22 results on '"Althaus, Karina"'
Search Results
2. Heparin-activated procoagulant platelet assay: a flow cytometry-based functional test for heparin-induced thrombocytopenia.
- Author
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Pelzl L, Uzun G, Marini I, Zlamal J, Trumpp PN, Karakuyu A, Bakchoul T, and Althaus K
- Subjects
- Humans, Flow Cytometry methods, Blood Platelets metabolism, Heparin, Platelet Activation, Platelet Factor 4, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Background: Functional platelet activation assays are required for the diagnosis of heparin-induced thrombocytopenia (HIT). Due to their sophisticated methodology, they are only available in reference centers., Objectives: To evaluate the diagnostic accuracy of the flow cytometry-based heparin-activated procoagulant platelet (HAPP) assay in the laboratory diagnosis of HIT., Methods: Procoagulant platelets (PCP), defined by the expression of phosphatidylserine and CD62-P, were evaluated by flow cytometry in platelet-rich plasma from healthy donors after incubation with patient sera in the absence and presence of heparin. A sample was considered positive in HAPP assay, if the following 3 criteria were met: 1) the percentage of PCPs was ≥10.3% after incubation with 0.2 IU/mL heparin, 2) the fold increase in presence of 0.2 IU/mL heparin compared with buffer was ≥1.5, and 3) 100 IU/mL of heparin resulted in ≥50% inhibition of PCP. HAPP assay was validated in a prospective cohort (n = 202) of consecutive specimens submitted to our laboratory for serologic diagnosis of HIT. Heparin-induced platelet activation (HIPA) assay was used as the reference standard., Results: HIT-positive sera induced PCPs in the presence of 0.2 IU/mL heparin, which was inhibited with 100 IU/mL of heparin. In the prospective validation cohort, there were 15 HIPA+ and 187 HIPA- sera. HAPP was positive in 20 samples in this cohort. Using optimized cut-offs, HAPP assay had a sensitivity of 93.3% and specificity of 96.8%., Conclusion: HAPP assay is promising as a simple and reliable functional assay for HIT; however, further studies are needed to confirm our results in larger cohorts., Competing Interests: Declaration of competing interests Tamam Bakchoul has received research funding from CoaChrom Diagnostica GmbH, DFG, Robert Bosch GmbH, Stiftung Transfusionsmedizin und Immunhämatologie e.V.: Ergomed, DRK Blutspendedienst, Deutsche Herzstiftung, Ministerium fuer Wissenschaft, Forschung und Kunst Baden-Wuerttemberg, has received lecture honoraria from Aspen Germany GmbH, Bayer Vital GmbH, Bristol-Myers Squibb GmbH & Co, Doctrina Med AG, Meet The Experts Academy UG, Schoechl medical education GmbH, Stago GmbH, Mitsubishi Tanabe Pharma GmbH, Novo Nordisk Pharma GmbH, Leo Pharma GmbH, Swedish Orphan Biovitrum GmbH, has provided consulting services to: Terumo, has provided expert witness testimony relating to HIT and non-HIT thrombocytopenic and coagulopathic disorders. All of these are outside the current work. Tamam Bakchoul, Karina Althaus and Jan Zlamal have a pending patent application for flow cytometry-based assays to detect HIT and VITT antibodies. Other authors declare no competing financial interests., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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3. Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.
- Author
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Zlamal J, Singh A, Weich K, Jaffal H, Uzun G, Pelzl L, Althaus K, and Bakchoul T
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- Humans, Phosphatidylserines adverse effects, P-Selectin metabolism, Thrombin, Heparin adverse effects, Antibodies, Platelet Factor 4 adverse effects, Thrombocytopenia metabolism, Thrombosis etiology, Thrombosis metabolism
- Abstract
Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.
- Published
- 2023
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4. Diagnostic Performance of a Particle Gel Immunoassay in Vaccine-Induced Immune Thrombotic Thrombocytopenia.
