Impact of thrombocytopenia-associated c.-118C>T and c.-140C>G ANKRD26 5'UTR variants in three-generational pedigree.

Inherited thrombocytopenias (ITs) encompass a group of rare disorders characterized by diminished platelet count. Recent advancements have unveiled various forms of IT, with inherited thrombocytopenia 2 (THC2) emerging as a prevalent subtype associated with germline variants in the critical 5' untranslated region of the ANKRD26 gene. This region is crucial in regulating the gene expression of ANKRD26, particularly in megakaryocytes. THC2 is an autosomal dominant disorder presenting as mild-to-moderate thrombocytopenia with minimal symptoms, with an increased risk of myeloproliferative malignancies. In our study of a family with suspected IT, three affected individuals harbored the c.-118C>T ANKRD26 variant, while four healthy members carried the c.-140C>G ANKRD26 variant. We performed a functional analysis by studying platelet-specific ANKRD26 gene expression levels using quantitative real-time polymerase-chain reaction. Functional analysis of the c.-118C>T variant showed a significant increase in ANKRD26 expression in affected individuals, supporting its pathogenicity. On the contrary, carriers of the c.-140C>G variant exhibited normal platelet counts and no significant elevation in the ANKRD26 expression, indicating the likely benign nature of this variant. Our findings provide evidence confirming the pathogenicity of the c.-118C>T ANKRD26 variant in THC2 and suggest the likely benign nature of the c.-140C>G variant.

What is the context?Inherited thrombocytopenias (ITs) are rare conditions characterized by low platelet counts. Inherited thrombocytopenia 2 (THC2) is caused by ANKRD26 gene changes leading to increased ANKRD26 expression as the main reason for subsequent thrombocytopenia. THC2 results in a mild-to-moderate decrease in platelet count and increases blood cancer risk. We focused on understanding two ANKRD26 variants in a family with a history of thrombocytopenia.What is new?We conducted functional analysis to understand the effect of variants on platelet function and gene expression. We identified three thrombocytopenic family members as carriers of ANKRD26 variant c.-118C>T. This variant is linked to increased expression of the ANKRD26 gene and confirmed as the likely cause of THC2. Another variant, c.-140C>G, was present in four healthy family members. Although it was considered causal for THC2 in the past, our study suggests that the c.-140C>G variant does not elevate ANKRD26 expression and does not cause thrombocytopenia.What is the impact?Understanding the genetic and functional implications of ANKRD26 gene variants is crucial for THC2 diagnosis and management. Our study emphasizes the necessity of conducting functional analyses to precisely evaluate the clinical significance of variants linked to inherited blood disorders. Carriers of the c.-118C>T variant should undergo vigilant monitoring for THC2 and potential cancer development. Conversely, the c.-140C>G variant does not pose a risk of THC2 or heightened cancer susceptibility.

Bleeding pattern and consumption of factor VIII concentrate in adult patients with haemophilia A without inhibitors in the Czech Republic between 2013 and 2021 (Czech National Haemophilia Programme registry data).

The manuscript provides an overview of treatment and its changes in adult patients with haemophilia A without inhibitors in the Czech Republic between 2013 and 2021 using data from the registry of the Czech National Haemophilia Programme (CNHP). Over a 9-year period, we focused on the reduction in the annual bleeding rate (ABR), joint bleeding rate (AJBR) and factor VIII consumption when patients with severe haemophilia A switched from on-demand treatment to prophylaxis. The ABR and AJBR include both patient-reported home treatment and treated hospitalisation episodes. All adult patients with severe haemophilia A were categorised into three groups according to the therapeutic regimen. The first group was patients on prophylaxis during the follow-up period, the second group consisted of patients on on-demand treatment, and the third group was patients who received both treatment regimens during follow-up. With an increase in the proportion of patients with severe haemophilia A on prophylaxis from 37 to 74% between 2013 and 2021, the ABR for all patients with severe haemophilia A decreased approximately 6.9-fold, and the AJBR decreased 8.7-fold. Expectedly, the factor consumption increased by approximately 68.5%. In the group of patients with severe haemophilia A who had switched from an on-demand to a prophylactic regimen, the total number of bleeding events decreased 3.5-fold, and the number of joint bleeding episodes decreased 3.9-fold. Factor VIII consumption increased by 78.4%. Our study supports a previously reported positive effect of prophylaxis on bleeding control. We believe that the substantial improvement in ABR justifies the increased treatment costs.

