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In the present study, humoral and T cell-mediated immune responses elicited by BBIBP-CorV (inactivated virus) and BNT162b2 (mRNA-based) vaccines against SARS-CoV-2 virus were compared. Convalescent volunteers were also investigated to evaluate adaptive immunity induced by live virus. Although both vaccines induced antibody- and T cell-mediated immune responses, our analysis revealed significant quantitative and qualitative differences between the two types of challenges. The BBIBP-CorV vaccine elicited antireceptor-binding domain (RBD) IgG, as well as anti-spike protein (S) IgG and IgA antibodies in healthy individuals, the levels of which were much lower than after BNT162b2 vaccination but still higher than in the convalescent patients. The cumulative IFNγ-positive T cell response, however, was only twofold higher in participants injected with BNT162b2 compared to those who were primed and boosted with BBIBP-CorV vaccine. Moreover, the inactivated virus vaccine induced T cell response that targets not only the S but also the nucleocapsid (N) and membrane (M) proteins, whereas the mRNA vaccine was able to elicit a much narrower response that targets the S protein epitopes only. Thus, the pattern of BBIBP-CorV-induced T cell response in virus-naive participants was similar to the cell-mediated anti-SARS-CoV-2 response observed in convalescent patients. Based on these data, we can conclude that the BBIBP-CorV inactivated virus vaccine is immunologically effective. However, the duration of BBIBP-CorV-induced integrated, antibody, and T cell-mediated, immune responses needs further investigation.

Providing evidence-based interpretative comments (IC) is an integral task of clinical laboratory professionals. It may be of special relevance for coagulation testing, where pathological first-line tests could trigger more specialized tests. Our aim was to evaluate the quality of ICs provided to the physician in two samples with activated partial thromboplastin time (APTT) prolongation.Two lyophilized plasma samples and their respective fictional clinical cases (case 1: heparin contamination and case 2: factor VIII deficiency) were sent to European laboratories for APTT and APTT mixing test measurement, and elaboration of ICs based on their results. The quality of ICs was evaluated in terms of analytical classification, laboratory interpretation, advice to physician, clarity, length and whether the clinical question was answered.A total of 214 laboratories were included. Classification of the analytical result was stated in 57 % of comments. Laboratory interpretation was found in 91 % of comments for case 1 and 83.3 % for case 2, among which 9.3 % and 6.5 % were considered wrong, respectively. Advice for the requesting physician was provided in 65.8 % of comments for case 1 and 61.2 % for case 2, among which 36 % and 4.7 % were considered wrong, respectively. More than 70 % of comments for both cases were evaluated as clear and of an adequate length.A significant number of laboratories provide clear interpretations and helpful advice for the management of altered coagulation results. Nevertheless, the finding of several confusing and misleading comments highlights the need for recommendations on elaboration of interpretative comments.

Unexpected prolongation of first-line coagulation tests, including activated partial thromboplastin time (APTT), should trigger further work-up by performing mixing tests to elucidate the underlying cause, direct further specific testing and clarify their possible clinical impact. The aim of our study was to assess whether methodological diversity has any impact on the APTT mixing test results and their interpretation.Two lyophilized plasma samples (case 1: heparin contamination [0.5 IU/mL]; case 2: factor VIII deficiency [0.13 IU/mL]) and their respective fictional clinical cases were sent to European laboratories for APTT measurement and performance of mixing tests. Participants were surveyed about the methodology (reagents, analytical platform, reference ranges), APTT results, mixing test conditions, their classification (normal, equivocal, prolonged) and categorization of the sample (factor deficiency, presence of inhibitor, anticoagulant, unknown).A total of 269 responses were included. For case 1, all participants reported a prolonged APTT, and 91% obtained no correction in the mixing test, without differences among reagents or analytical platforms. Only 15% of them selected the presence of an anticoagulant as the single cause for the prolongation. For case 2, 99% of participants reported a prolonged APTT, while some heterogeneity in the mixing test results was found. Eighty-six percent of participants selected factor deficiency as the cause for APTT prolongation.Most European laboratories obtained valid results for APTT and the subsequent mixing tests, despite using different methodologies. However, their classification could be improved. Therefore, more training and periodic evaluations are recommended to harmonize protocols and ensure proper result classification and categorization.

