A deep dive into lupus anticoagulant testing with Dr. Vittorio Pengo

• Lupus anticoagulant testing presents a uniquely demanding challenge for lab professionals
• General laboratories need experts in the area of coagulation to maintain quality and accuracy
• Automation should not be implemented at the expense of true expertise

Part two of a two-part series with Professor Vittorio Pengo

For laboratory professionals working in coagulation diagnostics, lupus anticoagulant (LA) testing presents a uniquely demanding challenge. Unlike most routine assays, it involves multiple sequential steps, two parallel test systems, and a complex interplay of reagent composition, patient-specific variables, and expert interpretive judgment. The analytical pitfalls are numerous: phospholipid instability, lack of reagent standardization across manufacturers, interference from anticoagulant medications, and phenomena such as the lupus cofactor effect can all produce misleading results if not recognized and understood.

The stakes are correspondingly high. An incorrect lupus anticoagulant result, whether a false positive or a false negative, directly affects the clinical management of patients with suspected Antiphospholipid Syndrome (APS) and can lead to dangerous consequences: unnecessary long-term anticoagulation on the one hand, or a missed diagnosis in a patient at high thrombotic risk on the other.

Professor Vittorio Pengo first described the Diluted Russell's Viper Venom Test (dRVVT) in 1986 and has spent decades studying the analytical challenges of lupus anticoagulant testing.¹ In the second part of this two part-series, he explains the reasoning behind current guideline recommendations and offers his perspective on how laboratories should work to improve lupus diagnostics.

LabLeaders: Why do guidelines recommend a three-step procedure: Screening, mixing, and confirmatory, rather than a simpler approach?

Prof. Pengo: Each step in the three-step process provides essential, non-redundant information, and confident diagnosis requires all three. The procedure cannot be safely abbreviated.

I want to emphasize the mixing step in particular, because in practice it is sometimes omitted. I recently reviewed a paper in which a laboratory reported patients as positive for lupus anticoagulant based on screening and confirmatory steps only, without performing a mixing test. When mixing was subsequently added, some of those cases turned out to have a negative mixing result. This raises a fundamental question: If the mixing test is negative, does the lupus anticoagulant truly exist?

My position is clear: If you mix the patient's plasma with normal pooled plasma and the coagulation time does not prolong, there is no inhibitor present. A true inhibitor will resist correction by normal plasma, and that prolongation is the hallmark of inhibitor activity. If the mixing test is normal, the extended clotting time seen at screening was the result of a factor deficiency, not an inhibitor, and there is no need to proceed to the confirmatory step. Conversely, if both screening and mixing are positive, you must follow through to confirmation. Always follow the full sequence: Screening, mixing, confirmation. You will be far more confident in your result, and you will avoid both over- and under-diagnosis.

LabLeaders: Why are two parallel assays required? Why not rely solely on the dRVVT or solely on an aPTT-based test?

Prof. Pengo: Relying on a single test is potentially misleading, and the direction of any discrepancy matters significantly.

Ideally, both the dRVVT and the Activated Partial Thromboplastin Time (aPTT)-based test should be positive before a confident diagnosis of lupus anticoagulant is made. When one test is positive and the other is negative, the clinical significance of that isolated positive depends on which test it is. From a mechanistic standpoint, when only one test is positive, there is often no associated activated protein C resistance, which means the mechanistic pathway that would explain hypercoagulability is absent. In those cases, the significance of the result is low.

If the dRVVT is positive and the aPTT-based test is negative, I have relatively less concern, though both results being positive remains the preferred finding. The situation I regard as most problematic is the reverse: An aPTT-based positive with a negative dRVVT. This configuration requires careful scrutiny before accepting it as a true positive.

I have been working with the dRVVT since I first described it in Philadelphia in 1986, and I can say with confidence that it is considerably more sensitive and specific for lupus anticoagulant than any aPTT-based test. The reasons are analytical.

LabLeaders: Why is the aPTT-based test more vulnerable to interference than the dRVVT?

