Platelet Neutralization Procedure Research Articles

Quality Analysis of Lupus Anticoagulant Testing: Indeterminate and Interference

Abstract Lupus anticoagulant (LA) testing is a commonly performed test within hemostasis laboratories. Criteria for diagnosing a LA based on the International Society on Thrombosis and Hemostasis are: prolonged phospholipid-dependent (PD) clotting test, evidence of an inhibitor, evidence the inhibitor is PD, and exclusion of specific inhibitors. At our academic institution, we analyzed Lupus anticoagulant results to establish common sources of drug interference (DI), identify causes of indeterminate results, and evaluate if repeat testing was performed on patients with an initial indeterminate test. At this institution, Lupus anticoagulant evaluation includes activated partial thromboplastin time (aPTT), Anti-Xa activity, Thrombin Time (TT), diluted Russell Viper Venom Time, hexagonal phase phospholipid assay, and Platelet Neutralization Procedure. Samples with Anti-Xa activity >0.1 IU/mL and/or elevated TT are treated with hepzyme; if the Anti-Xa activity or TT does not correct, testing is not completed. Samples with elevated aPTT undergo an immediate and incubated mixing study (MS). Samples are considered positive (POS) for LA if there is a positive screening test (+ST), +MS, and evidence of PD in at least one assay. Tests are resulted as indeterminate (IND) with a +ST and either +MS or +PD. LA results are reported as unlikely (UNL) if only a single phospholipid panel is positive without +ST or +MS, and negative (NEG) if the ST, MS, and PD assays are all negative. Patients with indeterminate LA testing are recommended to have repeat LA testing after 12 weeks. A total of 3025 cases were identified over the 18-month period (July 2020 – December 2021). The majority of cases were interpreted as NEG (2310; 76%) followed by 8% POS (229), 6% IND (174), and 1% UNL (34). While the majority of IND met the ST (99%) and PD (71%) criteria, only 32% were +MS, indicating failure of an inhibitor detection in the mixing study is the major factor preventing an IND from becoming positive. Within the IND group, only 25% (43) underwent the recommended repeat LA testing. The results of repeated 43 IND tests are as follows: 16 NEG, 4 POS, 19 IND, 3UNL, 1 DI. While a significant proportion of IND repeated testing became negative, a majority remained IND, arguing for more comprehensive LA testing. This quality study demonstrates the majority of patients at this institution who receive an interpretation of IND LA do not undergo the recommended repeat testing, underscoring the importance of follow-up and education to standardize care. Prospective studies maybe needed to elucidate the clinical significance of IND LAs. Additionally, 278 (9%) cases were canceled due to drug interference, the majority (192; 69%) due to direct oral anticoagulants (DOAC). Testing for LA in patients actively receiving DOACs should be discouraged. Alternatively, methods to remove DOAC interference should be standardized.

Nine-test panel has superior sensitivity to detect antiphospholipid antibody syndrome in patients with or without SLE

Anti-phospholipid antibodies (aPL) and lupus anticoagulant (LAC) represent diagnostic criteria for systemic lupus erythematosus (SLE) and underlie anti-phospholipid syndrome (APS) in patients with and without SLE. 526 healthy controls and 1633 SLE and 1835 primary APS (PAPS) patients were evaluated. LAC was assessed by hexagonal phase phospholipid neutralization assay (HPPNA), diluted Russell viper venom test (dRVVT), and platelet neutralization procedure (PNP). β2-glycoprotein-I and cardiolipin IgG, IgM, and IgA antibodies (aCL-IgG, aCL-IgM, aCL-IgA) were measured. 222/1633 SLE patients had APS based on the nine-test panel, which afforded the highest sensitivity (74%) and negative predictive value (90%) but lowest specificity (52%). HPPNA was the most sensitive individual test at 52%. The nine-test panel yielded the greatest sensitivity for aPL detection (70%) relative to HPPNA, the most sensitive individual test (36%) in PAPS. Superior sensitivity of a nine-test aPL panel has major implications for preventing potentially fatal thrombotic events in SLE and PAPS.