A Covid-19 betegség megjelenését követően rövid idő után világossá vált, hogy noha az elsősorban légúti tünetekkel jár, ezek mellett nagyon sok esetben egyéb szervek, szervrendszerek, így az idegrendszer károsodására utaló tünetek is megjelennek. Az első észlelés és közlés óta megjelent és naponta megjelenő nagy mennyiségű, a betegséggel, annak szövődményeivel foglalkozó közlemény között számos neurológiai komplikációkkal foglalkozó is megtalálható. Az adatok alapján viszonylag gyakori ideggyógyászati szövődménynek tartható az encephalitis kialakulása. Tünetei az esetek többségében a még zajló légúti kórfolyamattal egyidejűleg vagy a kialakult kritikus állapot során észlelhetők; többféle megjelenési formáját is közölték. Az általunk ismertetett esetben a központi idegrendszer érintettségét jelző tünetek több héttel a lezajlott Covid-19-pneumonia után, a beteg ebből a szempontból már gyógyult állapotában jelentek meg. A klinikum, a képalkotó, EEG- és liquorvizsgálatok eredményei alapján a tünetek hátterében encephalitis igazolódott. Az alkalmazott terápia mellett a klinikai szempontból rohamosan javuló folyamat hátterében az elvégzett vizsgálatok egyértelmű kórokot nem igazoltak. A korábban zajlott SARS-CoV-2-vírusinfekciót is figyelembe véve Covid-19 betegséget követő posztinfekciós központi idegrendszeri kórfolyamatot véleményeztünk, melyre vonatkozóan jelenleg csak elvétve található irodalmi adat.

A koronavírus-betegség (COVID–19) lefolyása rendkívül sokféle lehet. Az érintettek egy része gyakorlatilag tünetmentes marad, míg másoknál súlyos, esetenként halálos kimenetelű komplikációk alakulnak ki. Ennek a variabilitásnak a hátterében – bár kétségtelenül szükség van további kutatásokra is – minden jel szerint az immunrendszer egyes alkotóelemeinek eltérő, néha kifejezetten kóros működése áll. Összefoglalónkban azt szeretnénk bemutatni, hogy milyen kölcsönhatás alakul ki a természetes és az adaptív immunrendszer, valamint a súlyos akut légzőszervi szindrómát okozó koronavírus-2 (SARS-CoV-2) között az emberi szervezetben. Megvizsgáljuk, hogyan befolyásolja a betegség lefolyását az I-es típusú interferonoknak a genetikai hibákra vagy az antiinterferon-autoantitestek jelenlétére visszavezethető elégtelen aktivitása, a myeloid rendszer zavara, a hipergyulladásos állapot kialakulása, a lymphopenia és az adaptív immunrendszer egyénenként eltérő működése. Kitérünk azokra a kulcsfontosságú megfigyelésekre is, amelyek segítettek körvonalazni a SARS-CoV-2-specifikus humorális és sejtközvetített immunmemória legfontosabb jellemzőit. Orv Hetil. 2022; 163(20): 774–787.

Background Correct handling and storage of blood samples for coagulation tests are important to assure correct diagnosis and monitoring. The aim of this study was to assess the pre-analytical practices for routine coagulation testing in European laboratories. Methods In 2013–2014, European laboratories were invited to fill in a questionnaire addressing pre-analytical requirements regarding tube fill volume, citrate concentration, sample stability, centrifugation and storage conditions for routine coagulation testing (activated partial thromboplastin time [APTT], prothrombin time in seconds [PT-sec] and as international normalised ratio [PT-INR] and fibrinogen). Results A total of 662 laboratories from 28 different countries responded. The recommended 3.2% (105–109 mmol/L) citrate tubes are used by 74% of the laboratories. Tube fill volumes ≥90% were required by 73%–76% of the laboratories, depending upon the coagulation test and tube size. The variation in centrifugation force and duration was large (median 2500 g [10- and 90-percentiles 1500 and 4000] and 10 min [5 and 15], respectively). Large variations were also seen in the accepted storage time for different tests and sample materials, for example, for citrated blood at room temperature the accepted storage time ranged from 0.5–72 h and 0.5–189 h for PT-INR and fibrinogen, respectively. If the storage time or the tube fill requirements are not fulfilled, 72% and 84% of the respondents, respectively, would reject the samples. Conclusions There was a large variation in pre-analytical practices for routine coagulation testing in European laboratories, especially for centrifugation conditions and storage time requirements.

