David L. McGlasson, MS, MLS(ASCP)
Lupus Anticoagulants/Antiphospholipid Antibodies/Antiphospholipid Antibody Syndrome
Testing for Antiphospholipid Antibody Syndrome
Platelet-Poor Plasma (PPP)
The quality of the patient’s PPP greatly affects the integrity of the testing.
To obtain PPP, the capped citrated specimen is centrifuged at room temperature for at least 15 minutes at 1500–2500 Xg. Each lab must adjust the speed and time of centrifugation in order to achieve a PPP sample with a residual platelet count of less than 10,000/μL Coagulation testing can be performed by sampling the PPP directly from the centrifuged tube, or the PPP can be removed from the tube for later testing or storage. When removing the PPP, a small interface of PPP should be left over the packed cell layer. This ensures that the platelet layer is undisturbed and that platelets have not been resuspended into the PPP. If possible, use a horizontal-head centrifuge to obtain the supernatant PPP. This type of instrument produces a flat level plasma to blood cell layer. Angled-head centrifuges may cause platelets to “stick” to the side of the collection tube. If this type of system is used and the specimen not timely processed and in an upright position the platelets may slide back into the PPP and release granular properties that could interfere with the selected coagulation assays.
The use of PPP is essential for technical reasons. Platelets contain platelet factor 4 (PF4), which neutralizes heparin (thus affecting sample’s testing for the presence of heparin). Platelets contain phospholipids, which affect lupus anticoagulant and factor assay testing (especially if the sample is frozen and thawed). Platelets also contain proteases, which, when released during the thawing of a frozen sample, can alter results for von Willebrand factor testing.
A clot of any size in the sample renders it unacceptable. The whole blood specimen should be checked visually for clots while gently rotating the capped sample end-to-end several times. The specimen also can be checked for fibrin or clots after removal of the plasma or when testing is completed. After removing the plasma, wooden applicator sticks can be twirled inside the tube and pulled out to check for fibrin threads or clots. Coagulation testing is most reliable when performed on a fresh sample. If testing is delayed, the PPP can be stored at 18–24°C or 2–8°C for up to 4 hours before performing most tests. If the testing cannot be completed within 4 hours, the PPP can be stored at -70°C for 6 months to 1 year. A double-spin procedure should be employed on specimens that are going to be stored for later testing. Frozen samples must be thawed rapidly at 37°C because excessive heating (>5 minutes) can result in the loss of the labile factors V and VIII. Samples for coagulation testing should never be stored in self-defrosting freezers. The freeze–thaw cycles could rupture any residual platelets in the plasma, activate coagulation factors, and compromise sample integrity and stability.
Whenever possible, blood for LA detection should be collected in patients not receiving any anticoagulant treatment. If feasible briefly interrupt direct oral anticoagulants (DOAC). LA testing can be performed after checking the level of DOAC using the appropriate anti-IIa or anti-Xa method.
If necessary, check levels of acute phase reactants such as elevated FVIII and CRP which may give false-negative/false positive results depending on the clotting assay methods.
During acute thromboembolic occurrence, LA detection should be interpreted with care since subjects may be receiving UFH, LWMH, VKA, or DOACs.
Testing during pregnancy can result in false a positive/false negative test result and should be repeated after 3 months to verify results.3
No single test is sensitive for all LAs. Thus, at least two different screening tests based on different assay principles should be performed. If more than two types of screening tests are used, the risk of a false positive result increases to unacceptable levels.77 The dilute Russell viper venom time (dRVVT) test is recommended as the initial screening test. This assay picks up any deficiencies of the extrinsic portion of coagulation. The APTT has historically been used to screen for LA/APAs, although APTT’s sensitivity to LAs depends on the reagent. This assay is used in this setting to check for intrinsic factor issues. An APTT assay using silica as an activator with low phospholipid content is the second choice of screening methods. APTT reagents with ellagic acid activators are not recommended for detection of LA because they lack sensitivity for the presence of LA. Many modifications to the APTT, including the use of lower concentrations of phospholipids in the test reagent, have been recommended in an attempt to increase its sensitivity. Laboratory professionals must evaluate their APTT reagents as to the importance of LA/APA sensitivity versus the importance of sensitivity to specific factor deficiencies. In cases for which the screening APTT is not prolonged but the patient presents with a positive history of thrombophilia, the physician should proceed with designated testing for LAs. The presence of an LA should be considered if one of the two screening tests gives a positive result.
