Publications by authors named "Brian R Curtis"

91 Publications

Trastuzumab-Induced Thrombocytopenia Correlated by Drug-Dependent Platelet Antibodies.

JCO Oncol Pract 2020 Dec 3:OP2000734. Epub 2020 Dec 3.

NorthShore University HealthSystem, Evanston, IL.

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http://dx.doi.org/10.1200/OP.20.00734DOI Listing
December 2020

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis.

Blood 2021 Feb;137(8):1082-1089

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.
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http://dx.doi.org/10.1182/blood.2020008195DOI Listing
February 2021

Thrombocytopenia and Thromboses in Myocardial Infarction Associated with Eptifibatide-Dependent Activating Antiplatelet Antibodies.

Thromb Haemost 2020 Jul 29;120(7):1137-1141. Epub 2020 May 29.

Department of Medicine, Pathways Service, Massachusetts General Hospital, Boston, Massachusetts, United States.

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http://dx.doi.org/10.1055/s-0040-1712458DOI Listing
July 2020

Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Nucleic Acid Ther 2020 04 11;30(2):94-103. Epub 2020 Feb 11.

Ionis Pharmaceuticals, Carlsbad, California.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 10/L in 50% and ≥100 × 10/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 10/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR ( = 17 placebo;  = 31 inotersen) and OLE ( = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.
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http://dx.doi.org/10.1089/nat.2019.0829DOI Listing
April 2020

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia.

Transfus Apher Sci 2020 Feb 31;59(1):102712. Epub 2019 Dec 31.

Department of Medical Biology, UiT- The Artic University of Norway, Tromsø, Norway.

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies. Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.
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http://dx.doi.org/10.1016/j.transci.2019.102712DOI Listing
February 2020

A case of platelet transfusion refractoriness due to anti-CD36 with a successful treatment outcome.

Immunohematology 2019 Dec;35(4):139-144

Associate Professor, Pathology, University of Michigan.

Conclusions: Antibodies (Abs) against antigens on platelets (PLTs), including glycoprotein IV (CD36), can cause PLT refractoriness. Transfusing PLTs to patients with anti-CD36 is challenging because of the rarity of CD36-negative (CD36-) donors and the possibility of additional HLA Abs. We report a case of PLT refractoriness due to anti-CD36 and HLA Abs. A 21-year-old man (group O, D+) with assumed drug-induced aplastic anemia received multiple PLT transfusions and developed severe PLT refractoriness. He was found to have anti-CD36 as well as HLA class I Abs, with a CD36- phenotype on both PLTs and monocytes. He was diagnosed with type 1 CD36 deficiency and received intravenous immunoglobulin (IVIG) and rituximab to decrease future Ab production. The PLT corrected count increment (CCI) improved significantly with subsequent transfusions of flow crossmatchcompatible as well as uncrossmatched PLTs. He eventually received a bone marrow transplant and has been doing well since. The mean CCI before and after IVIG/rituximab treatment was 0.2 and 6.2, respectively. Soon after IVIG started, the patient's CCI after receiving CD36-, group AB, D+, and HLA untested PLTs was 0.8, but his CCI after receiving flow crossmatch-compatible PLTs was 12.6. Two months after IVIG was started, the mean CCIs for uncrossmatched apheresis PLTs and crossmatch-compatible PLTs were comparable (6.1 versus 6.0, respectively). Desensitization treatment with IVIG and rituximab lowered anti-CD36 and HLA Ab levels, and the CCI of PLT transfusion improved significantly. This case demonstrates that immune suppression is effective for successful PLT transfusion of patients with anti-CD36.
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December 2019

Severe Platelet Transfusion Refractoriness in Association with Antibodies Against CD36.

Lab Med 2020 Sep;51(5):540-544

The Platelet & Neutrophil Immunology Laboratory, Blood Center of Wisconsin (part of Versiti), Milwaukee, WI.