Highlights • Upregulated expression of ACE2 was recently reported in bronchoalveolar lavage fluid samples from COVID-19 patients. • We have first analyzed serum ACE2 activity in COVID-19 that increased about 40-fold over the normal range in the presence of 2-3-fold higher levels of endothelium biomarkers. • Soluble E-selectin followed the clinical status of our patient similarly to ferritin and IL-6 levels. • Based on the distinct time course of circulating ACE2, its dramatic rise may act as an endogenous nonspecific protective mechanism against SARS-CoV-2 infection that preceded the recovery of the patient., Endothelial cells express surface angiotensin converting enzyme 2 (ACE2), the main receptor for SARS-CoV-2 that promotes the infection of endothelial cells showing activation and damage. Bronchoalveolar lavage fluid from COVID-19 subjects showed a critical imbalance in the renin-angiotensin-aldosterone system with upregulated expression of ACE2. Recently, intravenous recombinant ACE2 was reported as an effective therapy in severe COVID-19 by blocking the viral entry to target cells. Here we present a case of a critically ill COVID-19 patient with acute respiratory distress syndrome where circulating ACE2 was first measured to monitor disease prognosis. ACE2 activity increased about 40-fold over the normal range and showed a distinct time course compared to 2-3-fold higher levels of endothelium biomarkers. Although the level of soluble E-selectin followed the clinical status of our patient similarly to ferritin and IL-6 levels, the dramatic rise in serum ACE2 activity may act as an endogenous nonspecific protective mechanism against SARS-CoV-2 infection that preceded the recovery of our patient.

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.

Abstract: The introduction of novel oral anticoagulants (NOAC) have long been expected drugs and they quickly became used widespread as their clinical effectiveness was as good as, or even better than the previously used only oral anticoagulant drug, the coumarins. Thus, the direct thrombin inhibitor dabigatran and the activated factor X inhibitors (rivaroxaban, apixaban, edoxaban) have become the part of daily therapeutic practice. Their permeation was facilitated by the guideline which suggested that no laboratory monitoring was required during NOAC treatment and this was very convenient for both patients and doctors. The clinical experience obtained in the past years, however have proved that the ‘one size fits all’ view is oversimplified and there are numerous situations when the determination NOAC levels is unavoidable or highly recommended. This review discusses the laboratory aspects of NOAC treatment, primarily summarizing their effect on the screening tests and special assays of hemostasis and we also describe the correct methods to determine their plasma concentrations. Orv Hetil. 2017; 158(49): 1930–1945.

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.

The introduction of novel oral anticoagulants (NOAC) have long been expected drugs and they quickly became used widespread as their clinical effectiveness was as good as, or even better than the previously used only oral anticoagulant drug, the coumarins. Thus, the direct thrombin inhibitor dabigatran and the activated factor X inhibitors (rivaroxaban, apixaban, edoxaban) have become the part of daily therapeutic practice. Their permeation was facilitated by the guideline which suggested that no laboratory monitoring was required during NOAC treatment and this was very convenient for both patients and doctors. The clinical experience obtained in the past years, however have proved that the 'one size fits all' view is oversimplified and there are numerous situations when the determination NOAC levels is unavoidable or highly recommended. This review discusses the laboratory aspects of NOAC treatment, primarily summarizing their effect on the screening tests and special assays of hemostasis and we also describe the correct methods to determine their plasma concentrations. Orv Hetil. 2017; 158(49): 1930-1945.

