Publications by authors named "James W Smith"

53 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

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

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

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

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

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

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

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

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

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

Development of immunohistochemistry services for cancer care in western Kenya: Implications for low- and middle-income countries.

Afr J Lab Med 2016 4;5(1):187. Epub 2016 May 4.

Department of Pathology, Indiana University School of Medicine, Indianapolis, Indiana, United States.

Background: Cancer is becoming a major cause of mortality in low- and middle-income countries. Unlike infectious disease, malignancy and other chronic conditions require significant supportive infrastructure for diagnostics, staging and treatment. In addition to morphologic diagnosis, diagnostic pathways in oncology frequently require immunohistochemistry (IHC) for confirmation. We present the experience of a tertiary-care hospital serving rural western Kenya, which developed and validated an IHC laboratory in support of a growing cancer care service.

Objectives Methods And Outcomes: Over the past decade, in an academic North-South collaboration, cancer services were developed for the catchment area of Moi Teaching and Referral Hospital in western Kenya. A major hurdle to treatment of cancer in a resource-limited setting has been the lack of adequate diagnostic services. Building upon the foundations of a histology laboratory, strategic investment and training were used to develop IHC services. Key elements of success in this endeavour included: translation of resource-rich practices to a resource-limited setting, such as using manual, small-batch IHC instead of disposable- and maintenance-intensive automated machinery, engagement of outside expertise to develop reagent-efficient protocols and supporting all levels of staff to meet the requirements of an external quality assurance programme.

Conclusion: Development of low- and middle-income country models of services, such as the IHC laboratory presented in this paper, is critical for the infrastructure in resource-limited settings to address the growing cancer burden. We provide a low-cost model that effectively develops these necessary services in a challenging laboratory environment.
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http://dx.doi.org/10.4102/ajlm.v5i1.187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436389PMC
May 2016

Triplets with neonatal alloimmune thrombocytopenia due to antibodies against human platelet antigen 1a.

Transfusion 2016 May 26;56(5):1166-1170. Epub 2016 Jan 26.

Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, ON.

Background: Neonatal alloimmune thrombocytopenia (NAIT) has been reported only rarely in twins and not at all, to our knowledge, in triplets.

Case Report: Nonidentical triplets were born with severe thrombocytopenia. Nadir platelet (PLT) counts were 17 × 10 , 12 × 10 , and 10 × 10 /L. NAIT was confirmed by an incompatibility for human PLT antigen (HPA)-1a and the presence of maternal anti-HPA-1a. The maternal genotype was HPA-1bb and the paternal genotype was HPA-1aa; thus, all children were affected.

Results: PLT counts for each infant improved with the administration of random-donor PLT transfusions. All three infants also received intravenous immunoglobulin. None had major bleeding. A small isolated subependymal hemorrhage was found incidentally in one infant; this remained stable on repeat imaging.

Conclusions: This is the first report of triplets with NAIT. Anti-HPA-1a is sufficiently potent to affect three infants simultaneously. Random-donor PLTs were effective in improving PLT counts in all three infants.
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http://dx.doi.org/10.1111/trf.13494DOI Listing
May 2016

Producing megakaryocytes from a human peripheral blood source.

Transfusion 2016 05 12;56(5):1066-74. Epub 2016 Jan 12.

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

Background: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs).

Study Design And Methods: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation.

Results: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions.

Conclusions: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.
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http://dx.doi.org/10.1111/trf.13461DOI Listing
May 2016

Pitfalls in the diagnosis of heparin-Induced thrombocytopenia: A 6-year experience from a reference laboratory.

Am J Hematol 2015 Jul 3;90(7):629-33. Epub 2015 May 3.

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

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic.
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http://dx.doi.org/10.1002/ajh.24025DOI Listing
July 2015

Antibody binding to megakaryocytes in vivo in patients with immune thrombocytopenia.

Eur J Haematol 2015 Dec 13;95(6):532-7. Epub 2015 Mar 13.

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

Objectives: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by increased platelet destruction and impaired platelet production. Antibody binding to megakaryocytes may occur in ITP, but in vivo evidence of this phenomenon is lacking.

