Summary
The laboratory diagnosis of heparin‐induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard ...for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet‐activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRApos), with relatively high levels of PF4‐specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4‐SRApos). Importantly, levels of PF4‐specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre‐test probability of HIT was intermediate/high in all ‘SRApos’ or ‘SRA‐PF4pos’ patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.
Background
Serotonin release assay (SRA) is considered as the “gold standard” for detecting pathogenic heparin‐induced thrombocytopenia (HIT) antibodies. However, this method is time consuming, ...expensive, and uses radioelements. Heparin‐induced multiple electrode aggregometry (HIMEA), light transmission aggregometry (LTA) with platelet rich plasma (PRP) or washed platelets (WP), adenosine triphosphate (ATP) release, and flow cytometry (FC) are available alternatives.
Objectives
To evaluate the performance of these assays, comparatively with SRA, for detecting HIT antibodies, using 5B9, a monoclonal IgG fully mimicking human HIT antibodies.
Patients/Methods
Heparin‐dependent platelet activation induced by 5B9 (50/20/10 µg/mL) was evaluated by all assays performed on the same day using platelets from 20 healthy donors. The three methods exhibiting the highest sensitivity to 5B9 were then assessed by testing samples from patients with either likely (n = 10), or indeterminate/unlikely HIT (n = 10).
Results
All methods exhibited good sensitivity for detecting 5B9 50 µg/mL, but only SRA and HIMEA were positive with 100% of donors using 5B9 20 µg/mL, followed by FC (83%). SRA detected 5B9 10 μg/mL with 90% of donors, while HIMEA and FC were positive in 45% and 44% of cases, respectively. Whereas SRA was positive with 9/10 samples from likely HIT, HIMEA and FC were positive with 6 and 7 of them, respectively. Neither SRA nor HIMEA was positive with indeterminate/unlikely HIT samples, while FC was positive or doubtful in three cases.
Conclusions
Serotonin release assay likely remains the most sensitive and specific assay for detecting platelet activating HIT antibodies, but HIMEA or FC are potential alternatives, despite being less performant.
Background
The diagnosis of heparin‐induced thrombocytopenia (HIT) requires functional assays to demonstrate that platelet factor 4 (PF4)‐specific antibodies activate platelets, typically when ...therapeutic heparin (H) concentrations are tested (“classical” pattern). Some HIT samples also activate platelets without heparin (“atypical” pattern), but with unclear clinical significance.
Objectives
We aimed to assess whether platelet activation pattern and some characteristics of PF4‐specific antibodies were associated with the severity of HIT.
Patients/Methods
Serotonin release assay (SRA) pattern of 81 HIT patients were analyzed and compared with their clinical and biological data, including levels of anti‐PF4/H immunoglobulin G (IgG) and anti‐PF4 IgG in 47 of them.
Results
Higher anti‐PF4/H IgG titers were measured in patients with an “atypical” SRA (optical density 2.52 vs. 1.94 in those with a “classical” pattern, p < .001). Patients of both groups had similar platelet count (PC) nadir and time to recovery, but those with an “atypical” SRA more frequently developed thrombotic events (69% vs. 34%, p = .037). Significant levels of anti‐PF4 IgG were detected in both groups (38% and 61%, respectively). Whatever the SRA pattern, a lower PC nadir (35 vs. 53 G/L, p = .006) and a longer PC recovery time (6 vs. 3 days, p = .015) were evidenced in patients with anti‐PF4 antibodies, compared with those with anti‐PF4/H IgG only.
Conclusions
An atypical SRA pattern with elevated anti‐PF4/H IgG titers seems associated with an increased risk of thrombosis in HIT. IgG antibodies to native PF4 may contribute to more severe and persistent thrombocytopenia, and their detection could be useful in clinical practice.
Abstract
Introduction/Objectives
Extracorporeal membrane oxygenation (ECMO) provides circulatory support in patients with severe heart failure, but the frequent use of unfractionated heparin exposes ...patients to high risk of heparin-induced thrombocytopenia (HIT). We prospectively evaluated the development and clinical impact of platelet factor 4 (PF4)-specific antibodies (Abs) during ECMO and whether specific biological characteristics could predict HIT.
Materials and Methods
From 2014 to 2018, we studied 57 adults who underwent an ECMO for at least 5 days. The plasma samples collected daily were tested for PF4-specific Abs using immunoassays to detect immunoglobulin (Ig) G, A, and M isotypes or only IgG. Serotonin release assay was performed without and with PF4 to detect pathogenic Abs.
Results
Twenty-nine patients (50%) were positive for PF4-specific Abs (IgG, A, M), with IgG in 17/57 (30%) and 16 of them (94%) were immunized within 10 days. PF4-specific IgG Abs did not affect the clinical or biological course of most patients. HIT was suspected in only two patients with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by a serotonin release assay, which was also more sensitive when exogenous PF4 was present.
Conclusion
PF4-specific Abs are common during ECMO but are mostly non-pathogenic and not associated with a less favorable prognosis. However, an abnormal platelet count evolution, in particular if associated with ECMO circuit dysfunction, should prompt the search for pathogenic PF4-specific IgG.
Drug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the ...most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, and vancomycin. Several different mechanisms have been identified in typical DITP, which is most commonly characterized by severe thrombocytopenia due to clearance and/or destruction of platelets sensitized by a drug-dependent antibody. Patients with typical DITP usually bleed when symptomatic, and biological confirmation of the diagnosis is often difficult because detection of drug-dependent antibodies (DDabs) in the patient’s serum or plasma is frequently not possible. This is in contrast to heparin-induced thrombocytopenia (HIT), which is a particular DITP caused in most cases by heparin-dependent antibodies specific for platelet factor 4, which can strongly activate platelets in vitro and in vivo, explaining why affected patients usually have thrombotic complications but do not bleed. In addition, laboratory tests are readily available to diagnose HIT, unlike the methods used to detect DDabs associated with other DITP that are mostly reserved for laboratories specialized in platelet immunology.
