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Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Letter to the Editors-in-Chief Introduction: In patients with suspected venous thromboembolism (VTE), it is recommended to estimate the pre-test probability of VTE, either by experience or by standardized scoring schemes (e.g. Wells or Geneva score), before performing a D-dimer test. Patients with a low probability or unlikely VTE should have D-dimer performed, and if negative, VTE can be excluded, without further investigations, while if positive, the patient should be referred to radiologic imaging to confirm or exclude VTE. Patients with a high pre-test probability or likely VTE should be referred directly to radiologic imaging without D-dimer testing. Thus, to estimate the pre-test probability of VTE before D-dimer testing is of uttermost importance since the diagnostic algorithm and the interpretation of the D-dimer result is dependent upon this. Further, the diagnostic accuracy of D-dimer can be expressed by the likelihood ratio (LR). LR is the ratio between the probability of a test result given that VTE is present, and probability of the same test result if VTE is absent. A LR of< 1 decreases the probability of disease and a LR of> 1 increases the probability of disease. When clinicians estimate the pre-test probability for VTE, the post-test probability (after D-dimer testing) can be calculated using the LR for D-dimer either for a positive or negative test (i.e. D-dimer above or below the cut-off level, respectively). Probably, most clinicians do not know the LR for D-dimer, but it is indirectly implemented in the above mentioned algorithm for investigating a patient with suspected VTE. The advice to rule out VTE in a patient if the pre-test probability of VTE is low and the D-dimer is below the cut-off level reflects that the post-test probability of VTE in this case is sufficiently low to rule out VTE (should be< 1-2%). However, if the pre-test probability of VTE is high or likely, a negative D-dimer cannot decrease the post-test probability enough to rule out VTE (the post-test probability will still be about 10%) [4,5]. Therefore, only imaging should be requested when pre-test probability of VTE is high. A positive D-dimer cannot increase post-test probability of VTE enough to confirm VTE diagnosis in any case, since D-dimer is a non-specific VTE marker and levels can be increased in several other clinical situations (e.g. infection, inflammation, cancer and pregnancy). The aims of this survey were 1) to study the relationship between pre-test probabilities estimated by clinicians using two case histories and how the same clinicians' categorized the patients into low, moderate or high probability of VTE or into unlikely or likely VTE, and 2) to examine the clinicians' perception of diagnostic accuracy of D-dimer by calculating the LRs based upon their estimated pre- and post-test probabilities. info:eu-repo/semantics/publishedVersion

Interpretive commenting (IC) is an integral part of postanalytical activities of laboratories when the clinical interpretation of laboratory results in the context of the clinical situation of a patient is provided. Harmonizing practices in IC can be an approach to ensure high-quality comments, which if followed by adequate clinical actions has a great potential in improving patient outcomes. This paper reviews basic work prior to harmonization of IC of common laboratory test results. Practices in IC are considerably diverse both within and between countries. The quality of comments is diverse and often clinically misleading in studies that characterize and estimate error prevalence in IC. Systems that can initiate, monitor, and maintain harmonization in IC are in an evolving state. Despite international initiatives, harmonized, implementable performance indicators and goals in IC are not yet available. External quality assurance (EQA) schemes are accessible mainly in English-speaking countries. A proposal for the standard structure of EQA schemes for interpretive comments in clinical chemistry and best practice recommendations for IC are available. Few studies that demonstrate evidence on the clinical utility of IC are available in the literature. To set a strategy on further steps toward harmonization in IC, well-controlled clinical studies need to be conducted, in collaboration with laboratories and their users on the clinical usefulness of IC. Until enough evidence on the value of IC in patient outcomes accumulates, standards of qualification and training for performing IC and more EQA schemes in native languages of the users are required to improve the quality of IC.