Internal Quality Control and External Quality Assurance in Testing for Antiphospholipid Antibodies: Part II—Lupus Anticoagulant

In addition to the presence of appropriate clinical features, the diagnosis of the antiphospholipid antibody syndrome (APS) fundamentally requires the finding of positive antiphospholipid antibody (aPL) test result(s), with these comprising clot-based assays for the identification of lupus anticoagulant (LA) and immunologic ("solid-phase") assays such as anticardiolipin antibodies (aCL) and anti-β2-glycoprotein I antibodies (aβ2GPI). This article is the second of two that review the process for, and provide recommendations to improve, internal quality control (IQC) and external quality assurance (EQA; or proficiency testing) for aPL assays. These processes are critical for ensuring the quality of laboratory test results, and thence the appropriate clinical diagnosis and management of APS. This article covers LA testing. We provide some updated findings from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and cover testing results for the past 3 years (2009 to 2011 inclusive). In brief: (1) essentially all laboratories currently perform LA testing using activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) methods, and about one-third also employ the kaolin clotting time (KCT); (2) KCT usage has dropped slightly, from around 50% of laboratories in 2009, to around 35% in 2011, presumably reflecting take up of the latest consensus recommendations; (3) other methodologies such as silica clotting time (SCT) and the platelet neutralization procedure (PNP) are only used by <5% of laboratories; (4) interlaboratory coefficients of variation (CVs) are in general moderate, and substantially better than those reported for solid-phase assays such as aCL and aβ2GPI, with median (range) values being 11.6% (9.2 to 25.5%) for APTT ratios, 16.7% (10.1 to 19.2%) for KCT ratios, and 11.7% (5.7 to 17.4%) for dRVVT ratios; (5) CVs increase slightly with increasing LA positivity; (6) most laboratories correctly interpreted test findings for LA, reporting normal samples as normal, and LA-positive samples as positive (albeit with varying gradings of positivity); and (7) however, some laboratories found interpretation to be challenging for some samples, namely a weak LA sample (which was reported as normal by around 50% of laboratories) and a very strong LA sample (which was reported as normal by around 10% of laboratories, primarily those that did not perform mixing studies).

Guidelines on the investigation and management of antiphospholipid syndrome

This guidance updates and replaces the previous guideline on the investigation and management of antiphospholipid syndrome (APS) published in 2000 (Greaves et al, 2000), though where there have not been changes we refer back to them when appropriate. The guidance is updated with reference to relevant publications since 2000. Publications known to the writing group were supplemented with additional papers identified by searching PubMed for publications in the last 11 years using the key words: lupus anticoagulant, anticardiolipin, antiphospholipid, b2–glycoprotein I, antiprothrombin and limits (clinical trial, randomized control trial, meta-analysis, humans, core clinical journals, English language). The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the Royal College of Obstetricians and Gynaecologists (RCOG), and the British Committee for Standards in Haematology (BCSH) Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMEN DATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with antiphospholipid syndrome though individual patient circumstances may dictate an alternative approach.

Detection of Lupus Anticoagulant In Women with Obstetric complications

AbstractAbstract 3185 Introduction:Lupus anticoagulant (LA) is one of the laboratory criteria for the diagnosis of antiphospholipid syndrome. To detect the presence of LA, ISTH recommends performing two assays based on different principles. Our LA profile includes dilute Russell viper venom time (dRVVT) and two different activated partial thromboplastin time (aPTT) reagents.The aim of the study was to evaluate sensitivity and specificity of aPTT and dRVVT in women with a history of pre-eclampsia, intra-uterine growth restriction, early recurrent abortion, late fetal loss and placental abruption collectively termed “placenta mediated complications”. Materials and Methods:We studied a total of 247 patients and 287 samples in a 10 months period. Based on consensus criteria, samples were retested within a period of three months. 62/247 plasmas samples were collected during pregnancy.Laboratory studies: Screening tests: aPTT was performed using PTT-LA (Stago) and an aPTT home made reagent with diluted cephalin. dRVVT was performed using Russel viper venom RVV (Stago) and diluted cephalin. Negative control plasma was prepared according to ISTH LA recommendations for the mixing tests. Confirmatory tests: We performed a home made reagent for platelet neutralization procedure (PNP)-aPTT and dRVVT; Rosner Index and % of Correction as criteria for mixing and confirmatory tests interpretation.Statistical evaluation was performed by ROC method using EP Evaluator 9.9 software. Results:172/287 samples (59.9%) were negative, while 115/287 samples (40.1%) were positive for LA. 44/115 samples (38.26%) were positive for both tests (aPTT and dRVVT), 60/115 samples (52.17 %) were positive only for dRVVT and 11/115 samples (9.57%) were positive only for aPTT.LA was confirmed in 9/62 pregnant women samples (14.5 %). 7 (11.3%) had abnormal dRVVT, the remaining 2 samples were positive for both tests.Table for PP–Assay(UCA)/Cut offSensitivity (SE)Specificity (SP)aPTT (Cephalin)0.812/43 seg57.494.8aPTT (PTT–LA, Stago)0.813/42 seg58.391.9dRVVT (Cephalin)0.964/45 seg87.0100 Conclusions:Our results demonstrated: 1- High prevalence of dRVVT positive plasmas 104/115 (90.43%); 2- Despite showing a low performance regarding sensitivity when compared to dRVVT, both aPTT tests were able to detect LA in 47.83% of the positive studies. 3- According to our experience, considering this initial cohort of selected patients, the use of dRVVT as the first and aPTT as the second screening test may help improve the performance for the diagnosis of LA. Disclosures:No relevant conflicts of interest to declare.