- Author
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Uzun G, Althaus K, Hammer S, Wanner Y, Nowak-Harnau S, Enkel S, and Bakchoul T
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- Adult, Aged, Female, Humans, Male, Middle Aged, COVID-19 prevention & control, COVID-19 Testing, Heparin adverse effects, Platelet Factor 4, Retrospective Studies, COVID-19 Vaccines adverse effects, Immunoassay, Purpura, Thrombocytopenic, Idiopathic chemically induced, Thrombocytopenia chemically induced
- Abstract
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of adenoviral vector-based COVID-19 vaccines. Similar to heparin-induced thrombocytopenia (HIT), antibodies reacting to platelet factor 4 (PF4) are responsible for platelet activation in VITT. The diagnosis of VITT includes the detection of anti-PF4 antibodies. Particle gel immunoassay (PaGIA) is one of the rapid immunoassays that is commonly used in the diagnosis of HIT to detect anti-PF4 antibodies. The aim of this study was to investigate the diagnostic performance of PaGIA in patients suspected of VITT. In this retrospective, single-center study, the correlation between PaGIA, enzyme immunoassay (EIA), and modified heparin-induced platelet aggregation assay (HIPA) in patients with findings suggestive of VITT was investigated. A commercially available PF4 rapid immunoassay (ID PaGIA H/PF4, Bio-Rad-DiaMed GmbH, Switzerland) and an anti-PF4/heparin EIA (ZYMUTEST HIA IgG, Hyphen Biomed) were used according to manufacturer's instructions. Modified HIPA was accepted as the gold standard test. Between March 8 and November 19, 2021, a total of 34 samples from clinically well-characterized patients (14 males, 20 females, mean age: 48.2 ± 18.2 years) were analyzed with PaGIA, EIA, and modified HIPA. VITT was diagnosed in 15 patients. Sensitivity and specificity of PaGIA were 54 and 67%, respectively. Anti-PF4/heparin optical density values were not significantly different between PaGIA positive and negative samples ( p = 0.586). The sensitivity and specificity of EIA, on the other hand, were 87 and 100%, respectively. In conclusion, PaGIA is not reliable in the diagnosis of VITT because of its low sensitivity and specificity., Competing Interests: T.B. has received research funding from CoaChrom Diagnostica GmbH, DFG, Robert Bosch GmbH, Stiftung Transfusionsmedizin und Immunhämatologie e.V.: Ergomed, DRK Blutspendedienst, Deutsche Herzstiftung, Ministerium fuer Wissenschaft, Forschung und Kunst Baden-Wuerttemberg; has received lecture honoraria from Aspen Germany GmbH, Bayer Vital GmbH, Bristol-Myers Squibb GmbH & Co., Doctrina Med AG, Meet The Experts Academy UG, Schoechl Medical Education GmbH, Stago GmbH, Mitsubishi Tanabe Pharma GmbH, Novo Nordisk Pharma GmbH, Leo Pharma GmbH, Swedish Orphan Biovitrum GmbH; has provided consulting services to Terumo; has provided expert witness testimony relating to heparin-induced thrombocytopenia (HIT) and non-HIT thrombocytopenic and coagulopathic disorders. All of these are outside the current work. Other authors declare no competing financial interests., (Thieme. All rights reserved.)
- Published
- 2023
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5. The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia.
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Singh A, Toma F, Uzun G, Wagner TR, Pelzl L, Zlamal J, Freytag V, Weich K, Nowak-Harnau S, Rothbauer U, Althaus K, and Bakchoul T
- Subjects
- Fondaparinux therapeutic use, Heparin therapeutic use, Humans, Immunoglobulin G, Platelet Factor 4, Anticoagulants therapeutic use, COVID-19 Vaccines adverse effects, Thrombocytopenia chemically induced, Thrombocytopenia drug therapy, Thrombosis chemically induced, Thrombosis drug therapy
- Abstract
Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT-Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation; however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made., (© 2022 by The American Society of Hematology.)
- Published
- 2022
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6. Organ Donation From a Brain Dead Donor With Vaccine-induced Immune Thrombotic Thrombocytopenia After Ad26.COV2.S: The Risk of Organ Microthrombi.
