Publications by authors named "Ishac Nazy"

31 Publications

Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia.

J Thromb Haemost 2021 Feb 27. Epub 2021 Feb 27.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Background: Thrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID-19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID-19 patients may be suspected of heparin-induced thrombocytopenia (HIT), given similar clinical features.

Objectives: We investigated the presence of platelet-activating anti-platelet-factor 4 (PF4)/heparin antibodies in critically ill COVID-19 patients suspected of HIT.

Patients/methods: We tested 10 critically ill COVID-19 patients suspected of HIT for anti-PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti-human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS-CoV-2 antibodies were measured using an in-house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) activity and the presence of anti-ADAMTS13 antibodies.

Results: Heparin-induced thrombocytopenia was excluded in all samples based on anti-PF4/heparin antibody and SRA results. Notably, six COVID-19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)-mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID-19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy.

Conclusions: Our study identifies platelet-activating ICs as a novel mechanism that contributes to critically ill COVID-19.
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http://dx.doi.org/10.1111/jth.15283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014456PMC
February 2021

A comparative study of platelet factor 4-enhanced platelet activation assays for the diagnosis of heparin-induced thrombocytopenia.

J Thromb Haemost 2021 Apr 19;19(4):1096-1102. Epub 2021 Feb 19.

Department of Medicine, Michael G. DeGroote School of Medicine, Hamilton, ON, Canada.

Background: Functional platelet activation assays, such as the serotonin release assay (SRA), are the gold standard for the diagnosis of heparin-induced thrombocytopenia (HIT). Recently, platelet activation assays using added platelet factor 4 (PF4) have been described and suggest improved sensitivity. Direct comparisons of these assays have not been performed.

Objective: We compare the performance characteristics of three PF4-enhanced platelet activation assays, the PF4/heparin-SRA (PF4/hep-SRA), the PF4-SRA, and the P-selectin expression assay (PEA), at a single reference laboratory.

Methods: Serum samples from two cohorts of patients were used. The referral cohort (n = 84) included samples that had previously undergone routine diagnostic testing for HIT and tested positive or negative using the SRA. The clinical cohort (n = 101) consisted of samples from patients with clinically confirmed HIT whose serum contained platelet-activating antibodies. We simultaneously tested all samples in PF4-enhanced SRA-based assays (PF4/hep-SRA, PF4-SRA) and the flow cytometry-based PEA.

Results: In the referral cohort, the three PF4-enhanced assays identified all samples that were previously determined to be positive in the SRA. However, specificity of the PF4/hep-SRA was 96.6%, the PF4-SRA was 84.7%, and the PEA was 67.8%. In the clinical cohort of samples, all SRA-based assays displayed high performance characteristics (>92.1% sensitivity, >98.4% specificity). Sensitivity and specificity of the PEA was the lowest, 65.8% and 63.5%, respectively; but improved to 92.1% and 96.8% using preselected platelet donors.

Conclusions: All PF4-enhanced assays demonstrated good performance characteristics when platelet donors were preselected. Further comparisons across multiple laboratories should be conducted for consensus on optimal HIT diagnostic testing.
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http://dx.doi.org/10.1111/jth.15233DOI Listing
April 2021

Serotonin-release assay-positive but platelet factor 4-dependent enzyme-immunoassay negative: HIT or not HIT?

Am J Hematol 2021 03 29;96(3):320-329. Epub 2020 Dec 29.

Department of Medicine, Michael G. DeGroote School of Medicine, Hamilton, Ontario, Canada.

