An in Silico Exploration of the Factors That Affect the Precision of the Bethesda Assay

Jansen N. Seheult, Laynalee Cardel, Julie I. Tange, Aneel Ashrani, William L. Nichols, Nahla Heikal, Dong Chen

Research output: Contribution to journalArticlepeer-review


Despite more than 40 years of experience performing the Bethesda assay (BA), poor intra- and interlaboratory precision remains the biggest laboratory challenge to date. Methods: The BA procedure was modeled using stochastic simulation techniques to determine the precision of the BA up to dilutions of 1:4,096, to estimate the minimum significant relative change at various inhibitor titers, and to understand the laboratory procedural variables that could significantly affect the performance of the BA at high dilutions. Results: Selecting the lowest dilution tube with a residual activity closest to 25% for calculating the reported Bethesda titer (BT), using a factor activity assay with a coefficient of variation less than or equal to 7.5% in the range of 15% to 50% factor activity level, performing the factor activity measurement in replicates, and minimizing pipette volumetric error resulted in the lowest imprecision in the reported BT. The factor neutralization kinetics of the inhibitor appear to have little impact on the precision of the assay if the incubation time is greater than 90 minutes. Conclusions: This in silico model will assist future laboratory efforts in standardizing the quantification of specific coagulation factor inhibitors and improving the precision of the reported results.

Original languageEnglish (US)
Pages (from-to)671-682
Number of pages12
JournalAmerican journal of clinical pathology
Issue number5
StatePublished - Nov 1 2020


  • Blood coagulation factor
  • Blood coagulation tests/methods
  • Computer models
  • Hemophilia
  • In silico
  • Inhibitor

ASJC Scopus subject areas

  • Pathology and Forensic Medicine


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