity of 91.7% and specificity of 97.1% for the IgA deficiency test. With regards to the detection of anti-IgA antibodies, the sensitivity was 89.3% and the specificity 100%. The reproducibility of the test was 100%. The ID-PaGIA screening assays are suitable for the investigation of transfusion-related anaphylactic reactions in a routine blood bank laboratory. Although the gel card technique does not quantify the level of anti-IgA antibodies, it is readily available, providing an effective and simple method for the diagnosis of anti-IgA related anaphylaxis and guidance for the appropriate transfusion practice in an emergency.

Long-Term Treatment and Transfusion of Normal Blood Components Following Tolerance Induction in Patients with Anti-IgA Anaphylactic Reactions

Background: In general, patients with significant anti-Ig-A do not tolerate intravenous (i.v.) administration of normal blood products. Here, we present our experiences in the induction of immune tolerance (IIT) and long-term treatment in a series of such patients affected in such a way. The question whether blood components from IgA-deficient donors are required will be discussed. Methods: Ten adult patients (4 females and 6 males; age ranging from 36 to 75 years) with anti-IgA were included in this study. All patients required long-term treatment with blood components. One patient had IgA deficiency and paroxysmal nocturnal hemoglobinuria (PNH), and all other patients had common variable immunodeficiency (CVID). The particle gel immunoassay was used for the detection of anti-IgA. Immune tolerance to IgA was induced by controlled subcutaneous (s.c.) and/or i.v. infusions of IgG preparations. Results: Prior to IIT, anti-IgA was detectable in plasma samples of all patients and significantly diminished or abolished by controlled s.c. and/or i.v. infusions of IgG. Multiple transfusions with normal blood components could be repeatedly performed with the patient suffering from PNH without any complications. As long as i.v. IgG (IVIgG) infusions were consequently administered as individually required (intervals 2-8 weeks), none of the patients developed reactions during observation (up to 10 years). However, interruption of treatment and re-exposure to IVIgG resulted in adverse reactions. Conclusion: Patients with significant anti-IgA can be safely desensitized and tolerate long-term IgG substitutions independent of the IgA concentration of the used blood component.

Unexpected reactions of the anti‐IgA antibody particle gel immunoassay

The cause of allergic transfusion reactions remains often unknown, but in rare cases anti-immunoglobulin A (IgA) antibodies in patients with IgA-deficiency can be found. We report on the use of the DiaMed particle gel immunoassay (PaGIA) for detection of anti-IgA antibodies in patients with allergic transfusion reactions. The examination of the suspected adverse reactions included an anti-IgA antibody test (ID-PaGIA Anti-IgA antibody test; DiaMed GmbH, Cressier , Switzerland) and measurement of IgA concentration in the patient's plasma. In the case of a discrepancy IgA subclasses were examined and neutralization of the anti-IgA antibodies by pure IgA was performed. Of 142 patients tested for IgA concentration and anti-IgA antibodies, 8 gave positive results for the anti-IgA antibody test. In seven of these cases (4.9% of the patients tested) IgA levels were found to be normal, and in four of five so tested, the positive result could not be neutralized with purified IgA. Only one patient had confirmed IgA deficiency with anti-IgA antibodies that were neutralized by addition of purified IgA. Cause and clinical relevance of a positive reaction of the anti-IgA antibody test in patients with normal total IgA and normal IgA subclasses remains unknown. Because of the high false positive rate we do not recommend this test as a screening test for anti-IgA antibodies when evaluating allergic transfusion reactions, but instead recommend measurement of total IgA in patient's plasma or serum as a primary screen for IgA deficiency with antibodies as a cause of allergic transfusion reaction.