IgG-specific and polyspecific PF4-dependent enzyme-immunoassays (EIAs) have exceptionally high sensitivity (≥99%) for diagnosis of heparin-induced thrombocytopenia (HIT), a drug reaction caused by platelet-activating antibodies detectable by serotonin-release assay (SRA). The IgG-specific EIAs are recommended for screening, as their high sensitivity is accompanied by relatively high specificity vis-à-vis polyspecific EIAs. We investigated the frequency of SRA-positive/EIA-negative (SRA+/EIA-) HIT, prompted by referral to our reference HIT laboratory of serial blood samples from a patient ("index case") with false-negative IgG-specific EIAs. Despite initial clinical suspicion for HIT, repeat negative IgG-specific EIAs prompted heparin resumption, which triggered recurrent thrombocytopenia and near-fatal cardiac arrest, indicating likely post-heparin HIT-associated anaphylactoid reaction. Further investigations revealed a strong-positive SRA, whether performed with heparin alone, PF4 alone, or PF4/heparin, with inhibition by Fc receptor-blocking monoclonal antibody (indicating IgG-mediated platelet activation); however, five different IgG-specific immunoassays yielded primarily negative (or weak-positive) results. To investigate the frequency of SRA+/EIA- HIT, we reviewed the laboratory and clinical features of patients with this serological profile during a 6-year period in which our reference laboratory investigated for HIT using both SRA and IgG-specific EIA. Although ~0.2% of 8546 patients had an SRA+/EIA- profile, further review of 15 such cases indicated clerical/laboratory misclassification or false-positive SRA in all, with no SRA+/EIA- HIT case identified. We conclude that while SRA+/EIA- HIT is possible-as shown by our index case-this clinical picture is exceptionally uncommon. Moreover, the requirement for a positive EIA is a useful quality control maneuver that reduces risk of reporting a false-positive SRA result.
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http://dx.doi.org/10.1002/ajh.26075DOI Listing
March 2021

The mega-importance of de novo lipogenesis in platelet production.

Nat Metab 2020 10;2(10):999-1000

Department of Medicine, McMaster University, Hamilton, Ontario, Canada.

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http://dx.doi.org/10.1038/s42255-020-00282-7DOI Listing
October 2020

High-dose IVIG plus cangrelor platelet "anesthesia" during urgent heparin-CPB in a patient with recent SRA-negative HIT-thrombosis with persisting platelet-activating antibodies.

Res Pract Thromb Haemost 2020 Aug 23;4(6):1060-1064. Epub 2020 Jul 23.

Department of Medicine Michael G. DeGroote School of Medicine McMaster University Hamilton ON Canada.

In a high-risk patient with subacute heparin-induced thrombocytopenia (HIT) type A (platelet count recovery following acute HIT but with persisting platelet-activating antibodies), in whom urgent cardiac surgery was required, a key clinical question arose: could intraoperative heparin be given safely with "platelet anesthesia" provided with high-dose intravenous immunoglobulin (IVIG) plus cangrelor (ultra-short-acting antiplatelet agent)? This approach proved successful, without unexpected postoperative thrombocytopenia or thromboembolism. In vitro studies confirmed that both IVIG and cangrelor contributed to perioperative inhibition of HIT antibody-induced platelet activation. Interestingly, despite the patient testing strongly positive in 4 HIT immunoassays (latex immunoturbidimetric assay and 3 enzyme-immunoassays), the serotonin-release assay (SRA) was consistently negative. Nevertheless, platelet-activating HIT antibodies were detectable using modified (platelet factor 4-enhanced) SRA. Our protocol of heparin rechallenge following IVIG/cangrelor provides both intraoperative and early postoperative inhibition of HIT antibody-induced platelet activation and is applicable to patients with circulating functional HIT antibodies requiring urgent heart surgery, including those with "SRA-negative HIT."
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http://dx.doi.org/10.1002/rth2.12348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443421PMC
August 2020

The role of fluid-phase immune complexes in the pathogenesis of heparin-induced thrombocytopenia.

Thromb Res 2020 10 6;194:135-141. Epub 2020 Jun 6.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada; McMaster Centre for Transfusion Research, Hamilton, Ontario, Canada. Electronic address:

Immune complexes assemble on the platelet surface and cause Fc-mediated platelet activation in heparin-induced thrombocytopenia (HIT); however, it is not known if fluid-phase immune complexes contribute to HIT. The objective of this study was to understand the role of fluid-phase immune complexes in platelet activation and HIT. Binding of wild-type and 15 platelet factor 4 (PF4) mutants to platelets was measured using flow cytometry. Platelet activation was measured using the PF4-dependent C-serotonin release assay (PF4-SRA) with KKO and a HIT-patient plasma in the presence of wild-type or PF4 mutants. To activate platelets, we found that a minimal level of wild-type PF4 is required to bind the platelet surface in the presence of KKO (2.67 relative MFI) or HIT-patient plasma (1.71 relative MFI). Only a subset of PF4 mutants was able to support platelet activation, despite having lower surface binding than the minimum binding required of wild-type PF4 (9 mutants with KKO and 2 mutants with HIT-patient plasma). Using individual PF4 mutants, we identified that HIT immune complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of fluid-phase HIT immune complexes in the development of thrombocytopenia and thrombosis associated with clinical HIT.
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http://dx.doi.org/10.1016/j.thromres.2020.06.012DOI Listing
October 2020

Dupilumab, severe asthma airway responses, and SARS-CoV-2 serology.

