Papers by Milena Velizarova
Proceedings of the Bulgarian Academy of Sciences
More than three years after the first wave of COVID-19, the biomarkers responsible for the course... more More than three years after the first wave of COVID-19, the biomarkers responsible for the course of this disease continue to be debated. Moreover, the new condition, long COVID, poses many unsolved questions. Therefore, we decided to perform a complex analysis of immune and biochemical parameters in patients with different severity of COVID-19. Eighty-five adult patients with RT-PCR confirmed SARS-CoV-2 infection hospitalized in the period April-November, 2020, were enrolled in the study. Extended immunological and biochemical parameters were analyzed in different time points after symptoms onset of COVID-19. Our results demonstrated that severe patients present with increased CRP, ferritin, D-dimer and NLR, decreased counts of lymphocytes, total T lymphocytes and major T-cell subsets, increased proportions of activated T cells, Tregs and Bregs. Markers of immune response dysfunction combined with hematological, inflammatory and coagulation indicators may be more informative for pr...
Biotechnology & Biotechnological Equipment
Clinical Laboratory
Background: Genetic polymorphisms of CYP2C9 and VKORC1 play a major role in pharmacokinetics and ... more 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/m 2. 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: Genetic polymorphisms of CYP2C9 and VKORC1 play a major role in pharmacokinetics and ... more 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/m 2. 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.
Veterinary clinical pathology, 2017
Reticulocyte hemoglobin content provided by the Siemens ADVIA (CHr) is an established marker of i... more Reticulocyte hemoglobin content provided by the Siemens ADVIA (CHr) is an established marker of iron deficiency. The IDEXX ProCyte Dx hematology analyzer now provides a similar variable, reticulocyte hemoglobin equivalent (RET-He). The objective was to evaluate RET-He and its diagnostic utility in dogs, and to calculate a cutoff value for diagnosing iron-deficient erythropoiesis (IDE). Furthermore, the prevalence of RET-He values below this cutoff value was established. One hundred and seventy-one CBCs of healthy dogs were used to establish a RI. Stability of RET-He was evaluated by repeated measurements over 48 hours (n = 10). The 25-run coefficient of variation (CV) was calculated, and correlation and bias between measurements of RET-He and CHr were assessed (n = 190). A cutoff value for diagnosing IDE was calculated. The utility of RET-He in the detection of IDE was evaluated in 123 dogs. The prevalence of low RET-He values was assessed retrospectively in a multicenter study (201...
Clinical and Transfusion Haematology
Cytogenetic study with G-banding techniques and fluorescent in situ hybridization was performed o... more Cytogenetic study with G-banding techniques and fluorescent in situ hybridization was performed on 5 patients with chronic and accelerated phase of chronic myeloid leukemia. Examinations with dual-color, dual fusion assay for detection of t(9;22) and DNA specific probes for detection of tumor suppressor genes ATM, p16 and p53 were carried out. Chromosome alterations of tumor suppressor genes were found in 4 cases: 4 with deletions of ATM, 2 with deletions of p16 and one with deletion of p53. In one patient with t(9;22;21) biallelic deletions of ATM and p16 was observed. In all patients the chromosome abnormalities were presented in minor clones (2-9%). In conclusion, our results suggest that genetic alterations of ATM, p16 and p53 may arising in chronic and accelerated phase of chronic myeloid leukemia and possibly are related with the clonal evolution of the disease. Key words: Chronic myeloid leukemia, chromosome aberration, ATM, p16, p53.
Journal of Medical Biochemistry, 2021
Background: DYMIND DH76 (DYMIND BIOTECH, China) is a new automated hematology system designed to ... more 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 wit...
Turkish journal of haematology : official journal of Turkish Society of Haematology, Jan 5, 2008
The majority of adults diagnosed with acute myeloid leukemia (AML) display acquired cytogenetic a... more The majority of adults diagnosed with acute myeloid leukemia (AML) display acquired cytogenetic aberrations at presentation. In this article, we present the major cytogenetic findings regarding AML and review their clinical significance for achievement of the first complete remission. We studied 71 adult patients with de novo AML, without previous myelodysplasia or alkylating therapy. Conventional cytogenetics and FISH were performed on bone marrow cells. The patients with AML were assigned to 12 subgroups according to established data for cytogenetic, molecular and general laboratory results. The selection of the analyzed parameters is consistent with internationally accepted "prognostic factors" in adult AML. Complete remission upon induction therapy was achieved in 40% of cases (in a mean period of 2.3 months from therapy initiation). The patients with t(15;17) PML-RARA and inv(16)/CBFbeta-MYH11ë demonstrated the highest frequency of complete remission. Patients with hy...
Genes and Immunity
Our study included 24 adult patients with newly diagnosed B-ALL. The diagnosis was conformed to m... more Our study included 24 adult patients with newly diagnosed B-ALL. The diagnosis was conformed to morphology, immunophenotyping, cytogenetic and molecular analyses. In 9/24 patients (37.5%) the presence of immunological markers for common B-ALL was found –CD10+(CALLA+) and cytoplasm light chains (cIgM).The average age in this group was 49 years, m/f=4/5, average WBC count of 8/9 was 10,6.109/l and 1/9 case with t(1;19) – 70.109/l. We report rare chromosome anomalies in adult CD10+ B-ALL. Cytogenetic and molecular aberrations were found in 8/9 patients: Ph+ or fusion gene bcr/abl –2 cases; t(8;14) or rearranged c-MYC oncogene– 2 cases; t(1;19) – 1 case; karyotype with del4p,del17p,mar14,inv(9) -1 patient; in 3 cases numerical changes were established: 2-with hyperdiploidy, one with trizomy 8. We ascertain that:1.CD10+ B-ALL has high frequency in childhood ALL and in this group of adults we found 37.5%; 2.The frequency of t(1;19) is high in pre-B-ALL, but this is a rare aberration in ...