- Author
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Uzun G, Bohnert BN, Althaus K, Nann D, Nadalin S, Heyne N, Fend F, Haap M, and Bakchoul T
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- Ad26COVS1, Brain Death, Humans, COVID-19, Thrombocytopenia chemically induced, Tissue and Organ Procurement, Vaccines
- Abstract
Competing Interests: T.B. has received research funding from CoaChrom Diagnostica GmbH, DFG, Robert Bosch GmbH, Stiftung Transfusionsmedizin und Immunhämatologie eV, Ergomed, Surrey, DRK Blutspendedienst, Deutsche Herzstiftung, Ministerium fuer Wissenschaft, Forschung und Kunst Baden-Wuerttemberg; has received lecture honoraria from Aspen Germany GmbH, Bayer Vital GmbH, Bristol-Myers Squibb GmbH & Co, Doctrina Med AG, Meet The Experts Academy UG, Schoechl Medical Education GmbH, Mattsee, Stago GmbH, Mitsubishi Tanabe Pharma GmbH, Novo Nordisk Pharma GmbH; has provided consulting services to Terumo; and has provided expert witness testimony relating to heparin-induced thrombocytopenia (HIT) and non-HIT thrombocytopenic and coagulopathic disorders. All of these are outside the current work. The other authors declare no conflicts of interest.
- Published
- 2022
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7. Multicentre evaluation of 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, as an internal quality control in HIT functional assays: Communication from the ISTH SSC Subcommittee on Platelet Immunology.
- Author
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Pouplard C, Rollin J, Vayne C, Charuel N, Ahmadi Z, Alberio L, Azjenberg N, Althaus K, Bakchoul T, Chong B, Curtis BR, Faille D, Gomez FJ, Gresele P, Morel-Kopp MC, Mullier F, Nazy I, Smith JW, Greinacher A, and Gruel Y
- Subjects
- Antibodies, Monoclonal, Anticoagulants adverse effects, Blood Platelets, Communication, Heparin adverse effects, Humans, Immunoglobulin G, Platelet Activation, Quality Control, Platelet Factor 4, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Background: Functional tests for the diagnosis of heparin-induced thrombocytopenia (HIT) exhibit variable performance., Objectives: We evaluated in a multicenter study whether 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, could be used as an internal quality control., Methods: 5B9 was sent to 11 laboratories in seven countries, and six initial concentrations ranging from 10 to 400 μg/mL were tested by heparin-induced platelet activation assay (HIPA), serotonin release assay (SRA), platelet aggregation test (PAT), flow cytometry (FC), or heparin-induced multiple-electrode aggregometry (HIMEA). Each method was evaluated in three different laboratories using experimental procedures identical to those usually applied for the diagnosis of HIT by testing platelets from 10 different healthy donors., Results: The procedures used varied among the laboratories, particularly when platelet-rich plasma and whole blood were used. Nevertheless, positive results were obtained with at least 100 μg/ml of 5B9 for most donors tested by all centers (except one) performing HIPA, SRA, or HIMEA. FC and PAT results were more heterogeneous. FC results from one center that used washed platelets preincubated with PF4 were positive with all donors at 50 µg/ml 5B9, but at least 200 μg/ml of 5B9 were required to activate cells with most donors tested using PAT., Conclusion: This study confirms that HIT functional tests are not well standardized and exhibit variable sensitivity for the detection of platelet-activating antibodies. However, 5B9 is a potentially useful tool to standardize functional tests, to select responding platelet donors, and consequently to improve the performance of these assays and comparability between laboratories., (© 2021 International Society on Thrombosis and Haemostasis.)
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- 2022
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8. Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia.
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Althaus K, Möller P, Uzun G, Singh A, Beck A, Bettag M, Bösmüller H, Guthoff M, Dorn F, Petzold GC, Henkes H, Heyne N, Jumaa H, Kreiser K, Limpach C, Luz B, Maschke M, Müller JA, Münch J, Nagel S, Pötzsch B, Müller J, Schlegel C, Viardot A, Bäzner H, Wolf M, Pelzl L, Warm V, Willinek WA, Steiner J, Schneiderhan-Marra N, Vollherbst D, Sachs UJ, Fend F, and Bakchoul T
- Subjects
- Adult, Autoantibodies, Blood Platelets, COVID-19 Vaccines, ChAdOx1 nCoV-19, Female, Humans, Male, Middle Aged, Pandemics, SARS-CoV-2, Vaccination adverse effects, Young Adult, COVID-19, Thrombocytopenia chemically induced
- Abstract
The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.
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- 2021
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9. Antibody-mediated platelet activation in COVID-19: A coincidence or a new mechanism of the dysregulated coagulation system?
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Althaus K, Zlamal J, and Bakchoul T
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- Antigen-Antibody Complex, Blood Platelets, Critical Illness, Humans, Platelet Activation, SARS-CoV-2, COVID-19, Thrombocytopenia
- Published
- 2021
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10. Real-life evaluation of an automated immunoassay for diagnosis of heparin-induced thrombocytopenia.