Allergy 2021 03 24;76(3):957-958. Epub 2020 Aug 24.

Firestone Institute for Respiratory Health, St Joseph's Healthcare & Department of Medicine, McMaster University, Hamilton, Ontario, Canada.

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http://dx.doi.org/10.1111/all.14534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436521PMC
March 2021

Platelet autoantibodies in the bone marrow of patients with immune thrombocytopenia.

Blood Adv 2020 07;4(13):2962-2966

Department of Medicine, Michael G. DeGroot School of Medicine and.

Autoantibodies cause platelet destruction in patients with immune thrombocytopenia (ITP); yet only 50% to 60% of patients have detectable platelet autoantibodies in peripheral blood. We hypothesized that in some ITP patients, platelet autoantibodies are sequestered in the bone marrow where pathological immune reactions target megakaryocytes or newly formed platelets. In this study, we modified the platelet glycoprotein-specific assay to test bone marrow aspiration samples for free platelet autoantibodies or antibodies bound to bone marrow cells in aspirate fluid from patients with ITP (n = 18), patients with nonimmune thrombocytopenia (n = 3), and healthy donors (n = 6). We found that 10 (56%) of 18 patients with ITP had autoantibodies in the bone marrow, including 5 (50%) of 10 with autoantibodies in bone marrow only, and 5 (50%) of 10 with autoantibodies in bone marrow and peripheral blood. In comparison, 6 (33%) of 18 ITP patients had autoantibodies in peripheral blood, most of whom (5 [83%] of 6) also had autoantibodies in bone marrow. Bone marrow autoantibodies were not detected in patients with nonimmune thrombocytopenia or healthy donors; however, peripheral blood autoantibodies were detectable in 1 (33%) of 3 patients with nonimmune thrombocytopenia. The sensitivity of platelet autoantibodies for the diagnosis of ITP increased from 60% (peripheral blood testing) to 72% (peripheral blood and bone marrow testing). Immune reactions limited to the bone marrow may be characteristic of certain subsets of ITP patients.
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http://dx.doi.org/10.1182/bloodadvances.2020001846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362381PMC
July 2020

Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and Assembly.

Biophys J 2020 10 21;119(7):1371-1379. Epub 2020 Apr 21.

Chemistry Department, University of Massachusetts-Amherst, Amherst, Massachusetts. Electronic address:

Association of platelet factor 4 (PF4) with heparin is a first step in formation of aggregates implicated in the development of heparin-induced thrombocytopenia (HIT), a potentially fatal immune disorder affecting 1-5% of patients receiving heparin. Despite being a critically important element in HIT etiology, relatively little is known about the specific molecular mechanism of PF4-heparin interactions. This work uses native mass spectrometry to investigate PF4 interactions with relatively short heparin chains (up to decasaccharides). The protein is shown to be remarkably unstable at physiological ionic strength in the absence of polyanions; only monomeric species are observed, and the extent of multiple charging of corresponding ions indicates a partial loss of conformational integrity. The tetramer signal remains at or below the detection threshold in the mass spectra until the solution's ionic strength is elevated well above the physiological level, highlighting the destabilizing role played by electrostatic interactions vis-à-vis quaternary structure of this high-pI protein. The tetramer assembly is dramatically facilitated by relatively short polyanions (synthetic heparin-mimetic pentasaccharide), with the majority of the protein molecules existing in the tetrameric state even at physiological ionic strength. Each tetramer accommodates up to six pentasaccharides, with at least three such ligands required to guarantee the higher-order structure integrity. Similar results are obtained for PF4 association with longer and structurally heterogeneous heparin oligomers (decamers). These longer polyanions can also induce PF4 dimer assembly when bound to the protein in relatively low numbers, lending support to a model of PF4/heparin interaction in which the latter wraps around the protein, making contacts with multiple subunits. Taken together, these results provide a more nuanced picture of PF4-glycosaminoglycan interactions leading to complex formation. This work also advocates for a greater utilization of native mass spectrometry in elucidating molecular mechanisms underlying HIT, as well as other physiological processes driven by electrostatic interactions.
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http://dx.doi.org/10.1016/j.bpj.2020.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567982PMC
October 2020

Management of major bleeds in patients with immune thrombocytopenia.

J Thromb Haemost 2020 07 6;18(7):1783-1790. Epub 2020 May 6.

Department of Medicine, McMaster University, Hamilton, ON, Canada.

