Biomedical subjects
Publications and source records attributed to D A Triplett.
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Antiphospholipid-protein antibodies (APA) are a family of immunoglobulins which have been defined by varying laboratory test systems. Lupus anticoagulants (LA) and anticardiolipin antibodies (ACA) are the two most prominent members of this family of antibodies. LA are detected utilizing various phospholipid (PL) dependent tests of coagulation (e.g., activated partial thromboplastin time [APTT], Kaolin Clotting Time [KCT], dilute Russell Viper Venom Time [dRVVT]). Originally, LA were thought to be a laboratory nuisance since the vast majority of individuals with LA did not bleed. Paradoxically, patients with LA were found to have an increased incidence of thromboembolic events and also recurrent spontaneous abortions (RSA). Thus, the laboratory detection of LA has become part of the work up of patients with thromboembolic disorders and RSA. ACA are detected using solid phase assay systems (radioimmunoassay or ELISA). The presence of ACA has the same clinical implications as that of LA. Although originally it was suggested ACA and LA were the same antibody, it is now well accepted that they, in many instances, are different antibodies. Therefore, it is critical for laboratories to evaluate patient samples for both LA and ACA. In approximately 60% of circumstances, both antibodies will be found. In the remaining cases, there will be discordance between the two test systems. The question of whether APA are causative, coincidental, or a consequence of the clinical complications of RSA and thrombosis remains controversial. Recent evidence based on prospective clinical studies and analysis of markers of in vivo coagulation suggests APA are causative.(ABSTRACT TRUNCATED AT 250 WORDS)
Lupus anticoagulants (LAs) are antibodies that interfere with phospholipid dependent coagulation reactions in vitro. This workshop was designed to provide the participants with an experience in identification of LAs, to evaluate different criteria for mixing studies, to assess the performance of recently introduced confirmatory studies and to assess the performance of two potential surrogate LA control plasmas. The results demonstrate that there continues to be significant variation in the sensitivity and responsiveness of APTT reagents to the presence of LAs, confirming the need for more than one screening assay before the presence of a LA can be ruled out. In this workshop, the best distinction between factor deficiency and inhibitors was obtained using a 1:1 mix of normal plasma with patient plasma and the criterion defining correction as shortening of the APTT to within 5 s of the APTT of pooled normal plasma. A 4:1 mix of patient to normal plasma did not work well in distinguishing factor deficiency from inhibitors. The platelet neutralization procedure, DVV confirm and StaClot LA all gave positive results with the LA samples. False positive platelet neutralization procedures were seen with the samples from patients on oral anticoagulants and a factor V inhibitor. False positive StaClot LA results were obtained with high titer factor VIII inhibitors. Both of the potential surrogate plasmas gave variable results with multiple assays; they can not be recommended for routine use at present.
Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.
The venom of P. textilis contains two different enzymes which convert human prothrombin into thrombin. Prothrombin activation by Textarin, a serine proteinase containing a calcium-binding molecule site, with a molecular mass of 50,000 to 53,000 Da and I.P. 5.5, separated from crude venom by either barium citrate adsorption or hydroxyl apatite chromatography, is strongly stimulated by phospholipid and calcium ions. A second activator, found in the supernatant of barium citrate adsorbed venom solution, activates prothrombin in the absence of any co-factor. Human plasma coagulation induced by Textarin, phospholipid and calcium ions is affected by lupus anticoagulants. Textarin may thus be used for the detection of lupus anticoagulants in patient plasma samples.
The incidence of antiphospholipid antibodies in patients with monoclonal gammopathy of undetermined significance (MGUS) was studied. Antiphospholipid antibodies were measured in the sera of 93 patients (49 women, 44 men; mean age 70 [+/- 21] years) with MGUS by using an enzyme-linked immunosorbent assay (ELISA). The phospholipids tested were cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Positive results were defined as a value higher than the number of multiples of the mean per phospholipid, which included 76 of 80 (95%; 20 age-matched) control individuals. The immunoglobulin-G (IgG) or IgM antiphospholipid antibodies isotype varied among the patients, as did the phospholipid specificity. For IgG, PI was found elevated in 32% of the MGUS samples, whereas the other phospholipid antigens ranged from 9% to 15%. The percentage of patients with IgM antiphospholipid antibodies was higher. The authors observed PS, PI, PA, and PC as positive in 45%, 35%, 25%, and 25% of patients, respectively. Of the 12 sera studied for IgA isotype, three (25%) were positive for PS, six (50%) for CL, and none for PE. Patients with MGUS manifested a significantly higher (P < .01) incidence of antiphospholipid antibodies in their blood than did the control persons. No difference in the incidence of antiphospholipid antibodies was seen between younger and older (age-matched) control patients. No correlation was found between serum levels of immunoglobulins and optical density reading of the blank plates used as ELISA controls.