Gabapentin, a structural analog of gamma-aminobutyric acid, is used to treat peripheral neuropathic pain. Here we report the first case of platelet function disorder associated with gabapentin ...treatment in a 44-year-old woman without a history of bleeding. She presented with mucocutaneous bleeding approximately 1 month after initiation of gabapentin and platelet function tests showed no aggregation to arachidonic acid and epinephrine, a defective response to ADP and a slightly decreased response to collagen. Gabapentin's imputability was supported by the fact that all platelet functions were normalized 6 days after drug discontinuation, with the simultaneous disappearance of bleedings.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Introduction
The accurate diagnosis of heparin‐induced thrombocytopenia (HIT) is essential to ensure adequate treatment and prevent complications. First step diagnosis test are immunoassays including ...enzyme‐linked immunosorbent assays (ELISAs) and rapid immunoassays.
Methods
Using a Bayesian approach, we prospectively evaluated the performance of the IgG PF4/polyvinylsulfonate ELISA and a chemiluminescent immunoassay (CLIA), which are specific for IgG and use the same antigenic target to detect HIT antibodies.
Results
One hundred and eighty‐four 184 consecutive patients with an intermediate (n = 159) or high (n = 25) clinical pretest probability of HIT based on the 4Ts score or platelet pattern were included. Both immunoassays (IAs) were performed on all 184 samples, and definite HIT was confirmed with a positive serotonin release assay in 29 patients (12.7%). The sensitivity (Ss) and negative predictive value (NPV) of ELISA were excellent (100%) allowing HIT to be excluded with good confidence when the test was negative. In addition, the Ss and NPV of the CLIA equalled 93.1% and 98.6%, respectively, as it was negative in two definite HIT. When the CLIA was negative, the post‐test probability of HIT was 0.7% in case of intermediate risk. Although there was excellent agreement between CLIA and ELISA results, the quantitative values provided by the two IAs were not correlated.
Conclusion
AcuStar HIT® detects more than 90% of HIT, as do all rapid IAs, and appears to be a good tool for excluding HIT when the pretest probability is intermediate. A chemiluminescent signal higher than 10 IU/mL is highly predictive of definite HIT with a PPV of 100%.
Introduction: The laboratory diagnosis of HIT is difficult and often requires to perform an enzyme immunoassay combined with a functional assay, either serotonin release assay (SRA) or ...heparin-induced platelet aggregation (HIPA), for detecting IgG antibodies that activate platelets in the presence of heparin (anti-PF4/H antibodies). SRA is today considered as the gold standard for HIT diagnosis, but its performances could be improved as suggested by a recent clinical history and subsequent experiments.
Case report: A 74 year-old man was hospitalized for a myeloma and treated with unfractionated heparin (UFH) for right atrium thrombosis. Thrombocytopenia and pulmonary embolism occurred the first day of heparin treatment and HIT was suspected. UFH was replaced by danaparoid sodium, and correction of platelet count was achieved within 3 days. Laboratory tests revealed the presence of significant levels of IgG to PF4/H (OD 1.2) with clearly positive HIPA. However, SRA was also performed to confirm the diagnosis of early onset HIT, but remained negative after testing platelets from two different donors. Unlike HIPA performed with PRP, SRA is done with washed platelets, and we thus hypothesized that PF4 concentration on cell surfaces might be too low for allowing heparin-dependent platelet activation when the patient's sample was tested. We thus performed a new SRA after adding exogenous PF4 (10 μg/ml) in the buffer and positive platelet activation pattern was obtained with a maximal release of 65% with 0.5 IU/ml UFH. This experience therefore prompted us to further evaluate whether addition of exogenous PF4 was beneficial or not on platelet activation induced by PF4/H antibodies when studied in SRA.
Patients and methods: Plasma samples from 45 patients with suspected HIT and significant levels PF4-specific IgG were studied. SRA was systematically performed without and with exogenous PF4 (10 μg/ml). This "optimal" concentration of exogenous PF4 was defined after evaluating the effects of variable concentrations of human PF4 on platelet activation induced by 5B9 a recently developed HIT monoclonal antibody.
Results: SRA was consistently negative when 5B9 was tested at concentrations lower than 5 μg/ml (1 or 2.5 μg/ml). In contrast, SRA was positive with these low 5B9 concentrations when 10 μg/ml of PF4 were present in the platelet buffer. When samples from suspected HIT were studied, conventional SRA (without PF4) was positive in 14 of 45 cases (Group SRA+), which all contained relatively high levels of PF4-specific IgG (median OD = 2.6). After addition of exogenous PF4, SRA was positive in 8 additional cases (Group SRAPF4+), with maximal release between 32% and 64% and a complete inhibition in the presence of 10 IU/ml UFH. Interestingly, levels of PF4-specific IgG were similar in this group and the 23 samples with whom SRA remained negative (median OD 1.6 vs. 1.3 respectively, p = 0.8). On the other hand, the pre-test probability of HIT (4Ts score) was always intermediate or high in 'SRA+' and 'SRAPF4+' groups, although it was low in more than 30% of patients with negative SRA.
Discussion: Addition of PF4 in the platelet buffer might improve the detection of pathogenic HIT antibodies using SRA. Our results are in agreement with a recent study (I Nazi et al, Journal of Thrombosis and Haemostasis, 2015; 13: 1900-7), although they were obtained with a lower concentration of exogenous PF4, but which may be observed in vivo.
No relevant conflicts of interest to declare.
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