Clinical Chemistry and Laboratory Medicine
Leukemic cells from a significant number of adults with acute lymphoblastic leukemia (ALL) expres... more Leukemic cells from a significant number of adults with acute lymphoblastic leukemia (ALL) express protein antigens characteristicof both lymphoid and myeloid cells. We studied incidence and the relations of myeloid-antigen (My) expression to cytogenetic features of ALL and to outcome. Leukemic blasts from 33 newly diagnosed adults with untreated ALL were examined for myeloid surface antigen expression. The simultaneous expression of lymphoid-associated antigens and at least one of three myeloid-associated antigens (CD33, CD13, and CD14) on cells were detected by a standard two-color direct immunofluorescent assay. My(+) ALL was established in 39% of cases, all of them B-lineage ALL. Immunologic subtyping of B-ALLs revealed an association between common B phenotype and coexpression of myeloid antigens-53% of My(+)ALL (P<0.05). Cytogenetic abnormalities that have been associated with My(+) ALLs were common, including t(9;22), 11q23 abnormalities, del 4p, and del 12p. The common AL...
Balkan Journal of Medical Genetics
The chimerism status after allogeneic bone marrow transplantation (BMT) provides substantial info... more The chimerism status after allogeneic bone marrow transplantation (BMT) provides substantial information about the replacement of host cells with donor cells after the transplantation. We studied six Bulgarian patients after sex-mismatched allogeneic BMT by fluorescent in situ hybridization (FISH) with alternatively labeled X and Y probes and three male-to-male donor-recipient pairs by genotyping of microsattellite loci. Complete hematopoietic chimerism (CC) was found in one patient who did not experience graft failure. Near-complete chimerism (NCC) was detected in three patients who experienced an autologous recovery over several months followed by relapse and graft failure. In one patient, low-level mixed chimerism (MC) that progressed to high-level MC, accompanied by full remission, was found. One patient showed low-level MC followed by complete loss of chimerism. In all patients the percentage of chimeric cells correlated with clinico-hematological data for remission or relapse....
Turkish Journal of Hematology, 2011
Objective: Treatment of acute lymphoblastic leukemia (ALL) in adults focuses on the initial asses... more Objective: Treatment of acute lymphoblastic leukemia (ALL) in adults focuses on the initial assessment of the prognostic relevant cytogenetic features as well as a response-guided therapy based on molecular data. We examined the importance of molecular-cytogenetic abnormalities for complete remission (CR) rates and the overall survival (OS) in adult ALLs. Materials and Methods: Conventional cytogenetics and fluorescence in situ hybridization were performed on bone marrow cells from 33 newly-diagnosed ALL adults. Two karyotype categories [standard-risk group-normal karyotype, hyperdiplody and other structural aberrations, and high-risk group-t(11q23)/MLL, t(9;22)/bcr-abl, t(1;19), t(8;14), C-MYC and complex karyotype] and the biologically and clinically relevant ALL ploidy subgroups were prospectively defined. Results: Chromosomal abnormalities were found in 52% of the cases with a high rate of poor-risk translocations-t(9;22), t(8q24), t(11q23), t(1;19). The total CR rate was 67% and the median time for achievement 2.33 months. Male sex, an age below 35 years and the absence of high risk translocations might have contributed to the high CR rates. Female patients, hyperdiplody, low white blood cells (WBC), and random cytogenetic aberrations had the longest OS. OS, 3-and 5-years survival periods were significantly shorter for poor-risk than standard risk group (p=.015, p=.001 and p=.005, respectively). Conclusion: This study emphasizes the lack of influence of cytogenetic aberrations on the CR and the time to achieve CR. However, our observations show that these aberrations are an independent prognostic factor in adult ALL-they allow predicting therapy resistance and the OS time after intense treatment.
International Journal of Health Sciences and Research, 2018
Although the modern haematology analyzers provide reliable full blood counts, there are interfere... more Although the modern haematology analyzers provide reliable full blood counts, there are interferences on the measurement of platelet counts, especially in patients which have a wide variety of abnormal erythrocytes. We report a patient with heterozygous beta-thalassaemia and pseudothrombocytosis (spurious elevation of platelet count). The case emphasizes, that impedance method is associated with spuriously high platelet counts in thalassemia patients. Optical platelet counts and blood smear review are recommended alternative platelet counting methods in patients with abnormal red blood cells.
Clinical Laboratory, 2015
BACKGROUND Hepcidin is a 25-amino peptide hormone that regulates iron homeostasis. Its serum quan... more 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.
Turkish journal of haematology : official journal of Turkish Society of Haematology, Jan 5, 2006
B-cell acute lymphoblastic leukemia (B-ALL) accounts for 20-30% of acute leukemias in adults. Com... more 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 establishe...
International journal of stroke : official journal of the International Stroke Society, Jun 5, 2016
International Journal of Health Sciences and Research, 2018
Although the modern haematology analyzers provide reliable full blood counts, there are interfere... more Although the modern haematology analyzers provide reliable full blood counts, there are interferences on the measurement of platelet counts, especially in patients which have a wide variety of abnormal erythrocytes. We report a patient with heterozygous beta-thalassaemia and pseudothrombocytosis (spurious elevation of platelet count). The case emphasizes, that impedance method is associated with spuriously high platelet counts in thalassemia patients. Optical platelet counts and blood smear review are recommended alternative platelet counting methods in patients with abnormal red blood cells.
Clinical Laboratory, 2015
BACKGROUND Hepcidin is a 25-amino peptide hormone that regulates iron homeostasis. Its serum quan... more 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.
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Papers by Milena Velizarova