A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Liver transplantation as potential curative method in severe hemophilia A: case report and literature review.

The authors present clinical case of orthotopic liver transplantation for cirhosis due to chronic viral hepatitis C in a subject with severe hemophilia A. Preoperatively performed pharmacokinetic study with recombinant F VIII confirmed satisfactory in vivo recovery of 2.1 %. A bolus application of 52 units F VIII/kg body weight with target F VIII activity over 100.0 % was administred shortly before the transplantation started. Totally, 30 000 units of recombinant F VIII, 3 thrombocyte concentrates, 2 erythrocyte concentrates, 5 units of virally inactivated plasma, 1 unit of fresh frozen plasma and 3 500 antithrombin units were used. There were no perioperative or postoperative bleeding complications, F VIII substitution was stopped on postoperative day 3. The patient was discharged on twentieth postoperative day.

A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome.

Mutations in the GP1BA gene have been associated with platelet-type von Willebrand disease and Bernard-Soulier syndrome. Here, we report a novel GP1BA mutation in a family with autosomal dominant macrothrombocytopenia and mild bleeding. We performed analyses of seven family members. Using whole-exome sequencing of germline DNA samples, we identified a heterozygous single-nucleotide change in GP1BA (exone2:c.176T>G), encoding a p.Leu59Arg substitution in the N-terminal domain, segregating with macrothrombocytopenia. This variant has not been previously reported. We also analysed the structure of the detected sequence variant in silico. In particular, we used the crystal structure of the human platelet receptor GP Ibα N-terminal domain. Replacement of aliphatic amino-acid Leu 59 with charged, polar and larger arginine probably disrupts the protein structure. An autosomal dominant mode of inheritance, a family history of mild bleeding episodes, aggregation pattern in affected individuals together with evidence of mutation occurring in part of the GP1BA gene encoding the leucine-rich repeat region suggest a novel variant causing monoallelic Bernard-Soulier syndrome.

One-Step Fabrication of a Microfluidic Device with an Integrated Membrane and Embedded Reagents by Multimaterial 3D Printing.

One of the largest impediments in the development of microfluidic-based smart sensing systems is the manufacturability of integrated, complex devices. Here we propose multimaterial 3D printing for the fabrication of such devices in a single step. A microfluidic device containing an integrated porous membrane and embedded liquid reagents was made by 3D printing and applied for the analysis of nitrate in soil. The manufacture of the integrated, sealed device was realized as a single print within 30 min. The body of the device was printed in transparent acrylonitrile butadiene styrene (ABS) and contained a 400 µm wide structure printed from a commercially available composite filament. The composite filament can be turned into a porous material through dissolution of a water-soluble material. Liquid reagents were integrated by briefly pausing the printing before resuming for sealing the device. The devices were evaluated by the determination of nitrate in a soil slurry containing zinc particles for the reduction of nitrate to nitrite using the Griess reagent. Using a consumer digital camera, the linear range of the detector response ranged from 0 to 60 ppm, covering the normal range of nitrate in soil. To ensure that the sealing of the reagent chamber is maintained, aqueous reagents should be avoided. When using the nonaqueous reagent, the multimaterial device containing the Griess reagent could be stored for over 4 days but increased the detection range to 100-500 ppm. Multimaterial 3D printing is a potentially new approach for the manufacture of microfluidic devices with multiple integrated functional components.

Comparing Microfluidic Performance of Three-Dimensional (3D) Printing Platforms.

Three-dimensional (3D) printing has emerged as a potential revolutionary technology for the fabrication of microfluidic devices. A direct experimental comparison of the three 3D printing technologies dominating microfluidics was conducted using a Y-junction microfluidic device, the design of which was optimized for each printer: fused deposition molding (FDM), Polyjet, and digital light processing stereolithography (DLP-SLA). Printer performance was evaluated in terms of feature size, accuracy, and suitability for mass manufacturing; laminar flow was studied to assess their suitability for microfluidics. FDM was suitable for microfabrication with minimum features of 321 ± 5 µm, and rough surfaces of 10.97 µm. Microfluidic devices >500 µm, rapid mixing (71% ± 12% after 5 mm, 100 µL/min) was observed, indicating a strength in fabricating micromixers. Polyjet fabricated channels with a minimum size of 205 ± 13 µm, and a surface roughness of 0.99 µm. Compared with FDM, mixing decreased (27% ± 10%), but Polyjet printing is more suited for microfluidic applications where flow splitting is not required, such as cell culture or droplet generators. DLP-SLA fabricated a minimum channel size of 154 ± 10 µm, and 94 ± 7 µm for positive structures such as soft lithography templates, with a roughness of 0.35 µm. These results, in addition to low mixing (8% ± 1%), showed suitability for microfabrication, and microfluidic applications requiring precise control of flow. Through further discussion of the capabilities (and limitations) of these printers, we intend to provide guidance toward the selection of the 3D printing technology most suitable for specific microfluidic applications.

In Silico Screening of Two-Dimensional Separation Selectivity for Ion Chromatography × Capillary Electrophoresis Separation of Low-Molecular-Mass Organic Acids.

A prerequisite for ordered two-dimensional (2D) separations and full utilization of the enhanced 2D peak capacity is selective exploitation of the sample attributes, described as sample dimensionality. In order to take sample dimensionality into account prior to optimization of a 2D separation, a new concept based on construction of 2D separation selectivity maps is proposed and demonstrated for ion chromatography × capillary electrophoresis (IC×CE) separation of low-molecular-mass organic acids as test analytes. For this purpose, 1D separation selectivity maps were constructed based on calculation of pairwise separation factors and identification of critical pairs for four IC stationary phases and 28 levels of background electrolyte pH in CE. The derived IC and CE maps were then superimposed and the effectiveness of the respective 2D separations assessed using an in silico approach, followed by testing examples of one successful and one unsuccessful 2D combination experimentally. The results confirmed the efficacy of the predictions, which require a minimal number of experiments compared to the traditional one-at-a-time approach. Following the same principles, the proposed framework can also be adapted for optimization of separation selectivity in various 2D combinations and for other applications.

[Is it necessary to revaccinate against hepatitis B virus when the titer of anti-HBs drops below 10 IU/L?]

Viral hepatitis B still represents a major epidemiological issue worldwide. After recombinant vaccine administration, a titer of anti-HBs antibodies ≥ 10 IU/L has long been considered to be seroprotective. Persons without this antibody level after complete vaccination are referred to as non-responders. A progressive decline in anti-HBs levels over years is also commonly seen in responders. Recently, there has been increasing evidence that the titer of anti-HBs ≥ 10 IU/L does not provide 100 % protection against infection and clinically manifest illness. Additionally, an important role of cellular immune memory has been demonstrated - without any correlation with anti-HBs titer. Based on current knowledge, there is no need for regular anti-HBs titer testing or booster vaccination in immunocompetent individuals with anti-HBs ≤ 10 IU/L. At present, regular serological testing and, if need be, revaccination to maintain anti-HBs ≥ 10 IU/L are recommended in immunocompromised persons and after liver transplantation.

[Treatment with rituximab as an opportunity for the prevention of infectious complications]. / Lécba rituximabem jako výzva k prevenci infekcních komplikací.

UNLABELLED: Rituximab, a monoclonal antibody against the surface antigen of B-lymphocytes CD20 is beeing used in the treatment of numerous hematological, hematooncological and autoimmune disorders. After administration of ritu-ximab, quick and almost complete depletion of B-lymphocytes with the exception of pre-B-lymphocytes and plasma cells occur. Neutropenia and low serum antibody levels in classes IgA, IgM and IgG may also develop. These changes usually persist for 6-12 months, rarely for several years. In the consequence, patients with the rituximab treatment are more prone to infections - usually of bacterial and viral origin. Concomitantly, rituximab treatment influences negatively postvaccination antibody production and therefore adequate preventive measures are necessary before the beginning of the treatment. The authors offer complex overview of actual literature, emphasize adequate education of patients as well as of healthcare providing staff and discuss the vaccination recommendation against preventable communicable diseases like influenza, pneumococcal diseases, tetanus, diphtheria and pertussis. KEY WORDS: autoimmune disease - immunosupression - infectious complications - prevention - rituximab - vaccination.

Novel Instrument for Automated pK(a) Determination by Internal Standard Capillary Electrophoresis.

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

Cost-effective three-dimensional printing of visibly transparent microchips within minutes.

