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317-841-0458

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Analytical Control Systems, Inc.

New Method for Quantifying Lupus Anticoagulant

By Roy Speck. June 1996.

Summary: A method is presented using a new reagent containing propyl gallate for the quantitative determination of lupus anticoagulant. The amount of an optimized phospholipid standard required by the clotting reaction was found to be 32-50 ug/ml at a 95% confidence level, with a mean of 41 ug/ml. This method eliminates the effect of heparin therapy, coumadin therapy, factor VIII inhibitor, factor IX inhibitor, and single-factor deficiencies that afflict presently used lupus anticoagulant screening and confirmatory procedures. Using this method, it should be possible to detect lupus anticoagulant in patients at a much lower level and follow the effect of therapy on lupus anticoagulant. Key Words: Lupus anticoagulant, phospholipid, activated plasma clotting time (APCT), activated partial thromboplastin time (APTT).

Lupus anticoagulant (LA) is characterized as anti-phospholipid antibodies (APA) that interfere with phospholipid-dependent clotting tests. LA is associated with arterial and venous thrombosis (1-4), as well as recurrent spontaneous abortion (5,6). The screening tests for LA are not entirely satisfactory. The procedures commonly in use lack sensitivity and exhibit a substantial amount of variation. Even the most recent studies do not show any general agreement regarding an acceptable procedure for detecting LA (Table 1).

Guidelines on testing for LA have been issued by the British Society for Hematology (14) and SSC Subcommittee for the Standardization for Lupus Anticoagulant (15):

There should be a prolongation of phospholipid dependent clotting tests, that is, kaolin clotting time, dilute Russell viper venom time, tissue thromboplastin inhibition test, plasma recalcification clotting time, or a sensitive activated partial thromboplastin time (APTT) test.

Clotting times of a mixture of test and normal plasma should be significantly longer than that of a mixture of non-LA plasma and normal plasma.

There should be a correction of the prolonged test result when there is an addition of lysed, washed platelets, phospholipid liposomes containing phosphatidyiserine, or hexagonal phase phospholipids.

The test results should be nonspecific for any individual clotting factor and include a positive enzyme-linked immunosorbent assay for antiphospholipid antibody.

Even when the confirmatory procedures are performed on plasma samples that demonstrate prolongation of the test results by the screening LA procedures, it is not always possible to identify an LA.

At best, LA can be identified as weak, moderate, or strong.

I have developed a new soluble chemical activator, propyl gallate, which rapidly activates the intrinsic pathway, as well as platelet factor 3 found in the platelet membrane. Removing the platelets by filtering plasma through a 0.22 um membrane filter and performing an activated plasma clotting time (APCT) that using this new reagent results in an increased sensitivity in detecting LA because of the low phospholipid present in the reaction. The platelet membrane microfragments in the filtrate supply the phospholipid used in the clotting reaction. I have also developed a purified optimized phospholipid that can be added in increasing known amounts to this reagent. By performing the APCT test using this reagent with different phospholipid content, the amount of LA present in the patient's plasma can be quantified. This study demonstrates the advantages offered by this new technique. The study plan is (a) determine normal phospholipid requirements for coagulation and (b) compare APCT, APTT, and phospholipid content while using normal and various abnormal plasmas.

Materials and methods: Materials

1. Analytical Control Systems (ACS) APCT Reagent (patented). Freeze-dried APCT Reagent containing propyl gallate activator buffer and stabilizer. Reconstitute with 3 ml distilled water. (Analytical Control Systems Inc., Fishers, IN, U.S.A.)
2. ACS APTT Reagent (patented). Freeze-dried APTT Reagent containing platelet substitute, propyl gallate activator, buffer, and stabilizer. Reconstitute with 5 ml distilled water.
3. ACS SpeckTin LA™:

   Freeze-dried SpeckTin-LA containing soluble chemical activator, buffer, and stabilizer, but no phospholipid. Reconstitute with 3 ml distilled water.

   Freeze-dried SpeckTin-LA (10) containing soluble chemical activator, buffer, stabilizer, and 10 fig/ml phospholipid. Reconstitute with 1 ml distilled water.

