Lupus Antibody Mimicking Reduced Plasmatic Coagulation in a Patient With Atrial Fibrillation and Ischemic Stroke

Background: Lupus anticoagulant (LA) owns procoagulant properties in vivo and prolongs phospholipid-dependent clotting times in vitro. The prolonged in vitro clotting time can be misinterpreted as a bleeding disorder. In some cases, it is necessary to differentiate LA-associated in vitro changes from in vivo coagulation factor deficiency. In this case, we used different laboratory testing in a patient with ischemic stroke and reduced prothrombin time (PT) to identify an in-vitro effect of LA excluding an in-vivo bleeding disorder. Methods: The activity of various coagulation factors was evaluated both with recombinant thromboplastin Innovin (Siemens Healthcare) and reagent tissue extracted thromboplastin Thromborel® (Siemens Healthcare). Moreover, a 1:1 plasma mixing test with standard plasma was performed. In order to exclude the interaction of tromboplastin and LA thromboplastin, an independent global coagulation test, thromboelastography, was used. Diluted-Russel-Viper-Venom (dRVVT) assay was applied to detect the presence of LA detection. Results: The activity of several coagulation factors measured with recombinant thromboplastin Innovin (Siemens Healthcare) showed a reduced activity of the following coagulation factors: Factor V (20.9%), Factor VII (23.8%), Factor X (19.7%) and international normalized ratio (INR) of 2.33. Re-assessment of the factor's activity with another reagent tissue extracted thromboplastin Thromborel® (Siemens Healthcare) showed a normalization of INR and factor's activity in comparison to thromboplastin reagent Innovin®: Factor V (77%), Factor VII (45.4%), Factor X (64.2%), and INR of 1.28. A plasma mixing study with 1:1 standard plasma revealed reduced (<50%) normalization of INR as well as coagulation factor's activity confirming a LA-inhibitor in the patient plasma. Diagnostic LA testing was also performed with dRVVT assay showing a significantly prolonged (112.8 s) test time. Thromboelastography revealed no abnormalities. Conclusions: Different thromboplastin reagents and plasma mixing tests as well as thromboplastin independent coagulation tests may be helpful to differentiate LA and in vitro changes from in vivo factor deficiency in patients with LA.

Background: Lupus anticoagulant (LA) owns procoagulant properties in vivo and prolongs phospholipid-dependent clotting times in vitro. The prolonged in vitro clotting time can be misinterpreted as a bleeding disorder. In some cases, it is necessary to differentiate LA-associated in vitro changes from in vivo coagulation factor deficiency. In this case, we used different laboratory testing in a patient with ischemic stroke and reduced prothrombin time (PT) to identify an in-vitro effect of LA excluding an in-vivo bleeding disorder.
Methods: The activity of various coagulation factors was evaluated both with recombinant thromboplastin Innovin (Siemens Healthcare) and reagent tissue extracted thromboplastin Thromborel ® (Siemens Healthcare). Moreover, a 1:1 plasma mixing test with standard plasma was performed. In order to exclude the interaction of tromboplastin and LA thromboplastin, an independent global coagulation test, thromboelastography, was used. Diluted-Russel-Viper-Venom (dRVVT) assay was applied to detect the presence of LA detection.

Results:
The activity of several coagulation factors measured with recombinant thromboplastin Innovin (Siemens Healthcare) showed a reduced activity of the following coagulation factors: Factor V (20.9%), Factor VII (23.8%), Factor X (19.7%) and international normalized ratio (INR) of 2.33. Re-assessment of the factor's activity with another reagent tissue extracted thromboplastin Thromborel ® (Siemens Healthcare) showed a normalization of INR and factor's activity in comparison to thromboplastin reagent Innovin ® : Factor V (77%), Factor VII (45.4%), Factor X (64.2%), and INR of 1.28. A plasma mixing study with 1:1 standard plasma revealed reduced (<50%) normalization of INR as well as coagulation factor's activity confirming a LA-inhibitor in the patient plasma. Diagnostic LA testing was also performed with dRVVT assay showing a significantly prolonged (112.8 s) test time. Thromboelastography revealed no abnormalities.