Background The association of plasma factor XIII (FXIII) level with venous thromboembolism (VTE) is still controversial and the effect of sex and FXIII B subunit (FXIII-B) polymorphisms in this respect have not been explored. Objectives 1/ To determine FXIII activity and antigen levels in patients with a history of VTE and how they are influenced by sex and FXIII-B polymorphisms. 2/ To explore the association of FXIII levels and FXIII-B polymorphisms with the risk of VTE. Methods 218 VTE patients and equal number of age and sex matched controls were enrolled in the study. FXIII activity was measured by ammonia release assay; FXIII-A2B2 and FXIII-B levels were determined by ELISAs. FXIII-B polymorphisms were identified by RT-PCR using melting point analysis. Results Adjusted FXIII activity and FXIII-A2B2 antigen levels were significantly higher in females with a history of VTE than in the respective controls. FXIII-B levels were significantly lower in male VTE patients than in controls. FXIII-A2B2 antigen levels in the upper tertile increased the risk of VTE in females (adjusted OR: 2.52; CI: 1.18–5.38). Elevated FXIII-B antigen level had a protective effect only in males (adjusted OR: 0.19; CI: 0.08–0.46). FXIII-B Intron K c.1952 + 144 C > G polymorphism significantly lowered FXIII activity, FXIII-A2B2 and FXIII-B antigen levels in both groups. FXIII-B polymorphisms did not influence the risk of VTE. Conclusions In VTE patients the changes of FXIII level and their effect on the risk of VTE show considerable sex-specific differences. Intron K polymorphism results in decreased FXIII levels, but does not influence the risk of VTE.

Apart from maintaining the highest quality of analytical test results, laboratories are now getting more focused on how to achieve the greatest impact of laboratory results on their patient's outcome. Laboratory professionals are now in the learning phase of implementing new practices at different steps of the extra-analytical phases of the testing process where laboratories used to contribute seldom, only sporadically. Recently, the achievable levels of harmonization and responsible contributors at various steps of the testing process have also been proposed. Based on this proposal some tasks of the extra-analytical phase should become primarily the responsibility of laboratories with the involvement of clinicians, like additive testing, individualized interpretative commenting and reporting results with clinical urgency in postanalytical (PA) phase. These tasks can be good targets to start with or to increase patient outcome-oriented extra-analytical activities of laboratories. The status of the present practice of the PA activities for which laboratories proposed to be primarily responsible in the testing process - laboratory-driven PA tasks - will be reviewed below. In addition, approaches of quality assessment (QA) with quality specifications of these laboratory-driven PA tasks and the available best practice recommendations in the light of the achievable level of harmonization will be discussed. Laboratory professionals are encouraged to improve their methodological, theoretical and communicational skills and take the lead and participate in the discussed PA activities that can assist in translating laboratory test results into clinical meaning and thereby lead to better clinical utilization of laboratory test results.

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.

Purpose: Recent studies strongly support the role of genetic factors in the aetiology of age-related macular degeneration (AMD). We investigated the frequency of Tyr402His polymorphism of the complement factor H (CFH) gene, Ser69Ala polymorphism at LOC387715, rs11200638 polymorphism of the HTRA1 gene and different apolipoprotein E (ApoE) alleles in Hungarian patients with AMD in order to determine the disease risk conferred by these factors. Methods: In a case-control study, we performed clinical and molecular genetic examination of 105 AMD patients (48 patients in the early and 57 in the late subgroup) and 95 unrelated healthy controls. Detailed patient histories were recorded with the use of a questionnaire focusing on known risk factors for AMD. Results: In the early AMD subgroup, homozygous CFH, LOC387715 or HTRA1 polymorphisms conferred a 4.9-fold (95% confidence interval [CI] 1.7–14.2), 7.4-fold (95% CI 2.1–26.2) or 10.1-fold (95% CI 2.5–40.8) risk of disease, respectively. In the late AMD subgroup, carriers of two CFH, LOC387715 or HTRA1 risk alleles were at 10.7-fold (95% CI 3.7–31.0), 11.3-fold (95% CI 3.2–40.4) or 13.5-fold (95% CI 3.3–55.4) greater disease risk, respectively. Two CFH and one LOC387715 risk alleles in combination conferred a 15.0-fold (95% CI 3.2–71.0) increase in risk, whereas two LOC387715 risk alleles combined with one CFH risk allele was associated with a 14.0-fold (95% CI 2.1–95.1) increased risk for late AMD. ApoE alleles neither increased disease risk nor proved to be protective. Conclusions: The CFH, LOC387715 and HTRA1 polymorphisms are strongly associated with the development of AMD in the Hungarian population. The association is particularly pronounced when homozygous risk alleles are present and in the late stages of the disease.

