Lupus Anticoagulant Assays Research Articles

Real-world practice of obstetricians in respect of assays for antiphospholipid antibodies

Objective. The international classification criteria (CC) for definite antiphospholipid syndrome (APS) recommend confirmation of the sustained presence, for at least 12 weeks, of both lupus anticoagulant (LA), as determined by aPTT and RVVT, and anti β2glycoprotein I (β2GPI) or anticardiolipin (aCL) IgG and/or IgM. However, it remains unclear whether obstetricians comply with the aforementioned CC for the diagnosis of APS in daily clinical practice. We performed a nationwide survey to examine the attitudes of Japanese obstetricians toward the use of assays for antiphospholipid antibodies (aPLs).Methods. A questionnaire was sent to 2,700 obstetric facilities where maternity checkups are carried out. The types of assays conducted for aPLs, ascertainment of persistence of the antibodies for at least 12 weeks, and the cutoff points used for the assays were examined.Results. Of the facilities surveyed, 61.5% carried out the assay(s) only once. In regard to the type of assay performed, 97.1% carried out the assay for aCL IgG and/or β2GPI-dependent aCL, while 67.9% performed the LA-aPTT and/or LA-RVVT assay. Only 8.8% carried out assays for both LA. As for the cutoff points used, 98% of the facilities used lower cutoff points described in the manufacturers’ manuals rather than the cutoff values recommended in the CC.Conclusion. Thus, only a limited number of facilities adhered precisely to the CC for the diagnosis of APS. Inappropriate treatment and unnecessary expense are potentially major concerns when facilities overdiagnose APS using lower cutoff points or without ascertaining the persistence of the antibodies for at least 12 weeks. On the other hand, some patients miss the opportunity to be treated for APS because of the absence of testing for LA.

Antiphospholipid antibody testing for the antiphospholipid syndrome: a comprehensive practical review including a synopsis of challenges and recent guidelines

The antiphospholipid (antibody) syndrome (APS) is an autoimmune condition characterised by a wide range of clinical features, but primarily identified as thrombotic and/or obstetric related adverse events. APS is associated with the presence of antiphospholipid antibodies (aPL), including the so-called lupus anticoagulant (LA). These aPL are heterogeneous in nature, detected with varying sensitivity and specificity by a diverse range of laboratory tests. All these tests are unfortunately imperfect, suffer from poor assay reproducibility (inter-method and inter-laboratory) and a lack of standardisation and harmonisation. Clinicians and laboratory personnel may struggle to keep abreast of these factors, as well as the expanding range of available aPL tests, and consequent result interpretation. Therefore, APS remains a significant diagnostic challenge for many clinicians across a wide range of clinical specialities, due to these issues related to laboratory testing as well as the ever-expanding range of reported clinical manifestations. This review is primarily focussed on issues related to laboratory testing for APS in regards to the currently available assays, and summarises recent international consensus guidelines for aPL testing, both for the liquid phase functional LA assays and the solid phase assays (anticardiolipin and anti-beta-2-Glycoprotein-I).

Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti‐FXa assays

Apixaban is an oral direct factorXa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information. To investigate the effects of apixaban on commonly used coagulation methods, and to evaluate anti-FXa assays for specific determination of the drug concentration. Apixaban was added to plasma from healthy subjects in the concentration range 0-1000μgL(-1) , and analyses were performed with different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, proteinC, and proteinS. A lupus anticoagulant assay and an APTT assay with varying phospholipid concentrations were used to study the phospholipid dependence. In general, apixaban showed fewer effects invitro than have been shown for rivaroxaban, another direct FXa inhibitor. The concentration needed to double the APTT varied between 2200 and 4700μgL(-1) , and the concentration needed to double the PT varied between 700 and 3900μgL(-1) . The effects on antithrombin, proteinC and proteinS assays were dependent on the type of reagent. Apixaban did not cause false-positive lupus anticoagulant results. Chromogenic anti-FXa assays showed linear dose-response curves with apixaban. Therapeutic concentrations of apixaban variably affect different assay groups, and even different reagents within an assay group. The effects were much smaller than with rivaroxaban. The use of APTT and/or PT assays to screen the anticoagulant activity of apixaban cannot be recommended. A chromogenic anti-FXa assay can be used for reliable measurements of apixaban concentration.

Both IgG and IgM anti-beta2 glycoprotein I antibodies assays are clinically useful to the antiphospholipid syndrome diagnosis

Objectives:The aim of our study was to evaluate the clinical values of anti-beta2 glycoprotein I antibodies (anti-beta2GPI) IgG and IgM comparing with lupus anticoagulant (LA), anticardiolipin antibodies (aCL) in the two clinical groups of antiphospholipid syndrome (APS), vascular thrombosis (VT) and pregnancy morbidity (PM).Methods:Eighty patients who fulfilled the APS clinical criteria, VT n = 34; PM n = 40, both VT and PM n = 6 were included. LA, aCL and three anti-beta2GPI ELISA kits were tested.Results:Sensitivities of LA, aCL and anti-beta2GPI assays were found respectively 62, 26 and 41% in VT, and 28, 28 and 30% in PM. The sensitivity for the APS diagnosis could reach to 63% using triple tests. The presence of LA (P<0·01, OR = 4·3) or anti-beta2GPI IgG alone (P<0·05, OR = 8·4) was significantly associated with VT. IgM isotype was found more frequent in PM (92%) than in VT (57%) among all positive anti-beta2GPI cases.Conclusion:Both IgG and IgM anti-beta2GPI assays were useful when clinical features of APS presented, even its standardization is ongoing. A decreased by half sensitivity of LA in PM compared with that in VT underlines the importance of adding anti-beta2GPI in PM of APS, especially IgM isotype although recent review questioned its significance.

Fulminant phlegmasia cerulea dolens with concurrent cholangiocarcinoma and a lupus anticoagulant

Phlegmasia cerulea dolens (PCD) is an aggressive and life-threatening form of venous thrombosis complicated by ischemic necrosis. Massive thrombosis extends to collateral veins resulting in venous congestion with fluid sequestration in the interstitium causing collapse of arterioles, which progresses to ischemia and, if severe, circulatory collapse and shock. The mortality rate for PCD is as high as 40%, especially when gangrene develops. PCD has been associated with acquired thrombophilias, including malignancy and antiphospholipid syndrome (APS). We present a unique case of a patient with PCD refractory to anticoagulant and thrombolytic therapy, whose fulminant course was attributed to concurrent cholangiocarcinoma and antiphospholipid antibodies identified by a positive lupus anticoagulant assay. This case highlights the importance of uncovering precipitating causes of thromboembolism, which may offer prognostic information and may necessitate therapy beyond anticoagulation and thrombolysis to reduce the morbidity of PCD. The current literature on PCD and APS, along with their associations with malignancy, is reviewed.

THU0029 Inflammation is Strongly Associated with Lupus Anticoagulant Positivity, Independent of Known Autoimmune Disease and Recent Venous or Arterial Thrombosis

BackgroundThe antiphospholipid syndrome (APS) is an auto-immune disorder defined by presence of antiphospholipid antibodies and either thrombosis or obstetric complications (1). Laboratory part of the diagnosis of APS comprise of...

C0360: Correlation of Chemiluminescent and Elisa Method for Determination of Anti-Cardiolipin Antibodies

Background: Despite lupus anticoagulant (LA) is one of the laboratory criteria for antiphospholipid syndrome (APS), it can be present in asymptomatic subjects or it can be associated with other clinical conditions like bleeding symptoms. We present a retrospective analysis (May 2011-July 2013) of LA positive tests not associated with APS. Methods: The results of 2000 consecutive LA assays, including both sensitive APTT and dRVVT, were analysed; 1501 of them were performed in patients with clinical criteria for APS and 499 in patients without criteria for APS. Results: At least a positive LA test, according to ISTH criteria, was found in 410/1501 (27.3%) and 216/499 (43.3%) of assays in the group with or without APS criteria respectively. The analysis of the clinical background in patients with positive LA but without APS showed that 18% (39/216) of cases were asymptomatic, 34.3% (74/216) had bleeding symptoms (epistaxis, gingivorrhagia, bruising, spontaneous hematomas) and 47.7% (103/216) had other clinical settings (infertility, chronic kidney disease, autoimmune disorders, haematological diseases, IHD, ITP, TIA, among others). Patients with bleeding symptoms showed platelet dysfunction (n =15); low VWF:RCo (not detectable-50 UI/dL) and/or VWF:Ag (9–50 UI/dL) (n = 9); 3 patients showed low Fibrinogen (one of them with low platelet count and one with low FX); hypoprothrombinemia (n =1); low FV (n =1); low FVII (n = 1); low FVIII (n = 6); low FXI (n = 1);a-FVinhibitor (n = 1); hyperfibrinolysis (n = 3) one of them with VWF:Ag and VWF:RCo in the normal limit; the other patients had normal laboratory phenotypes (n =33). Among the four patients with no detectable ristocetin cofactor, one of them had a severe von Willebrand disease. The other three displayed inhibitory effect against VWF:RCo; in these cases of acquired (AVWS) the alteration was associated with MGUS, MGUS and platelet associated immunoglobulins and Waldestrom macroglobulinemia. The patient with a-FV had multiple myeloma. Conclusions: These results showed that LA can be present not only in asymptomatic subjects without APS criteria, but also in patients with bleeding symptoms or other clinical settings. In those cases positive for LA and bleeding, an underlying bleeding defect should be suspected. A careful analysis of laboratory results should be done in order to achieve accurate diagnosis, essential for therapeutic decisions. C0360 CORRELATION OF CHEMILUMINESCENT AND ELISA METHOD FOR DETERMINATION OF ANTI-CARDIOLIPIN ANTIBODIES L. Slavik, J. Prochazkova, D. Janek, J. Ulehlova, M. Wijova, V. Krcova, A. Hlusi. University Hospital. Olomuc, Czech Republic

C0184: Lupus Anticoagulant in Patients without Thrombotic or Obstetric Complications

Background: Despite lupus anticoagulant (LA) is one of the laboratory criteria for antiphospholipid syndrome (APS), it can be present in asymptomatic subjects or it can be associated with other clinical conditions like bleeding symptoms. We present a retrospective analysis (May 2011-July 2013) of LA positive tests not associated with APS. Methods: The results of 2000 consecutive LA assays, including both sensitive APTT and dRVVT, were analysed; 1501 of them were performed in patients with clinical criteria for APS and 499 in patients without criteria for APS. Results: At least a positive LA test, according to ISTH criteria, was found in 410/1501 (27.3%) and 216/499 (43.3%) of assays in the group with or without APS criteria respectively. The analysis of the clinical background in patients with positive LA but without APS showed that 18% (39/216) of cases were asymptomatic, 34.3% (74/216) had bleeding symptoms (epistaxis, gingivorrhagia, bruising, spontaneous hematomas) and 47.7% (103/216) had other clinical settings (infertility, chronic kidney disease, autoimmune disorders, haematological diseases, IHD, ITP, TIA, among others). Patients with bleeding symptoms showed platelet dysfunction (n =15); low VWF:RCo (not detectable-50 UI/dL) and/or VWF:Ag (9–50 UI/dL) (n = 9); 3 patients showed low Fibrinogen (one of them with low platelet count and one with low FX); hypoprothrombinemia (n =1); low FV (n =1); low FVII (n = 1); low FVIII (n = 6); low FXI (n = 1);a-FVinhibitor (n = 1); hyperfibrinolysis (n = 3) one of them with VWF:Ag and VWF:RCo in the normal limit; the other patients had normal laboratory phenotypes (n =33). Among the four patients with no detectable ristocetin cofactor, one of them had a severe von Willebrand disease. The other three displayed inhibitory effect against VWF:RCo; in these cases of acquired (AVWS) the alteration was associated with MGUS, MGUS and platelet associated immunoglobulins and Waldestrom macroglobulinemia. The patient with a-FV had multiple myeloma. Conclusions: These results showed that LA can be present not only in asymptomatic subjects without APS criteria, but also in patients with bleeding symptoms or other clinical settings. In those cases positive for LA and bleeding, an underlying bleeding defect should be suspected. A careful analysis of laboratory results should be done in order to achieve accurate diagnosis, essential for therapeutic decisions. C0360 CORRELATION OF CHEMILUMINESCENT AND ELISA METHOD FOR DETERMINATION OF ANTI-CARDIOLIPIN ANTIBODIES L. Slavik, J. Prochazkova, D. Janek, J. Ulehlova, M. Wijova, V. Krcova, A. Hlusi. University Hospital. Olomuc, Czech Republic

Antiphospholipid syndrome in 2014: more clinical manifestations, novel pathogenic players and emerging biomarkers.

The clinical spectrum of the anti-phospholipid syndrome (APS) is not limited to vascular thrombosis or miscarriages but includes additional manifestations that cannot be explained solely by a thrombophilic state. Anti-cardiolipin, anti-beta2 glycoprotein I (anti-β2GPI) and lupus anticoagulant (LA) assays are not only the formal diagnostic and classification laboratory tools but also parameters to stratify the risk to develop the clinical manifestations of the syndrome. In particular, anti-β2GPI antibodies reacting with an immunodominant epitope on domain I of the molecule were reported as the prevalent specificity in APS patients, correlating with a more aggressive clinical picture. Several laboratory assays to improve the diagnostic and predictive power of the standard tests have been proposed. Plates coated with the phosphatidylserine-prothrombin complex for detecting antibodies represent a promising laboratory tool correlating with LA and with clinical manifestations. Anti-phospholipid antibodies can be found in patients with full-blown APS, in those with thrombotic events or obstetric complications only or in asymptomatic carriers. An inflammatory second hit is required to increase the presence of β2GPI in vascular tissues, eventually triggering thrombosis. Post-transcriptional modifications of circulating β2GPI, different epitope specificities or diverse anti-β2GPI antibody-induced cell signaling have all been suggested to affect the clinical manifestations and/or to modulate their occurrence.

The Challenge Of Russell's Viper Venom Time Interpretation For Lupus Anticoagulant Diagnosis: A Two-Year Retrospective Institutional Review

The UCSF hematology laboratory uses the Precision Biologic LA Check and LA Sure reagents for the Russell's viper venom time (RVVT), as part of the laboratory diagnosis of a lupus anticoagulant (LA). Currently, manufacturer's FDA-approved guidelines for interpretation differ from the recommended diagnostic criteria of the International Society for Thrombosis and Hemostasis (Pengo V et al. JTH. 2009, 7:1737). The manufacturer's guideline for positivity is patient results 1) >2 SD above the mean of normal donors for the screening test and 2), >2 SD above the mean of the ratio of the LA Check time to phospholipid-supplemented LA Sure time in normal donors. The ISTH guideline for positivity is patient results 1) >99th percentile of normal donors for the screening test, 2) >99th percentile of the ratio of mix of patient and pooled normal plasma (PNP) to the mix of normal donors and PNP with the screening reagent, and 3) >50thpercentile of the mean of the ratio of the LA Check time to phospholipid-supplemented LA Sure time in normal donors. Therefore, some cases meet one or the other definition of LA, but not both.We interpret the RVVT as positive when both manufacturer's and ISTH guidelines are met, negative when neither criteria are met, and equivocal when one set of criteria is met but not the other. Some authors have advocated using a similar “third” category of “indeterminate lupus anticoagulant” in order to highlight the potentially elevated risk for these patients to have pro-thrombotic events (Park SH et al. Korean J Hematol. 2012, 47:83).Here we report our two-year experience (7/6/2011-7/6/2013) with patients with prolonged screening RVVT (using LA Check). Those samples with prolonged screening RVVT greater than 2 standard deviations above the average of normal donors undergo additional testing: a 1:1 mix with PNP (using LA Check) and high phospholipid confirmatory testing (using LA Sure). We retrospectively reviewed all samples with a prolonged screening RVVT in a two year period to determine the frequency of deviations between the manufacturer's and ISTH guidelines, as well as the frequency of prolonged screening RVVT. We also correlate the RVVT results with the other lupus anticoagulant assay used at UCSF, the Staclot® LA 20 (utilizing hexagonal phase phosphatidylethanolamine)329 samples had a prolonged screening RVVT, representing 321 patients (24 patients had two RVVT performed, 2 patients had three RVVT). 1477 total RVVT were performed in the study period (i.e. 22.3% had a prolonged screen). Of the 329 samples with a prolonged screening RVVT, 116 were positive (35.3%), 139 were negative (42.2%), and 74 were equivocal (22.5%). Of the 74 cases with an equivocal RVVT, 36 (48.6%) had a positive result according to manufacturer's guidelines and 38 (51.4%) had a positive result according to ISTH guidelinesOf the 329 samples with a prolonged screening RVVT, 122 had a concurrent negative Staclot® LA 20 assay (37%), 16 had a concurrent equivocal Staclot® LA 20 assay (4.9%), 172 had a concurrent positive Staclot® LA 20 assay (52.3%), and 19 did not have a concurrent Staclot® LA 20 assay (5.8%).Of the 74 cases with an equivocal RVVT, 71 had a concurrent Staclot® LA 20 assay. Of those 71, 25 (35.2%) had a negative Staclot® LA 20, and 4 (5.6%) had an equivocal Staclot® LA 20, and 42 (59.2%) had a positive Staclot® LA 20. Of the 42 cases with an equivocal RVVT and a concurrent positive Staclot® LA 20 assay, 13 (31%) met RVVT criteria for manufacturer's guidelines and 29 (69%) met RVVT criteria for ISTH guidelines.Of the 329 samples with a prolonged screening RVVT, 47 were associated with identified concurrent anticoagulant treatment. 17 (36.2%) of these 47 had a negative RVVT, 13 (27.7%) had an equivocal RVVT, and 17 had a positive RVVT (36.2%).Overall, this study has shown that there are frequent cases (22.5%) where the RVVT assay meets one or the other guidelines for a positive LA. The equivocal diagnoses were approximately evenly split between those cases positive by manufacturer's guidelines or positive by ISTH guidelines. Compared to the overall sample, those cases with an equivocal RVVT did not more frequently have a positive Staclot® LA 20 assay. Those cases with an equivocal RVVT who were positive by ISTH guidelines did seem to enrich for a positive Staclot® LA 20 assay, potentially reflecting a better sensitivity of the ISTH guidelines for those patients with an LA as compared to the manufacturer's guidelines. Disclosures:No relevant conflicts of interest to declare.