Background: A standard approach to the recognition and management of major bleeding in immune thrombocytopenia (ITP) is lacking.

Methods: Retrospective cohort study of ITP patients presenting to the emergency department (ED) with severe thrombocytopenia (platelet count <20 × 10 /L) and bleeding in four academic hospitals from 2008 to 2016. We defined a major ITP bleed as a bleed at a critical site or causing hemodynamic instability.

Results: We identified 112 ITP patients (n = 141 visits) who presented to the ED with platelets <20 × 10 /L and bleeding. Twenty--nine patients (26%) had 32 ED visits with major bleeds. Risk factors for major bleeds were older age (odds ratio [OR] 1.03, 95% confidence interval [CI] 1.01-1.06), male sex (OR 3.25, 95% CI 1.22-9.32), and more prior ITP therapies (OR 1.42, 95% CI 1.10-1.87). Acute treatment of major bleeds required a median of three treatments (interquartile range [IQR] 2--4), which included intravenous immune globulin (91% of visits), corticosteroids (78% of visits), and platelet transfusions (75% of visits). Three patients (10%) died, nine (31%) developed recurrent bleeds, one (3%) developed arterial thrombosis, and one (3%) had permanent neurological disability. Six patients presented with minor bleeding and subsequently developed a major bleed after a median of 2 days (IQR 1-3). All six patients had oral purpura and four of six had gross hematuria preceding the major bleed.

Conclusions: Major ITP bleeds are associated with significant morbidity and mortality. Oral purpura and hematuria often preceded major bleeds. Further research is needed to refine the definition of a major ITP bleed and develop evidence-based treatment strategies.
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http://dx.doi.org/10.1111/jth.14809DOI Listing
July 2020

Combination of two complementary automated rapid assays for diagnosis of heparin-induced thrombocytopenia (HIT).

J Thromb Haemost 2020 06 27;18(6):1435-1446. Epub 2020 Apr 27.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Background: HIT diagnosis typically uses complementary diagnostic assays (eg, a PF4-dependent enzyme-immunoassay [EIA] and a platelet activation assay such as the serotonin-release assay [SRA]).

Objectives: To determine whether the combination of two automated assays-a latex immunoturbidimetric assay (LIA) that evaluates competitive inhibition of a HIT-like monoclonal antibody and a chemiluminescence immunoassay (CLIA) for detecting anti-PF4/heparin IgG-optimizes diagnostic sensitivity while also yielding good specificity, particularly at high assay reactivities.

Patients/methods: We determined operating characteristics using combined LIA/CLIA results from a HIT observational trial (n = 430; derivation cohort) and 147 consecutive patients with HIT (n = 147; supplementary derivation cohort). We also evaluated 678 consecutive samples referred for HIT testing (replication cohort). LIA/CLIA reactivities were scored individually as "negative" (<1.00 U/mL, 0 points), "weak" (1.00-4.99 U/mL, 1 point), "moderate" (5.00-15.99 U/mL, 2 points) and "strong" (≥16.00 U/mL, 3 points), thus contributing up to 6 points (maximum) when LIA/CLIA results were combined. We also examined whether higher LIA/CLIA scores predicted presence of platelet-activating antibodies by conventional and modified (PF4- or PF4/heparin-enhanced) SRA.

Results: Combined LIA/CLIA testing yielded high diagnostic sensitivity (~99%) similar to EIA. Interpretation of LIA/CLIA results using the 6-point scale indicated progressively greater likelihood for the presence of platelet-activating antibodies with increasing scores (semi-quantitative reactivity). A LIA/CLIA score ≥ 4 points predicted the presence of platelet-activating antibodies by SRA or PF4-enhanced SRA with high probability (~98%).

Conclusion: Combined LIA/CLIA testing optimizes diagnostic sensitivity, with progressively greater probability of detecting platelet-activating antibodies with higher assay reactivity that reaches 98% when both automated assays yield moderate or strong results.
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http://dx.doi.org/10.1111/jth.14794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317897PMC
June 2020

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor.

Br J Haematol 2020 08 4;190(4):594-598. Epub 2020 Mar 4.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.
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http://dx.doi.org/10.1111/bjh.16553DOI Listing
August 2020

Increased cytotoxic potential of CD8 T cells in immune thrombocytopenia.

Br J Haematol 2020 03 18;188(5):e72-e76. Epub 2019 Dec 18.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

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http://dx.doi.org/10.1111/bjh.16334DOI Listing
March 2020

Serotonin-release assay-negative heparin-induced thrombocytopenia.