Prof. Pengo: The aPTT-based test activates the contact phase of coagulation.² All aPTT activators, whether silica, ellagic acid, or kaolin, are charged substances that trigger this contact pathway, and this creates multiple additional sources of variability and interference. Factor VIII, for example, can suppress the lupus anticoagulant signal in the aPTT system, contributing to false negatives. The contact activation pathway introduces complexity that is simply not present in the dRVVT system, which bypasses this phase entirely.

As for the ongoing debate about which aPTT activator to use — silica versus ellagic acid versus others — my view is that the choice of activator is not the critical variable. The activator is not the primary driver of assay performance in this context.

LabLeaders: If the activator is not the key factor, what is?

Prof. Pengo: The phospholipids. This is the most important variable in lupus anticoagulant testing, and it is the one that receives the least attention from a standardization perspective.

To make this concrete: If you take plasma and add only silica, without any phospholipid, the coagulation time will be very long. The same is true for ellagic acid alone, or kaolin alone. The moment you add even a small amount of phospholipid, there is a sharp shortening of the clotting time. All aPTT-based tests incorporate a small amount of phospholipid in the reagent for precisely this reason: It is the phospholipid that lupus anticoagulant antibodies target, and it is the phospholipid that determines how well the assay can detect their presence.

The ratio and composition of phospholipids in the reagent are therefore critical for discriminating between positive and negative results. The reagent's sensitivity to lupus anticoagulant depends on the phospholipid formulation, not the activator. Reagent stability also depends primarily on phospholipids, not the activator. Phospholipids are chemically delicate, and they can oxidize over time and lose functionality. This is a pre-analytical concern that any laboratory running these assays should monitor carefully.

LabLeaders: How standardized are phospholipid formulations across manufacturers, and what are the implications?

Prof. Pengo: They are not standardized, and this is one of the most critical structural problems in lupus anticoagulant testing. Every manufacturer uses its own formulation: its own phospholipid compounds, its own concentrations, and its own ratios. There is no industry-wide standard. This means that reagents from different suppliers can behave very differently on the same patient sample.

Most formulations use a mixture of phosphatidylcholine and phosphatidylserine, but the proportions vary from manufacturer to manufacturer. Because of this lack of standardization, lupus anticoagulant results from different laboratories using different reagents are not directly comparable. This is one of the fundamental reasons why inter-laboratory variability in lupus anticoagulant testing is so high, and why results sent from one center to a reference laboratory so often do not agree.

LabLeaders: How should a laboratory handle weak positive results, especially when the presence of heparin or another interfering substance cannot be excluded?

Prof. Pengo: This is a very common and genuinely difficult scenario. My honest view is that weak lupus anticoagulant results should be interpreted with significant skepticism. A true, clinically meaningful lupus anticoagulant, particularly in a triple-positive patient, will generally produce a clear, unambiguous result. Weak results more often reflect analytical noise, interference, or isolated lupus anticoagulant without accompanying anti-cardiolipin or anti-β2GP1 antibodies, rather than a clinically significant APS profile.

When a weak positive is obtained and heparin exposure cannot be excluded, the first step is to perform a thrombin time. This allows you to determine whether heparin is present in the sample. If heparin is confirmed, the lupus anticoagulant result cannot be trusted.

For laboratories with specialized capability, which most routine clinical laboratories will not have, there is an additional verification method: Isolate the patient's immunoglobulin fraction and add it to normal pooled plasma. If the coagulation time prolongs compared to a buffer control, you have confirmed inhibitor activity originating from the antibody fraction. This is a technique confined to specialized reference centers and is not appropriate for routine use.

LabLeaders: What is the "lupus cofactor," and why is it important for laboratory professionals to understand it?

Prof. Pengo: The term "lupus cofactor" was introduced by Professor Loeliger, who worked in Leiden, Netherlands.³ He observed a paradoxical phenomenon during mixing tests for lupus anticoagulant. Instead of the coagulation time shortening upon addition of normal pooled plasma, as correction by normal plasma should produce, the time was actually prolonged. He asked why, and the question occupied researchers for many years.⁴

The answer, which we have now confirmed, is that prothrombin is the lupus cofactor. In patients with the lupus anticoagulant-hypoprothrombinemia syndrome, anti-prothrombin antibodies are present. When prothrombin, which is contained in the normal pooled plasma added during the mixing step, is introduced into the patient's plasma, the coagulation time is further prolonged rather than shortening it.⁴ This happens because the anti-prothrombin antibodies bind more effectively when their antigen is available to cover the phospholipid surface. The antibodies act as a more potent inhibitor when the target antigen is present.

