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Hex-phase Only for LA?

From Lorna Bogertman, Valley Health: Hello George, I have a question concerning lupus anticoagulant (LA) testing. We have had several requests from physicians to perform hexagonal phase LA testing only. Is it valid to perform this test without the rest of the LA profile? We have found that if we performed Stago’s LA-sensitive partial thromboplastin time (PTTLA) and the dilute Russell viper venom time (DRVVT), the LA would have been reported as negative, however in this case the hex phase test was positive. How should this be reported? If the profile is really negative, what are the other causes of a positive hex phase test other than LA?

Hello, Lorna, and thank you for your question. I’m in Rochester attending the Mayo Laboratories Clinical and Laboratory Update in Thrombosis, Anticoagulant, and Vascular Medicine, and had the opportunity to raise your question with several colleagues, who agree that the DRVVT and the hex-phase assay are not confirmatory, but rather must be employed in paraallel to test for LAs of different specificity. Thus, if the DRVVT is positive for LA and the hex-phase assay is negative, it is appropriate to report that LA is present, and likewise, if the hex-phase alone is positive, you can report LA. This of course assumes you’ve already ruled out a factor deficiency, factor-specific inhibitor, and heparin.

Stago’s StaClot LA kit is the system that is based on the hex phase reagent. The kit uses an LA-sensitive PTT reagent and a reagent that consists of phospholipids suspended in the hexagonal phase conformation. Hex-phase phospholipids bind and neutralize LA. You incubate the test plasma with the hex-phase phospholipid and compare the PTT performed on the mixture to the PTT performed on the plasma alone. If the mixture PTT result is shortened by a specified interval, usually 8 seconds, the test is positive for LA. DRVVT kits function similarly, but may detect LAs of slightly different specificity, which is why both are used.

The International Society on Thrombosis and Haemostasis guidelines for LA testing are available at Pengo V, Tripodi A, Reber G, et al.  Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost. 2009; 7:1737–40.

Comments (4)
Lupus Anticoagulant
Ali Sadeghi-Khomami
Sep 16, 2014 9:41am

Thanks George for the update, you are correct. If at least o
Thanks George for the update, you are correct. If at least one of two LA-screening tests is positive then positive result by a paired confirmation test is also required to call it LA-positive by the lab.

George Fritsma
Sep 16, 2014 2:58am

Thank you, Ali, for your comment on the LA profile, this is
Thank you, Ali, for your comment on the LA profile, this is very helpful. I’d like to add one update. When using two parallel LA detection systems such as the PTT-based and the DRVVT-based systems, as recommended by ISTH, you need demonstrate a positive result in only one of the two. The ISTH guideline states, “LA should be considered as positive if one of the two tests gives a positive result.” The two test systems are not considered to be confirmatory, rather, both are necessary because of LA heterogeneity. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemostas 2009: 7: 1737–40.

Ali Sadeghi-khomami
Sep 15, 2014 8:08pm

According to manufacturer instruction for use (IFU), Stago’s
According to manufacturer instruction for use (IFU), Stago’s PTT based assays Staclot LA and PTTLA are susceptible to interference from a number of analytes, including circulating anticoagulants, thrombin and other factor inhibitors. Additionally, interferences between C-reactive protein (CRP), a common acute phase reactant that elevates in inflammation for instance, and Staclot-LA have been reported with CRP levels over 5 mg/dL (Thrombosis Research. 2010: p102). For CRP interference with aPTT-based LA assay see also: ECAT Foundation International External Quality Assessment Programme in Haemostasis and Thrombosis. Exercise 2010-4. Print date 28 Feb 2011.

According to ISTH guidelines (2009), Lab diagnosis of LA requires two positive LA screening results. Considering the heterogeneity of antibodies involve in LA, these two tests should be based on different mechanisms of detection. For instance, intrinsic pathway: aPTT based screening (PTTLA) & common pathway: venom based assay (dRVVT) screening test. Then, samples found positive by screening tests need to be confirmed by a corresponding (paired) confirmatory test which contain extra phospholipid to by-pass LA effect, for instance confirmation by Staclot-LA delta correction or dRVVT ratio. Factor deficiencies (by mixing studies) and other coagulation abnormalities (anticoagulants: heparin, NOACs, Coumadin, etc) need to be over-ruled before finalizing lab diagnosis. Finally, in order to differentiate persistent LA from transient LA-like antibodies, lab also needs to re-confirm LA presence after 12 weeks in patients.

Stago’s Staclot-LA is an integrated mixing study, LA screen/confirm PTT-based assay used for LA confirmation. Application of Staclot-LA as the first line in LA assay without a sensitive screening test with a well established cut-off (e.g. PTTLA) is discouraged. This is mainly because of high risk of false positive and false negative results.

Typically for LA testing, manufacturers offer high sensitivity with screening tests and high specificity with confirmatory tests. Therefore it is important to stay compliant with LA-testing recommendation. This will mitigate interference risks and laboratory misdiagnosis.

McGlasson
Sep 15, 2014 7:19am

Back in 1995 two Scandavian investigators: Schjetlein R, Wis
Back in 1995 two Scandavian investigators: Schjetlein R, Wisloff F., published an article in AJCP 1995;103(1):108-11 titled An evaluation of two commercial test procedures for the detection of lupus anticoagulant. They looked at both the Staclot LA and the DRVVT/DRVVC assays. The sensitivity was 80% for the DRVV assays and 67% for the Staclot LA. However by applying both the commercial tests to all plasma samples tested, the LA positive samples showed a sensitivity of 97%. Remember both of these assays pick up differenct antibodies: prothrombin based and beta 2 glycoprotein I. if you do both you are going to find more positives. There were no false positive test results found in this study. When I performed the testing in the WARRS/APAAS study I used this logic and found a much higher number of LA positives than when I was only using the Staclot LA method. It shows you that one test just doesn’t fit all when it comes to LA testing.

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