Lupus Anticoagulant Testing

Introduction

"Lupus anticoagulants (LA) are a heterogeneous class of immunoglobulins that may develop spontaneously or as a consequence of autoimmune diseases" (Tripodi 2007:1629). In the laboratories, a lupus anticoagulant testing is done to establish the reason for unexplained thrombosis, persistent miscarriages or a delayed PTT test (Tripodi 2007). Presence of lupus anticoagulant in the body, after testing, requires a series of other tests to be conducted in order to determine its status; persistent or transient. Some of the tests used to detect lupus anticoagulant include APTT (Activated Partial Thromboplastin Time), KCT (Kaolin Clotting Time) and DRVVT (Dilute Russell Viper Venom Time) (Lupus Anticoagulants Testing 2011). Below is a review of primary research literature explaining the background of the above named tests and the general problems and challenges associated to lupus anticoagulant testing (Machin, Giddings, Greaves, Hutton & Malia 2006).

According to a survey conducted by association of Clinical Pathologists, APTT test is the most common test for detecting lupus anticoagulant. APTT test is a test usually conducted to establish the reason for unexplained bleeding or blood clotting. Different APTT reagents are used whereby; the sensitivity to lupus anticoagulant depends on the type of reagent used. According to results of this study, reagents with low phospholipids content are the most sensitive to lupus anticoagulant (Machin, Giddings, Greaves, Hutton & Malia 2006). APTT test is conducted through a screening process. If the first APTT test is prolonged then a second APTT test is done by collecting the patient's plasma and then mixing it pooled normal plasma (collected from normal donors). Then, the sample is taken through a screening process and the results are analysed. If the second APTT test is normal (not prolonged), then it indicates deficiency of one or more coagulant factors. If the second test is prolonged (not normal), then it indicates presence of an abnormal factor inhibitor also referred to as autoantibody.

During the test, a shortened aPTT is mostly related to high risks of thrombin related problems. Such problems include "increased levels of factor (F) VII and thrombin-antithrombin complex (TAT)" (Boekel, Bock, Vrielink, Liem, Hendriks & Kieviet 2007). In a study conducted by Boekel, Bock, Vrielink, Liem, Hendriks & Kieviet on 400 patients, Actin-Fs by Dade Behring among other reagents were used to measure aPTT. In the study, levels of VIII, IX, XI factors and TAT were measured in the patients. In the results, 59% of patients where shortened aPTT levels were carried using Actin-FS reagent had their results show a normal aPTT. The results of this test agreed with results of other tests which used Platelin-LS and automated-APTT reagents. It was concluded that even though many reagents used in detection of lupus anticoagulant have related results, Actin-FS is more sensitive in inducing shortened aPTT than automated  APTT and Platelin-LS reagents (Boekel, Bocka, Vrielink, Liem, Hendriks & Kieviet 2007:360).

In a different study conducted by Rolla, Vidali, Serino, Pergolini, Albano and Bellomo to detect lupus anticoagulant, Actin-FS was used to make the assay. In this study, Actin -FS was used due to its high sensitivity for lupus anticoagulant (2007). Reference time for aPTT-FS was set between 27 and 35 seconds. The results showed that all the 164 patients evaluated had their aPTT Actin-FS ratio with the normal range. In the same study, a standardized aPTT Actin-FS test by Dade Behring which was combined with screening test and a dRVVT test for confirmation was also conducted. The results of this test revealed that 52% of the patients had normal aPTT ratio, 145 had their aPTT ratio being above normal range, while 25% had abnormal aPTT ratio. The study concluded that aPTT Actin-FS by Dade Behring is a reliable test for detecting lupus anticoagulant.

However, due to lack of 100% positive results, the study concluded aPTT Actin-FS should be combined with other tests such as dRVVT test in detection of lupus anticoagulant should be used. However, according to the study, "aPTT Actin-FS is a reliable test for detecting lupus anticoagulant due to its sensitivity to defined oxidized phospholipids which are independent predictors of the response to LA detection" (Rolla, Vidali, Serino, Pergolini, Albano and Bellomo 2007:65).    In detecting presence of lupus anticoagulant, DRVVT test can be conducted if the initial APTT test was prolonged. DRVVT test is more sensitive to lupus anticoagulant that APTT.

A research conducted by the Association of Clinical Pathologists in 2006 using dilute phospholipids, dilute RVV and platelet rich plasma concluded that a ratio of 1:1 (50% normal to 50% test mixture) indicates presence of lupus anticoagulant (Machin, Giddings, Greaves, Hutton & Malia 2006). Dilute Russell viper venom is used in detecting lupus anticoagulant due to its ability to "induce thrombosis by activating factor X, which is turn changes Prothrombin into thrombin in the presence of factor V and phospholipids" (Thiagarajan, Pengo & Shapiro 2008).

When conducting DRVV test, clotting time usually ranges between 23 and 27 seconds (Pengo, Biasiole, Rampazzo, & Brocca 2008). This is when low concentrations of Russell's viper venom and phospholipids are used. With a clotting time of 30 seconds or more, it indicates that lupus anticoagulant antibodies are present and they are interfering with clotting. An initial DRVV test is usually followed by a confirmatory test. A confirmatory test is yet another DRVV test but more concentrated phospholipids are added to the assay in order to prevent the effect of lupus anticoagulants on phospholipids (Pengo, Biasiole, Rampazzo, & Brocca 2008).

