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Activated Plasma Clotting Time as a Simple Method to Detect Lupus Anticoagulant

By A Siegemund Centre of Internal Medicine, University of Leipzig, Germany Oct 1996.

In 110 patients with thrombotic events anticardiolipin antibodies (ACA) and lupus anticoagulant (LA) were measured. For ACA detecting we used the assays from elias, as sensitive methods for detecting LA the kits from Diagnostica Stago (Stago LA, PTT-LA, Staclot PNP and Platelin Excel LS (Organon Teknika). The results of these tests were compared with three new ones on the German Market: SpeckTin™ APCT (Spectra™ APCT in the US) activated plasma clotting time, SpeckTin™ APTT (Spectra™ APTT in the US) (purified soy extract) and SpeckTin-LA™ (phospholipid preparation, 4 concentrations) all Wak Chemie with the following results:

1. An increasing ratio of SpeckTin APCT/ SpeckTin APTT (2,7) is evidence of LA
2. In 30 patients with LA we found a sensitivity of 86% for Platelin Excel LS, 91% for PTT-LA, 96% for Staclot LA resp. Staclot PNP, 91% for SpeckTin APCT.
3. By titration with SpeckTin-LA™ we measured the activity of LA; we measured the amount of phospholipid until neutralization of LA and distinguished between high, low and moderate LA
4. The SpeckTins are simple and specific methods for LA detection and in contrast to the Stago methods adaptable on automatic coagulation systems.

Introduction: Lupus Anticoagulant (LA) is a group of auto-antibodies of different immunoglobin classes directed against negatively charged phospholipid (PL) complexes. As PL-interfering antibodies, the LA causes a prolongation of phospholipid-dependent tests of coagulation especially the APTT.

Due to the marked heterogeneity of antibodies, there exists no laboratory test which specifically and sensitively detects all subtypes of LA and it is often difficult to distinguish from other inhibitors. The relatively high incidence of LA and the relationship of thrombophilia requires screening tests which indicate promptly, at a moderate price and with high sensitivity the existence of LA

In accordance with the above described demands the aim of our investigations was testing two new reagents in the German market.

SpeckTin™ APCT- Activated Plasma Clotting Time reagent

SpeckTin™ APTT- Activated Partial Thromboplastin Time reagent

For use as LA screening test in the coagulation laboratory. As confirmatory test we select phospholipid titration with:

SpeckTin-LA™- phospholipid standards with different concentrations

Objectives: One of the most sensitive LA test presently in use is the kaolin clotting time without phospholipid. SpeckTin™-APCT also contains no phospholipid, which could be neutralized by the LA in the patient’s plasma. For this reason we mean the SpeckTin-APCT is a very sensitive reagent for detecting LA in platelet poor respectively platelet depleted plasma.

SpeckTin™-APTT with an optimized level of platelet substitute (purified soy extract) with an excellent sensitivity for heparin and deficiencies in coagulation factors was also used to discriminate between the APCT- prolongation due to deficiency of intrinsic coagulation factors, heparin or coumadin therapy or LA. As reference methods for LA-detection in our laboratory the following tests were used:

PTT-LA

Staclot LA

Staclot PNP

Platelin Excel LS

LUPO/LUCOR resp. LAC screen/ LAC confirm

Materials and Methods:

SpeckTin™ APTT: freeze-dried activated partial thromboplastin time reagent which contains purified soy phosphatide chemical activator. ACS, Inc. USA

SpeckTin™ APCT: freeze-dried activated plasma clotting time reagent which contains no phospholipid. ACS, Inc. USA

SpeckTin-LA™ set: four levels of freeze-dried phospholipid preparations in range of 10-500 ug/ml phospholipid. ACS Inc. USA

Platelin Excel LS (Organon Teknika GmbH, Eppelheim) with two incubation times (1 minute and 10 minutes).

PTT-LA: (Diganostica Stago) LA sensitive APTT reagent .

Staclot LA: (Diagnostica Stago) reagent system of hexagonal phase phospholipid molecules (phosphatidylethanolamin).

Staclot PNP: (Diagnostica Stago) Detection of LA by platelet neutralization procedure.

LUPO/LUCOR: (Gradipore Ltd., Australia) resp. since 1995 LAC screen/ LAC Confirm (Instrumentation Laboratory, Italy) the first is a Dilute Russell’s Viper Venom Tests. The second is a phospholipid-rich LA confirmatory reagent.

Test-Thrombin: (Behring) AG

Cardiolipin-antibody, IgG and IgM- ELISA (ELISA Medizintechnik)

Hepzyme®: (Dade) for neutralization of heparin

Membrane Filters 0,22 um (Macherey-Nagel)

All measurements were made on the ACL (Instrumentation Laboratory), the Stago tests were made on the KC 10 (Amelung GmbH).

Citrate blood samples were obtained by venipuncture. After centrifugation at 2500 g for 20 minutes (or double centrifugation) the plasma samples were stored in aliquots at 70 C or were kept at room temperature up to four hours.

Results: Normal range of SpeckTin™-APTT respectively SpeckTin™ APCT: Our own normal range was obtained from 127 individuals without therapy and without LA:

SpeckTin™-APCT: mean: 46,9 +/- 8,0 seconds (SD)

SpeckTin™-APTT: mean: 26,0 +/- 3,3 seconds (SD)

Ratio: APCT/APTT is always 1,0 to 2,3

The values in patients with a prevalence of LA differs significantly from individuals without LA:

SpeckTin™-APCT: mean: 170 seconds

SpeckTin™-APTT: mean: 33,5 seconds

Ratio: APCT/APTT range: greater than 2,8 for individuals without heparin, vitamin K antagonists and hepatic disorder.

In some problematic cases we suggest the following procedure:

• Apply heparin neutralizer like Hepzyme® for patients with heparin therapy
• In patients with hepatic disorders or congenital deficiencies in coagulation factors, the screening test should be completed with the PL standard titration by use of SpeckTin-LA™ set containing SpeckTin™ activator plus PL in different concentrations: see the results for two patients with factor XII-deficiencies (factor XII <1%), Figure 2 & Table 3.
• PL-standards can also be used for LA titration in normal patients to check the LA activity, Figure 1. According to the amount of PL which is necessary to neutralize the LA, we can differentiate the activity of the LAs in strongly, moderately or weakly present. On principle this result is comparable with LUPO/LUCOR (Figure 3); but there are differences because of the different measuring principle (Russell’s Viper Venom directly activates factor X; and is unaffected by contact factor abnormalities and factor VII deficiency or inhibitors).
• The coumarin effect can be estimated by additional determination of the APTT; to find the correlation to the INR further investigations are necessary.
• In more than 800 patients investigated in our laboratory with suspected LA (over 50 patients were positive for LA), we measured a specificity of 91,3%, this result is comparable with PTT-LA and Staclot LA with 91% resp. 95% (Table 4).

Conclusions:

• The prolongation of SpeckTin™ APCT using platelet depleted plasma is an excellent screening procedure for LA.
• Measurements of SpeckTin™ APTT gives further information about therapeutic treatment with anticoagulants.
• Very helpful is the ratio of SpeckTin™ APCT/SpeckTin™ APTT; by this ratio and under consideration of therapeutic drugs, we can distinguish between LA-positive and LA- negative patients.
• We can titrate the LA with PL standards to determine whether the LA is weakly, moderately or strongly present.
• Specificity is comparable with other methods for detection of LA.