Background The regulation of plasma factor XIII (FXIII) levels in healthy individuals has been only partially explored. The identification of major non-genetic and genetic regulatory factors might provide important information on the contribution of FXIII to the risk of cardio/cerebrovascular diseases. Objectives To determine the effect of age, smoking, BMI, fibrinogen concentration on plasma FXIII activity, complex FXIII antigen (FXIII-A 2 B 2 ) and total FXIII-B subunit (tFXIII-B) level, to correlate FXIII-B level with the other two FXIII parameters and to assess the variation of FXIII levels in carriers of major FXIII subunit polymorphisms. Methods 268 healthy individuals were enrolled in the study. FXIII activity was measured by the ammonia release assay; FXIII-A 2 B 2 and tFXIII-B were determined by ELISAs. FXIII-A p.Val34Leu, FXIII-B p.His95Arg and FXIII-B intron K c.1952 + 144 C > G polymorphisms were identified by RT-PCR using melting point analysis with fluorescence resonance energy transfer detection. Results All investigated FXIII parameters showed significant positive correlation with age and fibrinogen level; gender and BMI influenced only tFXIII-B. A highly significant positive correlation was demonstrated between tFXIII-B and the other FXIII parameters. FXIII-A p.Val34Leu polymorphism had only slight, if any effect on FXIII levels. The FXIII-B Arg95 allele moderately increased all three FXIII parameters, but the effect became statistically significant only after adjustment. The FXIII-B intron K G allele drastically decreased FXIII levels, and it seemed to be in synergism with the FXIII-A Leu34 allele. Conclusions Plasma FXIII levels are subjected to multifactorial regulation, in which age, fibrinogen level and FXIII-B intron K polymorphism are major determinants.

Introduction: Clinical algorithms consisting of pre-test probability estimation and D-dimer testing are recommended in diagnostic work-up for suspected venous thromboembolism (VTE). The aim of this study was to explore how physicians working in emergency departments investigated patients suspected to have VTE. Materials and methods: A questionnaire with two case histories related to the diagnosis of suspected pulmonary embolism (PE) (Case A) and deep venous thrombosis (DVT) (Case B) were sent to physicians in six European countries. The physicians were asked to estimate pre-test probability of VTE, and indicate their clinical actions. Results: In total, 487 physicians were included. Sixty percent assessed pre-test probability of PE to be high in Case A, but 7% would still request only D-dimer and 11% would exclude PE if D-dimer was negative, which could be hazardous. Besides imaging, a D-dimer test was requested by 41%, which is a “waste of resources” (extra costs and efforts, no clinical benefit). For Case B, 92% assessed pre-test probability of DVT to be low. Correctly, only D-dimer was requested by 66% of the physicians, while 26% requested imaging, alone or in addition to D-dimer, which is a “waste of resources”. Conclusions: These results should encourage scientific societies to improve the dissemination and knowledge of the current recommendations for the diagnosis of VTE. info:eu-repo/semantics/publishedVersion

Sample rejection due to preanalytical errors is very common in many medical laboratories worldwide, though the decision when and how to refrain from analyzing such samples is handled very heterogeneously. As a rational, it is mostly stated that it is done to prevent the patient from being harmed by wrong medical decisions based on such values. But when thinking of the consequences of laboratory results instead of their quality being important per se, the rejection of preanalytically altered samples might be harming the patient by the need of re- collection and timely delay. The importance of the result is never the result in itself, but the consequences the result will have for the treatment or monitoring of the patient. Then again, these consequences are only foreseeable if the specific clinical situation or the general clinical setting for the respective parameter is known. Based on this mindset it can be necessary to modify general performance specifications into "personalized" performance specifications for each laboratory parameter, which can only be achieved in close interaction with clinicians. In this opinion paper we want to present a pragmatic approach to the development of such performance specifications by extending the recommendations of the 1st Strategic Conference of the EFLM on 'Defining analytical performance goals 15 years after the Stockholm Conference on Quality Specifications in Laboratory Medicine', depending on the availability of clinical information, data on biological variation or state-of-the-art recommendations, thereby redirecting laboratory medicine to a more patient focussed profession.