One-step fabrication of transparent three-dimensional (3D) microfluidic to millifluidic devices was demonstrated using a commercial 3D printer costing $2300 with 500 mL of clear resin for $138. It employs dynamic mask projection stereolithography, allowing fast concept-to-chip time. The fully automated system allows fabrication of models of up to 43 mm × 27 mm × 180 mm (x × y × z) at printing speeds of 20 mm/h in height regardless of the design complexity. The minimal cross sectional area of 250 µm was achieved for monolithic microchannels and 200 µm for positive structures (templates for soft lithography). The colorless resin's good light transmittance (>60% transmission at wavelengths of >430 nm) allows for on-chip optical detection, while the electrically insulating material allows electrophoretic separations. To demonstrate its applicability in microfluidics, the printer was used for the fabrication of a micromixer, a gradient generator, a droplet extractor, and a device for isotachophoresis. The mixing and gradient formation units were incorporated into a device for analysis of nitrate in tap water with standard addition as a single run and multiple depth detection cells to provide an extended linear range.

Microfluidic isotachophoresis: a review.

Electromigration methods including CE and ITP are attractive for incorporation in microfluidic devices because they are relatively easily adaptable to miniaturization. After its popularity in the 1970s, ITP has made a comeback in microfluidic format (µ-ITP, micro-ITP) driven by the advantages of the steady-state boundary, the self-focusing effect, and the ability to aid in preconcentrating analytes in the sample while removing matrix components. In this review, we provide an overview of the developments in the area of µ-ITP in a context of the historic developments with a focus on recent developments in experimental and computational ITP and discuss possible future trends. The chip-ITP areas and topics discussed in this review and the corresponding sections include: PC simulations and modeling, analytical µ-ITP, preconcentration ITP, transient ITP, peak mode ITP, gradient elution ITP, and free-flow ITP, while the conclusions provide a critical summary and outlook. The review also contains experimental conditions for µ-ITP applications to real-world samples from over 50 original journal publications.

Health inequalities in post-COVID-19 outcomes among adults aged 50+ in Europe: has COVID-19 exposed divide between postcommunist countries and Western Europe?

BACKGROUND: COVID-19 affected people and countries disproportionately and continues to impact the health of people. The aim is to investigate protective health and socio-geographical factors for post-COVID-19 conditions in adults aged 50 years and older in Europe. METHODS: Using longitudinal data from the Survey of Health, Ageing and Retirement in Europe, collected from June to August 2021, protective factors against post-COVID-19 condition among 1909 respondents who self-reported a positive COVID-19 test result were investigated using multiple logistic regression models. RESULTS: Male adults living outside of Czechia, Poland, Hungary and Slovakia (Visegrad group, V4), who received the COVID-19 vaccination, tertiary or higher education, had a healthy weight (body mass index, BMI 18.5-24.9 kg/m2) and no underlying health condition/s, showed protective effects against post-COVID-19 condition. Health inequalities associated with BMI were observed in education attainment and comorbidities, with higher BMI having lower education attainment and higher comorbidities. Health inequality was particularly evident in individuals in V4 with higher obesity prevalence and lower attainment of higher education than those living in other regions in the study. CONCLUSION: Our study suggests that healthy weight and higher education attainment are predictors associated with a lower incidence of post-COVID-19 condition. Health inequality associated with education attainment was particularly relevant in V4. Our results highlight health inequality in which BMI was associated with comorbidities and educational attainment. To reduce obesity prevalence among older people with lower education, raising awareness about the risks of obesity and providing assistance in maintaining a healthy weight are needed.

Chemical vapour deposition in narrow capillaries: Electro-osmotic flow control in capillary electrophoresis.