Am J Hematol 2020 01 6;95(1):38-47. Epub 2019 Nov 6.

Department of Medicine, Michael G. DeGroote School of Medicine, Hamilton, Ontario, Canada.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Pathogenic HIT antibodies can be detected by the serotonin-release assay (SRA), a platelet activation test. We have regarded the SRA performed in our medical community ("McMaster" SRA) as having high sensitivity and specificity. Recently, the concept of "SRA-negative HIT" has been proposed for enzyme-immunoassay (EIA)-positive/SRA-negative patients with a HIT-compatible clinical picture, who test positive in a PF4-enhanced platelet activation assay. After identifying an index case of SRA-negative HIT, we estimated the frequency of this condition by performing the "PF4-SRA" (modified SRA using high concentrations of added PF4 rather than heparin) in EIA-positive patients from a cohort study evaluating clinical and laboratory diagnosis of HIT. We defined SRA-negative HIT as patients meeting three criteria: clinical picture compatible with HIT (4Ts ≥ 4 points); EIA-positive (≥1.00 units); and PF4-SRA-positive. Among 430 patients, 35 were EIA-positive/SRA-positive and 27 were EIA-positive/SRA-negative. Among these 27 SRA-negative patients, three were found to have subthreshold levels of platelet-activating antibodies by PF4-SRA, of whom one met clinical criteria for SRA-negative HIT. Thus, based on identifying one patient with SRA-negative HIT within a cohort study that found 35 SRA-positive HIT patients, we estimate the sensitivity of the McMaster SRA for diagnosis of HIT to be 35/36 (97.2%; 95% CI, 85.8-99.9%). Although the McMaster SRA is highly sensitive for HIT, occasional SRA-negative but EIA-positive patients strongly suspected of having HIT can have this diagnosis supported by a PF4-enhanced activation assay such as the PF4-SRA.
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http://dx.doi.org/10.1002/ajh.25660DOI Listing
January 2020

Timeline of heparin-induced thrombocytopenia seroconversion in serial plasma samples tested using an automated latex immunoturbidimetric assay.

Int J Lab Hematol 2019 Aug 3;41(4):493-502. Epub 2019 May 3.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

Introduction: HIT is caused by platelet-activating IgG that recognize multimolecular PF4/heparin complexes. HIT antibodies are generally detectable by PF4-dependent enzyme immunoassay (EIA) and by platelet serotonin-release assay (SRA) at the beginning of the HIT-related platelet count fall. We determined whether an automated immunoassay for HIT, the latex immunoturbidimetric assay (LIA), also detects antibodies early during the course of HIT. The LIA was also used to evaluate a patient with putative SRA-negative HIT.

Methods: We evaluated the timing and magnitude of LIA reactivity in serial plasma samples obtained from 19 SRA-positive patients (17 with abnormal platelet count changes indicating HIT; two with subclinical seroconversion) and one putative SRA-negative HIT patient, all obtained from patients who participated in a clinical trial of heparin thromboprophylaxis. We determined LIA status at the onset of the HIT-related platelet count fall.

Results: The LIA was positive in all 19 SRA-positive patients (median value, 7.3 U/mL [range, 1.2-35.5]; cutoff, 1.0 U/mL); for all 13 evaluable patients for whom an informative plasma sample was available at (or shortly before) the onset of the HIT-related platelet count fall, LIA reactivity was positive. Heterogeneity in seroconversion using the LIA was observed; some patients exhibited gradual increases in reactivity, whereas other patients showed rapid increase in reactivity over a few days. The single clinical trial patient who met clinical-pathological criteria for "SRA-negative HIT" tested LIA-positive.

Conclusion: The LIA detects HIT antibodies at the beginning of the HIT-associated platelet count fall. The LIA was also positive in a patient with SRA-negative HIT.
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http://dx.doi.org/10.1111/ijlh.13031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850468PMC
August 2019

The sensitivity and specificity of platelet autoantibody testing in immune thrombocytopenia: a systematic review and meta-analysis of a diagnostic test.

