Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1995-05-15
2001-07-17
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S962000
Reexamination Certificate
active
06261792
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to diagnostic assays, and in particular to diagnostic assays for lupus anticoagulants which employ phospholipids as assay reagents.
2. Description of the Related Art
A variety of diagnostic assays are known which include one or more phospholipids as assay reagents. For example, various blood coagulation tests, such as complete and partial thromboplastin times, prothrombin times, and the like, employ brain and other tissue extracts which include lipids. Similarly, the VDRL (Venereal Disease Research Laboratory) test for syphilis is based on the use of an antigen solution which includes cardiolipin, cholesterol and lecithin.
As with many assay systems, the foregoing assays suffer from the problem of false positives, i.e., for certain patients, the coagulation tests give results indicative of a coagulation problem, when, in fact, the patient's clotting mechanisms are normal, or, in the case of the VDRL test, a patient appears to have syphilis, but is, in fact, syphilis free.
Prior studies have established a correlation between these false positives and certain diseases. For example, blood samples from patients having the autoimmune disease systemic lupus erythematosus (SLE) often have prolonged coagulation times, even though clinically the patients do not exhibit bleeding tendencies and, indeed, in some cases may suffer from thrombotic episodes. The blood of such patients is said to contain “lupus anticoagulants”, “coagulation inhibitors”, “lupus inhibitors”, or “circulating inhibitors”. See T. Exner et al., “Studies on Phospholipids in the Action of a Lupus Coagulation Inhibitor,”
Pathology
. Vol. 7, pages 319-328 (1975); and P. Thiagarajan et al., “Monoclonal Immunoglobulin M-lambda Coagulation Inhibitor with Phospholipid Specificity,”
J. Clin. Invest
., Vol. 66, September 1980, pages 397-405.
It is presently believed that these “inhibitors” are in fact antibodies against phospholipids which are produced by the immune system of patients suffering from SLE. See P. Thiagarajan et al., supra. Similar anti-phospholipid antibodies have been found in the sera of patients suffering from other autoimmune diseases, such as connective tissues disease, Hashimoto's thyroiditis, rheumatoid arthritis, and the like. See P. F. Sparling, “Diagnosis and Treatment of Syphilis,”
New England Journal of Medicine
, Vol. 284, pages 642-653 (1971). Accordingly, patients with these diseases are also likely to give false positives when subjected to lipid-dependent diagnostic assays.
Efforts have been made in the past to solve the problem of false positives in lipid-dependent assays, and, in particular, lipid-dependent coagulation assays, but with only limited success. Thus, Exner et al., supra, reported that the effect of lupus inhibitor on the Russell viper venom coagulation test could be partially corrected by adding to the reagent mixture what Exner referred to as “partially characterized” phospholipids obtained from bovine cephalin using the Folch procedure. See J. Folch, “Brain Cephalin, A Mixture of Phosphatides. Separation from it of Phosphatidyl Serine, Phosphatidyl Ethanolamines, and a Fraction Containing an Inositol Phosphatide,”
J. Biol. Chem
., Vol. 146, pages 35-41 (1942).
Exner tested three phospholipid fractions identified as phosphatidyl ethanolamine, phosphatidyl serine, and inositol phosphatide. As reported by Exner, at low concentrations each fraction reduced somewhat the clotting times of plasma samples containing lupus inhibitor, but not to the levels observed for normal samples. At higher concentrations, the addition of these phospholipid fractions unfortunately changed both the clotting times of the inhibitor-containing samples and the clotting times of the normal samples. That is, rather than solving the false positive problem, the addition of these phospholipids to the reagent mixture resulted in a change in the overall response, including the baseline, of the assay. Of the three phospholipid fractions tested, Exner stated that the phosphatidyl ethanolamine fraction appeared to give the best corrective effect.
In addition to the Exner work, Thiagarajan et al, supra, studied the effects on coagulation assays of purified IgM-lambda paraprotein obtained from a patient whose response to lipid-dependent coagulation tests indicated the presence in the patient's blood of a lupus-type anticoagulant. The purified paraprotein, when added to normal plasma, was found to reproduce the abnormal coagulation times observed with the patients plasma. Studies using the paraprotein indicated that it reacted with phosphatidylserine and phosphatidic acid, but that it did not react with phosphatidylcholine or phosphatidylethanolamine.
A comparison of the results reported by Thiagarajan with those reported by Exner highlights the confusing state of the art at that time. Whereas Thiagarajan et al. found that their lupus anticoagulant would not react with phosphatidylethanolamine, Exner et al. found just the opposite. Moreover, in Exner's hands, phosphatidylethanolamine distorted the basic character of the assay, as evidenced by the fact that the presence of 0.05% phosphatidylethanolamine in the reagent mixture resulted in an over 40% increase in the clotting time of normal plasma, and only a 30% decrease in the clotting time of a mixture of 90% normal plasma and 10% patient plasma (see
FIG. 2A
of Exner et al.).
Janoff et al., U.S. Pat. No. 4,666,831, issued May 19, 1987, incorporated herein by reference, disclosed an improved lipid-dependent diagnostic assay in which the test sample to be assayed is pre-incubated with one or more phospholipids which have a hexagonal (H
II
) organization when dispersed in an aqueous phase. Alternatively, Janoff et al. pre-incubate with lipidic particles. As demonstrated by the test results presented in the Janoff patent, lipid-dependent diagnostic assays which include pre-incubation with such hexagonal (H
II
) phospholipids were shown to be less likely to exhibit false positives. Among the preferred phospholipids used in the Janoff patent were dioleoylphosphatidylethanolamine (DOPE) and egg phosphatidylethanolamine (EPE). A related patent, Janoff et al., U.S. Pat. No. 4,689,299, issued Oct. 6, 1987, discloses the use of bilayer-forming lysophospholipids as an alternative pre-incubation agent in such assays.
The use of such hexagonal (H
II
) phospholipids in assays is further discussed in Rauch et al., “Distinguishing Plasma Lupus Anticoagulants from Anti-Factor Antibodies Using Hexagonal (II) Phase Phospholipids,”
Thrombosis and Haemostasis
, 62(3) 892-896 (1989). In this article, also incorporated by reference into the present application, Rauch demonstrates in a series of tests that egg phosphatidylethanolamine (EPE), inhibits the anticoagulating effects of Lupus anticoagulant without affecting the anticoagulating effects of other tested anticoagulants, such as anti-factor antibodies or heparin. Thus, hexagonal (H
II
) phospholipids can be used in assays to specifically distinguish between Lupus anticoagulant and other anticoagulants.
In the assay process, as discussed in the Janoff et al. U.S. Pat No. 4,666,831 patent and the Rauch et al. article, a preliminary step is the preparation of a stock solution of the H
II
phospholipid suspended in a suitable aqueous carrier such as Hepes buffer. However, the hexagonal (H
II
) organization of such phospholipids which makes them suitable for use in such assays also makes them very difficult to suspend in aqueous media. This is particularly true of a pure synthetic phospholipid such as DOPE, because natural phospholipids, such as EPE, often have small amounts of impurities that may improve their suspendability. Thus when H
II
phospholipids are suspended in aqueous buffer, they have been found to precipitate quickly, thus causing problems in handling. Such mixtures need to be mixed continuously to maintain the suspensions. Furthermore, the hydrated phospholipids tend to stick to the sides of glass vessels, and are thus extr
Janoff Andrew S.
Rauch Joyce
Taraschi Theodore F.
Gitomer Ralph
Goodman Rosanne
The Liposome Company Inc.
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