Risk factors for thromboembolism and pulmonary artery hypertension following splenectomy in children with hereditary spherocytosis

The aim was to study risk-factors for vascular thrombosis and incidence of pulmonary artery hypertension (PAH) in splenectomized children with hereditary spherocytosis (HS) at a single center. Pre- and post-splenectomy hemoglobin and platelet counts were recorded. Post-splenectomy lipid-profile, fibrinogen, D-dimer, CRP and anti-coagulant-protein levels were compared to established controls. Echo-Doppler was performed for PAH. Twenty-six children with HS had undergone splenectomy; the mean age at surgery was 7.9 ± 3.7 years. Nineteen of the 26 were prospectively investigated at a median duration of 4.5 years (range: 4 months to 19 years) following splenectomy. Thrombocytosis was observed in 19 (73%), whereas no patient had erythrocytosis at the last follow-up visit. Total cholesterol, LDL-C, HDL-C, and triglyceride levels were not deranged (P ≥ 0.3). Mean CRP levels (males: 2.8 ± 0.5; females: 2.1 ± 0.5 mg/L) were significantly higher than described for normal children (P < 0.001). Six (23%) patients had a positive D-dimer assay. Protein S, anti-thrombin-III and fibrinogen were in range. A single patient had a borderline low protein C activity. Lupus anticoagulant and anti-cardiolipin antibody assays were negative. The mean tricuspid regurgitant jet velocity (TRJV) was 1.8 ± 0.55 meter per second (range: 0-2.4). None had a TRJV ≥2.5 meter per second to suggest PAH. There was no evidence of PAH, dyslipidemia, elevation of fibrinogen or a reduction in anti-coagulant proteins, at a median follow-up duration of 4.5 years following splenectomy in children with HS. However, elevated CRP level (42%), persistent thrombocytosis (73%) and elevated D-dimer levels (23%) were observed. These have been recognized as risk factors for cerebrovascular and coronary heart disease.

Occult pulmonary mucosa-associated lymphoid tissue lymphoma presenting as catastrophic antiphospholipid antibody syndrome

Catastrophic antiphospholipid antibody syndrome (CAPS) is characterized by fulminant thrombosis of the arterial and venous beds of multiple organ systems over a relatively short period of time and with a high mortality rate. Mucosa-associated lymphoid tissue (MALT) lymphoma of the lung has never been reported as a causative or precipitating factor for CAPS in the CAPS registry database. The present study describes a rare case of pulmonary MALT lymphoma of the lung that presented as CAPS. A 19-year-old Hispanic female presented with shortness of breath and abdominal pain. Computed tomography (CT) scans of the chest and abdomen revealed multiple portal vein thromboses and bilateral pulmonary nodules. Within one week of presentation, the patient developed a straight sinus thrombosis and upper extremity deep vein thrombosis, which led to shortness of breath. A biopsy of the lung nodule revealed MALT lymphoma. The present case illustrates a rarely reported pulmonary MALT lymphoma presenting as CAPS in a young female. The patient was successfully treated with 90 mg/m2 bendamustine on days one and two and rituximab 375 mg/m2 on day one of each 28-day cycle. Complete remission of the lung nodules was observed following three cycles of treatment, as visualized by positron emission tomography (PET)/CT scan. Fondaparinux was identified as a feasible anticoagulation drug of choice for this case. At seven months post-treatment, the patient continues to be stable with no further evidence of thrombosis and is currently undergoing rituximab maintenance therapy every six months for two years. A repeat lupus anticoagulant antibody assay turned and remained negative during the clinical follow-up period. A prompt diagnosis and early aggressive treatment is potentially curative and may dramatically decrease the mortality risk. Future studies should explore the role of rituximab in the management of CAPS-associated B-cell lymphoid malignancies.

Lupus anticoagulant, warfarin, and alternative laboratory monitoring of anticoagulation