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Althaus K, Westphal A, Strobel U, Bakchoul T, and Greinacher A
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- Anticoagulants adverse effects, Heparin adverse effects, Humans, Immunoassay, Platelet Factor 4, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
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- 2020
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11. Heparin-induced thrombocytopenia: Diagnostic challenges in intensive care patients especially with extracorporeal circulation.
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Althaus K, Straub A, Häberle H, Rosenberger P, Hidiatov O, Hammer S, Nowak-Harnau S, Enkel S, Riessen R, and Bakchoul T
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- Anticoagulants adverse effects, Critical Care, Extracorporeal Circulation, Humans, Platelet Factor 4, Heparin adverse effects, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Background: Heparin-induced thrombocytopenia (HIT) is a serious drug induced reaction that may be associated with life threatening complications. Platelet-activating antibodies directed against platelet factor 4 (PF4)/heparin complexes cause the disease. The diagnosis of HIT is challenging, as thrombocytopenia is a frequent finding in intensive care (ICU) patient population, especially during extracorporeal membrane oxygenation., Objective: To investigate the performance of a diagnostic algorithm for HIT in ICU patients., Methods: ICU patients who developed thrombocytopenia or thrombosis under heparin treatment were included in this study. The pretest probability for HIT was estimated using the 4Ts-score and patient's sera were tested using two rapid immunoassays (RA) LFI-HIT and PaGIA (from Milenia Biotec and DiaMed), and within 72 h using the IgG enzyme immunoassay (EIA) from Hyphen and the heparin induced platelet activation assay (HIPA)., Results: 392 consecutive ICU patients with suspected HIT were enrolled in this study, of whom 83/392 (21.2%) patients had extracorporeal circulation. Sera from 120/392 (30.6%) and 98/392 (25.0%) patients revealed positive results in RA and IgG EIA, respectively. The HIPA test revealed heparin-dependent platelet activation in a total of 15/392 (3.8%) ICU patients (3 medical and 12 surgical patients). In addition, sera from 7 patients revealed indeterminate HIPA results, of whom 2 patients had a clinical course compatible with HIT., Conclusions: Data from our study confirm the high frequency of IgG PF4/heparin antibodies in ICU patients under unfractionated heparin and shows that the combination of 4Ts-score and RA does not reduce the laboratory overinvestigation for HIT in these patients., Competing Interests: Declaration of competing interest TB reports receiving honorarium for a scientific talk from Aspen Germany, CSL Behring, Meet the Expert gGmbH, GSK gGmbH, Stago gGmbH German, and research Grants from the German Society of Research, the German Society for Transfusion Medicine and German Red Cross. The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
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- 2020
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12. Platelet dysfunction caused by a novel thromboxane A 2 receptor mutation and congenital thrombocytopenia in a case of mild bleeding.
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Bugert P, Fischer L, Althaus K, Knöfler R, and Bakchoul T
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- 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid pharmacology, Adult, Arachidonic Acid pharmacology, Blood Platelet Disorders metabolism, Blood Platelets drug effects, Blood Platelets metabolism, Child, Preschool, Female, Hemorrhage metabolism, Hemorrhagic Disorders genetics, Hemorrhagic Disorders metabolism, Heterozygote, Humans, Male, Mutation, Phenotype, Platelet Count, Receptors, Thromboxane A2, Prostaglandin H2 metabolism, Thrombocytopenia metabolism, Tubulin blood, Tubulin genetics, Blood Platelet Disorders genetics, Hemorrhage genetics, Receptors, Thromboxane A2, Prostaglandin H2 genetics, Thrombocytopenia congenital, Thrombocytopenia genetics
- Abstract
Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of TBXA2R and TUBB1 revealed heterozygosity for the novel TBXA2R*c.908T>C (p.L303P) mutation in D1 and D3. TUBB1 was either wild type (D2, D3) or heterozygous (D1, D4) for the common polymorphism TUBB1*c.920G>A (rs6070697; p.R307H). In conclusion, the bleeding phenotype of the index patient can be explained by a diminished platelet function caused by the TBXA2R*c.908T>C mutation inherited from the mother and a mild thrombocytopenia with unknown molecular basis that is inherited from the father.
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- 2020
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13. Evaluation of a flow cytometer-based functional assay using platelet-rich plasma in the diagnosis of heparin-induced thrombocytopenia.