   Freeze-dried SpeckTin-LA (50) containing soluble chemical activator, buffer, stabilizer, and 50 ug/ml phospholipid. Reconstitute with 1 ml distilled water.

   Freeze-dried SpeckTin-LA (200) containing soluble activator, buffer, stabilizer, and 200 jig/ml phospholipid. Reconstitute with 1 ml distilled water.

   Freeze-dried SpeckTin-LA (500) containing soluble chemical activator, buffer, stabilizer, and 500 jig/ml phospholipid.

4. Calcium chloride, 0.02 M (Sigma Chemical Company, St. Louis, MO, U.S.A.).
5. Vacutainer 6415, buffered 0.109 M sodium citrate (Becton-Dickinson Vacutainer Systems, Rutherford, NJ, U.S.A.)
6. Fibrometer coagulation instrument (Becton Dickinson Microbiological Systems, Hunt Valley, MD, U.S.A.)
7. Millipore membrane filter, 0.22-mm filter type: GS, Millipore GSWP 025 00 (Millipore Corporation, Bedford, MA, U.S.A.)
8. Normal and abnormal plasma samples (Universal Reagents, Indianapolis, IN, U.S.A.

Methods:

Preparation of platelet-depleted plasma

A clean venipuncture was performed without hemostasis using Vacutainer 6415 containing buffered 0.109 M sodium citrate. Samples were mixed immediately and centrifuged for 10 min at 2,000 RCF. The platelet poor plasma (PPP) was transferred to a plastic test tube with a plastic Pasteur pipette. The PPP was filtered through 0.22-um membrane filter into plastic test tube. The platelet-depleted plasma (PDP) samples were stored at room temperature for no longer than 2 hours. Hemolyzed samples were discarded.

Determination of activated plasma clotting time

APCT reagent (100 ul) and PPP (100 ul) were added to a reaction cuvette and mixed. After incubation at 37°C for exactly 3 min, 100 ul 0.02 m CaCl₂, which had been prewarmed at 37°C for at least 3 min, was added and the clotting time determined.

Determination phospholipid requirements for normal plasma

PDP samples were prepared from 40 adult donors who were taking no medication. The phospholipid requirement for each sample was determined and the 95% confidence limit was calculated. The procedure outline was as follows:

1. A series of SpeckTin LA vials with 0, 10, 50, 200, and 500 ug/ml phospholipid were reconstituted. Reconstituted reagents were mixed to give phospholipid intervals of 5 ug/ml for the appropriate range.
2. An aliquot of 0.02 M calcium chloride was warmed at 37°C for at least 5 min.
3. 100 ul PDP and 100 ul SpeckTin were added to the cuvette, mixed, and incubated at 37°C for exactly 3 min.
4. At the end of the incubation period, 100 ul 0.02 M calcium chloride was added and the clotting time was determined.
5. The results were recorded. The test was repeated with each of the reagents having phospholipid intervals of 5 ug/ml.
6. Phospholipid concentration versus clotting times was graphed.
7. The end point was determined as the point at which the baseline began. Note: When plasma levels of LA are elevated, larger concentrations of phospholipid are required for the titration (Fig. 1.)

Results: Phospholipid required for clotting reaction was determined (n = 40, mean = 41 ug/ml phospholipid, and 95% confidence limit = 32-50 ug/ml phospholipid), and APCT, APTT, and LA phospholipid assay results for normal and abnormal plasmas were contrasted (Table 2).

Discussion: LA neutralizes the phospholipid in the reaction mixture of in vitro phospholipid-dependent clotting tests. When the phospholipid content falls below that amount needed to support the clotting reaction,the test results become prolonged. However, a number of conditions can also prolong the test result. Among these are

1. Heparin therapy
2. Coumadin therapy
3. Factor-VIII inhibitor
4. Factor-IX inhibitor
5. Single intrinsic pathway factor deficiency

Because of these other possibilities for prolonged test results, additional procedures are necessary so that these other possibilities may be eliminated and the presence of LA may be confirmed.

