Sylvie Mulliez

Lupus anticoagulant-hypoprothrombinemia syndrome: report of two cases and review of the literature

Lupus anticoagulant-hypoprothrombinemia syndrome (LA-HPS) is a rare acquired disorder caused by prothrombin antibodies. The disease is most common in the pediatric age group (<16 years), and more prevalent in women. There are well-established clinical diseases associated with LA-HPS, most notably systemic lupus erythematosus (SLE) and viral infections. The clinical manifestation of LA-HPS varies greatly in severity and it may cause severe life-threatening bleeding diathesis. LA-HPS is to be suspected when a patient presents with bleeding and a prolonged activated partial thromboplastin and prothrombin time, in combination with a lupus anticoagulant. The diagnosis is confirmed in the laboratory by identification of reduced prothrombin levels. There are no standardized recommendations for treatment of the hemorrhage associated with the syndrome; corticosteroids are used as first-line treatment. This review summarizes what is currently known about the pathogenesis, clinical features, diagnosis, treatment and prognosis of LA-HPS, and presents two case reports.

Acquired hemophilia: a case report and review of the literature

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies against clotting factor VIII (FVIII). FVIII autoantibody is characterized as polyclonal immunoglobulin G directed against the FVIII procoagulant activity. This disease occurs most commonly in the elderly population and with preponderance of men in nonpregnancy‐related AHA. There are well‐established clinical associations with AHA such as malignancy, other autoimmune diseases and pregnancy. However, up to 50% of reported cases remain idiopathic. The clinical manifestation of AHA includes mostly spontaneous hemorrhages into skin, muscles and soft tissues, or mucous membranes. AHA should be suspected when a patient with no previous history of bleeding presents with bleeding and an unexplained prolonged activated partial thromboplastin time. The diagnosis is confirmed in the laboratory by the subsequent identification of reduced FVIII levels and FVIII inhibitor titration. There is a high mortality, making prompt diagnosis and treatment vitally important. The principles of treatment consist in controlling the bleeding and eradicating the inhibitor. Because of the overall high relapse rate (15–33%), it is also recommended to follow up these patients. The review summarizes what is currently known about the epidemiology, pathogenesis, clinical features, diagnosis, treatment and prognosis of AHA and starts with a case report.

Isolated acquired factor VII deficiency: review of the literature

OBJECTIVES Isolated acquired factor VII (FVII) deficiency is a rare haemorrhagic disorder. We report what is currently known about the pathogenesis, clinical features, diagnosis, treatment and prognosis of acquired FVII deficiency. METHODS We performed a literature search and included all articles published between 1980 and August 2015. RESULTS AND CONCLUSIONS Acquired FVII deficiency has been reported in 42 patients. There are well-established clinical diseases associated with acquired FVII deficiency, most notably infections, malignancy and haematological stem cell transplantation. The exact pathogenesis of the diseases is still unknown, but different pathophysiological hypotheses have been suggested. The clinical manifestation of acquired FVII deficiency varies greatly in severity; asymptomatic course as well as severe life-threatening bleeding diathesis and fatal bleedings have been described.

Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience.

*Corresponding author: Carolien Bonroy, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital (2P8), De Pintelaan, 185, 9000 Ghent, Belgium, Phone: +32 9 332 36 31, Fax: +32 9 332 49 85, E-mail: carolien.bonroy@uzgent.be Sylvie M.N. Mulliez and Thomas M. Maenhout: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Letter to the Editor

Influence of platelet clumps on platelet function analyser (PFA)‐200® testing

Sir, The platelet function analyser (PFA)-200 is widely used as a simple and rapid screening tool to measure global platelet haemostatic function to detect bleeding disorders as well as to monitor platelet inhibition by several antiplatelet drugs [1]. The PFA-200 simulates the process of platelet adhesion and aggregation triggered by either collagen/epinephrine (coll/EPI) or collagen/ADP (coll/ ADP) in vitro, reporting results as a ‘closure time’ (CT) [2]. The investigation of platelet function by PFA-200 is highly vulnerable to a broad series of pre-analytical variables. Sample collection and transportation may influence the results [3, 4]. Patient characteristics such as a platelet count <100 9 10/L and haematocrit <30% usually results in prolongation of the CT [5, 6], as well as the concentration of von Willebrand factor (vWF) in plasma [7]. The PFA-CT with coll/EPI test cartridge, but not the coll/ADP test cartridge, is usually prolonged by COX-1 inhibitors, such as aspirin [8]. According to manufacturer’s specification, the requirements for processing a specimen for PFA currently include the lack of microthrombi in the sample. Also haemolysed blood for PFA testing is not recommended [9]. Recently, we received a blood sample of a 56-year-old woman, to screen the platelet function before urgently needed neurosurgery; the aspirin (100 mg/day) therapy had been stopped since one day. PFA-200 was performed to evaluate the effect of antiplatelet therapy. The complete blood count revealed a platelet count of 500 9 10/L (normal range, 171–374 9 10/L) and haematocrit value of 32.8% (normal range, 35.8–43.7%). PFA-CT with coll/EPI and coll/ADP were both normal, respectively, 81 s (normal range, 82–150 s) with coll/EPI and 67 s (normal range, 62–100 s) with Coll/ADP in duplicate measurement. Because of the unexpected PFA result with normal CT, light transmission platelet aggregation (LTA) on platelet rich plasma with several agonists (epinephrine 10 lM, adenosine diphosphate (ADP) 2.5 lM and 5 lM, collagen 2.5 lg/mL and 5 lg/mL, ristocetin 0.5 mg/mL and 1.5 mg/mL, arachidonic acid, 0.25 mM and thromboxane A2 analogue U46619 10 lM) was performed on Chrono-log 700 (Chrono-Log, Havertown, PA, USA). The LTA showed normal aggregation with ADP, collagen, ristocetin and U46619. There was no aggregation with arachidonic acid, confirmed in repeated measurement. Although a normal coll/EPI CT the LTA with epinephrine was slightly reduced (an amplitude of 60%); this might be explained by the higher concentration of epinephrine used in the PFA cartridge. The LTA was compatible with the effect of aspirin (Figure 1). The complete blood count on citrated (0.109 mM or 3.2%) blood was performed on Sysmex KX-21N (Sysmex Corporation, Kobe, Japan) haematology analyser, and we noticed a flagging for platelet clumps. A microscopic review of the blood smear confirmed the presence of platelet clumps (Figure 2). This could explain the falsely shortened CT of the PFA with coll/EPI and the discrepancy with LTA. Other possible factors contributing to the short CT of the PFA could be the high platelet count (500 9 10/L) or an elevated vWF (not measured in this patient); however, we had little arguments to conclude to pronounced acute phase because the slightly elevated C-reactive protein (7.3 mg/L, normal range <5 mg/L). Platelet clumps are a frequent cause of flow obstructions and may cause cancellation of the test. We cannot exclude that the false short PFA-CT results in this patients may be due to platelet clumps not indicated as ‘flow obstruction’ by the instrument. In conclusion, we reported a discrepant result in a presurgery platelet function screening between PFA and LTA in a patient taking aspirin. Although variations in test results in regard to aspirin effect on PFA and LTA are described in the literature [10,11], we report another possible cause of false negative results with PFA. Considering the platelet count and haemotocrit is common practice in interpreting PFA-CT results. However, review for platelet clumps is not. Platelet clumps can falsely reduce the CT of the PFA and may lead to misdiagnosis of platelet function disorders, as well as inappropriate perceptions and clinical response related to antiplatelet therapy, as illustrated in this case.