A 53 year old woman was evaluated for recurrent native and prosthetic mitral valve thromboembolism in the setting of antiphospholipid antibody syndrome. In 1989, at age 30 years, she suffered a thromboembolic stroke and was diagnosed with antiphospholipid antibody syndrome for which long-term warfarin therapy was prescribed. She was subsequently assigned a goal international normalized ratio (INR) of 2.5–3.5. In 2008, at age 49 years, she was diagnosed with mitral valve stenosis and underwent mitral valve replacement (29 mm Carbomedics prosthesis). Pathology was consistent with nonbacterial thrombotic endocarditis involving a fibrotic and focally calcified mitral valve. After valve replacement, the target INR range was increased to 3.0–4.0. Nine months later, she developed thrombosis of the prosthetic mitral valve and required redo valve replacement surgery (27 mm St. Jude Biocor). Postoperatively, she developed heparin induced thrombocytopenia (HIT) for which she received argatroban while transitioning to warfarin. Her target INR range was subsequently increased to 3.5–4.5. In 2012, she developed recurrent prosthetic mitral valve thrombosis despite being mostly therapeutic(INR 3.5–4.5) and at times supra-therapeutic with INR monitoring. Transesophageal echocardiogram confirmed an 8 mm thick layer of organized thrombus encasing each of the prosthetic mitral valve leaflets with severe valvular stenosis. Laboratory testing upon admission revealed a prothrombin time (PT) of 101.7s (INR 11.1). Simultaneously measured chromogenic factor X activity was 20 % (therapeutic range 20–35 %) and factor II activity was 14 % (normal reference range 75–145 %), confirming adequate anticoagulation at that time. A third mitral valve replacement procedure was pursued (27 mm Hancock II bioprosthesis) and she was yet again restarted on warfarin postoperatively. Initial special coagulation testing done at our institution in 1989 established the presence of a lupus anticoagulant (LAC). This diagnosis was subsequently confirmed on four occasions (1995–2012). Prior to 2004, the LAC assays were performed using the dilute Russel viper venom time (DRVVT) assay kit from American Diagnostica (Stamford, CT). After 2004, the DRVVT assay was performed using the kit from Precision Biologics (Dartmouth, NS, Canada) following the manufacturer’s instructions. She had a persistently high titer of anticardiolipin antibodies (IgG and IgM) as well. Her baseline prothrombin PT (11.3s) and INR (1.1), were normal and remained so until 2012, when measured during brief interruptions of warfarin anticoagulation (six occasions; 1995–2011). Prior to 2009, the PT assay was performed with Innovin (Siemens, New York, NY formerly Dade Behring, Deerfield, IL). Beginning in 2009, the PT was performed with Recombiplastin 2G (Instrumentation Laboratories, Bedford, MA). The temporal trends in INR and chromogenic factor X activity are shown in Fig. 1. Since INR differences are much larger at low factor activities than at high activities, we compared the log INR with the factor activities. Log INR did not correlate well with chromogenic factor X activity (Fig. 2a, r = 0.32) and factor II (Fig. 2b, S. S. Ketha (&) R. D. McBane W. E. Wysokinski Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Gonda 4-130 (VM), 200 First St. SW, Rochester, MN 55905, USA e-mail: Ketha.Siva@mayo.edu

Unexpectedly Resolved Multiple Mobile Thrombi in a Normal Thoracic Aorta Associated With Colorectal Cancer and Protein C Deficiency

Staging System sixth edition. The patient did not have peritoneal or liver metastases. Based on the clinical presentation, TEE, and histopathology findings, the masses in the thoracic aorta were considered to be thrombi and thought to be the causative factor for the rectal perforation. Postoperative hematologic testing revealed protein C deficiency: Initial activity value, 32% (normal value, 64%-146%), and initial antigen value, 33% (normal value, 70%-150%). The patient did not have a medical history of protein C deficiency. Factor VIII, homocysteine and protein S activity, and antigen levels were within normal ranges. Anticardiolipin antibody, antinuclear antibody, proteinase-3-antineutrophilic cytoplasmic antibody, myeloperoxidase-antineutrophilic cytoplasmic antibody, and lupus anticoagulant assays all were negative. The patient did not undergo any additional surgery for the aortic thrombi, such as thrombectomy or segmental aortic resection, because it was thought to be too invasive for a patient with advanced cancer. The authors did not perform anticoagulant or thrombolytic therapy because of the significant risk of additional distal embolization. Only oral aspirin was given for aortic thrombus prophylaxis. The patient also received intensive treatment for peritonitis, including antibiotics, fluid management, and mechanical ventilation, after the surgery in the intensive care unit. His clinical course was good, and the aortic thrombi had disappeared completely on a followup CT scan performed on the 49th day of hospitalization (Fig 1B). Cyanosis of the bilateral digits, which was thought to be a sign of acute pedal arterial embolism, appeared during the clinical course, but it improved with intravenous alprostadil monotherapy. The patient was discharged to his home on the 61st day of hospitalization without any other serious distal embolism.

From Phenomenology to Mechanism: Resistance to Annexin A5 Anticoagulant Activity Identifies a Subset of Patients with Antiphospholipid Antibodies and Thrombosis

