Multiplex testing for Factor II and Factor V mutations in thrombophilia: technical verification and clinical validation of the cobas\xae Factor II and Factor V test

John W Longshore,Taraneh Tamaddon Rehage,Kelli Demartin,Partha Das,Karen Yu,Sylwia Karwowska,Guili Zhang,Deepa Jethwaney

doi:10.1007/s11239-018-1745-8

Abstract

Laboratory testing for thrombophilia is complicated but essential for diagnosis. In 2017, the cobas® Factor II and Factor V Test (cobas F2F5 test) was launched for use with the cobas z 480 analyzer. This qualitative polymerase chain reaction test enables multiplex Factor II and Factor V testing with flexible reporting and workflow efficiency. Here, we report the results from studies investigating the performance of the cobas F2F5 test. Technical performance verification, clinical validation, external laboratory performance, and workflow comparison studies were performed. Fresh and frozen whole-blood and genomic DNA (gDNA) samples were tested, and several manual and automated DNA isolation methods were used. Bidirectional Sanger sequencing was used to verify genotypes identified by the cobas F2F5 test. One hundred percent agreement between the cobas F2F5 test and Sanger sequencing was observed for all genotypes. An external laboratory using remnant clinical samples also yielded 100% agreement between cobas F2F5 test results and their routine testing method. The cobas F2F5 test reduced the total sample processing time compared with the LightCycler® 1.2 platform (98.6 vs 420.2 min; 96 samples). Hemoglobin, extraction buffer, and ethanol contamination of the gDNA sample can lead to invalid results. The cobas F2F5 test has a high degree of accuracy for identification of Factor II and Factor V genotypes. This multiplex testing with short sample processing time can reduce handling errors and increase efficiency. Both manual and automated DNA isolation methods can be used with the cobas F2F5 test.

Highlights

Thrombophilia, characterized by a predisposition to the development of thrombi in veins, arteries, or both, is the result of either inherited or acquired defects in the coagulation system [1, 2]
Compared with the LightCycler® 1.2 platform–based testing method, the cobas F2F5 test reduces the total processing time and number of steps required in amplification and detection. These results show that the new cobas F2F5 test is both time saving and cost-efficient and provides a high level of accuracy in Factor II and Factor V genotype identification
For Factor II testing (n = 300), the Overall percentage agreement (OPA) with bidirectional Sanger sequencing was 100%, with a 2-sided 95% lower confidence boundary (LCB; exact method) of 98.78%

Summary

Introduction

Thrombophilia, characterized by a predisposition to the development of thrombi in veins, arteries, or both, is the result of either inherited or acquired defects (or an interaction between the 2) in the coagulation system [1, 2]. Patients who test positive for Factor V Leiden should be considered for Factor II G20210A testing, pregnant women with a previous history of venous thromboembolism and recurrent pregnancy loss [3,4,5]. Based on internal data from market assessments and sales, we estimate that approximately 2.2 million Factor II and Factor V genotyping tests are performed annually worldwide (excluding Japan) [7]. The laboratory costs of thrombophilia testing are estimated to exceed $650 million (USD) annually [8]. There is an urgent need to optimize screening and diagnosis algorithms, make testing more cost-effective for laboratories, and reduce the frequency of inappropriate testing [9, 10]

Methods

Results

Conclusion