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Althaus K, Pelzl L, Hidiatov O, Amiral J, Marini I, and Bakchoul T
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- Aged, False Negative Reactions, Female, Flow Cytometry methods, Humans, Immunoenzyme Techniques, Male, Middle Aged, Platelet Aggregation drug effects, Platelet-Rich Plasma drug effects, Anticoagulants adverse effects, Heparin adverse effects, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Background: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT., Study Design: Sera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test., Results: 390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA., Conclusion: The Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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14. An international external quality assessment for laboratory diagnosis of heparin-induced thrombocytopenia.
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Eekels JJM, Althaus K, Bakchoul T, Kroll H, Kiefel V, Nazy I, Lee LS, Sachs U, Warkentin TE, and Greinacher A
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- Aged, Anticoagulants immunology, Biomarkers blood, Canada, Female, Germany, Heparin immunology, Humans, Laboratory Proficiency Testing, Male, Middle Aged, Observer Variation, Pilot Projects, Predictive Value of Tests, Reproducibility of Results, Singapore, Thrombocytopenia blood, Thrombocytopenia chemically induced, Thrombocytopenia immunology, Antibodies blood, Anticoagulants adverse effects, Heparin adverse effects, Immunoglobulin G blood, Immunologic Tests standards, Platelet Factor 4 immunology, Thrombocytopenia diagnosis
- Abstract
Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible., (© 2019 International Society on Thrombosis and Haemostasis.)
- Published
- 2019
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15. GNE variants causing autosomal recessive macrothrombocytopenia without associated muscle wasting.
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Revel-Vilk S, Shai E, Turro E, Jahshan N, Hi-Am E, Spectre G, Daum H, Kalish Y, Althaus K, Greinacher A, Kaplinsky C, Izraeli S, Mapeta R, Deevi SVV, Jarocha D, Ouwehand WH, Downes K, Poncz M, Varon D, and Lambert MP
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- Adolescent, Adult, Child, Female, Humans, Male, Mutation, Missense, Pedigree, Young Adult, Multienzyme Complexes genetics, Thrombocytopenia congenital, Thrombocytopenia genetics
- Published
- 2018
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16. Evaluation of a diagnostic algorithm for Heparin-Induced Thrombocytopenia.
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Farm M, Bakchoul T, Frisk T, Althaus K, Odenrick A, Norberg EM, Berndtsson M, and Antovic JP
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- Algorithms, Blood Platelets drug effects, Blood Platelets pathology, Humans, Likelihood Functions, Pilot Projects, Platelet Aggregation drug effects, Retrospective Studies, Thrombocytopenia pathology, Anticoagulants adverse effects, Heparin adverse effects, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Introduction: Heparin-Induced Thrombocytopenia (HIT) is a rare but serious immune-mediated complication of heparin treatment. HIT is characterized by an acute, transient prothrombotic state combined with thrombocytopenia and is caused by platelet-activating IgG antibodies that bind to complexes of heparin and platelet factor 4. The diagnosis of HIT relies on clinical presentation and the demonstration of HIT antibodies. One approach to improve the efficacy of laboratory analysis is to use a diagnostic algorithm., Aim: To evaluate one diagnostic algorithm for HIT where the 4 T's clinical risk score is combined with immunochemical and/or functional assays., Materials and Methods: The quality of the diagnostic algorithm was retrospectively evaluated in 101 patients with suspected HIT. Laboratory results obtained from the diagnostic algorithm were compared to Heparin-Induced Platelet Aggregation (HIPA) and clinico-pathological evaluation of patients' medical records., Results: We found that the algorithm had a diagnostic efficacy of 94%, with specificity of 94% and sensitivity 94%. Positive likelihood ratio (LR+) was 16.0, and the negative likelihood ratio (LR-) 15.5. The efficacy of PaGIA (n=95) was 0.46, and IgG-specific HPF4-abELISA (n=54) was 0.87., Conclusions: The diagnostic algorithm for HIT is sufficiently accurate and leads to in overall faster results and decreased cost of analysis. The weakest link of the algorithm is the risk of miscalculated 4T's scores, which is inevitably exacerbated by the insufficient experience most clinicians have with HIT. This highlights the importance of clear instructions from the laboratory and coagulation clinic., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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17. Alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a positive PF4/heparin-antibody test.
