Variability of anti-PF4/heparin antibody results obtained by the rapid testing system ID-H/PF4-PaGIA

Summary. Background: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin‐induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti‐PF4/heparin‐antibodies may assist clinical decision‐making. Objectives: To evaluate the performance of rapid ID‐H/PF4‐PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID‐H/PF4‐polymer lots (polystyrene beads coated with heparin/PF4‐complexes). Patients/Methods: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin‐induced thrombocytopenia between January 2000 and October 2008. Anti‐PF4/heparin‐antibodies were assessed by ID‐H/PF4‐PaGIA, commercially available ELISAs and heparin‐induced platelet aggregation test. Results: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low‐titre positive) in nine instances (1%; 95%CI, 0.4–1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty‐seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none – 82%; 95%CI, 66–98%), depending on the employed ID‐H/PF4‐polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT‐patients. Conclusion: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID‐H/PF4‐polymer lots, thus avoiding false negative results.     

Prospective evaluation of PF4/heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia

Summary.  Background:  Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies that recognize platelet factor 4-heparin (PF4/hep) complexes leading to platelet activation. Several methods are available for the identification of HIT antibodies. Objectives:  To evaluate the clinical usefulness of different antigen-binding assays for detection of antibodies against PF4/hep complexes in a prospective study. Patients/methods:  A prospective cohort of 500 surgical and medical patients suspected of having HIT was evaluated. The laboratory assessment included particle gel immunoassay (PaGIA), polyspecific ELISA recognizing IgG, IgM and IgA antibodies (Poly-ELISA), IgG-specific ELISA (IgG-ELISA) and the HIPA test. The pretest probability of HIT was determined using the 4T's model. Positive and negative predictive values (PPV, NPV) of each immunoassay were determined depending upon the heparin-induced platelet activation (HIPA) results and the clinical scoring. The operating characteristics of each immunoassay were determined using the receiver-operation characteristic (ROC) curve. Results:  Platelet-activating antibodies were identified in 35/500 patients, all of whom had intermediate to high clinical probability. PF4/hep antibodies were detected in 124, 86 and 90 sera using Poly-ELISA (PPV = 28), IgG-ELISA (PPV = 40.6) and PaGIA (PPV = 36.6). NPV was 100% for Poly- and IgG-ELISA, but only 99.5% for PaGIA. ROC analysis revealed that PaGIA is less informative than ELISA. The IgG-ELISA provides better diagnostic information than the other assays. In addition, there is a clear correlation between optical density (OD) value and the probability of having HIT. Conclusions:  Our observation indicates that an IgG-ELISA provides the best diagnostic information of all antigen-binding assays.     

Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity and clinical significance of antiplatelet factor 4/heparin antibodies of the IgG, IgM, and IgA classes

INTRODUCTION: Platelet-activating antiplatelet factor 4/heparin (anti-PF4/heparin) antibodies are the major cause of heparin-induced thrombocytopenia (HIT). However, the relative utility of functional (platelet activation) vs. antigen [enzyme-immunoassay (EIA)] assays, and the significance of assay discrepancies remain unresolved. METHODS: Consecutive patient sera (n = 1650) referred for diagnostic HIT testing were screened prospectively by both the heparin-induced platelet activation (HIPA) test and anti-PF4/heparin EIA - including individual classes (IgG, IgA, IgM) - with clinical correlations studied. Platelet microparticle and annexin-V-binding properties of the sera were also investigated. RESULTS: Only 205 (12.4%) sera tested positive in either the HIPA and/or EIA: 95 (46.3%) were positive in both, 109 (53.1%) were only EIA-positive, and, notably, only one serum was HIPA-positive/EIA-negative. Of 185 EIA-positive sera, only 17.6% had detectable IgM and/or IgA without detectable IgG. Among sera positive for EIA IgG, optical density values were higher when the sera were HIPA-positive (1.117 vs. 0.768; P < 0.0001), with widely overlapping values. Two HIPA-positive but EIA-IgG-negative sera became HIPA-negative following IgG depletion, suggesting platelet-activating antibodies against non-PF4-dependent antigens. Clinical correlations showed that HIPA-negative/EIA-positive patients did not develop thrombosis and had reasons other than HIT to explain thrombocytopenia. IgM/A antibodies did not increase microparticle penetration, but increased annexin-V binding. CONCLUSIONS: The anti-PF4/heparin EIA has high ( approximately 99%) sensitivity for HIT. However, only about half of EIA-positive patients are likely to have HIT. Anti-PF4/heparin antibodies of IgM/A class and non-PF4-dependent antigens have only a minor role in HIT.     

Prospective evaluation of the ''4Ts'' score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe disease that is often difficult to diagnose. A clinical scoring system, the '4Ts' score, has been proposed to estimate its probability before laboratory testing, and a particle gel immunoassay (H/PF4 PaGIA) has also been developed for rapid detection of HIT antibodies. AIM: To evaluate the performance of both methods when HIT is suspected clinically. METHODS: Two hundred thirteen consecutive patients were included in four centers. The probability of HIT was evaluated using the 4Ts score blind to antibody test results. HIT was confirmed only when the serotonin release assay (SRA) was positive. RESULTS: The risk of HIT was evaluated by the 4Ts score as low (LowR), intermediate (IR) or high (HR) in 34.7%, 60.6% and 4.7% of patients, respectively. The negative predictive value (NPV) of the 4Ts score was 100%, as the SRA was negative in all LowR patients. PaGIA was negative in 176 patients without HIT (99.4%, NPV) and the negative likelihood ratio (LR-) was 0.05. PaGIA was positive in 37 patients, including 21 with HIT (positive predictive value = 56.8%), with a positive LR of 11.4. A negative PaGIA result decreased the probability of HIT in IR patients from 10.9% before assay to 0.6%, whereas a positive result did not substantially increase the likelihood for HIT. CONCLUSION: The use of the 4Ts score with PaGIA appears to be a reliable strategy to rule out HIT.     

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.     

Platelet and monocyte antigenic complexes in the pathogenesis of heparin-induced thrombocytopenia (HIT)

Summary.  Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder that occurs in a small subset of patients receiving heparin. Twenty-five per cent (or higher) of affected patients develop limb or life-threatening thrombosis. The effectiveness of therapy is incomplete and may be complicated by bleeding. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAGs). However, antibodies with the same apparent specificity are found in many more patients without clinical disease and the reason why so few develop HIT is uncertain. We propose that HIT antibodies recognize cell surface PF4/GAG complexes on intravascular cells, including platelets and monocytes that are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications.     

血小板异常直方图在判断血小板计数结果中的应用价值

目的 探讨血小板异常直方图在判断血小板计数结果中的临床应用价值。方法 利用Sysmex K-4500血细胞分析仪对276例血液标本分析测定，并根据检测结果中血小板直方图、血小板平均体积（MPV）、血小板板分布宽度（PDW）、红细胞平均体积（MCV）和红细胞体积分布宽度（RDW）不同，分为正常血小板组、大血小板组、血小板聚集组、小红细胞干扰组，并对各组血小板进行显微镜手工法计数。结果 正常血小板组两种方法计数结果差异无统计学意义（P〉O．05）；大血小板组与血小板聚集组两种方法计数结果差异有统计学意义（P〈O．01），仪器法结果偏低；小红细胞干扰组两种方法计数结果差异有统计学意义（P〈O．01），仪器法偏高。结论 对于血小板直方图异常的标本应分析原因，重留标本复查或手工法计数进行纠正，为临床提供可靠检验数据。     

重组人白细胞介素-11治疗免疫性血小板减少症患者的临床效果

目的 研究重组人白细胞介素-11(rhIL-11)治疗免疫性血小板减少症患者的临床效果.方法 选取2017年1月至2020年1月本院收治的40例免疫性血小板减少症患者,依据治疗方法分为对症处理组(n=20,对症处理)、rhIL-11组(n=20,对症处理基础上rhIL-11治疗),比较两组的治疗效果.结果 rhIL-1...     

Use of plasma exchange in patients with heparin-induced thrombocytopenia: a report of two cases and a review of the literature

Heparin-induced thrombocytopenia (HIT), which is characterized by thrombocytopenia and potentially serious thromboses, may develop in patients exposed to heparin anticoagulation. HIT is caused by antibodies to the heparin/platelet factor 4 (PF4) complex. Management of HIT involves discontinuation of heparin and anticoagulation with a nonheparin alternative such as a direct thrombin inhibitor (DTI). This poses a challenge in the management of patients who need to undergo cardiopulmonary bypass surgery (CPB), because CPB requires anticoagulation with heparin and standardized protocols for use of DTIs are not widely available. We report two patients with HIT who underwent successful CPB with heparin anticoagulation following plasma exchange (PE) to reduce heparin/PF4 antibody titers. Case 1 is a 46-year-old male with cardiac amyloidosis who needed urgent placement of a left ventricular assist device. Case 2 is a 34-year-old woman with acute myocarditis who needed placement of a biventricular assist device. Both patients had positive enzyme-linked immunosorbent assay assays for heparin/PF4 antibodies and clinical evidence of HIT before PE. Following PE and subsequent CPB, neither patient had clinical or laboratory evidence of HIT. The literature regarding the use of PE for the treatment of complications of HIT and as prophylaxis before CPB is reviewed.     