BACKGROUND: In capillary electrophoresis (CE), the inner surface of fused-silica capillaries is commonly covalently modified with liquid silanes to control electroosmotic flow (EOF). This liquid phase deposition (LPD) approach is challenging for long and narrow-diameter capillaries (≥1 m, ≤25 µm ID) inhibiting commercial production. Here, we use chemical vapour deposition (CVD) to covalently modify capillaries with different silanes. Using a home-built CVD device, capillaries were modified with neutral (3-glycidyloxypropyl) trimethoxysilane (GPTMS), the weak base (3-aminopropyl) trimethoxysilane (APTMS), the weak acid 3-mercaptopropyltrimethoxysilane (MPTMS) and the neutral hydrophobic trichloro(1H,1H,2H,2H-perfluorooctyl) silane (PFOCTS). Gas-phase modification of GPTMS with acid and ammonia allowed further modification of the surface prior to molecular layer deposition (MLD) of poly(p-phenylene terephthalamide) (PPTA) using the self-limiting sequential reaction between terephthalaldehyde (TA) and p-phenylenediamine (PD) vapours. RESULTS: Capillaries coated with GPTMS by CVD showed a greater reduction in EOF at all pH values than the conventional LPD. APTMS showed a reduction of the EOF at pH 9, with EOF reversal observed below pH 6. MPTMS provided a slightly lower EOF than an unmodified capillary at high pH, and a slightly higher EOF at lower pH. PFOCTS provided the most consistent EOF as a function of pH. The deposition of successive layers of PPTA resulted in increased surface coverage of the polymer and a greater reduction in EOF at pH higher than 5. The stability of a 10 µm ID GPTMS coated capillary was tested at pH 8.8 in a 200 mM CHES/Tris BGE for the separation of inorganic anions. Over 1.5 months of continuous operation (≈4130 runs), the reproducibility of the apparent mobilities for chloride, nitrite, nitrate and sulfate were 2.43%, 2.56%, 2.63% and 3.05%, respectively. The intra-day and inter-day column-to-column reproducibility and batch-to-batch reproducibility for all the coated capillaries ranged between 0.34% and 3.95%. SIGNIFICANCE: The study demonstrates the superior performance of CVD coating for suppressing the EOF compared to LPD allowing the easy modification of long lengths of narrow capillary. The variation in silane, and the ability of MLD to modify and control the surface chemistry, provides a simple and facile method for surface modification. The stability of these coatings will allow long-term capillary electrophoresis monitoring of water chemistry, such as for monitoring fertiliser run-off in natural waters.

Isotachophoresis on a chip with indirect fluorescence detection as a field deployable system for analysis of carboxylic acids.

ITP with indirect fluorescence detection (IFD) was introduced three decades ago. Despite this fact, the method has never become widely adopted. The main aim of this work was to utilize the ITP-IFD for the separation of carboxylic acids by using a commercially available, portable, microfluidic chip electrophoresis system. On the 16.8-mm effective length separation channel, a maximum of eight carboxylic acids could be separated, with LOD values in a range from 0.12 to 0.4 mM. The commercial chips used for all experiments have multichannel structures important for analysis of more than one sample per a chip in case of standard use. This multichannel structure was used to investigate the possibility of multiple sample loading for ITP separation. Application of ITP-IFD was investigated for analysis of benzoate in diet soft drinks and the results were in good agreement with results of a CE method.

Determinants of Immune Response to Anti-SARS-CoV-2 mRNA Vaccines in Kidney Transplant Recipients: A Prospective Cohort Study.

BACKGROUND: Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has been recently shown to be impaired in kidney transplant recipients (KTRs), but the underlying factors affecting vaccine effectiveness need to be further elucidated. METHODS: In this prospective cohort study, antibodies against S1 and S2 subunits of SARS-CoV-2 were evaluated using an immunochemiluminescent assay (cutoff 9.5 AU/mL, sensitivity 91.2%, and specificity 90.2%) in 736 KTRs, who were previously either naive or infected with SARS-CoV-2 and vaccinated before or after transplantation. Cellular response was analyzed in a subset of patients using an interferon gamma release assay (cutoff 0.15 IU/mL, sensitivity 92%, and specificity 100%). RESULTS: Seroconversion was significantly more impaired in SARS-CoV-2-naive KTRs than in those previously infected (40.1% versus 97.1%; P < 0.001). Mycophenolate use (odds ratio, 0.15; 95% confidence interval, 0.09-0.24; P < 0.001) and depleting therapy in the past year (odds ratio, 0.19; 95% confidence interval, 0.05-0.8; P = 0.023) were found to be among independent factors associated with impaired humoral response. Similarly, the interferon gamma release assay tested in 50 KTRs (cutoff 0.15 IU/mL, sensitivity 92%, specificity 100%) showed that specific T-cell responses against spike protein epitopes are impaired in SARS-CoV-2-naive KTRs, as compared to previously infected KTRs (9.4% versus 90%, P < 0.001). All 35 KTRs vaccinated on the waiting list before transplantation exhibited sustained seroconversion persisting after transplantation. CONCLUSIONS: Survivors of coronavirus disease 2019 and those vaccinated while on the waiting list exhibited a marked immune response to mRNA vaccines, contrary to poor response in naive KTRs vaccinated after transplantation (NCT04832841).