Platelet-transfusion refractoriness (PTR) is common in patients with hematological malignancies. The etiology of immune PTR is typically human leukocyte antigen (HLA) antibodies (Abs) from pregnancy or previous transfusion. Herein, we report PTR in the setting of induction chemotherapy for acute myelogenous leukemia (AML) from Abs against CD36/glycoprotein (GP)IV. A 66-year-old African American woman presented with anemia and thrombocytopenia. She was found to have transfusion-dependent AML, and a 7 + 3 regimen (7 days of standard-dose cytarabine and 3 days of an anthracycline antibiotic or an anthracenedione, most often daunorubicin) was initiated. The patient developed profound thrombocytopenia, with platelet nadir of 0 by day 13. The results of HLA antibody screening were negative. However, the results of a screening test for platelet-specific antibodies screen showed Abs against cluster of differentiation (CD)36. The platelets of the patient lacked expression of CD36, and DNA analysis showed mutations in the CD36 gene. HLA Ab-mediated PTR is common in patients with hematological malignancies. However, once HLA Abs are excluded, other less-frequent Abs should be considered, particularly in patient populations of Asian, African, or Middle Eastern descent.
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http://dx.doi.org/10.1093/labmed/lmz091DOI Listing
September 2020

Post-Transfusion Purpura: Current Perspectives.

J Blood Med 2019 9;10:405-415. Epub 2019 Dec 9.

Versiti, Blood Center of Wisconsin, Milwaukee, WI, USA.

Post transfusion purpura (PTP) is an uncommonly reported post transfusion adverse event that can present with severe thrombocytopenia; sometimes resulting in significant bleeding and hemorrhage. Its diagnosis can be elusive given its substantial symptomatic overlap with other thrombocytopenic syndromes. Underdiagnosis and underreporting make the true incidence of disease difficult to define. While clinical suspicion is key, laboratory evidence of platelet-targeted antibodies and identification of the antigen(s) they recognize are necessary to confirm the diagnosis. A curious aspect of PTP is paradoxical destruction of both transfused and autologous platelets. Although the first case was reported over 50 years ago, this aspect of PTP pathogenesis is still not fully understood and is widely debated. Several theories exist, but conclusive evidence to support most is lacking. Despite limited understanding of disease incidence and etiology, treatment with IVIG (Intravenous Immunoglobulin) has become standard practice and can be highly effective. Although recurrence is rare, precautions should be taken if patients with a history of PTP require transfusions in the future.
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http://dx.doi.org/10.2147/JBM.S189176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910090PMC
December 2019

Bioengineered iPSC-derived megakaryocytes for the detection of platelet-specific patient alloantibodies.

Blood 2019 11;134(22):e1-e8

Blood Research Institute, Versiti, Milwaukee, WI.

Human platelet membrane glycoprotein polymorphisms can be immunogenic in man and are frequently the cause of clinically important immune reactions responsible for disorders such as neonatal alloimmune thrombocytopenia. Platelets from individuals carrying rare polymorphisms are often difficult to obtain, making diagnostic testing and transfusion of matched platelets challenging. In addition, class I HLA antibodies frequently present in maternal sera interfere with the detection of platelet-reactive alloantibodies. Detection of alloantibodies to human platelet antigen 3 (HPA-3) and HPA-9 is especially challenging, in part because of the presence of cell type-specific glycans situated near the polymorphic amino acid that together form the alloepitope. To overcome these limitations, we generated a series of HLA class I-negative blood group O induced pluripotent stem cell (iPSC) lines that were gene edited to sequentially convert their endogenous HPA-3a alloantigenic epitope to HPA-3b, and HPA-9a to HPA-9b. Subjecting these cell lines, upon differentiation into CD41+/CD42b+ human megakaryocytes (MKs), to flow cytometric detection of suspected anti-HPA-3 and HPA-9 alloantisera revealed that the HPA-3a-positive MKs specifically reacted with HPA-3a patient sera, whereas the HPA-3b MKs lost reactivity with HPA-3a patient sera while acquiring reactivity to HPA-3b patient sera. Importantly, HPA-9b-expressing MKs specifically reacted with anti-HPA-9b-suspected patient samples that had been undetectable using conventional techniques. The provision of specialized iPSC-derived human MKs expressing intact homozygous glycoprotein alloantigens on the cell surface that carry the appropriate endogenous carbohydrate moieties should greatly enhance detection of clinically important and rare HPA-specific alloantibodies that, to date, have resisted detection using current methods.
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http://dx.doi.org/10.1182/blood.2019002225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887112PMC
November 2019

Pes Anserinus: Anatomy and Pathology of Native and Harvested Tendons.