Lupus Anticoagulant Testing

Lupus anticoagulant (LAC) testing is important for evaluating patients with antiphospholipid syndromes and hypercoagulable states. We reviewed results of proficiency testing challenges (n = 5) distributed by the North American Specialized Coagulation Laboratory Association to examine LAC testing performed by participating laboratories. The activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) constituted major testing methods. In screening studies, LAC-sensitive APTT methods were more sensitive to weak LAC than dRVVT-based methods but less specific. In confirmatory testing, dRVVT methods performed better, but performance was LAC-dependent. The highest false-negative confirmatory test results were obtained for the platelet neutralization procedure. Noncompliance with recommendations for LAC testing by the International Society on Thrombosis and Haemostasis was high (8%-38%), with the majority of noncompliant laboratories failing to report results of mixing studies. These data provide new insights into LAC testing in North America and identify opportunities for standardization.

Increased Anti-Protein Z and Protein S Antibodies in Patients with Anti-Phospholipid Antibodies and/or Lupus Inhibitor.

Protein S (PS) and protein Z (PZ) are natural coagulation inhibitors that function as cofactors in mechanisms that decrease the generation of thrombin. Antibodies to PZ (aPZ) and PS (aPS) have been reported to be associated with adverse pregnancy outcomes (APO) and thrombosis (THRM). They have also been described in association with antiphospholipid antibodies (APLA) and the APLA syndrome (APLAS). We previously reported, ASH 2005, that there was an increase in aPZ and aPS in patients (pts) with serological and coagulation markers for APLA and lupus inhibitor (LI). In the current study we correlated the presence of aPZ and aPS in pts with APLA & LI with a THRM positive (+) or negative (−) history. We were not able to address the APO issue. The study group consisted of 26 male and 60 female, with ages ranging from 25 to 90, and mean age of 56. 33 pts are positive only for APLA (group 1) with 24 of them THRM(+), 16 pts are positive for LI only (group 2) with 7 of them THRM(+), and 37 pts are positive for both APLA and LI (group 3) with 21 of them THRM(+). The data was compared with 49 normal APLA/LI negative donors. We considered pts who have moderate to high levels of anticardiolipin and/or anti-β2 glycoprotein I (aB2GPI) as APLA positive. We define LI positive as pts with prolonged LI sensitive PTT, and positive for at least two of the confirmatory tests (Hexagonal phase assay, platelet neutralization procedure, and diluted Russel Viper Venom Time Ratio). THRM(+) included documented DVT, PE, stroke and TIA. We considered aPZ and aPS levels greater than 10 AU/ml as positive. Out data shows that in Group 1, statistically significant (SS) differences (p<0.05) between pts vs. the control group (CTR) were noted as increased (inc) aPZ IgG & IgM, and aPS IgM (aPZ IgG 5.1+/−9.3 vs 1.3+/−2.9AU/ml, aPZ IgM 20.6+/−43.9 vs 3.9+/−6.0AU/ml, aPS IgM 4.0+/−4.0 vs 1.0+/−1.2AU/ml). When compared THRM(+) vs CTR showed SS inc aPZ IgG, aPS IgM (aPZ IgG 6.0+/−10.7AU/ml, aPS IgM 3.9+/−3.0AU/ml). The 15 APLA patients with inc aPZ and/or aPS antibodies are all associated with positive aB2GPI IgM. In Group 2, SS differences (p<0.05) between pts vs. CTR were noted as inc aPS IgM (aPS IgM 3.2+/−4.0AU/ml). In Group 3, SS differences (p<0.05) between pts vs. CTR were noted as inc aPZ IgM and aPS IgM (aPZ IgM 16.5+/−25.5AU/ml, aPS IgM 3.0+/−5.3AU/ml). When compared THRM(+) vs CTR showed SS increased aPZ (aPZ IgM 19.8+/−31.9AU/ml). When compared THRM(−) vs CTR showed SS increased aPZ IgG & IgM and aPS IgM. (aPZ IgG 3.0+/−1.2AU/ml and IgM 12.5+/−15.5AU/ml, aPS IgM 2.1+/−1.6AU/ml). The 15 pts with elevated aPZ and/or aPS are also all associated with positive aB2GPI. However, two pts were positive for IgG subgroup instead IgM subgroup. Elevated aPZ IgG was only found in THRM(+) pts (11%). This would indicate that the inc aPZ IgG is associated with THRM. The 8 pts who had both inc aPZ and aPS antibodies were positive for THRM. Our data would indicate that aPZ and aPS antibodies might play a role in the prothrombotic state associated with APLAS. Our positive APLA group has a high % of inc aB2GPI-IgM. Does this affect the results in the THRM(+) group? Is the presence of IgM APLA more likely to be associated with aPS or aPZ? Does the combination of aB2GPI and inc aPS/aPZ have greater prognostic implications for THRM? Are there functional coagulation assays that might be used to determine the prothrombotic effect of the inc levels of aPZ and aPS antibodies?