Mixing studies and agarose gel electrophoresis are used to identify inhibitors. Confirmatory procedures using test systems with decreased phospholipids are tissue thromboplastin inhibition, dilute Russell viper venom test, and dilute phospholipid APTT test. Confirmatory procedures using test systems with increased phospholipids are platelet neutralization procedure, high phospholipid APTT, rabbit brain neutralization, phosphatidyiserineliposome APTT, and hexagonal phospholipid neutralization. Even when these other procedures are performed, the only thing that can be determined is whether the LA is weak or strong.

On the other hand, by determining the quantity of phospholipid needed to satisfy the requirements for the clotting reaction, these other interferences are eliminated. In fact, the results are essentially a quantitative measurement of the LA present in the patient's plasma. It may become necessary to redefine LA to include that antiphospholipid antibody which neutralizes the phospholipid that exceeds that which is required for the clotting reaction. With this LA-quantifying procedure, the true incidence of LA and the level of LA that is clinically significant can be determined. In addition, it is possible to follow the effect of therapy on the level of LA and perhaps assist in development of new pharmaceuticals for their effect on LA.

References:

1. Mintz G, Acevedo-Vazquez E, Gutierrez-Espinosa G, et al. Renal vein thrombosis and inferior vena cava thrombosis in systemic erythematosis. Arthritis Rheum 1984;27:539
2. Padmakumar K, Singh RR, Rai R, et al. Lupus anticoagulants in systemic erythematosis: prevalence and clinical associations. Ann Rheum Dis 1991; 498:986
3. Averbuch M, Koifman B, Levo Y. Lupus anticoagulant, thrombosis and thrombocytopenia in systemic lupus erythematosis. Am J Med Sci 1987;293:1
4. Santoro SA. Antiphospholipid antibodies and thrombotic disposition: underlying pathogenic mechanisms. Blood 1994; 83:2389
5. Hughes GRV. Thrombosis, abortion, cerebral disease and the lupus anticoagulant. BMJ 1983;287;1088
6. Hughes GRV An immune mechanism in thrombosis. Q J Med 1988,258:753
7. Barna LK, Triplett DA. A report on the first international workshop for lupus anticoagulant identification. Clin Exp Rheumatol 1991;9:557
8. Saxena R Saray AK, Kotte VK, et al. Evaluation of four coagulation tests to detect plasma lupus anticoagulants. Am J Clin Pathol 1991; 96:755.
9. Hemostasis Committe of the Societé Française de Biologie Clinique, Angeles-Cano E ed. Laboratory heterogeneity of the lupus anticoagulant: a multi-centre study using different clotting assays on a panel of 78 samples. Thromb Res 1992;66:349.
10. Triplett DA. Coagulation assays for lupus anticoagulant: a review and critique of current methodology. Stroke. 1992;23 (suppl 1):1-11.
11. Marshall LR. Detection of lupus anticoagulant: an Australian perspective. Pathology 1992; 24:360.
12. Messmore H, Fabbrini N, Hoppensteadt D, et al. Lupus anticoagulant assays. Semin Thromb Hemost 1994;20:79
13. Feng C. Sophia SF, Tsang SSF, et al. Evaluation of laboratory tests for lupus anticoagulant in a group of Chinese patients. Pathology 1994;26:40.
14. Lupus Working Party on behalf of the BCSH Hemostasis and Thrombosis Task Force: Guidelines on testing for the lupus anticoagulant. J Clin Pathol 44:885.
15. Exner T, Triplett DA, Taberner D, Machin SJ. Guidelines for testing and revised criteria for lupus anticoagulants, SSC subcommittee for the Standardization of Lupus Anticoagulants. Thromb Haemost 1991;65:320

APA, anti-phospholipid antibodies; APTT, activated partial thromboplastin time; DRVVT, dilute Russel viper venom time; KCT, kaolin clotting time; LA, lupus anticoagulant; PNP, platelet neutralization procedure; and TTIT, tissue thromboplastin inhibition test

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