Influence of vitamin K antagonist treatment on activated partial thromboplastin time

Vitamin K antagonists (VKA) perform their anticoagulant effect by inhibiting vitamin K epoxide reductase, an enzyme that recycles oxidized vitamin K to its reduced form. Vitamin K is a cofactor for the post-translational carboxylation of several vitamin K-dependent proteins. Consequently, VKA result in defective synthesis of the vitamin K-dependent clotting factors: factor (F) II, VII, IX, and X. The recommended method used for monitoring VKA is determination of the international normalized ratio (INR) which is derived from the prothrombin time (PT) [1]. This test is sensitive to the levels of FII, FVII and FX, but is not responsive to reduction of FIX, which can be detected by activated partial thromboplastin time (aPTT). APTT measures the efficacy of both the intrinsic and the common coagulation pathway and is also influenced by vitamin K-dependent clotting factors (FIX, FX and FII). Therefore, among patients treated with VKA, a prolonged aPTT is observed. The relationship between an elevated INR in a patient on VKA treatment with the aPTT is not known and probably depends on the FIX level. Very low FIX in patients on VKA treatment, due to mutation in the FIX gene, with prolonged aPTT have been described, but is very rare [2]. This relationship likely depends also on the combination of aPTT reagent and instrument utilized and aPTT reagents differ largely in sensitivity to coagulation factor deficiency [3]. Although aPTT measurement is not indicated in VKA monitoring, the aPTT is widely used as part of a coagulation screening panel. In case of VKA-treated patients a prolonged aPTT is often due to the VKA, although the measure of prolongation of aPTT in VKA-treated patients is unknown. Misinterpretation of a prolonged aPTT should be avoided and therefore the sensitivity of the aPTT reagent towards INR in AVK-treated patients can be informative. In this study, we have evaluated the relationship between INR and aPTT in patients on VKA anticoagulant treatment and the sensitivity of three different aPTT reagents, PTT-A (Diagnostica Stago, Asnières-sur-Seine, France), Cephascreen® (Diagnostica Stago) and C.K. Prest® (Diagnostica Stago). Patients from the Ghent University Hospital treated with VKA and monitored by PT/INR were collected. The study was approved by the Belgian regulatory agency (B670201419819). The trial was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Ghent University Hospital (registration no. 2014/101). Samples with an INR > 1.5 were prospectively and consecutively collected from January to April 2014 and frozen at –20 °C in different aliquots within 4 h after sampling. The aPTT with three different reagents (PTT-A, C.K. Prest® and Cephascreen®) and PT/INR (Neoplastine® CI plus, Diagnostica Stago) were measured. A simple linear regression analysis with 95% and 90% prediction limits between aPTT and INR was made using SPPS® Statistics 20 (IBM Business Analytics, Armonk, NY, USA). Samples with disproportionate aPTT (‘outliers’) were identified as those with an aPTT outside 90% prediction limits and were further analyzed. To measure clotting factor activity the one-stage clotting assay was used. STA®Thrombin 2 (Diagnostica Stago) and Biophen Heparin LRT (Hyphen BioMed, Andresy, France) were used to measure, respectively, thrombin time (TT) and anti-Xa activity. *Corresponding author: Katrien M.J. Devreese, Coagulation Laboratory, Laboratory for Clinical Biology Ghent University Hospital, De Pintelaan, 185 (2P8), 9000 Gent, Belgium, Phone: +32 9 3326567, Fax: +32 9 3324985, E-mail: katrien.devreese@uzgent.be Sylvie M.N. Mulliez and Julie Van den Bogaert: Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium