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The aim of our study was to quantify serum hepcidin levels in Bulgarian patients with chronic kidney disease (CKD). Expected high values of peptide hormones might provide a new therapeutic choice for anemia of chronic disease. We looked for correlation between serum hepcidin levels and some iron metabolism parameters in CKD patients. Results: The sandwich ELISA is highly specific for hepcidin-25. We found statistically significant differences in serum hepcidin levels in patients of the control group, with CKD stages II to IV and CKD stage V (on chronic dialysis): 12.7±8.7μg/L, 90.74±21.1μg/L, and 282.49±81.1μg/L, respectively. Significant correlation between serum hepcidin and transferrin saturation (r=0.340, P

Background: Copper (Cu) is a physiologically important trace element during pregnancy. The study aim is to assess the altered level of serum Cu and its association with some metabolic indexes in Gestational Diabetes Mellitus (GDM)., Methods: A total of 108 pregnant women (aged 18 - 40, second trimester) are included in the study and divided into two groups (GDM n = 54; pregnant with normal glucose tolerance (NGT), n = 54) after performing a 2-hour 75-g oral glucose tolerance test (OGTT). Maternal blood samples are collected at 26 - 28 gestational week. All biochemical parameters are measured in serum from fasting venous blood. Serum Cu levels are analyzed by flame atomic absorption spectrophotometry (Perkin Elmer AAnalyst 300, USA). Body Mass Index (BMI), insulin sensitivity/resistance, triglyceride-glucose (TyG), TyG-BMI (triglyceride glucose-body mass) indexes are calculated by formulas., Results: The following data were observed: significantly higher levels of serum Cu (p = 0.009), pre-pregnancy BMI (pre-pBMI), BMI at the GDM diagnosis (pBMI), TyG, pregnancy TyG-BMI (pTyG-BMI) p < 0.001, and triglycerides (Tgl) (p = 0.02) in GDM compared to NGT pregnancy. The study presents a positive correlation between serum Cu and pre-pBMI (p < 0.02), pBMI and pTyG-BMI (p < 0.001). Besides, pre-pBMI (mean ≥ 25 kg/m2), pBMI (mean ≥ 30 kg/m2), and pTyG-BMI are associated with 14.5% (OR 1.145, 95% CI: 1.064 - 1.232; p < 0.001), 15.3% (OR 1.153, 95% CI: 1.070 - 1.243; p < 0.001), and 5.9% (OR 1.059, 95% CI: 1.022 - 1.086; p < 0.001) increased risk for GDM development. No association is found between Cu and Tgl levels, fasting plasma glucose e(FPG) and TyG. ROC analysis suggests the serum Cu as a possible risk factor for GDM development. The analysis shows that at a cutoff point of ≥ 31.9 µmol/L, serum Cu presents a sensitivity and specificity of 64.8% and 66.7% in the prediction of GDM development (AUC = 0.659, p < 0.012). After adjustment for maternal age, gestational age, and family predisposition, the odds ratios (ORs) (95% CIs) still show association of Cu levels with increased GDM risk (OR 1.099, 95% CI 1.018 - 1.184, p = 0.013)., Conclusions: pTyG-BMI index exhibits a better interaction than TyG index, Tgl, and glucose separately with serum Cu levels where BMI has a mediator's role.

Background: Genetic polymorphisms of CYP2C9 and VKORC1 play a major role in pharmacokinetics and pharmacodynamics of coumarin anticoagulants. The purpose of our study was to assess the relative frequency of the above mutations in Bulgarian population in order to predict bleeding tendencies and precisely manage the anticoagulant therapy during the postoperative period after cardiac surgery with extracorporeal circulation., Methods: Genomic DNA samples from 200 Bulgarian patients subjected to cardiac surgery with extracorporeal circulation were analyzed for VKORC1 1639G>A and CYP2C9*2&*3 polymorphisms by real-time polymerase chain reaction (PCR), then allele frequencies of various genotypes were calculated by Hardy-Weinberg Equilibrium., Results: Median patients' age was 63.9 ± 10.8 years; 66.5% were male. Median BMI was 28.6 ± 5.4 kg/m2. Genotype distribution for CYP2C9 was *1/*1 - 51%, *1/*2 - 21%, *1/*3 - 13.5%, *2/*3 - 4%, *3/*3 - 2%, and *2/*2 - 1.5%. The calculated frequency of CYP2C9*1 allele was 74.25%, CYP2C9*2 allele was 13%, and CYP2C9*3 allele was 12.75%, and all allelic frequencies were in Hardy-Weinberg equilibrium (p-value = 0.358). The major VKORC1 genotype was G/A - 47%, followed by G/G - 35.5% and A/A - 17.5%). Based on Hardy-Weinberg Equilibrium, there was no significant difference between observed and expected frequencies (X - -3.779), presumably as a result of the homogeneity in the population., Conclusions: Analysis of the data obtained in the course of the study suggested that identification of homozygous carriers of VKORC1-1639 G>A (rs9923231) in Bulgarians may be useful in developing recommendations for personalized therapy. On the contrary, homozygous carriers of CYP2C9*2 or *3, included only 4.5% of the studied patients, thus indicating that this group would benefit less from dosing algorithms. Our results demonstrated good agreement with the results obtained in other studies conducted in the Caucasian population.