The practical consequence is that this mechanism can generate a false negative, or a paradoxical, difficult-to-interpret result, during the mixing test. Laboratory professionals need to be aware of this. If a mixing test produces a result that defies straightforward interpretation, or if a patient's clinical picture seems inconsistent with the mixing result, the lupus cofactor effect should be considered. This is precisely the kind of interpretive challenge that requires a dedicated coagulation specialist. A rotating generalist encountering this result would simply not have the context to recognize it for what it is.

LabLeaders: Should lupus anticoagulant retesting be performed even in confirmed triple-positive patients?

Prof. Pengo: Yes, always, even in triple-positive patients. According to the ACR/EULAR classification criteria, a single positive result earns only one point, which is insufficient to classify a patient as having APS. Retesting at 12 weeks is mandatory.⁵

Beyond initial confirmation, in patients with established APS we retest annually. Some patients do convert to negative over time. Sometimes this is associated with treatments such as hydroxychloroquine, which raises the question of whether anticoagulation can eventually be stopped. That remains an open question without a definitive randomized trial to guide practice. Whether a patient who converts to negative truly has a lower thrombotic risk is unknown, and any decision to stop anticoagulation must be made with great caution, thorough shared decision-making, and close follow-up.

LabLeaders: What is your vision for the future of coagulation laboratories?

Prof. Pengo:  My view may be uncomfortable to hear, but I believe it is necessary to say: Many general laboratories have become too large and too automated at the expense of the human expertise that coagulation testing specifically requires.

Automation in the solid-phase assay space has been a genuine advance. We moved from ELISA-based methods, which are operator-dependent, time-consuming, and prone to significant between-run variability, to automated solid-phase platforms that are more precise, faster, and produce much less variability. That progress is real and I welcome it. But in lupus anticoagulant testing specifically, automation cannot replace expert judgment. The three-step procedure requires interpretation at each step. Weak results require clinical context. Paradoxical mixing test findings require someone who understands what they mean.

One of the most damaging practices I see in large general laboratories is the rotation of personnel through the coagulation section. Coagulation is the most technically demanding area in the clinical laboratory. It is not chemistry, and it is not something a competent generalist can pick up on a rotating schedule. The consequence of inadequate expertise is not just an incorrect number on a report. It is a patient who may be wrongly diagnosed or denied a diagnosis that could change their treatment.

Let me give a concrete example of what can go wrong. A sample arrived at a laboratory with a normal prothrombin time but an aPTT that was too prolonged to report. The first laboratory concluded the sample was not coagulable. The second laboratory agreed — too long, unable to process. Only at a third laboratory, staffed with coagulation specialists, was the correct interpretation reached: The patient had hemophilia. Two laboratories had no framework for understanding what was in front of them.

My strong recommendation is that lupus anticoagulant testing should be centralized into fewer, more specialized centers with dedicated coagulation expertise, rather than being distributed across every general laboratory. A single center with a passionate, committed specialist will produce more reliable results than ten centers without one.

LabLeaders: What is your single most important message for laboratory professionals?

Prof. Pengo: Be an expert in coagulation, a true expert, not a generalist who rotates into hemostasis for a few months. The analytical, pre-analytical, and post-analytical demands of lupus anticoagulant testing require deep, dedicated knowledge. Know what a paradoxical mixing result might mean. Understand the lupus cofactor. Know when a weak positive should be verified with a thrombin time. Communicate with the clinician about what is in the blood.

If your laboratory does not have someone with that expertise, that is the problem that needs to be solved before the next complex sample arrives.

This is part two of a two-part series on lupus anticoagulant testing with Professor Vittorio Pengo. In part one, Professor Pengo addresses the clinical side of lupus anticoagulant testing. He focuses on how clinicians should approach testing in the emergency setting, when to rely on immunological surrogates instead of the LA assay, what the three clinical phenotypes of lupus anticoagulant mean for patient management, and more.