Possible incorrect genotyping of heterozygous factor V Leiden and Prothrombin 20210 gene mutations by the GeneXpert assay

• We analyzed 1648 samples using the GeneXpert HemosIL Factor II and Factor V assay.
• Two frozen/thawed heterozygous samples were incorrectly genotyped as homozygous.
• We suggest a 1:3 dilution of frozen/thawed samples before analysis.
• Inspection of amplification curves and Ct values is required to avoid artifacts.

BackgroundThe GeneXpert analyzer is a hands-off system for the detection of Factor V Leiden and of Prothrombin G20210A (GPRO) gene thrombophilic mutations. Although the system is efficient and easy to use, we report the rare possibility of incorrect genotyping.Methods1648 samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay: 1319 were freshly analyzed while 329 were frozen, thawed and diluted with saline prior to analysis to avoid clogging of the instrument syringe.ResultsTwo samples, both heterozygous, one for the factor V Leiden and the other for the GPRO gene, were incorrectly genotyped as homozygous for the relative mutation. Inspection of the Ct values and amplification curves and genotyping with PCR revealed the correct genotype as heterozygous for factor V Leiden and GPRO mutation.DiscussionThe GeneXpert HemosIL Factor II and Factor V assay is an automated, fast genotyping assay requiring almost no sample manipulation, advantageous characteristics if compared with other PCR-based methods. However, an inattentive use of it can generate incorrect diagnosis. A careful handling of the sample, in particular correct dilution of frozen/thawed samples before analysis, and the inspection of the amplification curves and Ct values are required to avoid artifacts.