The effect of repeated freeze-thaw cycles on anticardiolipin antibody levels was evaluated using an enzyme-linked immunosorbent assay. Normal human serum was spiked with known quantities of freeze-dried human polyclonal anticardiolipin antibody IgG and IgM (19 samples each) or IgA (11 samples). Each spiked sample was split into four identical aliquots; one aliquot was never frozen, and the remaining three were taken through successive freeze-thaw cycles. All aliquots from each sample were evaluated on the same day using the same plate and reagents. A significant decline in mean anticardiolipin IgG levels occurred between the aliquot which had never been frozen and the one which had been through three freeze-thaw cycles (Student's t-test, P = .04). Although mean IgM and IgA values declined as well, the differences were not significant. When individual samples were evaluated the decline appeared to occur most often between the second and third freeze-thaw cycle. Eight anticardiolipin IgG and three IgM-containing samples which had been positive initially became negative by the third freeze-thaw cycle. These data show that handling and storage of serum used to perform anticardiolipin antibody assays are important potential sources of assay variability.
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Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, IgA or a mixture) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, KCT, dilute Russell Viper Venom Time). LA are heterogeneous; consequently, the laboratory diagnosis is difficult and relies on multiple tests. We have developed a sensitive and relatively specific confirmatory test system based on fractions of two snake venoms. Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern brown snake), activates prothrombin in the presence of PL, factor V and calcium ions. Ecarin, a protein fraction of Echis carinatus venom, will activate prothrombin in the absence of any cofactors. The activation of prothrombin by Textarin yields thrombin while Ecarin yields meizothrombin. In the presence of LA, the Textarin time is prolonged and the Ecarin time is unaffected. The test results are reported as a ratio of Textarin/Ecarin times (abnormal greater than 1.3). We have evaluated this test system in the following patient populations: LA positive, therapeutically heparinized, stable oral anticoagulated, liver disease, routine preoperative, anticardiolipin antibody positive LA negative, hemophilia A, various other hereditary factor deficiencies or dysfunctional proteins, and specific inhibitors of factor V and factor VIII. The LA positive patients represented a mixed population of autoimmune disease, drug-induced and post-infectious states. Our findings indicate the sensitivity of the Textarin/Ecarin system in the confirmation of LA. In order to use the test system most effectively, it is recommended to incorporate polybrene with Textarin when evaluating heparinized samples. Factor V deficiency and specific inhibitors of factor V yielded, in some instances, false positive results.
Lupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e. g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.
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ARACHnase (Hemostasis Diagnostics International Co., Denver, CO) is a normal plasma that contains a venom extract from the brown recluse spider, Loxosceles reclusa, which mimics the presence of a lupus anticoagulant (LA). Seven activated partial thromboplastin time (APTT) reagents were used for platelet neutralization procedure (PNP) testing with ARACHnase: Automated APTT (Organon-Teknika, Durham, NC); Thrombofax and Thrombosil (Ortho, Raritan, NJ); Actin and Actin FSL (Dade, Aguado, PR); and Thromboscreen-Kontact and Thromboscreen-APTT LS (Pacific-Hemostasis, Ventura, CA). ARACHnase consistently displayed a positive PNP result of greater than 5 seconds correction of the initial baseline APTT. Thus, ARACHnase may provide a positive control for LA testing, regardless of the choice of APTT reagent and activator/phospholipid combination.
Plasma fibrinogen concentration appears to be an important risk factor for the development of atherosclerotic cardiovascular disease of a similar magnitude to cholesterol. The quality control of plasma fibrinogen assays has taken on new importance as a consequence of this potential role as an atherosclerotic risk factor. This article reviews the performance characteristics of 40,000 fibrinogen assays comprising the College of American Pathologists Proficiency Testing Program from 1988 through 1991. Instrument and reagent variables both play roles in the poor interlaboratory reproducibility documented by this study. The absence of either a national or international standard for plasma fibrinogen assays has been a major source of reagent variability. The validation and calibration of the College of American Pathologists lyophilized plasma reference preparation for fibrinogen determination is also reported in this study. The availability of this validated reference plasma should markedly improve interlaboratory reproducibility.
Antiphospholipid antibodies have been identified as a potential risk factor for both arterial and venous thromboembolic events. Whether these antibodies are causative or a consequence of a previous clinical event remains controversial. A variety of laboratory assays have been introduced to detect the two primary members of the antiphospholipid antibody family: (1) lupus anticoagulants and (2) anticardiolipin antibodies. There is considerable variability in the laboratory tests that have been utilized. A number of potential pathogenic mechanisms have been proposed for antiphospholipid antibodies. These mechanisms involve the interaction of antiphospholipid antibodies with endothelial cells, platelets, and various plasma regulatory proteins. Although venous thromboembolic events are more common, recent evidence suggests a high frequency of arterial events. The cerebral circulation is most commonly involved, followed by involvement of the coronary arteries. An interesting subset of young patients have peripheral vascular disease that often is relatively refractory to surgical treatment with a high incidence of graft occlusion.
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To evaluate sources of interlaboratory variation in factor VIII coagulant assays and the effects of uniform calibration on it, data were analyzed from Survey Set H2-C, 1987, of the College of American Pathologists, Northfield, Ill, into which the College of American Pathologists Reference Preparation for Factor VIII was incorporated. Peer group performance and its improvement by uniform calibration were evaluated by comparing means, precisions, and numbers of acceptable survey scores. Results showed a poorer performance by less experience laboratories and by those that used undesirable calibrators. Substantial improvement was seen with uniform calibration, especially among laboratories that had employed poorer calibrators. Although variance component analysis showed that the type of calibrator was the most important contributor to interlaboratory variation, it accounted for only 10% of total variability. Participant summaries and questionnaires that spanned several years revealed steady improvement in methodology but no striking changes in interlaboratory variation.