According to the guidelines developed by the International Society on Thrombosis (ISTH) for 2020, laboratory evaluation for the presence of a LA should include the following:
- Double centrifugation to ensure PPP, <10,000/μL, quick-freeze if testing is delayed; thaw at 37°C.
- Two clot-based assays employing separate clotting principles as screening tests. The dRVTT assay is the most robust. The other assay can be low-phospholipid APTT with silica activator. APTT with kaolin activator is problematic in automated photo-optic coagulometers; ellagic acid is insensitive to LAs; both are contraindicated. Integrated assays are recommended (e.g., DRVVT screen and confirm methods)
- Performance of a TT to detect therapeutic heparin and absorb heparin or collect sample at another time in the absence of anticoagulant therapy.
- Preparation of PPP for mixing studies is prepared “ad hoc” (homemade) by double centrifugation to ensure it is PPP. PNP (pooled normal plasma) must provide 100% activity for all clotting factors. Commercial lyophilized or frozen normal plasmas can be used if they fulfill these specifications. A 1:1 mixture of patient plasma with PNP should be tested without preincubation.
- Development of cutoff values using the local reagent/coagulometer combination on at least 40 adult healthy donors <50 years old.
- Performance of mixing studies on plasmas from healthy donors mixed with the PNP at a 1:1 proportion. The cutoff is the 99th percentile of the distribution of results from the healthy donors or an index of circulating anticoagulant (ICA) computed as follows:
ICA% = [(Clot time of patient-PNP mixture −
Clot time of PNP) / Clot time of patient] × 100
- Performance of the confirmatory test by increasing the concentration of PL in the screening test. Either bilayer- or hexagonal-phase phospholipid can be used. Frozen/thawed platelets are not recommended because of poor batch-to-batch consistency.
- Confirmatory testing that requires testing healthy donor plasma at both low PL concentrations (screen) and high PL concentrations (confirm) to obtain CTs. The confirmatory test cutoff is the value corresponding to the mean of the individual percentage corrections defined as follows:
[(Screen CT − Confirm CT)/Screen CT] × 100 =
The result is confirmation of LA if the percentage correction is above the cutoff value defined previously.
- Results should be normalized (ratio of result/PNP) and with accompanying interpretation.
- It should be noted that many manufacturers may suggest cut-off values for screening and confirm assays. However, there may be distinct differences between instrumentation that uses photo-optic versus mechanical end point technology. This is the reason each facility should establish their own cut-off values in a consistent manner.
Dilute Russell Viper Venom Time (dRVVT) Test
The dRVVT test uses a commercial preparation of the venom from the Russell viper (Daboia) to activate FX. The dRVVT test is based on the premise that LA activity is enhanced in the presence of reduced phospholipids. The screening reagent contains dilute Russell viper venom (dRVV), calcium chloride, and phospholipids. The reagent is added to patient PPP, and Russell viper venom (RVV) activates FX, resulting in clot formation. If LAs are present, the patient’s dRVVT is longer than that of the normal control.
The ratio of the patient’s clotting time (CT) to the CT of the normal control is determined. The normal ratio is usually less than 1.2. A confirmatory test also uses dRVV but with a higher phospholipid concentration. The final result is reported as a ratio of the two clotting tests (high and low phospholipid concentration), which is compared with the values of a reference population.4 The dRVVT appears to be more sensitive to aPL antibodies that react with anti-β2 glycoprotein I.5,6
The hexagonal-phase phospholipids (HPPs) test uses egg phosphatidylethanolamine in a hexagonal-phase configuration. The HPP assay is based on the fact that many LA antibodies specifically recognize the HPP configuration as an antigenic epitope. Addition of HPPs to the reaction mixture neutralizes the inhibitory effect of the LA antibodies but does not neutralize most factor-specific antibodies.
