Partial Thromboplastin Time - Lupus Anticoagulant Screen

Normal findings

Activated partial thromboplastin time (aPTT): 30-40 seconds ^[1]

PTT: 60-70 seconds ^[1]

Patients receiving anticoagulant therapy: 1.5-2.5 times the control value in seconds ^[1]

Possible critical values ^[1] :

• APTT: > 70 seconds
• PTT: > 100 seconds

In general, healthy subjects do not have a prolonged aPTT and do not have lupus anticoagulant (LA) activity.

LA activity is identified by in vitro prolongation of clotting time in coagulation assays. According to the International Society of Thrombosis and Hemostasis (ISTH), the following criteria must be met to confirm the presence of an LA. ^[2]

• Screening with 2 or more phospholipid-dependent coagulation tests and demonstrating prolongation of at least one.

• Evidence of inhibitory activity through a mixing study

• Demonstration of phospholipid-dependence of the inhibitor

• Exclusion of a coexisting specific coagulation factor inhibitor

In fulfillment of the first criteria, the aPTT is one of the most commonly used coagulation assays to screen for LA. However, sensitivity in standard commercial preparations varies. ^[3] This is likely due to the class and concentration of phospholipids because certain studies have reported abolishing the LA effect on the aPTT when reagents rich in phosphatidyl serine are used. ^[4] Thus, some authors recommend using a sensitive reagent with minimal phosphatidyl serine concentrations when using aPTT in the evaluation of a patient with suspected LA activity.

When such a technique is used, the test is referred to as the LA-sensitive aPTT or aPTT-LA. Other authors argue that a single aPTT using a reagent that is not optimally sensitive may suffice to diagnose LA, provided that its positivity is documented on 2 separate occasions. ^[5] At present, most laboratories have adopted increasing the sensitivities of their standard aPTT tests such that the two have become, in general, interchangeable. Regardless of the aPTT reagent used, interpretation of a prolonged aPTT in evaluation of LA activity must involve further tests to fulfill the aforementioned mentioned criteria to confirm the presence of LA.

Positive LA screening

If a prolonged aPTT or aPTT-LA is demonstrated, the patient’s plasma is then subjected to another phospholipid dependent coagulation assay of a different principle. This is based on evidence showing that no single test is sensitive for detecting all LA. However, the risk of false-positive results increases to an unacceptable level if more than 2 screening tests are performed. ^[2] The dilute Russel viper venom test (dRVVT) is widely used as another coagulation test for this purpose and is believed to be more specific for LA detection. ^[6] Other phospholipid-dependent coagulation tests include the Kaolin clotting test (KCT) and the dilute prothrombin time, both of which are not recommended by the ISTH due to poor reproducibility and reagent variability. ^[2]

Mixing study

Once an abnormal screening test result for LA is identified, the next step is to document the presence of an inhibitor by mixing the patient’s plasma with adding double-centrifuged normal plasma in a 1:1 proportion. A positive finding demonstrates persistence of a prolonged aPTT after the mixing study. In a hospitalized setting, the presence of heparin in the serum must be excluded prior to mixing and is usually done by performing a thrombin time (TT). However, less experience and literature is available on low molecular weight heparins (LMWH), factor Xa inhibitors, or direct thrombin inhibitors. ^{[2, 7]}

Confirmation of phospholipid dependence

Elucidating the character of the inhibitor is the next step after demonstrating its presence. For LA, demonstrating how its activity specifically depends on phospholipid concentration is key. This can be done in 2 ways.

The first method involves accentuating prolonged coagulation by reducing the amount of phospholipid, as is done in the tissue thromboplastin inhibition (TTI) test and the dilute aPTT test. The second and more sensitive method is to neutralize the inhibitor activity by increasing the amount of phospholipid, as is done in the platelet neutralization procedure (PNP) or the rabbit brain neutralization procedure (RBNP). The ITSH recommends the use of hexagonal phase phospholipids to increase phospholipid concentrations because studies have shown LA inhibitors are able to differentiate between lamellar and hexagonal phospholipid configurations. ^{[2, 8]}

Exclusion of a specific coagulation inhibitor

After confirming phospholipid dependence, the coexistence of a specific factor inhibitor, specifically factor VIII inhibitor, is excluded with an inhibitor assay. Although this occurrence is rare, failure to exclude such may result in catastrophic bleeding. ^[9]

Findings

Results of aPTT-LA testing can be subdivided into the following:

• Positive: All 4 criteria are met

• Intermediate: Less than 4 criteria met

• Negative: None of the 4 criteria are met

Patients who receive a positive result while being evaluated for antiphospholipid antibody syndrome (APS) must have repeat LA testing after 12 weeks to establish the diagnosis according to current criteria. Studies suggest that when LA is detected at 2 separate time intervals 12 weeks apart, a statistically significant association can be shown with prior thromboembolic events and fetal loss. ^{[10, 11]}

The specimen is blood. No specific patient preparation is needed. Optimally, the sample should not be collected after a meal because lipemia may interfere with photometric measurements of clot formation. Moreover, the sample should not be drawn from an arm with a heparinized catheter or a heparin lock.

Routine venipuncture is used for collection, with the following precautions:

• Avoid “digging” or over-eagerly searching for the vein with an embedded needle because a traumatic venipuncture may prematurely activate clotting factors and overwhelm the proportion of anticoagulant in the Vacutainer.

• Fill the tube to the indicated optimum amount and never under the minimum amount, allowing standardization of ratio of blood to anticoagulant.

• Gently mix the sample to distribute the anticoagulant throughout the sample and prevent premature clotting.

• Never combine two under filled tubes to make one tube

• If multiple blood specimens are to be obtained, aPTT must not be the first sample and should be ideally obtained as the second or third tube.

Blood is drawn into a light blue top tube containing 109nM (3.2%) buffered sodium citrate, which binds to serum calcium preventing coagulation. The tube has a specified filling volume to ensure sample collection in 9:1 ratio of blood to anticoagulant.

Samples should be tested within 4 hours of specimen collection. Sending the sample on ice will activate clotting and may result in falsely shortened clotting times.

Integral to aPTT-LA testing is the requirement of testing platelet poor plasma (platelet count of < 10x10⁹/L). This is accomplished by centrifugation at 1500 g for 15 min at room temperature. Most institutions, including the ISTH, recommend centrifuging twice to ensure the absence of platelet-factor-4, which can potentially activate clotting prematurely. ^[2] Once samples have been centrifuged, they can be stored at either 2-4^o C or 18-24^o C.

aPTT-LA may be part of the following panels:

• LA evaluation with reflex: This is a series of tests designed to detect the presence of antiphospholipid antibodies through coagulation studies.

• Antiphospholipid antibody panel: The previous panel may subsequently be included in a more comprehensive panel to screen for antiphospholipid antibodies. Other components of the panel include anti-cardiolipin antibodies (immunoglobulin [Ig]A, IgG, IgM), anti-beta-2-glycoprotein-1 antibodies (IgA, IgG, IgM) and antiphosphatidylserine antibodies (IgG, IgA, IgM).

Description

LA is an antibody that prolongs phospholipid dependent coagulation tests in vitro. The existence of this antibody was first demonstrated in 1952 by Conley and Hartmann when they observed prolongation of lipid dependent coagulation tests in patients with systemic lupus erythematosus (SLE). ^[12] For a lack of a better understanding of the underlying pathophysiology of this phenomenon, Feinstein and Rapaport coined the term “lupus anticoagulant” to describe the circulating antibody. ^[13]

Although the initial reports of the clinical manifestations of patients with circulating LA described bleeding tendencies, subsequent studies showed that bleeding only manifested when other hemostatic defects were present. ^{[14, 15]} It later became apparent that the presence of this antibody was not associated with a bleeding tendency but was instead associated with recurrent abortions and thromboses. ^{[16, 17, 18]} As such, LA has often been referred to as a “double-misnomer” because it is frequently absent in patients with SLE and is associated with in vivo thrombosis rather than bleeding.

In 1983, Hughes described in full detail a syndrome of thromboses or recurrent pregnancy losses occurring in patients with laboratory evidence of antibodies against anionic phospholipids. ^[19] Cardiolipin was used as the anionic phospholipid in a quantitative solid phase immunoassay developed to detect these antibodies. As such, he termed the syndrome as anticardiolipin syndrome. This was later renamed to antiphospholipid antibody syndrome (APS) when it became apparent that LA can be present in these patients and was also directed at these phospholipids. Gharavi et al later described induction of antiphospholipid antibodies with immunization with Beta-2-glycoprotein-1 (B2GP1). ^[20] Complexes of B2GP1 and phospholipid in vivo reveal epitopes that react with natural autoantibodies. High titers of these antibodies have also been linked to thrombosis and fetal wastage. ^{[21, 22]}

Three major antibodies have thus been linked to the clinical syndrome of APS and an international consensus statement on diagnostic criteria was proposed in 1998 and revised in 2006 and again in 2014. ^{[23, 24, 25]} APS is considered primary if no other underlying or concurrent autoimmune disease is present and is secondary if such conditions are present.