Absztrakt A szerzők heparin által kiváltott thrombocytopeniában szenvedő beteg billentyűhibája miatti harmadik szívműtétjének véralvadásgátlását hirudinnal végezték. A szoros aortaszűkület miatt prostacyclin-előkezelés nem jöhetett szóba, és a műtét utáni alvadásgátlás igénye a heparinoidok alkalmazását is kizárta. Rekombináns hirudint (Refludan®/Hoechst) adagoltak és a plazmaszintet ekarin alvadási idő segítségével ellenőrizték. Műtét után újraindították a cumarin hatású véralvadásgátló kezelést, de a hirudin adását az INR kívánt szintjének beállításáig folytatták. A műtét és az ezt követő időszak szövődménymentesen zajlott le, a beteg transzfúziót nem igényelt, és 6,5 évvel a legutóbbi szívműtétje óta panaszmentes és elégedett az életminőségével. Következtetés: Heparinindukált thrombocytopeniában szenvedő beteg nyitott szívműtétjének elvégzéséhez szükséges teljes fokú véralvadásgátlás hirudinnal biztonságosan végezhető, ha azt ekarin alvadási idővel ellenőrzik és vezérlik.

Due to a homozygous Gly204Arg mutation in the factor X (FX) gene no detectable FX antigen was found in the plasma of a one-year old patient with severe bleeding diathesis. The amino acid replacement destabilized the disulfide bond that holds the two FX chains together, decreasing the interaction between the Cys201-Cys206 loop region and the region connecting the EGF2 and serine protease domains. Both Gly204 FX and Arg204 FX were synthesized in transfected cells, but only the wild type protein became secreted. The mutant protein was diverted from the normal secretory pathway and retained at the trans Golgi-late endosome level.

PubMed ID: 26023801, Background: An unexpectedly detected prolonged activated partial thromboplastin time (APTT) can be a harmless laboratory finding, but can also reflect a thrombotic tendency or a bleeding disorder. The assistance of laboratory professionals in the interpretation of an unexpectedly detected prolonged APTT (uAPTT) is often required. The way in which uAPTTs are evaluated in laboratories was assessed in this international study with the aim of determining whether laboratory professionals are able to fulfill this need. Methods: Postanalytical practices after uAPTT were investigated and the mixing study methodology (if used) was studied by circulating a case report with a questionnaire to staff in the invited laboratories. In addition, the interpretations of those staff regarding the presence or absence of inhibitors in three APTT mixing study scenarios were examined. Results: Large within- and between-country variations were detected in both postanalytical practices and mixing study methodologies among the 990 responding laboratories, 90% of which were in 13 countries. Shortcomings regarding the investigation of uAPTTs leading to potentially incorrect or delayed clinical diagnoses were found in 88% of the laboratories. Of the laboratories to which the interpretative questions were sent, 49% interpreted all mixing study scenarios correctly. uAPTTs were investigated appropriately and all mixing study scenarios interpreted correctly in parallel in only 9.6% of the participating laboratories. Conclusions: The clinical requirement for the assistance of laboratory professionals in the interpretation of uAPTTs cannot be met at most of the participating laboratories. Laboratory professionals should be trained in the evaluation of ordinary laboratory tests, such as that for uAPTTs. © 2015 by De Gruyter., Acknowledgments: We thank all of the staff at the participating laboratories who responded to our survey. We are also thankful for EQALM and its member organizations for distributing invitations for this study to the participants. We thank especially those External Quality Assessment organizers who assisted this project in countries providing more than 20 responses each, which were as follows: Ö QUASTA (Austrian Society for Quality Assurance and Standardization of Medical Laboratory Tests), Vienna, Austria; CSMBLM (Croatian Society of Medical Biochemistry and Laboratory Medicine, CROQALM Programme), Zagreb, Croatia; DEKS (Danish Institute for External Quality Assurance for Laboratories in Health Care) Herlev, Denmark; CTCB (Centre Toulousain pour le Contrô le de qualité en Biologie clinique), Toulouse, France; RfB (Reference Institute for Bioanalytics), Bonn, Germany; Qualicont (In Vitro Diagnostic Quality Control Nonprofit Public Utility Ltd.), Szeged, Hungary; IEQAS (Irish EQA Scheme), Dublin, Ireland; Centro di Ricerca Biomedica of Veneto Region, Padova, Italy; NKK (Norwegian Clinical Chemistry EQA Program), Bergen, Norway; PNAEQ (Programa Nacional de Avalia ç ã o Externa da Qualidade), Lisbon, Portugal; CSCQ (Quality Control Center Switzerland), Ch ê ne-Bourg, Switzerland; SEKK, Pardubice, Czech Republic; SKML (Dutch Foundation for Quality Assessment in Medical Laboratories, Section Coagulation), Nijmegen in liason with ECAT Foundation, Voorschoten, The Netherlands. We are also grateful for Joseph Watine for his useful comments during the project and to Csilla P á l and S á ndor Kecskem é ti for their excellent technical assistance. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Financial support: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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.