Methods: We determined the proportion of megakaryocytes bound with immunoglobulin G (IgG) in bone marrow samples from primary patients with ITP (n = 17), normal controls (n = 13) and thrombocytopenic patients with myelodysplastic syndrome (MDS; n = 10). Serial histological sections from archived bone marrow biopsies were stained for CD61 and IgG. IgG binding and the number of bone marrow megakaryocytes were determined morphologically by a hematopathologist with four assessors after a calibration exercise to ensure consistency.

Results: The proportion of ITP patients with high IgG binding (>50% of bone marrow megakaryocytes) was increased compared with normal controls [12/17 (71%) vs. 3/13 (23%), P = 0.03]. However, the proportion of ITP patients with high IgG binding was no different than thrombocytopenic patients with MDS [12/17 (71%) vs. 7/10 (70%), P = 1.00]. IgG binding was associated with increased megakaryocyte numbers. Like platelet-associated IgG, megakaryocyte-associated IgG is related to thrombocytopenia but may not be specific for ITP.

Conclusion: Mechanistic studies in ITP should focus on antibody specificity and include thrombocytopenic control patients.
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http://dx.doi.org/10.1111/ejh.12528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851549PMC
December 2015

Management of infants born with severe neonatal alloimmune thrombocytopenia: the role of platelet transfusions and intravenous immunoglobulin.

Transfusion 2014 Mar 19;54(3):640-5. Epub 2013 Jul 19.

Institute for Immunology and Transfusion Medicine, University Children's Hospital, Universitätsmedizin Greifswald, Greifswald, Germany; Department of Neonatology, University Children's Hospital, Universitätsmedizin Greifswald, Greifswald, Germany; Department of Neonatology, University Children's Hospital, Tuebingen, Germany; Department of Transfusion Medicine, University of Rostock, Rostock, Germany; Department of Medicine, McMaster University, Hamilton, Ontario, Canada; Department of Pediatrics, Neonatal Division, McMaster University, Hamilton, Ontario, Canada; Canadian Blood Services, Hamilton, Ontario, Canada.

Background: Neonatal alloimmune thrombocytopenia (NAIT) is a fetomaternal incompatibility most commonly induced by maternal anti-HPA-1a alloantibodies. Transfusion of immunologically compatible platelets (PLTs) to prevent cerebral hemorrhage, the most severe complication in affected newborns, is usually recommended. Such PLT concentrates, however, are often not readily available.

Study Design And Methods: The efficacy of random-donor PLT transfusions and intravenous immunoglobulin (IVIG) for the management of 17 neonates across four centers with unexpected, severe NAIT was evaluated. Neonates were treated with random-donor PLTs alone (n=7), random-donor PLTs with IVIG (n=8), or matched HPA-1bb PLTs (n=2).

Results: All but one patient (treated with random PLTs and IVIG) achieved a posttransfusion PLT count of higher than 30 × 10(9) /L after the first PLT transfusion. The PLT count remained higher than 30 × 10(9) /L for longer than 24 hours in five of seven, seven of eight, and two of four newborns who received random-donor PLTs alone, random-donor PLTs with IVIG, or matched HPA-1bb PLTs, respectively. None of the newborns developed major bleeding or intracranial hemorrhage. IVIG did not appear to improve either posttransfusion PLT counts or total PLT transfusion requirements.

Conclusion: Transfusion of random-donor PLTs alone was effective at correcting critically low PLT counts and should be considered as first-line treatment of newborns with unexpected severe NAIT.
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http://dx.doi.org/10.1111/trf.12336DOI Listing
March 2014

Approach to the diagnosis and management of drug-induced immune thrombocytopenia.

Transfus Med Rev 2013 Jul 8;27(3):137-45. Epub 2013 Jul 8.

