Concomitant lupus anticoagulant and monoclonal IgMκ antibody in a patient with bleeding tendency: A case report and literature review
Authors: Marie‐Alexandra Alyanakian, Hikaru Okada, Christilla Bachelot‐Loza, Peggy Tournoux, Bruno Varet, Dominique Lasne
Journal: American Journal of Hematology (2011)

Antiphospholipid antibodies (aPL) are a heterogeneous population of autoantibodies including lupus anticoagulant (LAC), anticardiolipin antibodies and antiβ2-glycoprotein I antibodies. aPL may be associated with systemic lupus erythematosus, other autoimmune disorders, connective tissue disorders, malignancies, drug use and/or infections. They are usually associated with thrombotic complications (thromboembolism, spontaneous abortion, livedo reticularis). Bleeding is generally not a feature of aPL, but uncommonly, bleeding can occur if significant thrombocytopenia or prothrombin deficiency develop. An aPL-monoclonal gammopathy association has been described, particularly in patients suffering from lymphoid proliferations, including Waldenström's macroglobulinemia and lymphoma. Here we report a strong association between LAC and hemostasis abnormalities with monoclonal immunoglobulin Mκ in a 71-year-old woman who experienced excessive bleeding after dental extraction. Our findings suggest that the monoclonal component induced spontaneous platelet aggregation. Unlike previous reports, Waldenström's macroglobulinemia or lymphoma was not found in this patient. The patient was referred by her general practitioner to our Hematology Department because of abnormal bleeding after dental extraction, not requiring transfusion. Several years ago, the patient had undergone surgery, e.g. complete hysterectomy, cholecystectomy, and sclerotherapy for varicose veins, without complications. She had no personal history of abnormal bleeding or family history of coagulation disorders. No clinical abnormality was found. She was not taking any drug known to increase the hemorrhage risk. Complete blood count was normal (erythrocytes 4.25 × 1012/L, leukocytes: 3.9 × 109/L, platelets: 156 × 109/L). Her bleeding time was prolonged (11.5 min, normal range <8 min), as was the closure time measured with the Platelet Function Analyzer (PFA100) with the cartridge containing epinephrine (170 sec, normal range <160 sec), but normal with the ADP cartridge. Repeated coagulation tests showed normal prothrombin time, thrombin time, and fibrinogen level but a markedly prolonged activated partial prothrombin time (aPTT ratio 2.4, normal range <1.2). Mixing aPTT test of the patient's plasma with normal plasma did not correct the abnormal aPTT and the Rosner index was positive (50, normal range <15). The presence of a lupus anticoagulant (LAC), according to International Society on Thrombosis and Hemostasis criteria was screened by the dilute thromboplastin time index (dTT index: 2.70, normal range <1.2) and the dilute Russel viper venom time (dRVVT) (patient LA1 Screening Reagent/mean normal LA1 Screening reagent ratio: 2.17, normal range <1.2). The use of the LA2 Confirmation Reagent, which is similar to LA1 Screening Reagent but contains a high phospholipid concentration, largely corrects the clotting time, thus confirming the presence of the LAC (normalized ratio 2.2, normal range < 1.2). Factors II, V, VII and X were normal. Despite high dilutions (up to 1:640), factors VIII, IX, XI and XII levels could not be determined with a one-stage modified aPTT assay, because there was strong LAC interference in these assays. Factor IX tested with antigen assay was normal. Chromogenic or antigen assay was not available for other factors. A Bethesda assay showed no evidence for a factor VIII inhibitor. Von Willebrand factor (VWF) antigen (221%) and ristocetin-cofactor activity (207%) were elevated, consistent with an acute phase reaction and not suggestive of von Willebrand disease. Previous surgeries without any complications were reported in the patient's medical file. Thus, a congenital factor deficiency is unlikely. Her serum was positive for various antiphospholipid specificities with a low titer in enzyme-linked immunosorbent assay (ELISA anticardiolipin, antiprothrombin, phosphatidylinositol, and phosphatidylserine). The antinuclear antibodies, anti double stranded DNA, and autoantibodies to extractable nuclear antigens were negative. Owing to an accelerated erythrocyte sedimentation rate (ESR) without inflammatory syndrome, serum protein electrophoresis and immunofixation were performed and revealed the presence of a monoclonal IgMκ (9.5 g/L). The serum viscosity was normal (1.014 g/mL). There was a weak monoclonal kappa light chain in urine. Results of serum κ and λ free light chains were normal (16.1 and 14.1 mg/L respectively with a κ/λ ratio: 1.14, normal range 0.26–1.65). Total serum value of IgM was increased (8.2 g/L, normal range [0.22–2.93]); serum values of IgG and IgA were normal (10.1 and 0.80 g/L, respectively). Bone-marrow examination was normal, with no lymphoplasmacytoid infiltration. The monoclonal component was isolated and its potential activity on coagulation tests was assessed. When added to normal plasma, the purified monoclonal IgMκ caused a dose-dependent prolongation of LAC screening test (aPTT, dTT, dRVVT LA1 Screening test) and generated positive Rosner and dTT indexes, and dRVVT screening ratio. In the dRVVT confirmatory steps, we observed a shortening of the clotting time when excess phospholipids were added (positive dRVVT normalized ratio when 100 μg/mL of patient IgMκ was added to normal plasma) (see Fig. 1). The monoclonal IgMκ of 3 control patients with Waldenström's macroglobulinemia were studied, and no LAC activity was observed when they were tested under the same conditions. Such an association between monoclonal Ig and LAC activity has mainly been reported in patients with Waldenström's macroglobulinemia [1-3] or lymphoma [4-7] (Table I) without any clinical complications. Intriguingly, two myeloma patients were symptomatic, one with bleeding events [4] and the other with deep venous thrombosis [9]. But in comparison with those cases, no myeloma or lymphoma was found in our patient. Coexistence of LAC and acquired hemophilia was described in a monoclonal gammopathy of unknown significance (MGUS) patient [10]. Our patient had no detected specific factor VIII inhibitor in a Bethesda assay. Hemorrhagic symptoms associated with the presence of antiphospholipid antibodies have been mainly reported in the presence of decreased prothrombin levels [11, 12]. Our patient's factor II activity was normal but the purified monoclonal IgMκ had weak antiprothrombin specificity in ELISA. The other phospholipid-antigen specificities tested, i.e., β2-glycoprotein I, cardiolipin, cardiolipin + β2-glycoprotein I, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sphingomyelin, were negative (when tested using the purified monoclonal IgMκ protein). The lupus anticoagulant activity of the patient's IgMκ. Effect of increasing concentrations of the patient's (▴) or control (○) monoclonal IgMκ in normal plasma on the aPTT (A), dTT (B) and dRVVT (C). Index were calculated as follow: Rosner: (mixing aPTT test - normal plasma aPTT)/patient aPTT, dTT: mixing dTT test/normal plasma dTT, dRVVT normalized ratio: patient plasma LA1 Screening Reagent/mean normal LA1 Screening reagent) x (mean normal LA2 Confirmation Reagent/patient plasma LA2 Confirmation reagent). LAC is positive when its value is above the horizontal dotted line. Monoclonal immunoglobulins, high amounts of immunoglobulins or high viscosity can cause nonspecific interference with coagulation, causing PT and/or aPTT prolongations, prolongations in LAC screening assays (dilute phospholipid assays), noncorrection in mixing studies, difficulty in determining factor level, and if viscosity is increased, bleeding. However, viscosity is normal in this case, and the positive dRVVT confirmatory assay is consistent with LAC rather than nonspecific interference. Platelet dysfunctions were described in plasma-cell dyscrasia, particularly when associated with a monoclonal IgM [13]. The abnormal bleeding after dental extraction in the setting of normal VWF activity and prolonged bleeding time led us to study patient's platelet function by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP), as recommended for the diagnosis of bleeding disorders [14]. Spontaneous platelet aggregation (not shown) prevented any further evaluation of platelet sensitivity to various agonists. Therefore, we assessed the effect of adding our patient's IgMκ (100 μg/mL) to washed platelets from normal subjects. It induced delayed aggregation (see Fig. 2) whereas the IgMκ from the three controls tested under the same conditions had no impact on platelet aggregation. Surprisingly, the monoclonal component seems to have induced platelet-activation rather than inhibition, as reported previously [13, 15]. This effect of the monoclonal component on platelet could modify platelet sensitivity to physiological agonist. Thus, a link between patient's transient bleeding disorder and impaired platelet function induced by the monoclonal IgMκ cannot be excluded. Platelet aggregation in the presence of IgMκ. Optical transmission changes were recorded for 20 min after adding 100 μg/mL of IgMκ from our patient (a) or a control patient (b) to a washed control platelet suspension under stirring at 37°C. Representative of three experiments. Discovery of a LAC activity concomitant with the presence a monoclonal gammopathy, although usually asymptomatic or associated with thrombotic complications, does not exclude a bleeding risk. Careful clinical and laboratory evaluation is mandatory to evaluate that risk, especially when the patient is exposed to any kind of surgery. Patient and controls. Our 71-year-old female patient provided informed consent for this study. Four control subjects (blood donors from the Etablissement Français du Sang), who had not taken any medication during the 10 days preceding blood sampling, were enrolled with their written informed consent. Sample collection and biological assays. Blood was collected from the antecubital vein on EDTA with Vacutainer® system (Becton Dickinson, Franklin Lakes, NJ) for hematocrit and blood-cell counts determined on an XE2100 automatic cell counter (Sysmex corporation, Kobe, Japan), and on 0.129 M trisodium citrate (9:1) for PFA100 and clotting tests. Blood specimens were analyzed within 1 h after collection. The PFA100 system is a high-shear system that simulates primary hemostasis after injury to a small vessel [16]. Whole citrated blood