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Hron G, Knutson F, Thiele T, Althaus K, Busemann C, Friesecke S, Greinacher A, and Lubenow N
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- Aged, Critical Illness, Diagnosis, Differential, Enzyme-Linked Immunosorbent Assay, Femoral Neck Fractures complications, Humans, Methotrexate adverse effects, Pancytopenia chemically induced, Penicillanic Acid adverse effects, Penicillanic Acid analogs & derivatives, Piperacillin adverse effects, Platelet Count, Platelet Factor 4 immunology, Tazobactam, Transfusion Reaction, Heparin adverse effects, Platelet Factor 4 blood, Purpura, Thrombocytopenic chemically induced, Purpura, Thrombocytopenic diagnosis, Thrombocytopenia chemically induced, Thrombocytopenia diagnosis
- Abstract
Thrombocytopenia can cause diagnostic challenges in patients who have received heparin. Heparin-induced thrombocytopenia (HIT) is often considered in the differential diagnosis, and a positive screening can be mistaken as confirmation of the disorder. We present two patients who both received low-molecular-weight heparin for several days. In the first patient, clinical judgment rejected the suspicion of HIT despite a positive screening assay, and treatment for the alternative diagnosis of post-transfusion purpura was correctly initiated. In the second patient, the inaccurate diagnosis HIT was pursued due to a positive screening assay, while the alternative diagnosis of drug-dependent thrombocytopenia caused by piperacillin/tazobactam was rejected. This resulted in re-exposure to piperacillin/tazobactam which caused a second episode of severe thrombocytopenia. A positive screening assay for platelet factor 4/heparin-antibody should be verified by a functional assay, especially in patients with low pretest probability for HIT.
- Published
- 2013
- Full Text
- View/download PDF
18. Evaluation of automated immunoassays in the diagnosis of heparin induced thrombocytopenia.
- Author
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Althaus K, Hron G, Strobel U, Abbate R, Rogolino A, Davidson S, Greinacher A, and Bakchoul T
- Subjects
- Agglutination, Automation, Heparin chemistry, Humans, Immunoenzyme Techniques, Immunoglobulin G chemistry, Latex chemistry, Luminescence, Platelet Aggregation, Predictive Value of Tests, Reproducibility of Results, Sensitivity and Specificity, Thrombocytopenia chemically induced, Heparin adverse effects, Immunoassay methods, Thrombocytopenia diagnosis, Thrombocytopenia immunology
- Abstract
Background: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep) complexes. The in vitro demonstration of PF4/hep antibodies using functional and immunological methods is essential for optimal management of patients suspected to have HIT. Since functional assays are technically challenging and limited to specialized laboratories, antigen-binding assays are commonly used in routine laboratories., Study Design: Blood samples from 448 consecutive patients in whom HIT was suspected were investigated using a latex agglutination test HemosIL® HIT-Ab(PF4-H) (HemosIL-Ab), two chemiluminescence tests HemosIL AcuStar HIT-Ab(PF4-H) (HemosIL AcuStar-Ab) and AcuStar HIT-IgG(PF4-H) (HemosIL AcuStar-IgG), an in-house PF4/hep IgG enzyme immunoassay (EIA) and the heparin induced platelet aggregation (HIPA) test., Results: Antibodies against PF4/hep were detectable in 44 out of 119 samples using HemosIL-Ab among which 20 samples were also reactive in the HIPA; and in 122, 64 and 108 out of 448 sera using HemosIL AcuStar-Ab, HemosIL AcuStar-IgG and in-house PF4/hep IgG-EIA, respectively, among which 52 sera were also reactive in the HIPA. All assays had high sensitivities of >95% for platelet activating antibodies; however, they differed in their specificities. The highest specificity and positive predictive value was observed by HemosIL AcuStar-IgG (96% and 78%, respectively)., Conclusion: Automated immunoassays are useful in the laboratory investigations of HIT and present a potential improvement toward standardization of laboratory investigations of HIT. The high positive predictive capability may justify treating the patient with alternative anticoagulants without waiting for the results of a functional assay., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
19. Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay.
