Abstract
Introduction:
Factor V Leiden (FVL) is an autosomal dominant prothrombotic disorder, that impacts activated Factor V's (FVa) ability to be cleaved by activated Protein C (APC), due to an amino acid change at amino acid residue Arg506Gln. FVL is commonly screened for using aPTT clotting assays like the Chromogenix APC Resistance (ACPR) assay. The APCR assay produces a clotting ratio by comparing the clotting time in the presence and absence of APC. APCR ratios below 1.45 are considered to be homozygous while anything between 1.45 and 1.99 is considered heterozygous. However, this assay can be cofounded by Lupus Anticoagulants (LAs), that can cause false positive ratios, leading to false positives for FVL. The Hemoclot Quanti V-L assay on the other hand, is not susceptible to LAs, as it not an aPTT based clotting assay which are known to be impacted by LAs. The Quanti V-L assay also produces a clotting time that is inversely proportional to the FVL concentration present, allowing for a more accurate reading on an individual's FVL expression level. This assay was analyzed alongside FV activity to determine a FVL to FV activity (act.) ratio in order to analyze Quanti V-L accuracy and against the APCR in order to correlate it to the APCR.
Methods:
The Activated Protein C Resistance V (Chromogenix), FV activity (Stago), antiphospholipid antibodies using StaClotLA and/or dRVVT (Stago), and Hemoclot Quanti V-L (Hyphen Biomed) were performed on the STA R Max per manufacturer's instructions. PCR was used to confirm the FVL mutation.
Results:
Eighty-six unique individuals who were suspected of having FVL had an APCR, Hemoclot Quanti V-L, and FV act. result. A moderately strong correlation (r = -0.68, p = <0.001) was found between the APCR and the Quanti/FV act. ratio. For Homozygotes (N=6) the APCR ranged from 1.22-1.31 with a mean of 1.26; the Hemoclot Quanti V-L ranged from 87-139% FVL with a mean of 103% FVL, with Quanti/FV act. ratios that ranged from 0.91-1.15 with a mean of 1.07. For heterozygotes (N=74) the APCR ranged from 1.04-1.90 with a mean of 1.69, the Hemoclot Quanti V-L ranged from 33-85% FVL with a mean of 53% FVL, with Quanti/FV act. ratios ranging from 0.35-0.73 with a mean of 0.53. 5 individuals had an abnormal APCR, but a normal Hemoclot Quanti V-L. Their APCRs ranged from 1.75-1.99 with an average of 1.88; Hemoclot Quanti V-L ranged from 2-4% FVL with an average Quanti/FV act. ratio of 0.03. Three out of the five individuals were positive for an LA. The Hemoclot Quanti V-L had one false negative in which the Hemoclot Quanti V-L came back normal for an individual with a heterozygous PCR.
Discussion:
The Hemoclot Quanti V-L was able to screen effectively for FVL without susceptibility to LAs. Additionally, the Hemoclot Quanti V-L was able to run up to 40 samples from one set of reagents alone, while the Chromogenix APCR can only run around 20 per set of reagents, making the Hemoclot Quanti V-L a price effective choice for FVL screening. With a false negative rate of 1.1% (1/87) the Hemoclot Quanti V-L is an accurate screening assay. The one false negative had an APCR ratio of 1.95, a Quanti of 2% FVL, FV act. of 52 U/dL, and a ratio of 0.04, and was negative for an LA; this case highlights that borderline heterozygotes might illustrate the diagnostic thresholds of the Hemoclot Quanti V-L. The Hemoclot Quanti V-L provides quantitative %FVL assessment, which moderately (r = 0.40, p = <0.001) correlates to FV act. Further analysis of the quantitative aspect is needed to understand significance to other assays like Calibrated Automated Thrombogram with and without Thrombomodulin (CAT-TM). Compared to the APCR the Hemoclot Quanti V-L nonsusceptibility to LAs, high accuracy, and assay efficiency make it a practical and cost-effective option for FVL screening.
Conclusion:
The Hemoclot Quanti V-L is a more accurate screener for FVL and provides more specificity in FVL analysis than traditional APCR assays.