Expert opinion on current and future prophylaxis therapies aimed at improving protection for people with hemophilia A.

The next frontier in hemophilia A management has arrived. However, questions remain regarding the broader applicability of new and emerging hemophilia A therapies, such as the long-term safety and efficacy of non-factor therapies and optimal regimens for individual patients. With an ever-evolving clinical landscape, it is imperative for physicians to understand how available and future hemophilia A therapies could potentially be integrated into real-life clinical practice to improve patient outcomes. Against this background, nine hemophilia experts from Central European countries participated in a pre-advisory board meeting survey. The survey comprised 11 multiple-choice questions about current treatment practices and future factor and non-factor replacement therapies. The survey questions were developed to reflect current unmet needs in hemophilia management reflected in the literature. The experts also took part in a follow-up advisory board meeting to discuss the most important unmet needs for hemophilia management as well as the pre-meeting survey results. All experts highlighted the challenge of maintaining optimal trough levels with prophylaxis as their most pressing concern. Targeting trough levels of ≥30-50 IU/L or even higher to achieve less bleeding was highlighted as their preferred strategy. However, the experts had an equal opinion on how this could be achieved (i.e., more efficacious non-factor therapies or factor therapy offering broader personalization possibilities such as targeting trough levels to individual pharmacokinetic data). In summary, our study favors personalized prophylaxis to individual pharmacokinetic data rather than a "one-size-fits-all" approach to hemophilia A management to maintain optimal trough levels for individual patients.

Inexpensive portable capillary electrophoresis instrument for Monitoring Zinc(II) in remote areas.

A compact, inexpensive capillary electrophoresis instrument was developed for monitoring metal ions and evaluated for Zn(II) in remote contaminated locations in western Tasmania, Australia. The portable instrument, measuring 21 cm x 10 cm x 7 cm, was powered from the USB port of a laptop computer and built from off-the-shelf components costing ∼$1200 USD. Electrophoretic separations were conducted using a fused silica capillary (10-50 µm I.D.), applying 8.5 kV over capillaries ranging from 25 cm to 40 cm in length. The capillary inlet was connected with an electrically grounded cross-piece as flow-through injection interface. Automated fluidic management was achieved by controlling four mini peristaltic pumps and a solenoid valve. Detection was realised using a purpose-built visible LED absorption detector, optimised for the detection of Co(II), Cu(II) and Zn(II) after complexation with 4-(2-Pyridylazo) resorcinol (PAR). Limits of detection of sub-µM were obtained. The instrument was tested for continuous operation in the laboratory for up to 3 months, and relative standard deviations of <5.4% were found over 945 consecutive injections. In the field, the system was able to measure 106 samples within 11 h, the time it can be powered from the laptop computer. As Field measurement of Zn(II) in western Tasmania was demonstrated to show capability for on-site metal testing.

Analysis of von Willebrand Disease in the "Heart of Europe".

Background von Willebrand disease (VWD) is a genetic bleeding disorder caused by defects of von Willebrand factor (VWF), quantitative (type 1 and 3) or qualitative (type 2). The laboratory phenotyping is heterogenic making diagnosis difficult. Objectives Complete laboratory analysis of VWD as an expansion of the previously reported cross-sectional family-based VWD study in the Czech Republic (BRNO-VWD) and Slovakia (BRA-VWD) under the name "Heart of Europe," in order to improve the understanding of laboratory phenotype/genotype correlation. Patients and Methods In total, 227 suspected VWD patients were identified from historical records. Complete laboratory analysis was established using all available assays, including VWF multimers and genetic analysis. Results A total of 191 patients (from 119 families) were confirmed as having VWD. The majority was characterized as a type 1 VWD, followed by type 2. Multimeric patterns concordant with laboratory phenotypes were found in approximately 83% of all cases. A phenotype/genotype correlation was present in 84% (77% type 1, 99% type 2, and 61% type 3) of all patients. Another 45 candidate mutations (23 novel variations), not found in the initial study, could be identified (missense 75% and truncating 24%). An exon 1-3 gene deletion was identified in 14 patients where no mutation was found by direct DNA sequencing, increasing the linkage up to 92%, overall. Conclusion This study provides a cross-sectional overview of the VWD population in a part of Central Europe. It is an addition to the previously published BRNO-VWD study, and provides important data to the International Society of Thrombosis and Haemostasis/European Association for Haemophilia and Allied Disorders VWD mutation database with identification of novel causal mutations.