Disturbances of coagulation and fibrinolytic pathways were studied in 53 young patients with cerebral ischemia. Upon admission 26 of 53 patients had abnormality in at least one of the antithrombin-III, protein C, protein S activities or in activated protein C (APC) ratios. Three months after the first examination the majority of the previously detected abnormalities returned to normal values and the most frequent alterations were decrease in protein S activity (3 patients) and APC resistance (3 patients). Conditions resulting in impaired fibrinolysis were frequently detected upon admission. Elevation of plasminogen activator inhibitor-1, lipoprotein (a), and alpha-2-antiplasmin was present in 23, 10, and 4 cases, respectively. It is concluded that abnormalities of coagulation as well as of the fibrinolytic systems are prevalent in the acute phase of cerebral ischemia, however, the results may be significantly influenced by the disease process or the acute phase effect.

The Leiden mutation is a recent discovery. It is the main cause of inherited thrombophilia and has been found in 20-60% of deep vein thrombosis cases. More recently it has been found in a significant number of cases of obstetric complications attributable to placental thrombosis. Current patient management practice for dealing with the Leiden mutation is based mainly on information about deep vein thrombosis because there is little information on pregnancy complications. There are no prospective studies examining the risk of developing pregnancy complications for Leiden mutation carriers. The aim of this study is to do that by comparing the frequency of unfavourable pregnancy outcomes among carriers with those among controls. The number of women developing miscarriages, intrauterine deaths, or infertility problems among 128 Leiden mutation carriers was compared with the number among 461 controls. The risk of having at least one miscarriage or infertility problems was 1.5 times greater for Leiden mutation carriers than controls. This result was statistically significant (95% CI 1.2, 2.7). The risk of having at least two miscarriages or infertility problems was 2.5 times greater for Leiden mutation carriers than controls. This was also statistically significant (95% CI 1.2, 5.13).

The coagulation factor V Leiden mutation, leading to resistance to activated protein C (APC), is the most common inherited risk factor for venous thrombosis. In various systemic autoimmune diseases the hypercoagulable state was shown to be associated with the presence of antiphospholipid antibodies (aPL). Our aim was to determine the prevalence of both, Leiden mutation and aPL in autoimmune diseases and their impact on the occurrence of venous thrombosis. The dataset consists of results from 137 patients having Sjögren's syndrome (n = 50), progressive systemic sclerosis (n = 43) (PSS), undifferentiated connective tissue disease (n = 24) (UCTD) and mixed connective tissue disease (n = 20) (MCTD) with or without venous thromboembolic complications. The Leiden mutation was detected with polymerase chain reaction (PCR), aPL, such as lupus anticoagulant (LA) with screening and confirmatory procedures and others with enzyme linked immunosorbent assay (ELISA). The prevalence of mutation ranged between 8.3% and 18.0% (13.1%). The thromboembolic risk was found to be increased in the presence of aPL. Eight patients (5.84%) (4 heterozygous) experienced thromboembolic events and 3 out of 4 heterozygous showed aPL positivity, too. There were no difference between the frequencies of Leiden mutation in examined systemic autoimmune diseases and unselected populations.