网织血小板在系统性红斑狼疮患者的临床应用

目的：研究系统性红斑狼疮（SLE）患者合并血小板减少疾病的发病机制,探讨网织血小板（RP）在诊疗SLE合并血小板减少疾病中的临床应用价值。方法确诊为SLE患者55例和健康体检者50例,对SLE伴血小板减少的患者进行治疗前后检查,采用Sysmex XE-5000全自动血细胞分析仪检测网织血小板百分比（IPF）以及其他外周血细胞参数。结果 SLE患者组和正常对照组的IPF检测结果分别为（5．30＆#177;3．75）％、（2．74＆#177;1．05）％,两者比较差异有统计学意义（P＜0．05）;SLE伴血小板减少组的IPF[（10．14＆#177;3．66）％]、血小板计数（PLT）[（67．2＆#177;13．5）＆#215;109/L]、血小板平均体积（MPV）[（12．64＆#177;0．92）fL]、血小板分布宽度（PDW）[（18．24＆#177;1．70）fL]与正常对照组、SLE血小板未减少组比较,差异均有统计学意义（P＜0．05）;SLE伴血小板减少组治疗前后的IPF分别为（9．76＆#177;1．82）％和（5．86＆#177;0．96）％,两者比较差异有统计学意义（P＜0．05）。结论 SLE患者血小板减少的主要原因可能与外周血小板破坏有关,IPF的高低可以出反映骨髓血小板的生成情况,有助于疾病的辅助诊断和预后判断。     

南宁地区成人及儿童ITP患者体内血小板自身抗体特异性分布的研究

目的：研究血小板膜糖蛋白特异性自身抗体在成人及儿童原发免疫性血小板减少症（ITP）患者中分布的异同。方法应用酶联免疫吸附试验（PAKAUTO 试剂盒）检测 ITP 组（83例）及非 ITP 组（58例）患者血小板自身抗体,并分析成人组ITP（46例）,儿童组 ITP（37例）的抗 GP Ⅱ b／Ⅲ a 、抗 GP Ⅰ b／Ⅸ及抗 GP Ⅰ a／Ⅱ a 自身抗体特异性分布规律。结果 ITP 组血小板自身抗体阳性率为66．27％,高于非 ITP 组的6．90％,差异有统计学意义差异有统计学意义（P＜0．05）。成人组女性 ITP 患者占60．87％,儿童组女性 ITP 患儿占64．86％,差异无统计学意义（P ＞0．05）,但两组女性发病率均高于男性,差异有统计学意义（P＜0．05）。成人组 ITP 患者血小板自身抗体阳性率为63．04％,儿童组 ITP 患儿抗体阳性率为70．27％,差异无统计学意义（P＞0．05）;且成人组与儿童组 ITP 患者血小板自身抗体特异性分布差异无统计学意义（P ＞0．05）,均以抗 GP Ⅱ b／Ⅲ a 和抗 GPⅠ b／Ⅸ抗体多见。结论血小板自身抗体检测对 ITP 诊断、鉴别诊断及治疗均有重要的参考价值,GP Ⅰ a／Ⅱ a 抗体介导的 ITP很值得深入研究。     

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Summary. Background: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource‐limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. Methods: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin‐9 in granulocytes and platelet thrombospondin‐1 and molecular screening of candidate genes. Results: Thirty‐one patients from 14 pedigrees were included; their median age was 32 (4–72) years and platelet count 72 (4–147) × 10⁹ L^?1. Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9‐related disease in four, while classic and monoallelic Bernard–Soulier syndrome, gray platelet syndrome, X‐linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. Conclusions: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.     

Prospective observational evaluation of the particle immunofiltration anti-platelet factor 4 rapid assay in MICU patients with thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) results from antibodies to PF4/heparin complexes and clinical diagnosis is difficult. We evaluated the particle immunofiltration anti-platelet factor 4 (PIFA) rapid assay, in conjunction with a clinical risk score, in the diagnosis of HIT.

