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Haematology, Including Haemostasis Clinica Chimica Acta Clinica Chimica Acta 493 (2019) S379–S433 Contents lists available at SciVerse ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim Haematology, including haemostasis cases of homozygous mutants T677 T, 2 cases of homozygous W034 mutants C1298C and 1 case heterozygous normal C677T with mutation in C1298C homozygosis. Prevalence of the different genotypes of the MTHFR gene performed in a South Spain health area ^ Conclusions R. Coca Zuñigab, A. González Rayaa, G. Callejón Martína, E. Martín The study shows that 9.3% of the studied population presents Sálidoa, A. Lendinez Ramireza, M. Cantero Sancheza, M.L. Hortas the heterozygous combination of both mutations C677T + A1298C, Nietoa in these patients the activity of MTHFR will be significantly aHospital Costa del Sol, Marbella, Spain reduced. The heterozygous forms of the two alterations separately bHospital Universitario Virgen de las Nieves, Granada, Spain represent 48%, which do not present associated risk of hyperho- mocysteinemia. 6.9% and 16.2% of the cases correspond to Background-aim homozygous mutant forms for T677 T and C1298C respectively, these mutations should be considered as factors of high indepen- Methylenetetrahydrofolate reductase (MTHFR) is an enzyme dent thrombotic risk for causing high homocysteine levels. The that participates in the regulation of intracellular folate, an results of the study show that 67.3% of the studied population is essential compound for the synthesis of proteins and nucleic considered asymptomatic, because they do not present any of the acids. This enzyme catalyzes the transformation of 5,10-methy- mutation in homozygosis. It should therefore be considered to lenetetrahydrofolate to 5-tetrahydrofolate, a donor of methyl establish a right protocol to be followed in which the determination groups necessary for the conversion of homocysteine to methio- of the mutation of the MTHFR gene should be subsequent to a state nine. Deficiency in its activity can lead to hyperhomocysteinemia, of hyperhomocysteinemia. which is considered as a risk factor for both atherogenesis and thrombogenesis. A genetic polymorphism has been detected doi:10.1016/j.cca.2019.03.809 frequently in 677 nucleotide (C677T), which involves the substi- tution of alanine for valine, this variant is associated with a low enzymatic activity (up to 50%). A second polymorphism has been located at position 1298 (A1298C) which involves the substitution ^W035 of glutamine for alanine. The aim of this work is to study the prevalence of mutations in Understanding antiphospholipid antibody (APLA) syndrome in the MTHFR gene in patients from a south of Spain health area during the laboratory – Diagnostic challenges the years 2017 and 2018. S.N. Kamatham, P. Suryadeep, K. Azmathullah, S.S. Gorji, B. Jupalle, Methods B.V.L.N. Murthy, H.A. Syed CARE Hospital, Road No.1, Banjara Hills, Hyderabad, India We performed a genomic DNA study to 43 patients referred to our laboratory with clinical suspicion of hyperhomocysteinemia and Background-aim family study to the confirmed cases with genotype of high risk. To carry out the genotyping, an automated DNA extraction was ^Laboratory evidence of antibodies against phospholipids or performed on a maxwell®16 (promega) device. DNA amplification phospholipid binding protein co-factor, results in an acquired and detection of mutations with FRET probes by real-time polymer- autoimmune thrombophilia and hence essential in diagnosis of APLA. ase chain reaction (RT-PCR), in a Light 2.0 Cycler autoanalyzer (Roche Diagnostic ®). The test determines the presence or absence of Methods the mutation and distinguishes between the homozygous and heterozygous genotypes. The laboratory has algorithm approach, using both coagulation and serological studies, the former done by automation and later by Results ELISA 1. Screen for Prothrombin time and APTT with mixing studies leading to 2. identification of inhibitors by temperature incubation 3. Of the 43 cases analyzed, there were: 8 cases of normal Dilute Russel Viper Venom time 4. assays of IgG, IgM & IgA for both homozygotes C677C/A1298A, 7 cases of heterozygotes C677T, 14 anticardiolipin and antiphospholipid 5. Followed by IgG & IgM for ®2 heterozygotes A1298C, 4 cases of heterozygotes C677T/A1298C, 7 glycoprotein. 