J Thromb Haemost 2019 05 20;17(5):787-794. Epub 2019 Mar 20.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

Essentials The diagnosis of ITP is based on a platelet count < 100 × 10  L and exclusion of other causes. There are no standard tests or biomarkers to diagnose ITP. The sensitivity of platelet autoantibody testing is low (53%). The specificity is high (> 90%). A positive autoantibody test can be useful to rule in ITP but a negative does not rule out ITP. SUMMARY: Background Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a low platelet count and an increased risk of bleeding. The sensitivity and specificity of platelet autoantibody tests is variable and their utility is uncertain. Objective The purpose of this study was to perform a systematic review and meta-analysis of platelet autoantibody tests in the diagnosis of ITP. Methods Ovid Medline, PubMed, and Web of Science were searched from inception until 31 May 2018. Two reviewers independently assessed studies for eligibility and extracted data. Studies that reported testing results for antiplatelet autoantibodies on platelets (direct tests) or in plasma/serum (indirect tests) for 20 or more ITP patients were included. Results Pooled estimates for sensitivity and specificity were calculated using a random effects model. Pooled estimates for the sensitivity and specificity of direct anti-platelet autoantibody testing for either anti-glycoprotein IIbIIIa or anti-glycoprotein IbIX were 53% (95% confidence interval [CI], 44-61%) and 93% (95% CI, 81-99%), respectively. For indirect testing, the pooled estimates for the sensitivity and specificity were 18% (95% CI, 12-24%) and 96% (95% CI, 87-100%), respectively. Conclusions Autoantibody testing in ITP patients has a high specificity but low sensitivity. A positive autoantibody test can be useful for ruling in ITP, but a negative test does not rule out ITP.
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http://dx.doi.org/10.1111/jth.14419DOI Listing
May 2019

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia.

Platelets 2019 29;30(8):1017-1021. Epub 2019 Jan 29.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University , Hamilton , Ontario , Canada.

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = -0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.
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http://dx.doi.org/10.1080/09537104.2018.1562169DOI Listing
February 2020

An international external quality assessment for laboratory diagnosis of heparin-induced thrombocytopenia.

J Thromb Haemost 2019 03 20;17(3):525-531. Epub 2019 Feb 20.

Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible.
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http://dx.doi.org/10.1111/jth.14383DOI Listing
March 2019

Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin-induced thrombocytopenia.

J Thromb Haemost 2019 02;17(2):389-399

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

Essentials Many patients produce antibodies but few lead to heparin-induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non-pathogenic antibodies. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti-PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. Objectives To identify PF4 amino acids essential for binding pathogenic HIT antibodies. Methods Alanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet-activating murine monoclonal anti-PF4/heparin antibody (KKO) and HIT patient sera (n = 9). Results and Conclusions We identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n = 45) and non-pathogenic antibodies (EIA-positive but not platelet activating, n = 28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.
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http://dx.doi.org/10.1111/jth.14369DOI Listing
February 2019

High sensitivity and specificity of an automated IgG-specific chemiluminescence immunoassay for diagnosis of HIT.

Blood 2018 09 31;132(12):1345-1349. Epub 2018 Jul 31.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

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http://dx.doi.org/10.1182/blood-2018-04-847483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202723PMC
September 2018

Megakaryocyte apoptosis in immune thrombocytopenia.

Platelets 2018 Nov 22;29(7):729-732. Epub 2018 May 22.

a Department of Medicine, Michael G. DeGroote School of Medicine , McMaster University , Hamilton , Canada.

The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.
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http://dx.doi.org/10.1080/09537104.2018.1475637DOI Listing
November 2018

Statins for high cholesterol … and for low platelets?

Blood 2018 03;131(11):1159-1161

University of Pennsylvania.

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http://dx.doi.org/10.1182/blood-2018-01-824888DOI Listing
March 2018

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Br J Haematol 2018 04 13;181(2):234-241. Epub 2018 Mar 13.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.
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http://dx.doi.org/10.1111/bjh.15165DOI Listing
April 2018

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure.

Eur J Haematol 2018 May 30;100(5):502-510. Epub 2018 Mar 30.

Canadian Blood Services, Centre for Innovation, Hamilton, ON, Canada.

Background: Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis.

Methods: Using flow cytometry and/or fluorescence microscopy, we analyzed desialylation patterns and eryptotic alterations in erythrocytes exposed to Clostridium perfringens-derived neuraminidase.

Results: Exogenous bacterial neuraminidase significantly augmented membrane PS exposure and cytosolic Ca levels in a dose- and time-dependent manner. Neuraminidase treatment significantly reduced fluorescence-tagged agglutinin binding, an effect temporally preceding the increase in PS externalization. Neuraminidase-induced PS exposure was significantly curtailed by pretreatment with the pan-sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. Neuraminidase treatment further induced hemolysis but did not significantly impact erythrocyte volume, ceramide abundance, or the generation of reactive oxygen species.