Abstract 631 Background:The antiphospholipid (aPL) syndrome (APS) is a diagnostic category that is defined by non-mechanistic empirically-derived assays - the lupus anticoagulant (LA) assay and aPL-antibody immunoassays for anticardiolipin and anti-ß2 glycoprotein-I antibodies. APS patients have heterogeneous antibodies and it is likely that several mechanisms are involved in the disease process. Annexin A5 (AnxA5) is a potent anticoagulant protein that forms 2-dimensional crystals over membranes that express phosphatidyl serine and shields the phospholipid from availability for phospholipid-depended coagulation reactions. We previously demonstrated that aPL antibodies can disrupt this crystallization and accelerate coagulation reactions. We therefore investigated whether disruption of AnxA5 anticoagulant activity might mark patients with histories for aPL antibody associated thrombosis. Design:We investigated samples from 966 adult patients from a large urban academic medical center with the following categories of histories: thrombosis, including venous thromboembolism (VTE) and stroke (n=433); pregnancy complications attributable to placental insufficiency (n=218); autoimmune disease (n=279); cancer (n=63); renal disease (n=101); liver disease (n=51); HIV (n=23); and free of known disease (n=140) (total >966 because of concurrent diagnosis). An assay that detects annexin A5 resistance (A5R) was performed on all samples. Results were expressed as “% AnxA5 anticoagulant activity”. A quintile analysis was performed to obtain cutoffs for A5R values. Values among groups were compared using student t-test, one way ANOVA and odds ratio analysis. Results:aPL-antibody positive patients with thrombosis (VTE and/or stroke) have significantly lower mean A5R values (194±57, n=76) than aPL-antibody negative patients with a thrombotic event (238±41, n=175; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 54% (41/76) of patients in the aPL-antibody positive and thrombosis group had A5R values within the lowest quintile of results (<199%), in contrast to 15% (26/175) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and thrombosis (OR: 13.7, p<0.001). aPL-antibody positive patients with pregnancy complications (PCs) have significantly lower A5R values (218±45, n=111) than aPL-negative patients with pregnancy complications (248±43, n=107; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 28% (31/111) of patients in the aPL-antibody positive and pregnancy complication group had A5R values within the lowest quintile of results (<199%), in contrast to 8% (9/107) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and pregnancy complications (OR: 4.6, p<0.001). There were no significant differences in AnxA5 anticoagulant activity in patients with cancer, HIV, and renal disease, however there were significantly lower A5R values in patients with autoimmune disease and liver disease. The latter appeared to correlate with severity of liver disease. Patients with triple aPL-antibody positivity or greater had significantly lower mean A5R values (155±48%, n=23, p<0.001), than patients with double positivity (193±55%, n=44, p<0.01), or single positivity (214±47, n=111, p<0.05). All aPL-positive groups had significantly lower A5R values than patients who were negative for aPL antibodies (240±43%, p<0.001). [Display omitted] Conclusion:A subset of ∼50% of aPL-antibody positive patients with thrombosis and ∼30% with pregnancy complications showed evidence for this mechanism - resistance to annexin A5 anticoagulant activity. The identification of specific mechanisms for aPL-antibody associated thrombosis and pregnancy complications offers the possibilities of improving diagnosis and developing targeted treatments for this enigmatic disorder. Disclosures:No relevant conflicts of interest to declare.

Antiphospholipid antibodies: Evaluation of the thrombotic risk

The laboratory diagnosis of the antiphospholipid syndrome (APS) via antiphospholipid antibody (aPL) tests, including lupus anticoagulant (LAC), anti-cardiolipin (aCL), or anti-beta2 glycoprotein I (aβ2GPI) antibodies remains a challenge. Coagulation tests for LAC as well as solid phase assays for aCL and aβ2GPI have methodological shortcomings, although for LAC large progress have been made in standardization. All assays are associated with clinical APS-criteria (thrombotic and/or pregnancy complications) but with limited specificity. Besides, clinical studies demonstrating the association between the presence of aPL and thrombosis are not always well designed and result in wide ranges of odds ratio with large variation between studies. The best association between thrombotic complications and aPL is found for LAC. The association between thrombosis and aCL or aβ2GPI is at least inconsistent. The inclusion of more specific assays, such as the domain-I-β2GPI.antibodies is too premature and depends on further investigation in large clinical studies and the commercial availability.The search for new assays should proceed to identify patients with aPL with increased risk for thrombosis, preferable in large prospective studies. Meanwhile, with the current available LAC, aCL and aβ2GPI assays it is strongly recommended to make antibody profiles. Multiple positivity of tests seems clinically more relevant. The strengths and weaknesses of the current laboratory criteria for APS are discussed in view of their role in risk stratification of patients with thrombotic events.

Cooperation experience in a multicentre study to define the upper limits in a normal population for the diagnostic assessment of the functional lupus anticoagulant assays

Phospholipid-dependent coagulation tests for lupus anticoagulant (LA) are considered an important step for the diagnosis of anti-phospholipid syndrome; however, LA laboratory detection is difficult because of many variables. Five hospital laboratories, located in a North-Italy area and using the same method for LA testing, cooperated to standardise sample treatment and analytical procedure in order to define the upper values for LA negativity. In total, 200 normal subjects (40 for each centre) were studied for six LA functional assays, using the same procedure, reagent lot and analyser type. The first tests done were LA screen and LA confirm assays, based on diluted Russell's Viper Venom Time, with low and high phospholipid content, respectively. The second tests performed were silica clotting time screen and confirm assays, based on activated partial thromboplastin time, with low and high phospholipid content, respectively. Finally, two mixing assays were executed for both screening assays, diluting patient sample with a pool prepared with plasma collected from the study population. Data analysis demonstrated a difference between centres for all assays when results were expressed in seconds; the difference disappeared when results are normalised with the local mean normal value of each centre and are expressed as a normalised ratio. The study population was normally distributed; so the value corresponding to 99th percentile was used as limit value for LA negativity. Values expressed as normalised ratio, for LA and silica clotting time screenings were 1.22 and 1.23, respectively. The study allowed us to define a uniform approach to LA testing and evaluation for laboratories employing the same methods.