Introduction Standardisation of treatment with vitamin K antagonists (VKAs) is still an issue after 60 years of use. The study aimed to explore aspects of VKA monitoring in primary and secondary care. Methods Two case histories were distributed to physicians in 13 countries. Case history A focused on a patient with atrial fibrillation on stable anticoagulation (latest INR 2.3). Physicians were asked about frequency of INR measurement, when to change the VKA dose, and the patient's annual risk of ischemic stroke and bleeding. Case history B focused on a patient with an unexpected INR of 4.8, asking for the patient's 48-hour bleeding risk, the immediate dose reduction and time until a repeat INR. Results Altogether, 3016 physicians responded (response rate 8 – 38%), of which 82% were from primary care and 18% from secondary care. Answers varied substantially within and between countries regardless of level of care and VKA used. Median number of weeks between INR measurements was 4 – 6 weeks. Median threshold INR for increasing or decreasing the VKA dose was 1.9 and 3.1, respectively. Risk of ischemic stroke and bleeding were overestimated 2 – 3 times. In case history B, the median dose reduction the two first days was 75% for GPs and 55% for specialists, irrespective of estimates of bleeding risk; with one week to a repeat INR. Conclusion Variation in VKA monitoring is substantial implying clinical consequences. Guidelines seem either unknown or may be considered impracticable. Further efforts towards standardisation of VKA management are needed.

Background The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. Methods We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. Results The Elucigene CF29 Tm v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. Conclusion We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program.

Development of autoantibody against coagulation factor V (FV) is a rare clinical condition with hemorrhagic complications of varying severity. The aim of this study was to establish the pathomechanism of an acquired FV deficiency and characterize the FV inhibitor responsible for the clinical symptoms. A 78-year-old female was admitted to hospital with severe gastrointestinal bleeding. General clotting tests and determination of clotting factors were performed by standard methods. FV antigen and FV containing immune complexes were measured by ELISA. The FV molecule was investigated by Western blotting and by sequencing the f5 gene. The binding of patient's IgG to FV and activated FV (FVa) was demonstrated in an ELISA system and its effect on the procoagulant activity of FVa was tested in clotting tests and in a chromogenic prothrombinase assay. Localization of the epitope for the antibody was performed by blocking ELISA. FV activity was severely suppressed both in plasma and platelets. FV antigen levels were normal by ELISA using polyclonal anti-FV antibody or monoclonal antibody against the connecting region of FV, but depressed when HV1 monoclonal antibody against the C2 domain in the FV light-chain was used as capture antibody. The FV molecule was found intact. An IgG reacting with both FV and FVa was present in the patient's plasma and its binding to FV was inhibited by HV1 antibody. FV-containing immune complexes were detected in the patient's plasma and platelet lysate. The patient's IgG inhibited the procoagulant function of FVa. An anti-FV IgG was present in the patient's plasma and platelets. The autoantibody reacted with an epitope in the C2 domain of FV light chain and neutralized the procoagulant function of FVa.

MINI REPORT Comparison of PFA-100 Closure Time and Template Bleeding Time of Patients with Inherited Disorders Causing Defective Platelet Function Adrienne Kerenyi1, Agota Schlammadinger2, Eva Ajzner1, Istvan Szegedi3, Csongor Kiss3, Zoltan Pap4, Zoltan Boda2 and Laszlo Muszbek1 1Department of Clinical Biochemistry and Molecular Pathology, 2Second Department of Medicine, 3Department of Pediatrics, University Medical School of Debrecen, Debrecen, 4City Health Service, Ophthalmology Unit, Debrecen, Hungary.

5 Complete screening of CFTR gene mutations in cystic fibrosis patients from Eastern Hungary G. Ivady1, L. Madar1, B. Nagy2, F. Gonczi3, M. Macek4, E. Ajzner5, E. Gombos1, J. Kappelmayer1, I. Balogh1. 1University of Debrecen, Clinical Biochemistry and Molecular Pathology, Debrecen, Hungary; 2University of Debrecen, Department of Pediatrics, Debrecen, Hungary; 3Kenezy Gyula County Hospital, Department of Pediatrics, Debrecen, Hungary; 4Charles University, Institute of Biology and Medical Genetics, Prague, Czech Republic; 5Josa Andras County Hospital, Central Laboratory, Nyiregyhaza, Hungary