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

Drug-induced immune thrombocytopenia (DITP) is a challenging clinical problem that is under-recognized, difficult to diagnose and associated with severe bleeding complications. DITP may be caused by classic drug-dependent platelet antibodies (eg, quinine); haptens (eg, penicillin); fiban-dependent antibodies (eg, tirofiban); monoclonal antibodies (eg, abciximab); autoantibody formation (eg, gold); and immune complex formation (eg, heparin). A thorough clinical history is essential in establishing the diagnosis of DITP and should include exposures to prescription medications, herbal preparations and even certain foods and beverages. Clinical and laboratory criteria have been established to determine the likelihood of a drug being the cause of thrombocytopenia, but these criteria can only be applied retrospectively. The most commonly implicated drugs include quinine, quinidine, trimethoprim/sulfamethoxazole and vancomycin. We propose a practical approach to the diagnosis of the patient with suspected DITP. Key features are: the presence of severe thrombocytopenia (platelet nadir <20×10(9)/L); bleeding complications; onset 5 to 10days after first drug exposure, or within hours of subsequent exposures or after first exposure to fibans or abciximab; and exposure to drugs that have been previously implicated in DITP reactions. Treatment involves stopping the drug(s), administering platelet transfusions or other therapies if bleeding is present and counselling on future drug avoidance. The diagnosis can be confirmed by a positive drug re-challenge, which is often impractical, or by demonstrating drug-dependent platelet reactive antibodies in vitro. Current test methods, which are mostly flow cytometry-based, must show drug-dependence, immunoglobulin binding, platelet specificity and ideally should be reproducible across laboratories. Improved standardization and accessibility of laboratory testing should be a focus of future research.
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http://dx.doi.org/10.1016/j.tmrv.2013.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728035PMC
July 2013

Irritable bowel syndrome: a review and update.

Clin Colon Rectal Surg 2012 Mar;25(1):46-52

Department of Gastroenterology, Ochsner Clinic Foundation, New Orleans, Louisiana.

The understanding of irritable bowel syndrome (IBS) has undergone a rapid evolution with scientific advancement. IBS is a common functional bowel disorder that generates a significant health care burden and is the most commonly diagnosed gastrointestinal condition. There are well-established diagnostic criteria and algorithms for the initial evaluation of patients presenting with the symptoms of IBS. The symptoms can be targeted for therapy with a variety of pharmaceutical and nonpharmaceutical agents. Therapy should be individualized for the patient, and the cornerstone for any effective treatment strategy should be the solid patient-physician relationship.
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http://dx.doi.org/10.1055/s-0032-1301759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348735PMC
March 2012

Management of cyclosporine overdose in a hematopoietic stem cell transplant patient with sequential plasma exchange and red blood cell exchange.

J Clin Apher 2011 13;26(3):156-8. Epub 2010 Dec 13.

Department of Pharmacy, Clinical and Administrative Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73126, USA.

Cyclosporine is commonly used as an immunosuppressive agent in both solid organ and bone marrow transplant. While used for graft rejection in organ transplantation, cyclosporine has been used to enable tolerance and for prevention of acute graft-versus-host disease in bone marrow transplant [Ratanatharathorn et al., Blood 1998;92:2303-2314]. Cyclosporine has a narrow therapeutic window, and many patients develop some level of toxicity even within the therapeutic range. Common toxicities include hypertension, nephrotoxicity, electrolyte abnormalities, hyperglycemia, and neurotoxicity [Woo et al., Bone Marrow Transplant 1997;20:1095-1098]. Management of cyclosporine toxicity is not clearly defined and is primarily supportive in nature. In cases of significant elevations of cyclosporine levels, limited data are available but suggest that whole blood exchange may be effective [Kwon et al., J Heart Lung Transplant 2006;25:483-485; Leitner et al., Transplantation 2003;75:1764-1765]. We present a case of successful rapid clearance of cyclosporine utilizing a combined approach of red cell exchange and plasma exchange.
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http://dx.doi.org/10.1002/jca.20277DOI Listing
November 2011

Increased all-cause and cancer mortality in HTLV-II infection.

J Acquir Immune Defic Syndr 2010 Jul;54(3):290-6

Blood Systems Research Institute, San Francisco, CA 94118, USA.

Background: Human T-lymphotropic virus (HTLV)-I and HTLV-II cause chronic human retroviral infections, but few studies have examined the impact of either virus on survival among otherwise healthy individuals. The authors analyzed all-cause and cancer mortality in a prospective cohort of 155 HTLV-I, 387 HTLV-II, and 799 seronegative subjects.

Methods: Vital status was ascertained using death certificates, the US Social Security Death Index or family report, and causes of death were grouped into 9 categories. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazards models.