AJR Am J Roentgenol 2019 11 30;213(5):1107-1116. Epub 2019 Jul 30.

Division of Musculoskeletal Radiology, University of California San Diego Health System, San Diego, CA.

The purpose of this article is to review the anatomy and pathology of the pes anserinus to increase the accuracy of imaging interpretation of findings affecting these medial knee structures. The pes anserinus, consisting of the conjoined tendons of the sartorius, gracilis, and semitendinosus muscles and their insertions at the medial aspect of the knee, is often neglected during imaging assessment. Common pathologic conditions affecting the pes anserinus include overuse, acute trauma, iatrogenic disorders, and tumors and tumorlike lesions.
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http://dx.doi.org/10.2214/AJR.19.21315DOI Listing
November 2019

Immune neutropenia mediated by micafungin.

Am J Hematol 2019 07 14;94(7):830-832. Epub 2019 Apr 14.

Division of Hematology, Massachusetts General Hospital Cancer Center, Boston, Massachusetts.

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http://dx.doi.org/10.1002/ajh.25483DOI Listing
July 2019

Drug-induced thrombocytopenia: 2019 Update of clinical and laboratory data.

Am J Hematol 2019 03 27;94(3):E76-E78. Epub 2018 Dec 27.

Department of Biostatistics & Epidemiology, College of Public Health, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.

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http://dx.doi.org/10.1002/ajh.25379DOI Listing
March 2019

High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (Pl).

Blood Adv 2018 11;2(21):3001-3011

Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI.

Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as Pl) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents.
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http://dx.doi.org/10.1182/bloodadvances.2018023341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234362PMC
November 2018

Autonomous conformational regulation of β integrin and the conformation-dependent property of HPA-1a alloantibodies.

Proc Natl Acad Sci U S A 2018 09 12;115(39):E9105-E9114. Epub 2018 Sep 12.

Blood Research Institute, BloodCenter of Wisconsin, Part of Versiti, Milwaukee, WI 53226;

Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The β integrins, including αβ and αβ, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the αβ from αβ and affect their functions with unknown mechanisms. Here we designed a single-chain β subunit that mimics a separation of α/β heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the β in the bent state vs. the extended state and in the complex with α vs. α This study provides deeper understandings of integrin conformational activation on the cell surface.
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http://dx.doi.org/10.1073/pnas.1806205115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166792PMC
September 2018

Investigation into the Mechanism(s) That Leads to Platelet Decreases in Cynomolgus Monkeys During Administration of ISIS 104838, a 2'-MOE-Modified Antisense Oligonucleotide.

Toxicol Sci 2018 08;164(2):613-626

Nonclinical Development, Ionis Pharmaceuticals Inc, Carlsbad, California, 92010.

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.
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http://dx.doi.org/10.1093/toxsci/kfy119DOI Listing
August 2018

Non-chemotherapy drug-induced neutropenia: key points to manage the challenges.

Authors:
Brian R Curtis

Hematology Am Soc Hematol Educ Program 2017 12;2017(1):187-193

Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI.

Non-chemotherapy idiosyncratic drug-induced neutropenia (IDIN) is a relatively rare but potentially fatal disorder that occurs in susceptible individuals, with an incidence of 2.4 to 15.4 cases per million population. Affected patients typically experience severe neutropenia within several weeks to several months after first exposure to a drug, and mortality is ∼5%. The drugs most frequently associated with IDIN include metamizole, clozapine, sulfasalazine, thiamazole, carbimazole, amoxicillin, cotrimoxazole, ticlopidine, and valganciclovir. The idiosyncratic nature of IDIN, the lack of mouse models and diagnostic testing, and its low overall incidence make rigorous studies to elucidate possible mechanisms exceptionally difficult. An immune mechanism for IDIN involving neutrophil destruction by hapten (drug)-specific antibodies and drug-induced autoantibodies is frequently suggested, but strong supporting evidence is lacking. Although laboratory testing for neutrophil drug-dependent antibodies is rarely performed because of the complexity and low sensitivity of tests currently in use, these assays could possibly be enhanced by using reactive drug metabolites in place of the parent drug. Patients typically experience acute, severe neutropenia, or agranulocytosis (<0.5 × 10 neutrophils/L) and symptoms of fever, chills, sore throat, and muscle and joint pain. Diagnosis can be difficult, but timely recognition is critical because if left untreated, there is an increase in mortality. Expanded studies of the production and mechanistic role of reactive drug metabolites, genetic associations, and improved animal models of IDIN are essential to further our understanding of this important disorder.
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http://dx.doi.org/10.1182/asheducation-2017.1.187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142577PMC
December 2017

Severe thrombocytopenia in a patient following liver transplantation caused by HPA-1a antibodies produced by the liver donor.