The test is performed by incubating the test plasma at 37°C with and without the HPP reagent. An APTT is performed on both of these incubations using an LA-sensitive reagent. If LA antibodies are present in the test plasma, the HPPs would neutralize them, resulting in a shortened CT for the tube containing it compared with the tube without HPPs. By comparing the two CTs, the presence of aPL antibodies can be identified.
This assay has two advantages: (1) The LA-sensitive reagent contains a heparin inhibitor that makes the test system insensitive to heparin levels up to 1 IU/mL and (2) the hexagonal phase procedure adds normal plasma to the test system to correct any prolongation of CT from factor deficiencies that might be present. The correction of the APTT result varies, depending on the instrument used. Photo-optical systems may have shorter times than electromechanical devices with different reagent/instrument combinations. Each laboratory should establish its own cutoff by determining the range from the mean of a normal population. Some vendors list a cutoff of 8 seconds for electromechanical systems. Unfortunately, many laboratories use the 8 second cut-off for their facility without validating it. The correction is consistent with the presence of a phospholipid-dependent inhibitor.7,8
When the dRVVT or APTT are prolonged, further testing can be performed to identify the specific cause of the abnormality. To assist in selecting the appropriate diagnostic test after the screening tests, an algorithm can be followed. As each of the following specific tests is described, its sensitivity to decreased levels of clotting factor(s) or acquired anticoagulants and whether these levels would be detected in the specific screening test are discussed.
- Mixing studies (also known as circulating anticoagulant screen or screening test for circulating inhibitor) are performed to differentiate a factor deficiency from the presence of a circulating inhibitor. These studies repeat the screening tests that have abnormal results (APTT, dRVVT) using a 1:1 dilution of the patient’s PPP mixed with a pooled normal plasma (PNP). Factor levels of at least 50% of normal levels (100% activity is desired) are generally sufficient to produce a normal correction of APTT or dRVVT result, repeating the APTT and dRVVT with dilution of 1:1 mix of patient and PNP corrects the prolonged patient result if it is caused by a deficiency of one or more procoagulant factors. If the results are prolonged a LA presence or a suspected factor inhibitor may be suspected. The testing is performed immediately after mixing patient and PNP and again after incubating the mixture at 37°C.9
Three step procedure summation for determining the presence of LAs in clotting assays
Prior recommendations in the ISTH 2009 guidelines discussed integrated paired test systems paired performance with the (1) screening methods of the APTT and the dRVVT, (2) confirmation step with low and high phospholipid contents with the comments that they do not require the mixing steps. Subsequently the newer CLSI guidelines emphasized that all three steps of (1) screening, (2) mixing and (3) confirmation are essential. The current ISTH guidelines 2020 recommend mixing studies to be performed. I would recommend an excellent paper by Favaloro E1,6,9
Interpretation of the mixing test can be difficult leading to improper diagnosis of the presence of an LA. The ISTH 2020 guidelines give two alternatives: a local cut-off value of the CT (not clearly indicated that the value should be expressed as ratio) of the index of circulating anticoagulant known as the Rosner index. They also stated that mixing tests should be interpreted with a mixing test-specific cutoff expressed as a normalized ratio because recent studies suggested it has better sensitivity than the alternatively recommended index of circulating anticoagulant.
Normalization of the results requires an extra calculation of the former screen/confirm ratio. PNP is used to reduced intra-laboratory variability.
