Presentation Details
| Normalization of thrombin generation results using within-run plasma normalizator simultaneously. Loic J.Letertre1, 2, Pall T.Onundarson1, Jon T.Bergthorsson1, 2. 1Landspitali - The National University Hospital, Reykjavik, Iceland.2The University of Iceland, Reykjavik, Iceland |
Abstract
Background: Automated thrombin generation (ATG) assessment theoretically has an almost limitless potential for assessing anticoagulation as well as hypercoagulable and bleeding phenotypes. However, ATG assessment has been limited by high intra- and especialy inter-assay as well as reagent-dependent variation. This has complicated ATG comparison from one center or study to another and has deterred clinical usability of the assay. Objectives: We hypothesized that within-run normalization of thrombin generation results using a suitable pooled normal plasma (PNP) in addition to applying standardized manufactured reagents and calibrators would reduce such variations. If successful, normalization would provide an improved ATG method and also a clinically meaningful unit that might bring ATG closer to clinical use. Methods: A single user measured ATG and standard clinical laboratory coagulation tests in pooled normal plasmas (PNPs) of different origin using a single manufacturer´s instrument and different lots of standard reagents. The tested PNPs included an in-house prepared double-centrifuged PNP frozen plasma, different lots of commercial frozen PNPs and a single lot of lyophilized PNP. ATG was also assessed in double-centrifuged plasma samples from 30 healthy individuals. Particle debris present in the plasmas was assessed using flow cytometric methods. Results: The raw calibrated ATG intra-assay variation performed on the PNPs was very low, i.e. coefficient of variation (CV%) below 6% for Peak TG and below 3.7% for ETP. Raw data inter-assay variation was within 10.7% for the Peak TG and within 3.1% for the ETP. The manufactured frozen plasma PNP GK7370 fitted best the median ATG in our normal cohort and was therefore selected as normalizator plasma. The in-house and lyophilized PNPs were less suitable for normalization. Using the selected normalizator plasma and standardized reagents, inter-assay variation remained low (below 12% for peak TG and 4.1% for ETP). Furthermore, following normalization, reagent lot-to-lot variation (CV%) was markedly reduced, i.e. 74% for ETPs, 78% for Peak TG and 92% for time to peak TG. Flow cytometry did not identify tissue factor in the PNPs although some debris of platelet membrane origin was present. Conclusions: Normalization of ATG using a frozen PNP that had ATG results fitting the median ATG of a normal cohort led to a marked reduction in reagent lot-to-lot variation. Normalization of ATG results by reducing reagent-dependent variation may help open the door for ATG use in clinical practice and comparative studies.
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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.