Announcement
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Laboratory Validation of ADAMTS-13 Antibody Assay Melissa S.Stuart, Rajiv K.Pruthi, M.B.B.S, Dong Chen, M.D., Ph.D.. Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA |
Abstract
Background: Immune thrombotic thrombocytopenic purpura (iTTP) is characterized by presence of IgG antibody to ADAMTS-13. Options for laboratory testing includes determination of a functional antibody/inhibitor. An ELISA based assay, the ADAMTS-13 antibody assay (ADMAB) may be used to detect both clearing and non-clearing antibody. ADMAB identifies IgG antibodies that may not demonstrate functional clearance in a modified Bethesda assay or can not be determined accurately due to gross icterus interference with FRET ADAMTS-13 activity. Methods: Technoclone Technozym® INH ELISA Kit was performed on the Janus G3 liquid handling system (Revvity, Waltham, MA) and BioTek Synergy H1M microplate reader (Agilent Technologies, Winooski, VT). Accuracy was performed on 50 de-identified waste plasmas and 50 contrived from diluted waste plasma, supplied by an external reference laboratory. Linearity panels performed included two elevated waste plasmas with 11 concentrations that encompassed the analytical measuring interval. Precision studies utilized three levels of pooled plasmas (4 assayed in duplicate, once daily for ten days). The limit of detection (LOD) and lower limit of quantification (LLOQ) were determined using five kit diluent or blank samples and five low-level patient samples, each assayed 20 times over three days, across two different kit lots. The reference interval contained 120 normal donor samples (60 male/60 female, aged>18 years). Analytical specificity assessed hemolysis (H), icterus (I), and lipemia (L) interference indices from the ACL TOP 750 preanalytical sample integrity check module (Werfen, Bedford, MA) or Cobas 8000 (HITACHI, Tokyo, Japan). Clinical sensitivity and specificity review was comprised of 49 authorized patient plasma waste samples. Results: Accuracy demonstrated R2=0.924, slope=0.975, and overall average difference 2.98U/mL. Linearity samples ranging from 2.1-80.1U/mL, obtained R2=0.980/0.987 and slope=0.970/1.038 with recovery within ±10% of assayed levels >10U/mL and within ±2SD for values <10U/mL. Repeatability and within-laboratory coefficient of variation (CV) attained 6.5% and 8.5% at 82.6U/mL, 9.6% and 14.7% at 20.7U/mL, and SD of 0.8 and 1.3 at 6.1U/mL. LOD on two lots achieved 2.2 and 3.3U/mL, and LLOQ of 3.0U/mL was established. Reference interval limits: Negative <12 U/mL, Borderline 12-15 U/mL, Positive >15 U/mL. H-index levels up to 900mg/dL and I-index up to 45mg/dL did not result in loss of recovery. Figure 1 demonstrates the utility of ADMAB for detecting IgG antibody in the presence of elevated bilirubin for laboratories using FRET-based methods for ADAMTS-13 activity assays. Lipemia resulted in false positive/spurious results at L-index levels >9000mAbs. Clinical sensitivity 83%, clinical specificity 96%, positive predictive value 95%, negative predictive value 86%. Analysis of clinical sensitivity and specificity ROC in Figure 2 additionally support an optimal threshold limit for the assay at 12U/mL with natural rounding. Conclusions: ADMAB assay meets analytical performance needs and offers additional evidence in the ADAMTS-13 battery of tests for understanding and management of patients with TTP. This method offers a reliable confirmatory tool for IgG antibody in sample types that preclude functional Bethesda testing by the FRET-method and in patients who do not present with functional clearing IgG antibodies. Continued evaluation in clinical scenarios will refine its utility in the laboratory.
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.
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.