A dRVVT mix ratio: dRVVT screen 1:1 mix of patient:PNP /
dRVVT screen PNP
The cutoff for a positive mix ratio is greater than 1.2 or 2 times the SD of the mean of the ratio of the in-house values. The dRVVconfirm (dRVVC) is performed if both the dRVVT screen and mixed ratio are abnormal:
dRVVT confirm ratio: dRVVT confirm (patient plasma) / dRVVT confirm PNP
The next step is to perform a normalized dRVVT ratio using the screen ratio in the numerator and the confirm ratio in the denominator.
Normalized ratio: dRVVT screen ratio / dRVVT confirm ratio
The cutoff for the normalized ratio is >2.0 the SD of the mean of the normalized ratio of a reference population. Each commercial dRVVT manufacturers vary in their sensitivity for detecting LA.
Solid Phase Testing
We have currently discussed the preferred coagulation testing to determine the presence of LA/APAs. The next part of this topic will cover the other important phase in the ability to classify the diagnosis of APS.
Anticardiolipin antibodies (ACA)
will recognize a mixture of a complex of a phospholipid called cardiolipin which is coupled to β2GP1 proteins. Cardiolipin is a negatively charged phospholipid first discovered by Pangborn in 1942. It is an important component of the inner mitochondrial membrane. The name cardiolipin was derived because it was discovered in animal hearts. The procedure may now be performed by ELISA, flow cytometry and chemiluminescence. The standards are quantitative but not uniform. There are also methods that target β2GPI bound to cardiolipin or those that detect purified β2GPI. There is great variability between laboratories in determining what levels are positive and negative. Many studies have led to researchers conceding that elevated levels of IgG, IgM and in some select populations IgA the ACA and β2GPI antibodies (>99th percentile) are considered pathologic for indications of thrombotic states presence.
LAs and ACA antibodies can be found in 60% of cases of APS. The ACA is not affected by anticoagulant therapy as are the clottable LA testing may be. Nor are they compromised in the presence of an ongoing thrombosis episode or deficiency of coagulation factors or inhibitors.
ACA levels are the most sensitive for the presence of APS but are non-specific for thrombotic processes. Infectious diseases can elevate transient ACA positive values. Disorders such as subjects with a positive VDRL, HIV, Lyme disease, Epstein-Barr and cytomegalovirus, Gulf War syndrome, Hepatitis C and others can be ACA positive. If they don’t have a thrombotic disorder, they can mistakenly be given a diagnosis of APS. Subjects with APS have β2GPI bound to cardiolipin as antigenic site. Many of the new assays for ACA antibodies include a combination of both the ACA+β2GPI.
Most laboratories now run only the IgG and IgM ACA and β2GPI assays as part of the APS profile work-up. An elevated β2GPI level is more predictive of a thrombotic disorder than the ACA alone. Together with a positive LA, ACA, and a β2GPI you have a triple positive situation that is the most predictive of a high thrombotic risk APS patient. The highest odds ratio for thrombosis is reached for those subjects who have all three of the above criteria. IgM positive findings for the solid phase assays without an elevated IgG finding may not have clinical value.
Subjects that have positive test results after 3 months will differentiate between a transient antibody from a chronic autoantibody and will usually require anticoagulation.
Further testing may be required if the LA, ACA and β2GPI are negative the requesting physician still may suspect the present of an APS situation. In consultation with the pathologist a request may be generated for testing for anti-phosphatidylserine/antiprothrombin immunoassays. The presence of these antibodies is also highly suggestive for the presence of an APS particularly anti-phoshatidylserine are predictive in recurrent fetal loss.10-16
Therefore, a true APS subject with a triple positive group finding is highly suggestive for a thrombotic episode in the presence of an LA, ACA/ β2GPI at the same time.
- Clinical Laboratory and Standards Institute (2014). Laboratory Testing for the lupus anticoagulant. CLSI [Document No. H60A] Wayne PA. Author.
- Miyakis S, Lockshin MD, Atsumi T et al: (2006). International Consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). Journal Thrombosis and Haemostasis. 4(4);295-306.