Conclusion: Collectively, our data reveal that alteration of erythrocyte sialylation status by bacterial neuraminidase favors eryptotic cell death, an effect potentially contributing to reduced erythrocyte lifespan and anemia in sepsis.
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http://dx.doi.org/10.1111/ejh.13047DOI Listing
May 2018

Platelet-Activating Antibodies Are Detectable at the Earliest Onset of Heparin-Induced Thrombocytopenia, With Implications for the Operating Characteristics of the Serotonin-Release Assay.

Chest 2018 06 8;153(6):1396-1404. Epub 2018 Jan 8.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada; McMaster Centre for Transfusion Research, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. It is unknown whether platelet-activating antibodies are detectable at the onset of the HIT-related platelet count fall.

Methods: Available blood samples from 18 patients obtained at onset of HIT were tested using the serotonin-release assay (SRA), a test for platelet-activating antibodies, and a PF4-dependent enzyme-linked immunosorbent assay (ELISA). Patient samples showing a delay of > 2 days between ELISA and SRA seroconversion were tested for subthreshold levels of platelet-activating antibodies using two modifications of the SRA that amplify detection of HIT antibodies. We also estimated SRA sensitivity and specificity in two postorthopedic surgery clinical trials (633 samples), including assessing whether a positive SRA influenced platelet count recovery in the absence of thrombocytopenia.

Results: Platelet-activating HIT antibodies were detected in all 18 patients at the beginning of the HIT-related platelet count fall. Although ELISA seroconversion usually preceded SRA seroconversion by only 1 day (median), subthreshold levels of platelet-activating antibodies were detected in both patients who exhibited a lag between ELISA and SRA seroconversion. SRA sensitivity was 100% (18/18), and its specificity was 97% (597/615). Nonthrombocytopenic SRA-positive patients with ongoing heparin treatment exhibited blunted platelet count recovery vs control subjects, suggesting even higher SRA specificity for detecting abnormal platelet count profiles.

Conclusions: Platelet-activating HIT antibodies are detectable at the onset of the HIT-related platelet count fall. The SRA has high sensitivity and specificity for HIT, and indicates that presence of HIT antibodies can blunt postoperative platelet count recovery.
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http://dx.doi.org/10.1016/j.chest.2018.01.001DOI Listing
June 2018

Misdiagnosis of primary immune thrombocytopenia and frequency of bleeding: lessons from the McMaster ITP Registry.

Blood Adv 2017 Nov 28;1(25):2414-2420. Epub 2017 Nov 28.

Department of Medicine, Michael G. DeGroote School of Medicine, and.

Nonspecific diagnostic criteria and uncertain estimates of severe bleeding events are fundamental gaps in knowledge of primary immune thrombocytopenia (ITP). To address these issues, we created the McMaster ITP Registry. In this report, we describe the methodology of the registry, the process for arriving at the diagnosis, and the frequency of bleeding. Consecutive patients with platelets <150 × 10/L from a tertiary hematology clinic in Canada were eligible. Patients completed a panel of investigations and were managed per clinical need. Two hematologists initially determined the cause of the thrombocytopenia using standard criteria and reevaluated the diagnosis over time, which was adjudicated at regular team meetings. Bleeding was graded from 0 (none) to 2 (severe) prospectively using an ITP-specific tool. Data were validated by duplicate chart review and source verification. Between 2010 and 2016, 614 patients were enrolled. Median follow-up for patients with >1 visit was 1.7 years (interquartile range, 0.8-3.4). At registration, 295 patients were initially diagnosed with primary ITP; of those, 36 (12.2%) were reclassified as having a different diagnosis during follow-up. At registration, 319 patients were initially diagnosed with another thrombocytopenic condition; of those, 10 (3.1%) were ultimately reclassified as having primary ITP. Of 269 patients with a final diagnosis of primary ITP, 56.5% (95% confidence interval [CI], 50.4-62.5] experienced grade 2 bleeding at 1 or more anatomical site, and 2.2% (95% CI, 0.8-4.8) had intracranial hemorrhage. Nearly 1 in 7 patients with primary ITP were misdiagnosed. Grade 2 bleeding was common. Registry data can help improve the clinical and laboratory classification of patients with ITP.
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http://dx.doi.org/10.1182/bloodadvances.2017010942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729626PMC
November 2017

Development of a high-yield expression and purification system for platelet factor 4.