INTRODUCTION
Lupus anticoagulant (LA) is an antiphospholipid antibody that binds phospholipids and proteins associated with the cell membrane (1). Phospholipids are essential in vivo because of their biological role in cell membranes and are used in vitro to accelerate coagulation. LA have procoagulant properties in vivo, but since they bind phospholipids in the testing assay, they prolong phospholipid-dependent clotting times in vitro (2). The presence of LA and arterial and/or venous thrombosis and/or pregnancy morbidity is one of the characteristics of antiphospholipid syndrome (3). The diagnosis and management of this disease can be challenging, as increased in vitro clotting time can be misinterpreted and may delay anticoagulation.

CASE PRESENTATION
A 77-year-old female patient presented to the emergency department with an acute onset of aphasia, dysphagia and rightsided hemiplegia. The patient had a history of hypertension as well as cutaneous lupus erythematosus without any bleeding events. Prior medication was only verapamil (Ca-channel blocker). She did not take any other medication during the last few weeks. The neurological investigation revealed global aphasia, right-sided homonymous hemianopsia, rightsided hemiplegia and hemihypoalgesia. As an acute stroke was suspected, the patient underwent multinodal cranial CT scanning within the first 15 min of arrival in the emergency department to obtain the necessary information needed for further acute treatment.
There were no signs of an acute intracerebral bleeding but a proximal occlusion of the M1 segment of the left middle cerebral artery (MCA) was detected as well as corresponding perfusion deficit in the territory of the left MCA. As the presentation was within 30 min symptom onset, intravenous thrombolysis with a recombinant tissue plasminogen activator, in combination with a mechanical thrombectomy, where a complete recanalization of the MCA could be achieved, was immediately performed. New onset atrial fibrillation, which was detected in the emergency room, was suspected as possible cause of the stroke. The routine blood coagulation tests sampled at admission showed normal platelet count (171/nl) and reduced plasmatic coagulation. Marburg, Germany). As no coagulation disorder was known and the test results were completed within 30 min of arrival in the emergency, the thrombolysis therapy was performed.

CLINICAL COURSE AFTER ADMISSION
The patient was referred to the neurological intensive care unit. A subsequent up cranial CT scanning revealed full demarcation of a large frontoparietal infarction in the territory of the left MCA and no evidence of bleeding despite in-vitro reduced plasmatic coagulation (Figure 1).
Because of the repeated reduced plasmatic coagulation test, haemostaseology specialists were involved. Further laboratory investigation and the analysis of the activity of separate coagulation factors revealed a reduced activity of coagulation Factor V (20.9%), Factor VII (23.8%), Factor IX (66%), Factor X (19.7%), Factor XI (53%), Factor XII (26%), and Factor XIII (65%) (Citrate blood, using reagents Innovin R /ActinFS R and SYSMEX 2100i Siemens Health Care Diagnostics, Marburg, Germany. FXIII activity was measured by ammonia released chromogenic assay (Berichrom R F XIII, Siemens Sysmex System CS 5100). Von Willebrand Factor antigen (VWF Ag R , Siemens System BCS R XP) and platelet agglutination caused by ristocetin cofactor assay (INNOVANCE R VWF, Siemens System BCS R XP), fibrinogen (Dade R , Siemens Sysmex System CS 5100), and Factor VIIIc (ActinFS R , Siemens Sysmex System CS 5100) as well as platelet function tests (Platelet Function Assay (PFA-100) und Platelet Aggregation by Born) were normal. The global in vitro coagulation test -thromboelastography showed normal test results. The LA test showed pathological results: the diluted-Russel-Viper-Venom (dRVVT) assay was significantly prolonged (112.8 S). The antiphospholipid antibodies were slightly increased in the screening test, however the further IgM and IgG Testing showed results in the RR ( Table 1). 1:1 Plasma mixing study with standard plasma revealed a normalization of INR as well as of coagulation factors activity ( Table 2) as effect of inhibitory activity of patient plasma. Re-assessment of the factor's activity with another reagent Thromborel R (Siemens Healthcare) showed a normalization of INR and factor's activity in comparison to the previously used thromboplastin reagent Innovin R (   of dysphagia without any bleeding complication and the patient was discharged to a neurological rehabilitation center.

