David L. McGlasson, MS, MLS(ASCP)

Several years ago, I was the lead author of a manuscript titled: Comparison of six dilute Russell Viper Venom Time (DRVVT) lupus anticoagulant (LA) screen/confirm assay kits.1 The purpose of the protocol was to see if using normalized ratio gave our laboratory any better inter-method consistency when performing the DRVVT method. This question whether laboratories need to employ the normalization calculation correction was raised again in a new manuscript, To normalize or not?: Dilute Russell viper venom time testing.2

Therefore, a review of studies is warranted. Eleven years ago, we compared commercially available normalized DRVVT LA screens and confirmed reagent systems ratios (SCR) for inter-method consistency. In total, we gathered reagents from 6 different manufacturers. We had technical issues with data from the lots of 1 of the manufacturers so that data was not used in comparison with the data from the other 5 manufacturers.

We used an automated coagulometer for all testing,  and we followed each manufacturer’s instructions for reagent testing. LA-positive and LA-negative controls were obtained as well as a commercial pooled normal plasma (PNP) for use in daily precision testing. Our laboratory assayed 43 aliquots of locally sourced plasmas with previously determined LA-positivity using low-PL and DRVVT screen/confirm methods. Aliquots of 42locally provided LA-negative plasmas were used to establish the mean reference interval (MRI) and standard deviation (SD). We used MRI + 3SD to provide action limits, which approximates the 99th percentile as specified by the ISTH while using a reasonable number of normal subjects’ aliquots. We assayed all controls, daily PNPs and positive and negative LA plasma aliquots using identical sample volumes on the automated coagulometer.

Screen and confirm intervals in seconds were recorded for all 5 DRVVT screen/confirm kits. and screen/confirm ratios were computed for each of the positive and negative plasmas. We also used the following formula to normalize screen/confirm ratios to the MRI and the PNP screen/confirm results.

Subject screen /MRI screen
MRI-normalized ratio =
Subject confirm/MRI confirm

Or…

Subject screen /PNP screen
PNP-normalized ratio =
Subject confirm/PNP confirm

 

Where…

  • MRI-normalized ratio is the screen/confirm ratio normalized to the mean of the reference interval.
  • PNP-normalized ratio is the screen/confirm ratio normalized to the value of the pooled normal plasma performed daily.

Formula for Normalizing the DRVVT Screen/confirm Ratio:

The normalized ratio was calculated by dividing the raw DRVVT screen/confirm values in seconds by the laboratory’s means of the reference interval (MRI) for each reagent.

The analysis of variance (ANOVA) was used to compare all results.

The MRI + 3SD positive cut-off for all 5 kits was >1.2, correlating with the action limits with all tested kits using local normal plasmas.

The mean/screen confirm ratios p value was 0.14, showing no statistically significant differences. LA+ specimens had a range of 1.91-2.04 with all kits.

When combining LA+ and LA- screen / confirm specimen results, a range of 1.59-1.67was generated. The within-run coefficient of variation (CV%) was less than 5.0% for all specimens assayed. DSI LA+ and LA- controls yielded a CV% of less than 5.5%.

In our study, the normalization added little to the clinical result interpretation on 420 individual assays on 5 commercial kits tested.

A new manuscript titled: To normalize or not?: Dilute Russell viper venom time testing was recently published.This study was conducted 11 years after our initial study and it questioned the need for all laboratories to employ the normalization calculation correction.

This protocol compared ratios of the non-normalized DRVVT SCR in subjects’ specimens and the normalized SCR that resulted using normal reference pooled plasma (NRPP). The purpose of this study was to determine if the normalization calculation had any effect on the difference between a positive or negative ratio of the LA+ or LA- assayed patient specimens.

This study also used an automated coagulation analyzer to perform all the DRVVT testing.  All their specimens ruled out the presence of direct oral anticoagulants (DOACs) in all positive specimens. The DRVVT of commercial PNP was also measured. Initially this study was conducted with single lots of the DRVVT reagents and PNP.  Subsequent studies have been performed after validation using ISTH guidelines based on the 99th percentile with no changes of the cutoff of >1.2.3,4

This study group used a retrospective analysis of 464 subjects in which DRVVT testing was performed.  Confirmatory testing was performed on assays with positive screens. DRVVT testing of the NRPP was analyzed.  A positive non-normalized DRVVT test or a normalized DRVVT SCR was a ratio greater than 1.2.

Of the 464 specimens evaluated, 26 (5.6%) tested positive for the presence on an LA using the conventional screen/confirm ratio and using the normalized ratio calculation. Only 12 had a history of thrombosis.  No subjects had a clinical history had a pregnancy loss. The 26 positives had a mean SD ratio of 1.53 +/- SD of 0.29 using the non-normalized ratio (1.41-1.65) vs. 1.49 +/-0.30 using the normalized calculation (1.37-1.61).

This did give a statistical difference of (P=0.0096) using the paired t-test. SCR bias was 0.04 (2.51%). The total error was 0.64. (<3SD of the CAP 2021-2022 proficiency testing surveys. (2021-CGS1A-CFGS1B, and 2022-CGS1A)2. No sample status changed between the two methods of calculating the SCR. Using the normalized ratio did not change clinical significance of any specimen analyzed. There was 100% agreement in the results between both calculations used.

The ISTH suggests that a normalization calculation for DRVVT testing be performed to reduce variations of studies with different reagent/instrument combinations. This communication points out that this step in determining the ratio may not be necessary for within-laboratory LA testing.

However, nonnormalized ratios may show a disparity among different methods. Therefore, normalized ratios are important for reducing inter-method differences and for cross-laboratory studies. This may be evident when conducting proficiency testing.

DRVVT SCR discriminates between LA+ and LA- specimens and provides reproducible results.  In both of these studies, normalization added no clinical differences in interpretation of final results.1,2

 

REFERENCES

  1. McGlasson DL, Fritsma GA. Comparison of Six Dilute Russell Viper Venom Time Lupus Anticoagulant Screen/Confirm Assay Kits. Semin Thromb Hemost 2013;39:315-319.
  2. Zhang Y, Creer M, Olajumoke O Oladipo. To normalize or not?: Dilute Russell viper venom time testing. Am J Clin Pathol 2024;XX:1-5.
  3. Pengo V, Tripodi A, Reber G et al: Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009;7(10):1737-1740.
  4. Devreese KM, DeGroot PG, deLaat B et al: Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis. J Thromb Haemost. 2020;18:2828-2839.

 

ML-00-01167Rev01