Changes in Components of the Natural Coagulation Inhibitor System, in Patients with Antiphospholipid Antibodies and/or the Lupus Inhibitor.

The cell surface glycoproteins thrombomodulin (TM) and endothelial protein C receptor (EPCR) are major intermediaries in the down-regulation of thrombin and are noted to be shed from endothelial cells (ECs) due to the actions of proinflammatory cytokines, thrombin and other agents. Antiphospholipid antibodies (APLA) have been described as perturbing ECs. The level of protein S (PS) and protein Z (PZ) have been reported to inconsistently relate to the presence of antibodies and clinical outcomes. We previously reported using a coagulation assay system to demonstrate that adverse pregnancy outcomes (APO) are associated with a greater incidence of resistance to the anticoagulant effects of TM. In the current study we measured the plasma levels of soluble TM (sTM), soluble EPCR (sEPCR) as a possible indicator of APLA-EC interaction. We measured levels of free PS, PZ, sTM and sEPCR in patients (pts) with APLA and/or lupus inhibitor (LI), and correlated the findings with a positive or negative history for thrombosis [THRM(+) or (−)]. APO were not assessed at this time. The study group consisted of 26 males and 60 females, ages ranging from 25 to 90 years; mean of 56. GROUP 1: 33 pts with increased (inc) APLA and 24 of them THRM(+), GROUP 2: 16 pts positive for LI and 7 of them THRM(+), GROUP 3: 37 pts positive for both APLA & LI and 21 of them THRM(+). The pts were compared to 49 normal APLA negative donors (CTR). Pts who have moderate to high levels of anticardiolipin and/or anti-β2 glycoprotein I are APLA positive. LI positive pts have prolonged LI sensitive PTT, and are positive for at least two of the confirmatory tests (Hexagonal phase assay, platelet neutralization procedure, and diluted Russel Viper Venom Time Ratio). GROUP 1 has statistically significant (SS) (p<0.05) decreased (dec) sTM, sEPCR, and free PS when compared to CTR (sTM 1.0+/−1.4 vs 5.1+/−3.3ng/ml, sEPCR 43+/−53 vs 145+/−140ng/ml, free PS 63+/−25 vs 76+/−14%). Free PS is SS lower in the THRM(−) vs. THRM(+). PZ levels do not demonstrate SS differences in the subset comparisons. 8% of THRM(+) pts have PZ deficiency (PZ <1.0ug/ml) with none noted in the THRM(−) pts. Free PS is dec in a majority of the THRM(−). This might be related to the much higher % of females in the THRM(−). Therefore issues such as use of OCP, estrogens, current or recent pregnancy might influence PS. The incidents of dec sTM levels (<2.0ng/ml) are surprisingly higher in this group of patients (82%). This is strikingly higher than the CTR (4%). There is no apparent difference in the THRM(+) to THRM(−). The number of patients with dec sEPCR (<5ng/ml) is much lower however; none of the THRM(−) has dec levels. Inc sTM (>8.0ng/ml) nor inc sEPCR (>284ng/ml) is noted in any of the Pts. In GROUP 2, there is SS dec sTM & sEPCR (sTM 1.6+/−1.8ng/ml, sEPCR 42+/−32ng/ml) vs. CTR with no SS difference in the THRM(+) vs. THRM(−). In GROUP 3, sEPCR is SS dec in pts (sEPCR 68+/−100ng/ml) vs. CTR and CTR vs THRM(+) (sEPCR 29+/−25ng/ml). sTM is SS dec in the CTR vs. THRM(+) (sTM 1.3+/11.4ng/ml). Our data shows low levels of sTM and sEPCR in the APLA patients. This might indicate that the levels are consumed by antibody interactions and could indicate that the APLA and associated other antibodies might interfere with their functions. Previous reports indicating a resistance to TM in a coagulation assay method in patients with THRM and APO raises the possibility of an inhibiting substance present in the APLA. Further work in larger patient groups is necessary to clarify these issues.

High Prevalence of Anti-Protein Z and Anti-Protein S Antibodies in Patients with Other Serological Markers for Antiphospholipid Antibodies.