Am J Hematol 2018 Jan 31;93(1):150-153. Epub 2017 Oct 31.

Northwestern University, Chicago, Illinois, 60611.

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http://dx.doi.org/10.1002/ajh.24944DOI Listing
January 2018

A Platelet Factor 4-Dependent Platelet Activation Assay Facilitates Early Detection of Pathogenic Heparin-Induced Thrombocytopenia Antibodies.

Chest 2017 10;152(4):e77-e80

Medical Sciences Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Pathology, Medical College of Wisconsin, Milwaukee, WI. Electronic address:

Heparin-induced thrombocytopenia (HIT) is a dangerous complication of heparin therapy. HIT diagnosis is established by recognizing thrombocytopenia and/or thrombosis in an affected patient and from the results of serological tests such as the platelet factor 4 (PF4)/heparin immunoassay (PF4 ELISA) and serotonin release assay (SRA). Recent studies suggest that HIT antibodies activate platelets by recognizing PF4 in a complex with platelet glycosaminoglycans (and/or polyphosphates) and that an assay based on this principle, the PF4-dependent P-selectin expression assay (PEA), may be even more accurate than the SRA for HIT diagnosis. Here, we demonstrate that the PEA detected pathogenic antibodies before the SRA became positive in two patients with HIT studied serially, in one case even before seropositivity in the PF4 ELISA. In one of the patients treated with plasma exchange, persistent dissociation between the PEA and SRA test results was observed. These results support a role for the PEA in early HIT diagnosis.
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http://dx.doi.org/10.1016/j.chest.2017.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812761PMC
October 2017

Severe neonatal alloimmune thrombocytopenia caused by maternal sensitization against a new low-frequency alloantigen (Dom ) located on platelet glycoprotein IIIa.

Transfusion 2017 07 18;57(7):1847-1848. Epub 2017 May 18.

Platelet & Neutrophil Immunology Lab.

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http://dx.doi.org/10.1111/trf.14160DOI Listing
July 2017

Detection and identification of platelet antibodies using a sensitive multiplex assay system-platelet antibody bead array.

Transfusion 2017 07 25;57(7):1724-1733. Epub 2017 Apr 25.

Platelet and Neutrophil Immunology Laboratory, Milwaukee, Wisconsin.

Background: Tests for platelet-specific antibodies are important in the diagnosis of immune platelet disorders. Monoclonal antibody glycoprotein capture assays have been the gold standards in platelet antibody detection for almost 30 years. However, such assays are complex and cumbersome to perform, which limits their routine use. We report the performance of a newly developed, easy to perform platelet antibody bead array (PABA) for the detection of platelet-specific antibodies.

Study Design And Methods: PABA is the equivalent of the monoclonal antigen capture enzyme-linked immunosorbent assay (ELISA) (MACE) on a bead and instead with fluorescent detection of immunoglobulin (Ig)G platelet antibodies by Luminex. Antibodies against platelet glycoproteins (GP) GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIV, and class I human leukocyte antigen (HLA) could be detected in a patient's serum simultaneously in a single well of a microplate. Results from 80 patient samples and 20 normal donor samples were compared using PABA, MACE, and a commercial ELISA kit.

Results: PABA detected all antibodies, with specificity for human platelet antigens (HPAs) HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b, HPA-15b, and HLA. Overall, compared with MACE, the sensitivity and specificity of PABA were 99% and 97%, respectively, and both were significantly better than those of the commercial ELISA. PABA had greater analytic sensitivity than MACE for HPA-1a, HPA-3a, and HPA-5b antibodies. In addition, PABA detected HPA-5b and HPA-3b antibodies that were missed by MACE. The overall false-positive rate of PABA compared with MACE was 2.7%.