0009-8981/$ – see front matter Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 Results glomerulonephritis) was observed. In addition, the patient presented episodes of paroxysmal atrial fibrillation. She was given enoxaparin. Over the past 10 years there has been an annual increase in Since its administration, a downward trend in platelet values was referral to laboratories for investigating the APLA syndrome from appreciated. The following platelet values were obtained (range of various clinical specialities. The mechanisms and pathophysiology of normality 140.00–400.00 10^3/mL): 215.00 (11/22/2018), 154.00 the clinical symptoms are highly heterogeneous. (11/24/2018), 49.00 (11/27/2018). Upon suspicion by the laboratory, At our tertiary care hospital laboratory, the referrals have been anti heparin antibodies were determined (normal range 0.00–1.00 U/ abundant and varied, with increasing numbers every month. The mL): 12.40 (04/12/2018). incidence of numbers being as mentioned in the etiologies referred below: Results 1. Autoimmune disorders Heparin-induced thrombocytopenia was diagnosed, confirmed 2. Young strokes after positivity of the anti-heparin antibodies, so treatment with 3. Deep vein thrombosis enoxaparin was suspended until the recovery of platelet numbers for 4. Bad obstetric history subsequent initiation of oral anticoagulation. 5. Infertility 6. APLA associated with renal, neurological, cardiac presentations Conclusions 7. Catastrophic APLA (having definite criteria of clinical and laboratory) The presence of anti-heparin antibodies is an underdiagnosed Conclusions complication and should be suspected in any patient with anticoag- ulant treatment and thrombocytopenia and/or thrombotic events. After the diagnosis, treatment should be suspended and the The APLA syndrome is both a diagnostic and clinical challenge. administration of other anticoagulants such as heparinoids or direct The Global Anti Phospholipid Syndrome score (GAPPS) computation thrombin inhibitors should be evaluated. Because these incidental makes risk stratification understandable for better therapeutic findings are so rare, the laboratory must be alert to prevent possible approach. complications that put the patient's life at serious risk. Acknowledgement. All clinical specialities. doi:10.1016/j.cca.2019.03.811 doi:10.1016/j.cca.2019.03.810 ^W037 ^W036 Impact of doxycycline on platelet activation during platelet Contribution from the laboratory in the diagnosis of heparin- concentrates storage – Preliminary research induced thrombocytopenia. A case study A. Rak-Pasikowskaa, A. Wrzyszczb, K. Wisniewskab, I. Bil-Lulaa J. Paco Ferreira, T. HernÁndez Lemes, M. Kassih Ibrahim, A.M. aDivision of Clinical Chemistry, Department of Medical Laboratory SÁnchez De Abajo, M. Navarro Romero, G. Garcia Aguilar, T. Dorta Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnos- Ramos, A. Cabrera Argany tics, Wroclaw Medical University, Wroclaw, Poland Servicio de Análisis Clínicos y Bioquímica Clínica, Complejo Hospitalario bDivision of Laboratory Haematology, Department of Medical Labora- Universitario Insular Materno Infantil (CHUIMI), Las Palmas de Gran tory Diagnostics, Faculty of Pharmacy with Division of Laboratory Canaria, Spain Diagnostics, Wroclaw Medical University, Wroclaw, Poland Background-aim Background-aim Heparin is the most commonly used anticoagulant treatment. During platelet concentrates' (PCs) storage platelets lose their Bleeding is the most common complication, however another functions and their activation increases. The reasons of these changes possible side effect is heparin-induced thrombocytopenia (HIT), a are not fully investigated. Plasma, erythrocytes and leukocytes life-threatening complication of exposure to heparin that occurs in a present in PCs may be a source of matrix metalloproteinases small percentage of patients exposed, regardless of the product, dose, (MMPs). MMPs may be one of the factors which are presumably schedule, or route of administration. HIT results from an autoanti- responsible for lesions of platelets during storage. Doxycycline is one body directed against endogenous platelet factor 4 (PF4) in complex of the MMPs inhibitor. We hypothesized that doxycycline can delay with heparin. This antibody activates platelets and can cause serious or decrease platelet activation. The aim of the study was to evaluate arterial and venous thrombosis with a mortality rate up 20%; platelet activation during storage of PCs with and without doxycy- although with improved recognition and early intervention, mortal- cline addition. ity rate have been reported as below 2%. Methods Methods Four PCs were divided into two parts and doxycycline was added Patient 79 years old woman. Generalized asthenia was reported to one of them (PC + D) (10 μM final concentration). They were from three weeks ago, analytical was performed observing acute stored simultaneously in the same conditions. Samples were renal failure with creatinine 6.60 (0.67–1.17 mg/dL). The patient was separated from main concentrate in the day of preparation (0 h), admitted as a matter of urgency at the hospital. After study, focal after 24, 48, 72 and 144 h of storage. In every sample, an expression
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