Anti-protein Z (aPZ) and protein S (aPS) antibodies have been reported to be associated with adverse pregnancy outcome and thrombosis. We have studied the prevalence of aPZ and aPS antibodies in 63 patients who were referred to our laboratory for antiphospholipid antibody (APA) and lupus inhibitor (LI) evaluation, and 46 normal controls. We considered patients who have increased levels of anticardiolipin (ACL), anti-ß2 glycoprotein I (aB2GPI), or antiphosphatidyl-serine antibodies (aPSer) as APA positive. We define LI positive as patients with prolonged LI sensitive PTT (PTT-LA), and positive for any one of the confirmatory tests (Hexagonal phase assay, platelet neutralization procedure, and diluted Russel Viper Venom Time Ratio). 27 of the 63 patients have APA, 24 of the 63 patients have LI, and 12 of the 63 patients have both APA and LI. Table 1 shows the means and standard deviation (SD) of aPS and aPZ levels of the 63 patients compared to the 46 normal controls. The means aPZ IgM for the total 63 patients group and the 27 patients with APA alone are statistically significantly greater than the control group (p<0.05). We considered aPZ and aPS levels greater than the normal control mean plus 2 SD as positive (aPZ IgG>2.32, IgM>2.95, aPS IgG>3.18, IgM>5.8). Of the 63 patients, 7 (11%) are positive for aPZ IgG, 22 (35%) positive for aPZ IgM, 4 (6%) positive for aPS IgG, and 4 (6%) positive for aPS IgM. In the aPZ, IgG and IgM, positive patients (N=28), 9 (32%) are LI only, 16(57%) are APA only, and 3 (11%) have both LI and APA. Of the 16 APA only, 13 have ACL, 11 have aPSer, and 8 have aB2GPI. For the aPS positive group, 4 positive IgG patients have APA only, and 4 positive IgM patients have LI only. We also have three additional patients other than the 63 patient, who have increased anti-prothrombin antibodies present. These three patients have high aPS IgM antibody (mean 4.50 +/− 3.16). In conclusion, we have found that aPZ IgM antibody are higher in the patients with serological markers for APA and LI compared to our normal controls. We also found that a substantial minority of our patients have increase aPZ (46%) and aPS (13%) antibodies. Since these antibodies have been reported to be associated with thrombosis and adverse pregnancy outcome they may have prognostic implications in those patients with serological markers for APA and LI. We also noticed that high percentage of our aPZ and aPS positive patients have LI. Our data would suggest the inclusion of these antibodies in the evaluation for immune related prothrombotic states and adverse pregnancy outcome. However, larger population study with detailed clinical information and patient outcomes are needed to make appropriated recommendations as to the efficacy for this type of laboratory testing.Mean aPS and aPZ levels of the 63 patients and 46 controlsCTR (N=46)All patients (N=66)APA only (N=30)LI only (N=24)APA + LI (N=12)aPZ IgG0.50 +/− 0.911.01 +/− 2.280.92 +/− 1.081.20 +/− 3.520.84 +/− 0.58aPZ IgM0.99 +/− 0.982.83 +/− 2.96*3.07 +/− 2.0*2.21 +/− 3.663.52 +/− 3.24*aPS IgG0.64 +/− 1.270.84 +/− 1.081.22 +/− 1.470.35 +/− 0.290.98 +/− 0.6aPS IgM2.02 +/− 1.892.51 +/− 3.522.22 +/− 1.302.93 +/− 5.452.30 +/− 1.77* vs CTR P<0.05

Presence of Direct Thrombin Inhibitors Can Affect the Results and Interpretation of Lupus Anticoagulant Testing

In vitro experiments were conducted to determine whether the direct thrombin inhibitors argatroban and lepirudin can interfere with the results of lupus anticoagulant (LA) testing. Concentration-response curves were generated to calculate the concentration of anticoagulant that prolongs the activated partial thromboplastin time (aPTT) to 75 seconds (2.5 times the baseline average). Corresponding concentrations of anticoagulant were added to plasma samples before running dilute Russell viper venom time (DRVVT) and LA-sensitive aPTT (PTT-LA) tests. Because the DRVVT test system contains an antiheparin agent, DRVVT results were not prolonged in the presence of heparin. With argatroban added to normal plasma samples, neither the DRVVT percent correction of ratio nor the DRVVT test/confirm ratio was elevated, but when added to LA-positive plasma, some false-negative results were observed. Lepirudin increased the DRVVT percent correction of ratio and the DRVVT test/confirm ratio into a range that could lead to false-positive identifications of LAs. In sharp contrast to the DRVVT test system, distinction between LA-positive and LA-negative plasma samples was maintained and possibly even enhanced in the platelet neutralization procedure correction phase of the PTT-LA test system.

Presence of Direct Thrombin Inhibitors Can Affect the Results and Interpretation of Lupus Anticoagulant Testing

In vitro experiments were conducted to determine whether the direct thrombin inhibitors argatroban and lepirudin can interfere with the results of lupus anticoagulant (LA) testing. Concentration-response curves were generated to calculate the concentration of anticoagulant that prolongs the activated partial thromboplastin time (aPTT) to 75 seconds (2.5 times the baseline average). Corresponding concentrations of anticoagulant were added to plasma samples before running dilute Russell viper venom tests (DRVVTs) and LA-sensitive aPTTs (PTT-LAs). Because the DRVVT test system contains an antiheparin agent, DRVVT results were not prolonged in the presence of heparin. With argatroban added to normal plasma samples, neither the DRVVT percent correction of ratio nor the DRVVT test/confirm ratio were elevated, but when added to LA-positive plasma, some false-negative results were observed. Lepirudin increased the DRVVT percent correction of ratio and the DRVVT test/confirm ratio into a range that could lead to false-positive identifications of LAs. In sharp contrast to the DRVVT test system, distinction between LA-positive and LA-negative plasma samples was maintained and possibly even enhanced in the platelet neutralization procedure correction phase of the PTT-LA test system.

A Case of IgM- λ Waldenström's Macroglobulinemia with Activity Against Phospholipids, Masquerading as Factor V Inhibitor.

Background: The Lupus anticoagulant is known to prolong coagulation tests, however the source of antibody causing this prolongation is not clear in the majority of patients. We are reporting a patient with prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT) secondary to a lupus anticoagulant like activity of an IgM paraprotein.Case report: An 82 years old male patient was referred in January 1996 to our clinic for abnormal PT and aPTT. He was asymptomatic. There was no lymphadenopathy or organomegaly. The hemoglobin was 10.4 (g/dl) with normal white cell and platelet count. Renal and liver function tests were normal. Coagulation studies revealed aPTT 145.4 seconds, the PT 38.8 seconds, thrombin time 24.9 seconds, fibrinogen level of 336 mg/dl and D Dimer <500. In-vitro mixing studies with normal plasma failed to correct the prolongation of the clotting tests without and with incubation for 1 hour. This suggested the presence of an inhibitor. Factor inhibitor assays were done that suggested the presence of a factor V inhibitor (14.8 Bethesda units). The apparent lower level inhibitory activity against other factors II, VII and X was presumed to be due to inhibition of prothrombinase complex by factor V inhibitor. Agarose affinity column chromatography with factor V bound to the agarose did not remove the PT inhibitory activity. A lupus anticoagulant was suspected when the PT and aPTT of normal pooled plasma was prolonged by the addition of patient's plasma. The patient's plasma with pooled normal plasma gave a positive dilute thromboplastin inhibition test, 1:50 dilution >3.1 and 1:500 dilution >2.9 (normal <1.3) as well as a positive PNP (platelet neutralization procedure) as evidenced by a shortening of aPTT from >106 seconds to 48 seconds upon the addition of platelet phospholipids (a difference of >5 sec is taken as positive for LA). Expanded antiphospholipid antibodies test were done. Anti-cardiolipin IgM (>100MPL), anti-phosphotidyl serine IgM (7.3SD) were detected. A serum protein electrophoresis revealed monoclonal gamma spike of approximately > 4gm/dl and quantitative immunoglobulin by radial immuno-diffusion (RID) showed IgG- 1691 mg/dl, IgA - 185 mg/dl, IgM >192 mg/dl. The IgM >192 mg/dl is limited by the linearity of the test. IFE (Immunoelectrophoresis) demonstrated IgM-λ spike. Serum viscosity was 3.4. We suspected that the IgM paraprotein might be the inhibitor. IgM was purified by Euglobulin flocculation test. The PT and aPTT of normal pooled plasma was prolonged on its addition. The conclusion was that antiphospholipid activity was in IgM. Bone marrow biopsy in January 1996 showed <10% of plasmacytoid lymphocytes. Repeat bone marrow biopsy in December 1997 showed increased plasmacytoid cells to 60%. In view of increased serum viscosity to 4.3 and bone marrow biopsy findings as above, a diagnosis of progressive Waldenström's Macroglobulinemia was established. In addition to intermittent plasmapheresis, an oral chlorambucil (0.25mg/kg) q21 day was given for 14 months from December 1997, with a decrease in paraproteins, serum viscosity and PT, aPTT. Patient remained asymptomatic till March 2002 when he developed clinically progressive disease as evidenced by hepatosplenomegaly. He refused all investigations and interventions at this time and was lost to follow up. During the entire follow up period lasting 6 years he did not demonstrate any bleeding or thrombotic tendency.

The prevalence of lupus anticoagulant in normal pregnancy and in women with recurrent fetal loss—recommendations for laboratory testing for lupus anticoagulant

BACKGROUNDThere is wide disagreement in the literature on the rate of detection of lupus anticoagulant (LA) in women with recurrent fetal loss (RFL). The aim of this study was to determine the prevalence of LA using four phospholipid-dependant coagulation tests in a large population of Saudi women.PATIENTS AND METHODSWe determined the prevalence of LA in women with RFL (n=925), normal pregnancy (n=663), and in healthy blood donors (n=204), at the King Khalid University Hospital, Riyadh. The following coagulation tests were employed: the activated partial thromboplastin time (APTT), platelet neutralization procedure (PNP), kaolin clotting time (KCT) and the dilute Russel's viper venom test (dRVVT).RESULTSIn RFL patients, positive APTT was 10.2%, APTT+PNP 3.6%, KCT 10.5%, and dRVVT 10.9%. In normal pregnancy, the corresponding figures were 12.8%, 3.1%, 10.8%, and 5.6%. Three positive tests occurred in 2.3% of RFL patients, including APTT+KCT 3.5%, APTT+dRVVT 3.9%, and KCT+dRVVT 4.1%. The corresponding figures for normal pregnancy were 1.6% for three positive tests, and 3.0%, 1.8%, 2.4%, respectively. The dRVVT was the only test that showed a rate of positive results almost double that seen in normal pregnancy.CONCLUSIONSIf only one or even two screening tests were performed, a significant number of LA positive cases would have been missed. This could make a difference to treating physicians as to the possible etiology and management of RFL. It is therefore advisable to routinely use the three tests (APTT, KCT and dRRVT) when screening for LA.

Improved confirmation of weak lupus anticoagulants by employing sensitive and insensitive reagents to the lupus anticoagulant.

Detection of a lupus anticoagulant (LA) is of major importance to detect a thrombotic tendency. Confirmation of its phospholipid dependency may represent a tricky step in the diagnostic algorithm for LA, as several tests may yield borderline results of little diagnostic help. To improve on this point, we had previously employed a procedure comparing sensitive [rabbit brain kaolin (RBK)] and insensitive [soy bean phosphatides (SBP)] reagents to LA to screen and confirm LA at the same time in activated partial thromboplastin systems (aPTT). Here we compared its performance against a platelet neutralization procedure (PNP). To allow comparisons of our procedures with the PNP, a percentage ratio correction was calculated according to the following formula: [(RBK ratio - SBP ratio) x 100]/ RBK ratio. Similarly for the PNP: percentage ratio correction = [(buffer ratio - platelet phospholipid ratio) x 100]/buffer ratio. On 44 known LA plasmas, the PNP, expressed in seconds, yielded 15 (34%) negative or borderline results. After ratio transformation, the PNP still yielded 10 of 15 (66%) uncertain results, whereas the RBK/SBP procedure did not give any uncertain results (P = 0.0002). The mean percentage ratio correction was far superior for the RBK/SBP procedure than for the PNP (53.24 +/- 15.93 versus 20.28 +/- 12.15%, P < 0.0001). The use of sensitive and insensitive reagents to the lupus anticoagulant increases the confirmatory yield of LA in aPTT systems and may deserve inclusion amongst the confirmatory procedures for its diagnosis.

Antiphospholipid antibodies and preeclampsia: a case-control study

Objective: To assess the association between the occurrence first of preeclampsia and antiphospholipid antibodies. Methods: We conducted a prospective case-control study of 180 pregnant women with their first incidents of preeclampsia and no histories of thrombosis or systemic autoimmune diseases. Preeclampsia ( n = 180) was defined as blood pressure (BP) at least 140/90 mmHg after 20 weeks’ gestation and proteinuria at least 0.3 g per 24 hours. Two control subjects were matched to each case ( n = 360). They were pregnant women without hypertension or proteinuria and without histories of thrombosis or systemic autoimmune disease. Lupus anticoagulant (activated partial thromboplastin time, diluted thromboplastin time, platelet neutralization procedure) and anticardiolipin antibodies (immunoenzymatic assays) were assessed in both groups, and the coagulation state (levels of thrombin-antithrombin III complexes, fragments 1 + 2 of prothrombin) was also evaluated. The analysis design was a sequential plan with 5% type I error and 95% power. Results: There was no association between antiphospholipid antibodies and preeclampsia. The odds ratio for the association was 0.95 (95% confidence interval 0.45, 2.61). Antiphospholipid antibodies were detected in eight of 180 preeclamptic women and in 19 of 360 controls. In contrast, there was a clear, confirmed activation of coagulation during preeclampsia. Conclusion: Despite evidence of a prothrombotic state during preeclampsia, it is unlikely that antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies) represent risk factors for preeclampsia among women with no previous preeclampsia and no histories of thrombosis or systemic autoimmune disease.