Results: After a median follow-up of 15.9 years, there were 105 deaths: 22 HTLV-I, 41 HTLV-II, and 42 HTLV-seronegative. Cancer was the predominant cause of death, resulting in 8 HTLV-I, 17 HTLV-II, and 15 HTLV-seronegative deaths. After adjustment for confounding, HTLV-I status was not significantly associated with increased all-cause mortality, though there was a positive trend (HR: 1.6, 95% CI: 0.8 to 3.1). HTLV-II status was strongly associated with increased all-cause (HR: 2.4, 95% CI: 1.4 to 4.4) and cancer mortality (HR: 3.8, 95% CI: 1.6 to 9.2).

Conclusions: The observed associations of HTLV-II with all-cause and cancer mortality could reflect biological effects of HTLV-II infection, residual confounding by socioeconomic status or other factors, or differential access to health care and cancer screening.
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http://dx.doi.org/10.1097/QAI.0b013e3181cc5481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891114PMC
July 2010

Diagnosis and management of neonatal alloimmune thrombocytopenia.

Transfus Med Rev 2008 Oct;22(4):255-67

The McMaster Platelet Immunology Diagnostic Laboratory, McMaster University, Hamilton, Ontario, Canada.

Neonatal alloimmune thrombocytopenia (NAT) is a life-threatening bleeding disorder caused by maternal platelet antibodies produced in response to fetal platelet antigens inherited from the father. Antiplatelet antibodies cross the placenta and cause destruction of fetal platelets, leading to severe thrombocytopenia, and potentially bleeding, including fatal intracerebral hemorrhage. Incompatibilities between maternal and fetal platelets for the human platelet antigen 1a (previously called PL(A1)) account for most of the patients with NAT, but other antigens are commonly implicated. Diagnostic testing for NAT involves genotyping of maternal, paternal, and sometimes fetal DNA; platelet antigen phenotyping; and maternal platelet antibody investigations using specialized platelet glycoprotein specific assays. The management of women and infants at risk for NAT remains largely empiric; and mounting evidence points to prohibitive risks of invasive procedures such as fetal blood sampling and intrauterine platelet transfusions, except in rare circumstances. Improvements in our understanding of the pathophysiology of NAT, and of clinical and laboratory predictors of severity, may help develop better treatments and improve our ability to identify mothers at risk.
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http://dx.doi.org/10.1016/j.tmrv.2008.05.003DOI Listing
October 2008

Long-term increases in lymphocytes and platelets in human T-lymphotropic virus type II infection.

Blood 2008 Nov 28;112(10):3995-4002. Epub 2008 Aug 28.

School of Public Health, University of California, Berkeley, USA.

Human T-lymphotropic viruses types I and II (HTLV-I and HTLV-II) cause chronic infections of T lymphocytes that may lead to leukemia and myelopathy. However, their long-term effects on blood counts and hematopoiesis are poorly understood. We followed 151 HTLV-I-seropositive, 387 HTLV-II-seropositive, and 799 HTLV-seronegative former blood donors from 5 U.S. blood centers for a median of 14.0 years. Complete blood counts were performed every 2 years. Multivariable repeated measures analyses were conducted to evaluate the independent effect of HTLV infection and potential confounders on 9 hematologic measurements. Participants with HTLV-II had significant (P < .05) increases in their adjusted lymphocyte counts (+126 cells/mm(3); approximately +7%), hemoglobin (+2 g/L [+0.2 g/dL]) and mean corpuscular volume (MCV; 1.0 fL) compared with seronegative participants. Participants with HTLV-I and HTLV-II had higher adjusted platelet counts (+16 544 and +21 657 cells/mm(3); P < .05) than seronegatives. Among all participants, time led to decreases in platelet count and lymphocyte counts, and to increases in MCV and monocytes. Sex, race, smoking, and alcohol consumption all had significant effects on blood counts. The HTLV-II effect on lymphocytes is novel and may be related to viral transactivation or immune response. HTLV-I and HTLV-II associations with higher platelet counts suggest viral effects on hematopoietic growth factors or cytokines.
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http://dx.doi.org/10.1182/blood-2008-05-155960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581993PMC
November 2008