We studied the prevalence and the effect of coagu lation factor V Leiden mutation on the occurrence of throm botic episodes in 120 Hungarian patients having systemic lupus erythematosus (SLE) with or without antiphospholipid anti body. The frequency of the factor V Leiden mutation in Hungarian SLE patients was 13%, which is comparable with those found previously in a healthy Caucasian population. The incidence of venous thrombosis among factor V Leiden carriers has been found to be higher (odds ratio [OR] 1.7) than it is in patients without Leiden mutation (38% vs 29%). In addition, the fre quency of venous thrombosis in the heterozygous SLE patients (OR 8.4 [confidence interval (CI) 0.8-83.9] P=0.06) is depen dent on the coexistence of other risk factors, such as antiphos pholipid antibody. Moreover, among heterozygous factor V SLE patients, the Leiden mutation could explain the tendency to have significantly higher prevalence of fetal losses (OR 3.9 [CI 1.2-12.0] P = 0.02) and higher prevalence of cerebrovascu lar lesions, cardiac valvular abnormalities, and Raynaud's syn drome than that found in individuals without factor V Leiden mutation or those having antiphospholipid antibody. Systemic lupus erythematosus patients with combined de fects suffer more severely from thrombosis than those with a single risk factor do, suggesting that thrombophilia is a multi- factorial disorder in SLE, also. Although, the factor V Leiden mutation does not seem to be a significant risk factor for ve nous thrombosis in SLE, these data demonstrate that Leiden mutation can be regarded as an additive thrombogenic factor providing higher predisposition to several vasoocclusive disor ders in SLE.

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A2B2 complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.

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.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26-0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30-393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98-12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD. © 2012 Losonczy et al., This study was supported by by the Hungarian Research Fund OTKA K68616 (to A.B.) and by the Social Renewal Operational Programme TAMOP 4.2.1./ B-091/1/KONV-2010-0007 project (to A.B. and I.B.).

Congenital thrombophilia is responsible for thromboembolic complications despite prolonged low-molecular-weight heparin (LMWH) prophylaxis following hip and knee endoprosthesis surgery.A series of 86 patients with hip or knee endoprosthesis surgery were assessed 1 year after operation. Antithrombin III, protein C, and protein S were determined, and the activated protein C sensitivity ratio was measured. We screened for the presence of lupus anticoagulant, factor V Leiden mutation, and polymorphism of prothrombin G20210A. The lower limb venous circulation was monitored by color Doppler ultrasonography. Pulmonary embolism (PE) was diagnosed using ventilation and perfusion scintigraphy.In all, 33 patients had thromboembolic complications, 18 with thrombophilia (7 with combined form). Of the 53 patients without complications 12 had thrombophilia (2 with combined form). The differences were statistically significant. The risk score, the prevalence of FV Leiden and prothrombin G20210A mutations, and lupus anticoagulant were also significantly higher in the symptomatic group. Deep vein thrombosis (DVT) developed preoperatively in 15 patients; DVT and PE in 4 patients; thrombophilia was diagnosed in 53% and 75% of these cases. In all, 17 patients had postoperative thromboembolic complications: DVT developed in nine and PE in one patient (all with thrombophilia); DVT + PE developed in seven patients (all but one had thrombophilia).Significant differences were found in the incidence (Por = 0.01) of thrombophilia and the risk score (Por = 0.02) between symptomatic and asymptomatic patients. We recommend preoperative thrombophilia screening for patients with a history or familial prevalence of thromboembolism and/or with a high risk score (or =15). In cases of thrombophilia, the form and duration of anticoagulant treatment must be decided individually.

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.