Lupus anticoagulant detection: out of control?

Expert guidelines indicate that normalised ratios are preferred to clotting times for lupus anticoagulant (LA) assays to mitigate analytical variation. We investigated the effects of deriving normalised ratios from the reference interval (RI) mean or different normal pooled plasmas (NPP). Screen, confirm and mixing tests for dilute Russell's viper venom time and APTT were converted to normalised ratios and interpreted for LA. Of 1000 clinical samples, 824 generated identical interpretations using RI mean or NPP-derived ratios and 57 identified LAs in one or both assays via either denominator. Separate RIs were applied for normalised ratios derived from the NPP or RI mean. Applying percentage correction index (PCI) to screen and confirm assays irrespective of screen elevation increased agreement to 92.5%. Two frozen and one lyophilised NPP were then used to derive ratios for 204 samples and 130 generated identical interpretations with all NPPs, 14 had overall interpretation parity and 19 overall agreement via PCI, giving 79.9% overall agreement. The results derived from each NPP were interpreted against RIs derived from RI means to reflect differences resulting from NPPs with clotting times dissimilar to RI means. Disparities were largely a function of closeness of NPP clotting times to test RI means and not owing to clotting factor level differences and likely related to manufacturing variables. Diagnostic benefit of normalised ratios can be maximised by matching NPP values to RI means. If RI mean is employed, and it likely requires re-establishing with new reagent batches.

Current International Initiatives in Antiphospholipid Antibody Testing

Positive results in the anticardiolipin antibody (aCL), anti-β2-glycoprotein I antibody (aβ2GPI), and/or lupus anticoagulant (LA) assays constitute the laboratory criteria for the definite diagnosis of the antiphospholipid syndrome (APS). These tests became available from as early as the1980s, and since then several novel assays have become available that have varied usefulness in the diagnosis and prognosis of APS patients. For almost three decades there has been an ongoing effort to standardize the aCL, aβ2GPI, and LA assays, but there are still reports of significant intra- and interlaboratory variation in the results of all three assays. There have also been numerous issues with the implementation of novel (noncriteria) antiphospholipid antibody (aPL) tests in standard testing panels, due to either lack of standardized testing methods or limited evidence of their clinical utility in APS patients. At the recent 13th International Congress on Antiphospholipid Antibodies (APLA 2010, 13-16 April 2010, Galveston, TX), two task forces were set up to address these problems. This review gives a general description of current problems hindering the standardization of aPL tests and the implementation of novel assays as standard components of aPL testing panels. It also highlights the approach used by APLA 2010 Task Forces to address these problems and presents their recommendations.

Internal Quality Control and External Quality Assurance in Testing for Antiphospholipid Antibodies: Part I—Anticardiolipin and Anti-β2-Glycoprotein I Antibodies

Over 30 years since it was first described as a discrete clinical entity, the antiphospholipid antibody syndrome (APS) remains a challenge for both laboratory workers and clinicians in a wide range of specialties. In addition to the presence of appropriate clinical features, the diagnosis of APS also fundamentally requires the finding of positive antiphospholipid antibody (aPL) test result(s), which comprise 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 first of two that review the process for, and provides 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 aCL and aβ2GPI testing. In brief, IQC is a process that helps control the quality of laboratory test results on a test-by-test basis; IQC should include samples that provide values around the assay critical cut-off values, and there is added value in the inclusion of non-kit assay controls. EQA is a process that helps laboratories assess their performance against those of their peers. For aCL and aβ2GPI testing, we provide some updated findings from the Royal College of Pathologists of Australasia Immunology Quality Assurance Program, and covering testing for the past 3 years (2009 to 2011 inclusive). Findings show similar trends to past years, indicating limited improvement in cross-laboratory test results and interpretations. In summary: (1) EQA participants reported greatly varying numerical test data for both aCL and aβ2GPI, with interlaboratory coefficients of variation > 50% with most test challenges; (2) there was considerable overlap in the interpretation (negative, positive, low positive, moderate positive, strong positive) that different participants ascribed to identical numerical test results; and (3) there was limited consensus among participants as to whether test results for individual EQA specimens were either positive or negative for aCL and/or aβ2GPI.