DISCUSSION
Both, atrial fibrillation and antiphospholipid syndrome (APS) are associated with cerebrovascular events (4,5). The state of the art treatment of an acute stroke is thrombolytic therapy with the thrombolytic drug -tissue plasminogen activator alteplase (6). However, impaired coagulation can be a contraindicated for fibrinolytic therapy. Thus, INR values above 1,7 are associated with increased risk of intracranial bleeding. Therefore, thrombolytic therapy is contraindicated in these cases (7). In addition hemorrhagic disorders and a deficiency of coagulation factors, lupus antibodies impair global coagulation tests (8).
In the present case, the patient did benefit from thrombolytic therapy and further secondary prevention of thromboembolic events due to APS or atrial fibrillation. However, medical therapy in this case was challenging: on the one hand, there was the need of fibrinolytic therapy and anticoagulation as secondary prevention of thromboembolic events and, on the other hand, in vitro signs of hemorrhagic coagulation disorder due to impaired INR and reduced activity of separate coagulation factors. The fact that there were no signs of impaired liver function and no history of bleeding or any hemorrhagic disorders, gave suspicion that the laboratory results do not depict in-vivo hemostasis. Furthermore, thrombolytic therapy did not cause any bleeding complications, which could be typical for a case of severe coagulation factor deficiency. Therefore, indirect methods, such as a plasma mixing test and testing with other less lupus sensitive reagents were used.

LUPUS ANTICOAGULANT
LA is one of the most mysterious antibodies in patients with an antiphospholipid syndrome. On the one hand, an antiphospholipid antibody may cause a phospholipid-dependent prolongation of the clotting time in vitro and, on the other hand, it is associated with an increased risk of thrombosis (9). This ambivalence is based on the feature of building complexes which slow down coagulation reactions in vitro by forming stable complexes on coagulation active phospholipids (8). Laboratory diagnostic tests for the LA are heterogeneous and include phospholipid-dependent clotting assays, plasma mixing studies, and demonstration of the phospholipid-dependency of the inhibitory activity. Due to the heterogeneity of antibodies, reagents and the variability of the analyser, the general algorithms of LA detection are missing (3). LA is usually detected with clotting assays based on aPTT and dRVVT. Both can be performed at low and high phospholipid concentration, or on 1:1 mixtures of tested sample and a normal plasma pool (10). Some prothrombin assays have demonstrated to be sensitive to the effects of LA, leading to a prolonged PT and consequently false INR values. Thromboplastin reagents are widely used as clotting activator in vitro and they contain either recombinant tissue factor or tissue factor extracted from the brain and placental sources. Recombinant thromboplastins have been discussed to be more sensitive to variation in the presence of LA. Accordingly, tissue extracted thromboplastins are more suitable for measurements (11). The reason for such correlations is not known. Possible explanation is an interaction of LA either with recombinant replicated tissue factor or a different phospholipid composition (12). In summary, the fact of different results using different reagents could be a good evidence of the existence of LA in the present case. Both, plasma mixing test and the use of different reagents were helpful to choose the anticoagulation strategy for this patient. Furthermore, thromboplastin independent coagulation tests, such as thromboelastography, should be used to rule out a severe bleeding coagulation disorder.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
AH, TB, MB, and UH contributed to the conception and design of the paper. VH and MK contributed to preparing laboratory data. MN and AA contributed to developing the idea of the study and revising the article critically for important intellectual content. All authors contributed to the article and approved the submitted version.