- Author
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Althaus K, Strobel U, Warkentin TE, and Greinacher A
- Subjects
- Antibody Specificity, Densitometry methods, Heparin blood, Humans, Immunoenzyme Techniques standards, Immunoglobulin G immunology, Pilot Projects, Platelet Factor 4 blood, Sensitivity and Specificity, Thrombocytopenia blood, Thrombocytopenia immunology, Heparin immunology, Immunoenzyme Techniques methods, Immunoglobulin G blood, Platelet Factor 4 immunology, Thrombocytopenia diagnosis
- Abstract
Background: IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain., Objective: To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined., Methods: We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test., Results: Platelet-activating antibodies were rarely detected (<0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83=17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD≤1.0). However, decreased sensitivity was observed with strong-positive sera (OD>1.0): 11/69 such sera (16%) that did not show >40% inhibition by high heparin nevertheless contained platelet-activating antibodies., Conclusion: Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5-≤1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (>1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
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20. Platelet dysfunction caused by a novel thromboxane A2 receptor mutation and congenital thrombocytopenia in a case of mild bleeding.
- Author
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Bugert, Peter, Fischer, Lars, Althaus, Karina, Knöfler, Ralf, and Bakchoul, Tamam
- Subjects
BLOOD platelet disorders ,BLOOD platelets ,THROMBOCYTOPENIA ,CONGENITAL disorders ,PLATELET count ,HEMOPHILIA - Abstract
Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of TBXA2R and TUBB1 revealed heterozygosity for the novel TBXA2R*c.908T>C (p.L303P) mutation in D1 and D3. TUBB1 was either wild type (D2, D3) or heterozygous (D1, D4) for the common polymorphism TUBB1*c.920G>A (rs6070697; p.R307H). In conclusion, the bleeding phenotype of the index patient can be explained by a diminished platelet function caused by the TBXA2R*c.908T>C mutation inherited from the mother and a mild thrombocytopenia with unknown molecular basis that is inherited from the father. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
21. Blood donor‐derived buffy coat to produce platelets in vitro.
- Author
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Marini, Irene, Rigoni, Flavianna, Zlamal, Jan, Pelzl, Lisann, Althaus, Karina, Nowak‐Harnau, Stefanie, Rondina, Matthew T., and Bakchoul, Tamam
- Subjects
BLOOD platelets ,PROGENITOR cells ,BLOOD cells ,BLOOD ,BLOOD platelet transfusion - Abstract
Background and objectives: Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor‐derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution. Materials and methods: CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested. Results: Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5·3 x 106 vs. 3·0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coat‐derived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7·2 vs. 2·7, respectively). Importantly, platelets produced from buffy coat‐derived cells could be activated by agonists. Conclusion: Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood‐derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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22. MYH-9 Related Platelet Disorders: Strategies for Management and Diagnosis.
- Author
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Althaus, Karina and Greinacher, Andreas
- Subjects
- *
BLOOD platelet disorders , *EPSTEIN-Barr virus diseases , *THROMBOCYTOPENIA , *MYOSIN , *GRANULOCYTES , *GENETIC code , *CYTOSKELETAL proteins , *IMMUNOFLUORESCENCE - Abstract
MYH-9 related platelet disorders belong to the group of inherited giant platelet disorders. The MYH-9 gene encodes the non-muscular myosin heavy chain IIA (NMMHCIIA), a cytoskeletal contractile protein. Several mutations in the MYH-9 gene lead to macrothrombocytopenia, and cytoplasmic inclusion bodies within leukocytes, while the number of megakaryocytes in the bone marrow is normal. Four overlapping syndromes, known as May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome and Sebastian platelet syndrome, describe different clinical manifestations of MYH9 gene mutations. Macrothrombocytopenia is present in all affected individuals, whereas only some develop additional clinical manifestations such as renal failure, hearing loss and presenile cataracts. The bleeding tendency is usually moderate, with menorrhagia and easy bruising being most frequent. The biggest risk for the individual is inappropriate treatment due to misdiagnosis of chronic autoimmune thrombocytopenia. More than 30 mutations within the 40 exons of the MYH-9 gene leading to macrothrombocytopenia have been identified, of which the upstream mutations up to amino acid ∼1400 are more likely associated with syndromic manifestations than the downstream mutations. Diagnosis is based on identification of the granulocyte inclusion bodies using blood smears and immunofluorescence and is finally confirmed by identifying the mutation. Treatment is supportive and should be aimed to prevent iron deficiency anemia. Beside renal failure, the biggest risk for patients affected by a MYH-9 disorder are the adverse effects resulting form treatment based on the misdiagnosis of immune thrombocytopenia. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
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