Conclusions: The PABA is a rapid, highly sensitive and specific, multiplex bead-based assay for detecting human platelet antibodies. Such a simple yet high-throughput platform will facilitate practical, routine testing for the identification of platelet-specific antibodies.
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http://dx.doi.org/10.1111/trf.14122DOI Listing
July 2017

IVIg for Treatment of Severe Refractory Heparin-Induced Thrombocytopenia.

Chest 2017 09 17;152(3):478-485. Epub 2017 Apr 17.

Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Medicine, Medical College of Wisconsin, Milwaukee, WI.

Background: Heparin-induced thrombocytopenia (HIT) complicated by severe thrombocytopenia and thrombosis can pose significant treatment challenges. Use of alternative anticoagulants in this setting may increase bleeding risks, especially in patients who have a protracted disease course. Additional therapies are lacking in this severely affected patient population.

Methods: We describe three patients with HIT who had severe thromboembolism and prolonged thrombocytopenia refractory to standard treatment but who achieved an immediate and sustained response to IVIg therapy. The mechanism of action of IVIg was evaluated in these patients and in five additional patients with severe HIT. The impact of a common polymorphism (H/R 131) in the platelet IgG receptor FcγRIIa on IVIg-mediated inhibition of platelet activation was also examined.

Results: At levels attained in vivo, IVIg inhibits HIT antibody-mediated platelet activation. The constant domain of IgG (Fc) but not the antigen-binding portion (Fab) is required for this effect. Consistent with this finding, IVIg had no effect on HIT antibody binding in a solid-phase HIT immunoassay (platelet factor 4 enzyme-linked immunoassay). The H/R131 polymorphism in FcγRIIa influences the susceptibility of platelets to IVIg treatment, with the HH131 genotype being most susceptible to IVIg-mediated inhibition of antibody-induced activation. However, at high doses of IVIg, activation of platelets of all FcγRIIa genotypes was significantly inhibited. All three patients did well on long-term anticoagulation therapy with direct oral anticoagulants.

Conclusions: These studies suggest that IVIg treatment should be considered in patients with HIT who have severe disease that is refractory to standard therapies.
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http://dx.doi.org/10.1016/j.chest.2017.03.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812774PMC
September 2017

Low yield of ventilation and perfusion imaging for the evaluation of pulmonary embolism after indeterminate CT pulmonary angiography.

Emerg Radiol 2017 Oct 12;24(5):525-530. Epub 2017 Apr 12.

Department of Radiology, Thomas Jefferson University Hospital, 132 South 10th Street, Philadelphia, PA, 19107, USA.

Purpose: Ventilation and perfusion (VQ) imaging is common following suboptimal CT pulmonary angiogram (CTPA) for pulmonary embolism (PE) evaluation; however, the results of this diagnostic pathway are unclear. The purpose of our study is to determine the incidence of PE diagnosed on VQ scans performed in patients with suboptimal CTPAs.

Methods: One hundred twenty-two suboptimal CTPAs with subsequent VQ scans within 1 week were retrospectively identified. VQ reports utilizing modified ​prospective investigation of pulmonary embolism diagnosis (PIOPED) and prospective investigative study of acute pulmonary embolism diagnosis (PISAPED) criteria were evaluated for presence of PE; intermediate probability, high probability, and PE present were considered PE positive. Three hundred consecutive reports of each diagnostic CTPA and diagnostic VQ studies were reviewed to estimate baseline PE positive rates at our institution. These were compared to the positive VQ scan rate after suboptimal CTPA by Fisher's exact test. Reported reason for suboptimal CTPA was noted. When contrast bolus timing was suboptimal, we measured main pulmonary artery (mPA) Hounsfield units (HU). Potential alternative diagnoses in CTPA reports were noted.

Results: 97.5% (119/122) of VQ scans following suboptimal CTPA were negative for PE, and 2.5% (3/122) were positive for PE. This was significantly lower than baseline PE positive rate of 10.7% (32/300, p < 0.01) for VQ imaging, and 10.3% (31/300, p < 0.01) for CTPA at our institution. Most (79.5%) CTPAs were suboptimal due to contrast timing. Average mPA density in these cases was 164 ± 61 HU. Most of these studies ruled out central PE. Potential alternative diagnosis was reported in 34/122 (28%) of suboptimal CTPAs, for which pneumonia accounted 59%.