samples were analyzed with both PFA100 cartridges containing a nitrocellulose membrane with a central aperture (Siemens, Marburg, Germany). One cartridge is coated with collagen and 10 μg of epinephrine, the other is coated with collagen and 50 μg of adenosine 5-diphosphate. The system measures closure time, i.e., the time required to obtain full occlusion of the membrane aperture. This time is dependent on platelet activation and aggregation. Bleeding time was determined with the Surgicutt device (International Technidyne Edison, NJ). For clotting tests, blood samples were centrifuged at 2,500 g for 20 min at 12°C to obtain platelet-poor plasma (PPP). Coagulation tests (prothrombin time, activated thromboplastin time and fibrinogen, and measurement of coagulation factor II, V, VII, and X levels) were performed on an STA analyzer using standard techniques. Factors VIII, IX, XI, and FXII were determined on KC10μ (Amelung, North Rhine-Westphalia, Germany) using a one-stage modified aPTT assay with CK-Prest cephalin (dilution 1:8) and factor-depleted plasmas (Diagnostica Stago, Asnières, France). Factor IX antigen was determined using Asserachrom IX: AG (Diagnostica Stago). After a second centrifugation, PPP was aliquoted and stored at −80°C; it was used later to perform LAC assays, VWF antigen (STA-liatest VWF; Diagnostica Stago) and VWF ristocetin-cofactor activity (VWF: RCo) on an optical aggregometer (Regulest Aggregometer, AFFI-Bio, Nancy, France). For LAC testing, mixing aPTT test with 1:1 dilution of the patient's plasma was performed with normal lyophilized plasma (CoagNorm, Diagnostica Stago). The Rosner index was calculated as followed: (mixing aPTT test—normal plasma aPTT)/patient aPTT. The dTT index was determined with 1:500 diluted Neoplastin CI (Diagnostica Stago) and calculated as followed: mixing dTT test/normal plasma dTT. The dRVVT (LA1/LA2, Siemens, Marburg, Germany) was carried out according to the manufacturer's recommendations. Briefly, the LA1 Screening Reagent clotting time being more than 2 SD longer than the mean of normal plasma, the LA2 Confirmation Reagent time was measured. LA2 Confirmation Reagent was similar to LA1 Screening Reagent but contains a high phospholipid concentration, which counteracts the LAC antibody and largely corrected the clot time. Results were expressed in normalized ratio [(patient LA1 Screening Reagent/mean normal LA1 Screening reagent) × (mean normal LA2 Confirmation Reagent/patient LA2 Confirmation reagent)]. If ratio was greater than 1.2, LAC was present. For platelet function, PRP was obtained after a 10-min centrifugation of whole blood at 100 g at 20°C. The platelet count was adjusted to 2.5 × 108/mL with autologous PPP and then studied by LTA. Platelet aggregation was quantified as the percentage of maximal optical transmission change. Spontaneous platelet aggregation in PRP under stirring (1,100 rpm) at 37°C prevented any further evaluation of platelet sensitivity to the different agonists usually tested [14]. Control subjects' platelets were isolated from acid citrate dextrose (BD Vacutainer®) anticoagulated whole blood. Briefly, PRP were obtained as described above and then consecutively subjected to differential centrifugations in presence of 0.2 μM prostaglandin E1 and 0.06 IU/ml of apyrase (Sigma Aldrich, Saint Louis, MO). The platelet pellets were resuspended at 2.5 × 108 platelets/mL in 10 mM Hepes pH 7.35, 140 mM NaCl, 3 mM KCl, 5 mM NaHCO3, 0.5 mM NaHCO3, and 10 mM glucose. Platelet stability was controlled under stirring at 37°C in the absence of any inducer at the beginning and the end of each experiment. IgMκ 100 μg/mL from our patient or a control were added to 0.250 mL of platelet suspension in the aggregometer under stirring (1,100 rpm) at 37°C. Platelet aggregation was quantified as the percentage of maximal optical transmission change recorded 20 min after addition of IgMκ. Primary platelet-aggregation slopes and maximal aggregation were analyzed. The monoclonal component was isolated using the euglobulins property to precipitate in low ionic force medium [17]. Purity of isolated paraprotein was assessed by electrophoresis and by immunofixation, which demonstrated that the purified protein had the same migratory properties as the original. The antiprothrombin activity of the purified monoclonal component was detected with Asserachrom anti-prothrombin, (Diagnostica Stago). The other phospholipid antigen specificities, i.e. β2-glycoprotein I, cardiolipin + β2-glycoprotein I, phosphatidylcholine, phosphatidyl inositol, phosphatidylserine and sphingomyelin were determined with AESKULISA Phospholipid-8PRO-GM (Aesku Diagnostics, Wendelsheim, Germany). The authors are grateful to Dr A. Appert-Flory for patients' platelet-function studies by light-transmission aggregometry in platelet-rich plasma. Marie-Alexandra Alyanakian* , Hikaru Okada* , Christilla Bachelot-Loza* , Peggy Tournoux§, Bruno Varet* ¶, Dominique Lasne* §, * Université Paris-Descartes, Paris, France, Laboratoire d'Immunologie, Hôpital Necker, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France, Inserm U765, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France, § Laboratoire d'Hématologie, Hôpital Necker, AP-HP, Paris, France, ¶ Service d'Hématologie Adultes, Hôpital Necker, AP-HP, Paris, France.