Antiphospholipid Antibodies and Preeclampsia

In Brief Objective To assess the association between the occurrence first of preeclampsia and antiphospholipid antibodies. Methods We conducted a prospective case-control study of 180 pregnant women with their first incidents of preeclampsia and no histories of thrombosis or systemic autoimmune diseases. Preeclampsia (n = 180) was defined as blood pressure (BP) at least 140/90 mmHg after 20 weeks' gestation and proteinuria at least 0.3 g per 24 hours. Two control subjects were matched to each case (n = 360). They were pregnant women without hypertension or proteinuria and without histories of thrombosis or systemic autoimmune disease. Lupus anticoagulant (activated partial thromboplastin time, diluted thromboplastin time, platelet neutralization procedure) and anticardiolipin antibodies (immunoenzymatic assays) were assessed in both groups, and the coagulation state (levels of thrombin-antithrombin III complexes, fragments 1 + 2 of prothrombin) was also evaluated. The analysis design was a sequential plan with 5% type I error and 95% power. Results There was no association between antiphospholipid antibodies and preeclampsia. The odds ratio for the association was 0.95 (95% confidence interval 0.45, 2.61). Antiphospholipid antibodies were detected in eight of 180 preeclamptic women and in 19 of 360 controls. In contrast, there was a clear, confirmed activation of coagulation during preeclampsia. Conclusion Despite evidence of a prothrombotic state during preeclampsia, it is unlikely that antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies) represent risk factors for preeclampsia among women with no previous preeclampsia and no histories of thrombosis or systemic autoimmune disease Antiphospholipid antibodies were not a risk factor for preeclampsia among women who have never had preeclampsia.

Antiphospholipid antibodies: an epiphenomenon in Tourette syndrome.

Antiphospholipids (aPLAs) have been previously identified in children with Tourette syndrome (TS), which has led to the speculation that these antibodies might have a pathophysiologic role in this disorder. Therefore, 21 healthy children and adolescents with TS, whose ages ranged from 7 to 17 years, underwent laboratory studies designed to diagnose the lupus anticoagulant, anticardiolipin (aCL) antibodies [immunoglobulin (Ig) G, IgA, and IgM], and antinuclear antibodies. Although five subjects had at least one value that differed from accepted laboratory standards, the changes were marginal in four of them. Lupus anticoagulant was identified in one patient, based on a minimal requirement of a prolonged dilute Russell viper venom time, clotting studies that did not correct after mixture with normal plasma, and an abnormal platelet neutralization procedure. A prolonged (but correctable) activated partial thromboplastin time was found in one individual, and aCL IgG was marginally increased in three subjects. Two (10%) of a control population of 20 same-age children also had low positive aCL IgG levels. There were no differences in tics (onset, type, frequency, severity, and family history) or comorbid features between children with normal or "abnormal" laboratory study results. Our data suggest that the presence of aPLAs in TS represents an epiphenomenon rather than a pathophysiologic mechanism.

An Approach to Factor Assays in Patients with Strong Lupus Anticoagulants

Higb-titer lupus anticoqgulants (LACs) may present technical and diagnostic dffficulties in the evaluation of patients with specific factor deficiencies or inhibitors. These potent LACS, although infrequently encountered, may be very confusing because they do not generate the characteristic pattern of rising factor levels with increasing sample dilutions. In addition, they may prevent assessment of individual factor defects by lowering activities below detectable or evaluable levels (&lt;1%). To help resolve these problems, our taboratory has developed an approach that incorporates a variation of the platelet neutralization procedure (PNP), wherein we add phospholipid (PL) to adsorb the LAC in each of the dilutions of the individual factor assays. Using factor VIII for the prototypic studies, we characterized three basic result patterns that might arise, depending on the amount of factor activity recovered. As observed in patients exhibiting two of these patterns, an appreciable increase in activity agrees with the neutralization of a LAC and provides evidence against a coexistent severe deficiency of that factor and presumptive evidence against a coexistent, strong specific factor inhibitor. Key .Words: Lupus anticoagulants-Factor assays-Factor inhibitors.

Laboratory Identification of Lupus Anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants

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.

Evaluation of laboratory tests for lupus anticoagulant in a group of Chinese lupus patients

The sera of 69 Chinese patients with systemic lupus erythematosus were tested for the presence of lupus anticoagulant (LA) by a panel of laboratory tests: Kaolin clotting time (KCT), dilute Russell viper venom time (DRVVT) and platelet neutralization procedure (PNP). The prevalence of LA varied among the 3 tests (10-19%), and was 10% when LA was considered present if either KCT or DRVVT and the PNP were positive. Concordance was fair between KCT and PNP, but was poor for DRVVT with either of the other 2 tests. Only 2 of our lupus patients had a history of thrombo-embolic disease, and neither were serologically positive for LA. The incidence of thrombo-embolic diseases and that of LA were both too low in this group of Chinese lupus patients for their association to be evaluated.