Conclusion: There is very low incidence of PE diagnosed on VQ imaging performed after suboptimal CTPA. This may be attributed to the ability of most suboptimal CTPAs to rule out central PE.
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http://dx.doi.org/10.1007/s10140-017-1503-9DOI Listing
October 2017

An unexpected development after surgery-post-transfusion purpura!

Am J Hematol 2016 Aug 3;91(8):848-51. Epub 2016 Jun 3.

Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045246PMC
http://dx.doi.org/10.1002/ajh.24414DOI Listing
August 2016

Update on the nomenclature of human neutrophil antigens and alleles.

Transfusion 2016 06 4;56(6):1477-9. Epub 2016 Apr 4.

Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.

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http://dx.doi.org/10.1111/trf.13575DOI Listing
June 2016

A Novel PF4-Dependent Platelet Activation Assay Identifies Patients Likely to Have Heparin-Induced Thrombocytopenia/Thrombosis.

Chest 2016 09 19;150(3):506-15. Epub 2016 Feb 19.

Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Medicine, Medical College of Wisconsin, Milwaukee, WI.

Background: Almost without exception, patients with heparin-induced thrombocytopenia/thrombosis (HIT) have antibodies that recognize platelet factor 4 (PF4) in a complex with heparin; however, many heparin-treated patients without HIT are also antibody-positive. A platelet activation test, the serotonin release assay (SRA), is useful for identifying a subset of antibodies that are platelet-activating and most likely to cause HIT. However, this "gold standard" assay for HIT diagnosis is technically demanding and is routinely available only through referral laboratories, limiting its availability for timely diagnosis and management.

Methods: We compared the diagnostic performance of the SRA with that of a technically simple platelet activation assay, the PF4-dependent P-selectin expression assay (PEA), which uses platelets pretreated with PF4 as targets for antibody detection. Archived serum samples from 91 patients for whom clinical information (HIT 4Ts [thrombocytopenia, timing of platelet count fall, thrombosis, and other causes of thrombocytopenia] score) was available were used. Patients with an intermediate 4Ts score and a PF4 ELISA (enzyme-linked immunosorbent assay) optical density ≥ 2.0, or a high 4Ts score and a PF4 ELISA optical density ≥ 1.0, were considered HIT positive; others were designated HIT negative.

Results: The PEA had higher diagnostic accuracy (area under the curve, 0.92 vs 0.82; P = .02) than the SRA, using this definition of HIT. Eleven of 16 serum samples that were PEA positive and SRA negative were HIT positive. Studies done with identical target platelets and serially diluted samples from patients with HIT showed that the PEA is inherently more sensitive than the SRA for the detection of platelet-activating antibodies.

Conclusions: The PEA is technically less demanding than the SRA and may be more accurate for the diagnosis of HIT.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028397PMC
http://dx.doi.org/10.1016/j.chest.2016.02.641DOI Listing
September 2016

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.

Blood 2016 Feb 3;127(6):675-80. Epub 2015 Dec 3.

Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Pharmacology and Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI.

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.
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http://dx.doi.org/10.1182/blood-2015-10-675751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751021PMC
February 2016

Recent progress in understanding the pathogenesis of fetal and neonatal alloimmune thrombocytopenia.

Authors:
Brian R Curtis

Br J Haematol 2015 Dec 7;171(5):671-82. Epub 2015 Sep 7.

Platelet & Neutrophil Immunology Laboratory and Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs in c. 1 in 1000 births and is caused by maternal antibodies against human platelet alloantigens that bind incompatible fetal platelets and promote their clearance from the circulation. Affected infants can experience bleeding, bruising and, in severe cases, intracranial haemorrhage and even death. As maternal screening is not routinely performed, and first pregnancies can be affected, most cases are diagnosed at delivery of a first affected pregnancy. Unlike its erythrocyte counterpart, Haemolytic Disease of the Fetus and Newborn, there is no prophylactic treatment for FNAIT. This report will review recent advances made in understanding the pathogenesis of FNAIT: the platelet alloantigens involved, maternal exposure and sensitization to fetal platelet antigens, properties of platelet Immunoglobulin G antibodies, maternal-fetal antibody transport mechanisms and efforts to develop an effective FNAIT prophylaxis.
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http://dx.doi.org/10.1111/bjh.13639DOI Listing
December 2015