It has recently been postulated that thrombophilia may have a role in the aetiology of Perthes’ disease. The published reports, however, remain conflicting. In this study a retrospective analysis of the coagulation parameters was made in 47 patients with Perthes’ disease and the results compared with the clinical data. Five patients with Factor V Leiden mutation were found (10.6%) and surprisingly four of them had a homozygous pattern. These four patients showed the most severe form of the disease, Catterall group IV, with flattening of the entire epiphysis, involvement of the metaphysis, shortening and broadening of the femoral neck, trochanteric overgrowth and developed mushroom-shaped aspherical laterally displaced femoral heads in dysplastic acetabula. We would like to suggest that the homozygous form of Factor V Leiden mutation has some role in the clinical course of Perthes’ disease and particularly its most severe form.

Recent reports seem to support the role of the thrombophilia and decreased fibrinolysis in the aetiopathogenesis of aseptic necrosis of bone. In the present study, haemostatic disturbances were analysed in adults (n = 49) and patients in childhood (Perthes disease) (n = 47) with aseptic necrosis of the femoral head. Fibrinolytic parameters (in vitro clot lysis, plasminogen, plasmatic plasminogen activator inhibitor-1 activity, D-dimer) along with lipoprotein (a) [Lp(a)] and fibrinogen were measured. von Willebrand factor, platelet activation and some thrombophilic factors (activated protein C resistance and factor V Leiden mutation, protein C, protein S activity) were also determined. Impaired fibrinolysis, an increased Lp(a) level along with slow clot lysis and increased platelet activation were found in adult cases. We detected five cases of factor V Leiden mutations (one heterozygotic and four homozygotic) among patients with Perthes disease. The clinical course of the heterozygous case was similar to the usual form of Perthes disease. The most severe form of Perthes disease has been observed in homozygous factor V Leiden mutation cases. The mutation of factor V Leiden per se probably does not induce the development of aseptic necrosis of bone tissue in childhood, but it does play a role in its acceleration. Homozygous factor V Leiden mutation definitely runs a more severe course. On the other hand, in adult cases, the disturbances of haemostasis, impaired fibrinolysis, elevated Lp(a) level, increased platelet activation and slight elevation of fibrinogen might have clinical relevance. Further studies should focus on proving the role of the haemostatic alterations in the pathogenesis of severe forms of aseptic bone necrosis. The use of antithrombotic drugs in order to slow the process of aseptic necrosis also has to be addressed in future surveys.

A male infant with severe bleeding tendency had undetectable factor V activity. Sequence analysis of the proband's DNA revealed one base deletion in exon 13 (2952delT) and one base insertion in exon 16 (5493insG) in heterozygous form. Both mutations introduced a frameshift and a premature stop at codons 930 and 1776, respectively. The proband's father and mother were heterozygous for 2952delT and for 5493insG, respectively. Both mutations would result in the synthesis of truncated proteins lacking complete light chain or its C-terminal part. In the patient's plasma, no factor V light chain was detected by enzyme-linked immunosorbent assay. The N-terminal portion of factor V containing the heavy chain, and the connecting B domain was severely reduced but detectable (1.7%). A small amount of truncated factor V–specific protein with a molecular weight ratio of 236 kd could be immunoprecipitated from the plasma and detected by Western blotting. This protein, factor VDebrecen, corresponds to the translated product of exon 16 mutant allele.

A new one-step ELISA was developed for the determination of the concentration of blood coagulation factor XIII subunit A (FXIII-A) in plasma and in cell lysates. Monoclonal antibodies directed against different epitopes on FXIII-A were used for the assay. The capture antibody was biotinylated on its carbohydrate moiety and the detection antibody was labelled with horseradish peroxidase. The antigen-antibody reaction was carried out in the well of a streptavidin-coated microplate. Complex formation with FXIII subunit B (FXIII-B) and association to fibrinogen did not influence the accessibility of the antibodies to FXIII-A. The method could be performed within 2 h and demonstrated good reproducibility, recovery and sensitivity. Plasma samples could be assayed after storage at -20 degrees C for at least 6 months. However, in the case of platelet lysates freezing and rethawing resulted in a significant loss of FXIII-A. FXIII-A concentrations measured in the plasma samples of healthy individuals and patients correlated well with the concentrations of complexed plasma FXIII (A2B2) and with the results of FXIII activity measurements. A reference range of 46-82 fg/platelet was established for platelet FXIII-A.

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