The updated international consensus statement on classification criteria for definite APS diagnosis requires one of the following clinical criteria and one of the laboratory criteria to be met: ^[23]

Clinical criteria are as follows:

• Vascular thrombosis: One or more clinical episodes of arterial, venous, or small vessel thrombosis is observed in any tissue or organ. Thrombosis must be confirmed by objective validated criteria (ie, unequivocal findings of appropriate imaging studies or histopathology). For histopathologic confirmation, thrombosis should be present without significant evidence of inflammation in the vessel wall.

• Pregnancy morbidity: Any of the following meets the criteria: One or more unexplained deaths of a morphologically normal fetus at or beyond 10 weeks’ gestation, with normal fetal morphology documented by ultrasound or by direct examination of the fetus; one or more premature births of a morphologically normal neonate before the 34 weeks’ gestation because of eclampsia or severe preeclampsia or recognized features of placental insufficiency; 3 or more unexplained consecutive spontaneous abortions before the 10 weeks’ gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded.

Laboratory criteria are as follows:

• LA present in plasma on 2 more occasions at least 12 weeks apart

• Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (ie, >40 GPL or MPL units or >99th percentile) on 2 or more occasions

• Anti-beta2-glycoprotein I antibody of IgG and/or IgM isotype in serum or plasma (in titer >99th percentile) present on 2 more more occasions, at least 12 weeks apart, measured by a standardized enzyme-linked immunoassay

Between LA and anticardiolipin, the former has been shown to be more specific and predictive for the occurrence of thromboses and pregnancy morbidity. A meta-analysis of the risk of venous thrombosis in patients with antiphospholipid antibodies without a previous thrombotic episode or an underlying autoimmune disorder showed that the mean odds ratio was 11 for LA and 3.2 for anticardiolipin. ^[26] Moreover, positivity for both LA and anticardiolipin, but not anticardiolipin alone, was predicative of a higher risk of recurrent thrombo-occlusive events in patients in APS with a first ischemic stroke. ^[27]

Indications/Applications

The International Society of Thrombosis and Hemostasis (ISTH) recommend the following grades of appropriateness to search for LA based on clinical characteristics and scenarios:

• Low - Venous thromboembolism (VTE) or arterial thromboembolism in elderly patients

• Moderate - Incidentally found prolonged aPTT in asymptomatic subjects, recurrent and spontaneous early pregnancy loss, provoked VTE in young patients

• High - Unprovoked VTE and unexplained arterial thrombosis in patients younger than 50 years of age, thrombosis at unusual sites, late pregnancy loss, any thrombosis or pregnancy morbidity in patients with autoimmune disease

No contraindications are noted; however, the ISTH discourages nonspecific testing in asymptomatic individuals not otherwise described above.

Considerations

Perhaps the single most important variable in the detection of LA activity is the preparation of platelet poor plasma. With the exception of the clotting time, the sensitivity of all other screening tests is inversely proportional to the residual platelets in the plasma tested. Platelet poor plasma with significant residual platelets may demonstrate shortened clotting times on freezing and thawing due to inadvertent platelet neutralization effect. ^[28] Optimum preparation of platelet poor plasma may be achieved using double centrifugation or filtered plasma.

Interpretation of LA testing results can be difficult because of prolonged baseline clotting times while the international normalized ratio (INR) is therapeutic. If determination of LA activity is an indication to continue vitamin K antagonist (VKA) therapy, some authors attest brief discontinuation of a VKA for diagnostic procedures is not a high-risk option in most instances. ^[7] The ISTH recommends testing for LA 1-2 weeks after discontinuation or when the INR is less than 1.5. ^[2] If bridging with LMWH is desired, blood for LA testing can be obtained 12 hours after the last dose. If the INR is 1.5-3, a 1:1 dilution of patient plasma and PNP is an alternative, although interpretation may still be difficult due to dilution. ^[2]

Evaluating for LA in proximity to an acute thrombotic event can be problematic for two reasons. First, most patients are anticoagulated either by therapeutic doses of heparin or VKAs. Second, acute phase reactants like factor VIII may be increased during and also interfere with interpretation of results. The ITSH advises caution in interpretation of results under these circumstances. ^[2]