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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Discrepant results in von Willebrand testing - An algorithmic approach to address heterophilic antibody interference in the INNOVANCE �VWF GPIbM activity assay(THSNA �Travel Awardee) Karen E.Flint1, Jeremy D.Williams1, Joshua L.Rhodes1, Kenneth D.Friedman2, Andrea Staum2, Sandra L.Haberichter2, Bailey Hutchison2, Andrew Podd2, Lori Luchtman-Jones1, 3. 1Hemostasis and Thrombosis Lab, Hematology Division, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH, USA.2Diagnostic Laboratories, Versiti/Blood Center of Wisconsin, Milwaukee, WI, USA.3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA |
Abstract
Background: Current von Willebrand (VWD) disease guidelines favor a Ristocetin-independent, platelet-dependent activity assay, von Willebrand factor (VWF) GPIbM, over the classic VWF Ristocetin cofactor activity, for its improved precision, especially for patients with benign VWF variants that poorly bind Ristocetin. However, in clinical use of the only commercially available assay kit, a turbidimetric latex-bead-based VWF GPIbM kit (T-GPIbM) utilizing mouse monoclonal antibodies against human recombinant GPIb fragments, a subset of patient results was much higher than, and discrepant with, VWF antigen (Ag). Results varied by kit lot. Accurate VWD diagnosis relies on absolute VWF activity (and VWF Ag), but also on the activity:antigen ratio. Although the kit reportedly contains a heterophile antibody (HA) blocking reagent, we hypothesized that HA non-specific agglutination explains false elevations, that interference should persist if T-GPIbM assay is repeated without the GPIbM reagent 3, and would disappear in a lab-developed VWF GPIbM ELISA assay (E-GPIbM). Objectives: To demonstrate that HA interference explains falsely elevated T-GPIbM activity and to develop an algorithm to identify interference in the T-GPIbM. Methods: Patient samples included 12 with disproportionately high VWF T1-GPIbM activity:VWF Ag ratios (>1.2) and 3 normal ratios (0.7-1.2). Split-sample, frozen (70oC)-thawed platelet poor plasmas (n=15) were analyzed by T1-GPIbM (our lab) per kit instructions and repeated as the same assay without reagent 3 - “Blank” (INNOVANCE® VWF Ac, Siemens Healthineers, USA) on Stago STA R Max (Diagnostica Stago, USA). Shared samples were also evaluated by T2-GPIbM on Siemens CS-5100 (Versiti, Milwaukee, WI, USA), and also by the E-GPIbM (Versiti). In our laboratory, plasmas were also incubated in HA blocker tube (Scantibodies Laboratory, Inc, Santee, CA, USA) and analyzed as HA-T1-GPIbM. Percent differences within and between labs, and percent differences between testing methods were calculated. Mean ratios were calculated to determine differences between the testing methods. Results: Of 15 tested samples, 6 had minimal results for the Blank (<4%) and excellent agreement of results for T-GPIbM assays, even with HA blocking or E-GPIbM. Three of these plasmas were normal-ratio samples. The other 9 demonstrated discrepant Blank activity of 15-44%. By HA-T1-GPIbM, 8/9 (89%) sample activity/antigen ratios normalized (≤ 1.2); all had normal ratios by E-GPIbM. Compared to T1-GPIbM, ratios dropped by 75% (E-GPIbM) and 76% (HA blocking). Conclusion: The commercially available T-GPIbM VWF activity assay is affected by HA, non-specific agglutination that varies by patient and kit lot. Awaiting kit improvement, our laboratory now screens samples by running both T1-GPIbM and Blank assay, and, for VWF GPIbM activity:VWF Ag ratio ≥ 1.5, or a Blank result >10% of the T1-GPIbM, then HA blocking is performed. If the HA-blocked T1-GPIbM result decreases by >20%, no VWF GPIbM activity value is reported, with a comment entered that there is possible heterophile antibody interference. If the result changes <20%, results are reported.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.