Platelets 2018 May 27;29(3):249-256. Epub 2017 Nov 27.

a Department of Medicine , Michael G. DeGroote School of Medicine, McMaster University , Hamilton , Ontario , Canada.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.
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http://dx.doi.org/10.1080/09537104.2017.1378808DOI Listing
May 2018

The effect of rituximab on anti-platelet autoantibody levels in patients with immune thrombocytopenia.

Br J Haematol 2017 07 25;178(2):302-307. Epub 2017 Apr 25.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case-control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet-bound anti-glycoprotein (GP) IIbIIIa and anti-GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti-GPIIbIIIa levels (P = 0·02) but not anti-GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.
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http://dx.doi.org/10.1111/bjh.14664DOI Listing
July 2017

Performance characteristics of an automated latex immunoturbidimetric assay [HemosIL HIT-Ab] for the diagnosis of immune heparin-induced thrombocytopenia.

Thromb Res 2017 May 11;153:108-117. Epub 2017 Mar 11.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada; McMaster Centre for Transfusion Research, Hamilton, Ontario, Canada.

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-PF4/heparin antibodies. Given time-sensitive treatment considerations, a rapid and accurate laboratory test for HIT antibodies is needed.

Aims: To determine operating characteristics for the HemosIL HIT-Ab, a rapid, on-demand, fully-automated, latex immunoturbidimetric assay (LIA), for diagnosis of HIT.

Methods: We evaluated LIA sensitivity, specificity, negative (NPV) and positive predictive value (PPV), negative (LR-) and positive likelihood ratio (LR+), using citrated-plasma from 429 patients (prospective cohort study of 4Ts scoring; HIT, n=31), and from consecutive HIT patients (n=125), using reference standard serotonin-release assay (SRA). Comparators included two PF4-dependent enzyme-immunoassays (EIAs). We used stratum-specific likelihood ratios (SSLRs) to determine how differing magnitudes of LIA-positivity influenced post-test probability of HIT.

Results: LIA operating characteristics were: sensitivity=97.4% (152/156); specificity=94.0% (374/398); PPV=55.6% (30/54); and NPV=99.7% (374/375). At manufacturers' cutoffs, LIA specificity and PPV were superior to the EIAs. Although a negative LIA pointed strongly against HIT (LR-, 0.034), the post-test probability was ~2% with high 4Ts score. The LIA's LR+ was high (16.0), with SSLRs rising substantially with greater LIA-positivity: 5.7 (1.0-4.9U/mL), 31 (5.0-15.9U/mL), and 128 (≥16U/mL). A LIA-positive result (at 1.0 cutoff) indicated at least 24% HIT probability (low 4Ts score), rising to 90% with high 4Ts score.

Conclusions: Although approximately 1 in 40 SRA-positive patients tested LIA-negative, the LIA's high NPV and PPV indicate that this rapid assay is useful for the diagnostic evaluation of HIT, including in low pre-test situations.
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http://dx.doi.org/10.1016/j.thromres.2017.03.010DOI Listing
May 2017

The unique immunological features of heparin-induced thrombocytopenia.

Br J Haematol 2017 04 3;177(2):198-207. Epub 2017 Apr 3.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Heparin-induced thrombocytopenia (HIT) is a serious drug reaction that leads to a decrease in platelet count and a high risk of thrombosis. HIT patients produce pathogenic immunoglobulin G (IgG) antibodies that bind to complexes of platelet factor-4 (PF4) and heparin. HIT immune complexes crosslink Fc-receptors resulting in platelet and monocyte activation. These events lead to the release of procoagulant chemokines and tissue factor, which together create an intensely prothrombotic state. HIT represents an atypical immune response because it has features of both T cell-dependent and T cell-independent mechanisms. The disorder is characterized by newly formed anti-PF4/heparin IgG antibodies, which are characteristic of a T cell-dependent mechanism; however, re-exposure to heparin, months after HIT, does not lead to a memory response, which is consistent with a T cell-independent mechanism. In this review, we discuss the immunobiological events that can explain these features, including the role for T cell-dependent and T cell-independent mechanisms in HIT antibody generation, the immunogenic characteristics of the PF4/heparin antigen, and the concept of a temporary loss in immune regulation contributing to the onset of HIT. We also present a novel immunobiological model to explain the atypical immune response that is characteristic of HIT.
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http://dx.doi.org/10.1111/bjh.14603DOI Listing
April 2017