Methods

We performed a prospective observational study in all patients admitted to the medical intensive care unit (MICU) in a large academic medical center. Patients were screened daily for thrombocytopenia defined as either a platelet count that decreased by at least 33% or an absolute platelet count less than 150,000/μL. Patients with suspected HIT underwent PIFA and ELISA testing for anti-PF4/heparin antibodies. Available residual frozen sera were sent to a reference laboratory for serotonin release assay (SRA) testing.

Results

During the study period, 340 patients were admitted to the MICU, of which 143 patients met criteria for thrombocytopenia. Forty-three patients had no evidence of recent heparin exposure. PIFA and ELISA testing were performed on 100 patients, of which 92 had samples available for SRA analysis. PIFA results were negative in 62, positive in 28 and inconclusive in 2 patients. The 4Ts score showed low to intermediate risk in 57 of the PIFA negative patients. The ELISA results were negative in 86 and positive in 6 patients. SRA testing identified 3 patients with a positive SRA test and 89 patients with a negative result. All patients with a negative PIFA result also had a negative SRA result. In the one patient deemed to have clinical HIT, the pretest probability was high (4Ts score of 6) and the anti-PF4/heparin antibody testing revealed a positive SRA, inconclusive PIFA and a negative ELISA result.

Conclusions

While thrombocytopenia in our population is common, the prevalence of HIT is low. The combination of a low to intermediate pretest probability with a negative PIFA test can rapidly exclude the presence of platelet activating anti-PF4/heparin antibodies and, therefore, HIT as the cause of the thrombocytopenia. Since a positive PIFA result has a low positive predictive value, a positive PIFA is not diagnostic of HIT and additional evaluation is warranted.     

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

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

Lepirudin in patients with heparin-induced thrombocytopenia – results of the third prospective study (HAT-3) and a combined analysis of HAT-1, HAT-2, and HAT-3

OBJECTIVES: To assess efficacy and safety of lepirudin in patients with heparin-induced thrombocytopenia (HIT) in a prospective study (HAT-3) as well as in a combined analysis of all HAT study data. PATIENTS/METHODS: Patients with laboratory-confirmed HIT were treated with lepirudin in three different aPTT-adjusted dose regimen and during cardiopulmonary bypass (CPB). Endpoints were new thromboembolic complications (TEC), limb amputations, and death and major bleeding. A historical control group (n = 120) was used for comparison. RESULTS: After start of lepirudin in 205 patients treated in HAT-3, the combined endpoint occurred in 43 (21.0%). Thirty (14.6%) patients died, 10 (4.9%) underwent limb amputation, and 11 (5.4%) new TECs occurred. Major bleeding occurred in 40 patients (19.5%) (seven during CPB surgery). Combining all prospective HAT trials (n = 403), after start of lepirudin treatment, the combined endpoint occurred in 82 patients (20.3%), with 47 deaths (11.7%), 22 limb amputations (5.5%), 30 new TECs (7.4%), and 71 (17.6%) major bleedings. Compared with the historical control group (log-rank test), the combined endpoint after start of treatment was reduced (29.7% vs. 52.1%, P = 0.0473), primarily because of reduction in new thromboses (11.9% vs. 32.1%, P = 0.0008). Mean lepirudin maintenance doses ranged from 0.07 to 0.11 mg kg(-1) h(-1). Major bleeding was more frequent in the lepirudin-treated patients (29.4% vs. 9.1%, P = 0.0148). CONCLUSIONS: The rate of new TECs in HIT patients is low after start of lepirudin treatment. The rate of major bleeding of 17.6% might be reduced by reducing the starting dose to 0.1 mg kg(-1) h(-1).     

Sensitive enzyme immunoassay for the measurement of platelet factor 4 in blood plasma

A sandwich enzyme immunoassay method for the measurement of platelet factor 4 (PF4) was developed with the use of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E. coli. The measurable range was 30 pg to 3 ng of PF4 per tube. Within-run and between-run coefficients of variation were less than 10%. The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.952, slope = 0.954, gamma-intercept = 2.43 ng/ml). Platelets contained large amounts of PF4 (7.21 +/- 1.97 ng/10(6) cells or 2.51 +/- 1.13 ng/mg protein), whereas the PF4 levels in red blood cells and lymphocytes were negligible, confirming the specific localization of PF4 in platelets. The applicability of the immunoassay method was tested to determine the in vitro release of PF4 during preparation and storage of platelet concentrates.