- McGlasson DL, Gosselin RA. Hemostasis: Laboratory Testing and Instrumentation. Clinical Laboratory Hematology. Pearson 4th ed. Boston, MA. 2019
- McGlasson DL, Fritsma GA (2013). A comparison of six dilute Russell Viper Venom Time. Seminars in Thrombosis and Hemostasis 39;:315-319.
- Favaloro EJ,, Wong RCW. (2014). Antiphospholipid antibody testing for the antiphospholipid syndrome: a comprehensive practical review including a synopsis of challenges and recent guidelines. Pathology 46;6:481-495.
- Devreese KM, de Groot PG, de Laat B et al: (2020) Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis. .J Thromb Haemost 18:2828-2939.
- Devreese KMJ. (2020). How to Interpret Antiphospholipid laboratory Tests. Current Rheumatology Reports 22:38.
- Charles LA, McGlasson DL, Hawksworth BA, Ashcraft JH, Ortel TL. Evaluation of a Modified Procedure for STACLOT-LA for the Confirmation of LUPUS Anticoagulants. In Press, Blood Coagulation and Fibrinolysis, Vol. 5, Oct 1994, pg. 601-604.
- Favaloro E (2020): Mixing studies for lupus anticoagulant: mostly yes, sometimes no. Clin Chem Lab Med58;487-491.
- Galli M, Luciani D, Bertolini G. (2003) Anti-beta-2-glycoprotein I, antiprothrombin antibodies and the risk of thrombosis in the antiphospholipid syndrome. Blood. 102;27:17-23.
- Nash MJ, Camilleri RS, Kunka D, Mackie IJ et al: (2004) The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies. J Thromb Haemost. 2;7:1077-81.
- Willis R, Papalardo E and Nigel Harris (2017) Solid Phase immunoassay for the detection of anticardiolipin antibodies. Methods Mol Biol 1646; 185-199.
- Cohen D, Berger, SP. Steup-Beekman GM et al: (2010) Diagnosis and management of the antiphospholipid syndrome. BMJ340:254
- Shi H, Zheng H, Yin YF et al: (2018) antiphosphatidylserine/prothrombin antibodies as potehntial diagnostic markers and risk predictors of venous thrombosis and obstetric complications in antiphospholipid syndrome. Clin Chem Lab Med.56;614-624.
A group that has only ACA present but not have thrombotic potential is an example of an infectious issue or inflammatory response. An example of this is of a study that I performed several years ago on Gulf War Veterans that had a high level of positive ACA presence.
We initiated a study to assess the presence of three types of aPL’s (aCL, anti-β2GP1, and aPS), in a group of Gulf War Veterans. These veterans had high incidences of chronic headaches/migraine headaches, (54.6%), memory loss (52.7%), chronic fatigue (61.5%), joint pain (59.8%), muscle aches (43.5%), attention deficit problems ((43.1%), and sleep problems (53.8%).
These results suggest that the frequency of aCL but not aPS or anti-β2GP1 are higher in gulf War Veterans than fibromyalgia/CFS patients with similar symptoms. The groups were also age matched. All participants were between 18-45 years in age. The presence of aPS and anti-β2GP1, antibodies are more strongly associated with thrombosis than the presence of aCL alone.
The presence of aCL alone has been described in patients exposed to a variety of infections, medication and other medical conditions. Although this group of Gulf War Veterans did not have thrombosis, the clinical significance of aCL antibodies in this group is not clear and requires further study.
The group of individuals from the Rheumatology Department at Georgetown who had similar symptoms had very low positives in all antibody studies compared to the Gulf War Group. Previous studies have observed positive aCL levels in normal blood bank donors (IgG: 4.6-6/5%), IgM 4.6-9.4%). Levels of aPS and anti-β2GP1 antibodies are extremely low in subjects that have no personal of family history of thrombotic events.1
1McGlasson DL, Heron EA, Doe RH, Brey RL, Clauw DJ, Harris MD. The Presence of Antiphospholipid Antibodies in Gulf War Veterans Evaluated at Wilford Medical Center. Clin Hemostasis Rev. June 1999.