Background: DYMIND DH76 (DYMIND BIOTECH, China) is a new automated hematology system designed to provide CBC count, including a 5-part WBC differential count, and its analytical performance should be assessed before adoption for clinical use., Methods: The analyzer was evaluated according to the International Council for Standardization in Haematology guideline. The purposes of this study were to assess its analytical performance in comparison to SYSMEX XN 1000 hematology analyzer currently used in our laboratory, as well as to compare the automated and manual WBC differential., Results: Within-run precision in all concentration ranges was very good with coefficients of variation (CVs) between 0.02% and 2.5% except for platelets over 500×109/L (CV 9.5%). Within-batch imprecision showed CVs lower the declared deviation ranges. Accuracy (defined as trueness) was excellent for all CBC and white cell differential parameters, compared with the state of the art%. Linearity was confirmed with excellent regression coefficients (0.999-1.000), even in the lowest values, and carryover was ≤ 1%. Comparison between DYMIND DH76 and SYSMEX XN 1000 was also very good with correlation coefficients (R2) for WBC (1.000), RBC (0.999), hemoglobin (0.999) and PLT over 50×10 9 /L (0.994) and R2 was lower but still acceptable (0.910) for PLT<50×10 9 /L. R2 for neutrophils, lymphocytes, eosinophils, basophils, and monocytes were 0.974, 0.982, 0.957, 0.625, and 0.836, respectively, in the comparison between the manual and DYMIND DH76 automated differential WBC counts., Conclusions: With excellent analytical performance and acceptable comparative analysis, DYMIND DH76 hematology analyser covered the predefined international standards and requirements and is fully appropriate for clinical application., Competing Interests: Conflict of Interest: The authors stated that they have no conflicts of interest regarding the publication of this article., (2021 Milena Velizarova, Teodora Yacheva, Mariana Genova, Dobrin Svinarov, published by CEON/CEES.)

Epigenetic changes, in particular DNA methylation processes, play a role in the pathogenesis and progression of type 2 diabetes mellitus (T2DM) linking genetic and environmental factors. To clarify this role, we have analyzed in patients with different duration of T2DM: (i) expression levels of methyl-CpG-binding domain protein 2 ( MBD2 ) as marker of DNA methylation, and ii) methylation changes in 22 genes connected to cellular stress and toxicity. We have analyzed MBD2 mRNA expression levels in16 patients and 12 controls and the methylation status of stress and toxicity genes in four DNA pools: (i) controls; (ii) newly-diagnosed T2DM patients; (iii) patients with T2DM duration of <5 years and (iv) of >5 years. The MBD2 expression levels were 10.4-times increased on average in T2DM patients compared to controls. Consistent increase in DNA methylation fraction with the increase in T2DM duration was observed in Prdx2 and SCARA3 genes, connected to oxidative stress protection and in BRCA1 and Tp53 tumor-suppressor genes. In conclusion, increased MBD2 expression in patients indicated general dysregulation of DNA methylation in T2DM. The elevated methylation of Prdx2 and SCARA3 genes suggests disturbance in oxidative stress protection in T2DM. The increased methylation of BRCA1 and Tp53 genes unraveled an epigenetic cause for T2DM related increase in cancer risk.

Background: Hepcidin is a 25-amino peptide hormone that regulates iron homeostasis. Its serum quantification helps to provide the right therapeutic choice in iron-deficiency anemia and anemia in chronic diseases. Diurnal levels of serum iron might affect hepcidin secretion during the day. Blood collection time is an important part of the preanalytical phase of its quantification., Methods: During the period 2013 - 2014, we collected blood samples for serum hepcidin quantification in 100 healthy controls. The samples were collected in vacuettes with serum separator gel at three different times during the day: 07:30 - 08:30, 12:00 - 13:00, and 16:00 - 17:00 hours. Hepcidin levels were measured with an ELISA method., Results: We found a significant difference in serum hepcidin levels during the chosen three blood taking times. The normal range for Bulgarian population is 3.05 μg/L - 37.75 μg/L. The measured levels were: at 07:30 - 08:30 hours 12.2 μg/L (5.5 μg/L - 23.6 μg/L), 12:00 - 13:00 hours 14.1 μg/L (7.1 μg/L - 27.2 μg/L), and 16:00 - 17:00 hours 16.5 μtg/L (9.9 μg/L - 29.6 μpg/L) 10.7 < r < 1.0; p < 0.5 between 07:30 - 08:30 and 12:00 - 13:00 hours and p < 0.05 between 07:30 - 08:30 hours and 16:00 - 17:00 hours and 12:00 - 13:00 hours and 16:00 - 17:00 hours]. No significant differences were found for transferrin saturation between measured groups [0.1 < r < 0.3; p > 0.5]., Conclusions: In order to obtain the most correct results for serum hepcidin quantification (especially in border to referent range levels) in the preanalytical phase, it is important to consider the time of blood sampling.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for 20-30% of acute leukemias in adults. Combined application of data from immunophenotyping, karyotyping and molecular analyses allows a better understanding of this heterogeneous disease. We studied 30 adult patients with newly diagnosed B-ALL by conventional cytogenetics, fluorescent in situ hybridization (FISH) and immunophenotyping analyses. We report statistically significant prevalence of structural aberrations (43%) over numerical changes (17%) (p=0.02). The most frequent structural changes were t(9;22)(q34;q11)/bcr-abl-17%, t(8q24)/C-MYC-10%, t(11q23)/MLL-6%, del 4p-6%, del12p-3%, and t(1;19)-3%. Complex karyotype was found in 17% and normal karyotype in 30%. The most frequent immunophenotype was of common B-ALL (43%), and cytogenetic and/or molecular abnormalities were found in 78% of them. We distinguished a relatively high incidence (17%) of mature B-ALL and 60% of them were associated with t(8;14)/C-MYC. We established association of cytogenetic aberrations with immunophenotype only in mature B-ALL. The other immunophenotypes are characterized by genetic heterogeneity and the presence of cytogenetic abnormalities unusual for adult B-ALL - trisomy 8 and t(1;19)(q23;p13).