If the clotting time for the confirmatory test is normalized with that of the initial test, then a determination ratio is derived by comparing the times with or without excess phospholipids (Thiagarajan, Pengo & Shapiro 2008). A ratio of 1:2 (time without excess phospholipids to time without excess phospholipids) indicates presence of lupus anticoagulants. According to many researchers, DRVV test is the most recommended test for detecting lupus anticoagulant (compared to aPTT) since it is more sensitive to lupus anticoagulant and it is not affected by deficiencies or inhibitors of clotting factors such as VII, IX or XI like in the case of APTT (Thiagarajan, Pengo & Shapiro 2008).

Kaolin clotting time (KCT) is yet another test used to detect lupus anticoagulant. KCT is normally an activated PTT test which does not contain phospholipids (Loscalzo & Schafe 2008). KCT relies on residual cell membrane fragments and plasma lipids to provide a phospholipids surface for coagulation reaction (Galli, Finazzi, Bevers & Barbu 2006:619). KCT has been ranked as the most sensitive test for detecting circulating anticoagulants. This is because it is sensitive to presence of unfractionated heparin and it detects all types of inhibitors (Loscalzo & Schafe 2008).

When conducting KCT, normal plasma from patient is diluted with Kaolin. The calcium is added to the mixture to instigate coagulation. Kaolin clotting time is the time taken after calcium has been added and when clotting occurs (Ferro et al 2006). Unlike in the previously discussed tests, KCT does not use phospholipids in the correction step. As an alternative, prolonged Kaolin clotting test is corrected by using more quantities of normal plasma. Prolonged KCT indicates presence of deficient factors while normal KCT indicates presence of coagulation inhibitor. Normal clotting time in KCT ranges between 60 and 120 seconds. A KCT average ratio of 20:80 (mixture of patient plasma to normal plasma) is the normal ratio for KCT in detecting lupus anticoagulant (Ferro et al 2006).

Detecting lupus anticoagulant using APTT, DRVVT and KCT has been met with various challenges and problems by the different practitioners. One of the general challenges of testing lupus anticoagulant is encountered in the KCT test (Galli, Finazzi, Bevers & Barbu 2006). As earlier mentioned, KCT test used Kaolin to dilute normal plasma and then calcium is added to instigate coagulation. This Kaolin suspension is usually muddy. Therefore, it is opaque. As a result, use of optical density to record the measurements required to establish the endpoint becomes difficult. This renders the whole process to be conducted manually, a thing which proves to be very tedious (Galli, Finazzi, Bevers & Barbu 2006). However, recent research has revealed automation of KCT which is made possible by use of less opaque Kaolin suspension (O'Neill & Parkin 2005).

Another general challenge facing lupus anticoagulant testing is lack of ability by the tests to give true results in just one test (Tripodi 2007). The challenge here is that, lupus anticoagulant test has a possibility of giving false positive results. For instance, in an APTT test, the initial test maybe prolonged or not. If the initial test is prolonged, then it may be due to two possibilities. That is due to presence of anticoagulant therapy by the patient or contamination of the heparin, or due deficiency of one or more coagulant factors.

A second APTT test is required in order to determine the real cause of prolonged PTT. The results of the second test may be prolonged or normal. If they are prolonged, it means presence of autoantibody and this requires even further tests to be done (Tripodi 2007). The level of phospholipids reagent in APTT test interferes with the results of the test. Low concentration in a sample may indicate positive results while the truth is, positive results were due to the level of phospholipids in the sample and not due to lupus anticoagulant. Due to such complications in the test, positive results maybe obtained causing wrong diagnosis to be administered to a patient.

In addition, for positive results to be obtained in an APTT test, some other tests such as platelet count, fibrinogen testing and thrombin time testing need to be included (Forero, Escoba, & Vernot 2009). Otherwise, reliance on the results of APTT test only may not be sufficient in making a decision concerning diagnosis to a patient. When choosing which of the named tests should accompany APPT test, careful analysis of the patient's history is required too, therefore complicating the procedure of conducting a single test to detect lupus anticoagulant more and more tedious.

In testing lupus anticoagulant, presence of anticoagulation treatment affects the results of the tests. Testing lupus anticoagulant is difficult in patients under anticoagulant therapy or those receiving heparin (Dragona, Minotti, Bongarzoni, & Avvisati 2001). This is because the current laboratories lack a reliable method of testing lupus anticoagulant in such patients. According to Dragona, Minotti, Bongarzoni, & Avvisati, patients being given anti-coagulant therapy such as vitamin K antagonists may show prolonged coagulation times due to their treatment. This can make the detection of lupus anticoagulant activity in serum difficult (2001:49).

Finally, with the current available laboratory methods, exact quantification of lupus anticoagulants is difficult (Forero, Escoba, & Vernot 2009). Therefore, once a lupus anticoagulant test has tuned positive, it becomes difficult to determine the exact quantity of lupus anticoagulant which has been detected in the body. Therefore, lack of LAs quantifying methods is yet another problem encountered in detection of lupus anticoagulant.