Associations of variants of folate cycle genes with features of the clinical course of severe intraventricular hemorrhages in premature infants.

BACKGROUND: Summary data indicate that it has increased attention to the study of the role of the folate cycle and the genes encoding its key components in the complicated course of the neonatal period in premature infants. Therefore, the aim of our study was to investigate the relationship of folate cycle gene variants with the features of the neonatal course in premature infants with severe intraventricular hemorrhages (IVH). METHODS: The study included 24 preterm infants with with IVHs of 3d and 4th degree that received standard clinical, laboratory and instrumental examination. RESULTS: Apgar scores at 1 and 5 minutes were significantly lower in patients with AA genotype according to variant A1298C of the MTHFR gene. The concentration of total protein on 6th day after birth was negatively correlated with the A66G variant of the MTRR gene. The mean concentration of ionized calcium in the first day after birth was higher in the subgroup of patients with the AA genotype (according to variant A1298C of the MTHFR gene). In the subgroup of patients requiring mechanical ventilation, the frequency of AA genotype according to variant A2756G of the MTR gene was significantly increased. The presence of respiratory disorders and oxygen dependence was negatively correlated with variant A1298C MTHFR. The day of surfactant administration was positively correlated with variant A1298C of the MTHFR gene. CONCLUSION: The results of this study indicate that gene variants MTHFR (C677T, A1298C), MTRR (A66G), MTR (A2756G), RFC1 (G80A) may affect the neonatal course in premature infants with severe IVH.

Readiness of clinical laboratory diagnostics and cardiologists to implement the recommendations of the fourth universal definition of myocardial infarction.

This study describes the problems of the implementation of the fourth universal definition of myocardial infarction in the medical institutions of four cities: Volgograd (with Volzhsky), Yekaterinburg, Perm, Ufa, and districts of the Volgograd region. The multicenter study was conducted in the form of a questionnaire of specialists in cardiology and laboratory services. After a survey of cardiac specialists, it was found that a third of them did not see the benefits of the hs-cTn test recommended for the diagnosis of myocardial infarction, and almost half of the specialists surveyed believed that myoglobin was a necessary test for detecting myocardial infarction. Probably, this is due to the fact that 16 clinical diagnostic laboratories from the 5 above regions still perform the determination of myoglobin in patients with suspected myocardial infarction. The material and technical support of medical and diagnostic institutions generally meets the requirements of the fourth universal definition of myocardial infarction. However, there is a problem of «qualitative¼ equipment of the regions of the Volgograd region, since only 3 out of 31 districts declared the possibility of carrying out a quantitative determination of hs-cTn , and qualitative analysis was carried out on platforms that are not monitored by the IFCC. It is worrying that almost half of the specialists of the clinical and diagnostic laboratories of the central district hospitals of the Volgograd region did not indicate the manufacturer of reagents for determining troponins. Thus, in the educational programs of advanced training of specialists in cardiology and laboratory services, it is necessary to include aspects related to the explanation of analytical characteristics, the characteristics of the technology for performing troponin tests and the related interpretation options for the results.

[THE COMPARISON OF RESULTS OF DETECTION OF MINIMAL RESIDUAL DISEASE IN PERIPHERAL BLOOD AND MARROW IN CHILDREN OF THE FIRST YEAR OF LIFE WITH ACUTE LYMPHOBLASTIC LEUCOSIS].

The occurrence of minimal residual disease is an important prognostic factor under acute lymphoblastic leucosis in children and adults. In overwhelming majority of research studies bone marrow is used to detect minimal residual disease. The comparative characteristic of detection of minimal residual disease in peripheral blood and bone marrow was carried out. The prognostic role of occurrence of minimal residual disease in peripheral blood and bone marrow under therapy according protocol MLL-Baby was evaluated. The analysis embraced 142 pair samples from 53 patients with acute lymphoblastic leucosis and various displacements of gene MLL younger than 365 days. The minimal residual disease was detected by force of identification of chimeric transcripts using polymerase chain reaction in real-time mode in 7 sequential points of observation established by protocol of therapy. The comparability of results of qualitative detection of minimal residual disease in bone marrow and peripheral blood amounted to 84.5%. At that, in all 22 (15.5%) discordant samples minimal residual disease was detected only in bone marrow. Despite of high level of comparability of results of detection of minimal residual disease in peripheral blood and bone marrow the occurrence of minimal residual disease in peripheral blood at various stages of therapy demonstrated no independent prognostic significance. The established differences had no relationship with sensitivity of method determined by value of absolute expression of gene ABL. Most likely, these differences reflected real distribution of tumor cells. The results of study demonstrated that application of peripheral blood instead of bone marrow for monitoring of minimal residual disease under acute lymphoblastic leucosis in children of first year of life is inappropriate. At the same time, retention of minimal residual disease in TH4 in bone marrow was an independent and prognostic unfavorable factor under therapy of acute lymphoblastic leucosis of children of first year of life according protocol MLL-Baby (OO=7.326, confidence interval 2.378-22.565).

[PREDICTIVE MODEL OF EARLY BACTERIAL INFECTIONS DEVELOPMENT IN PREMATURE INFANTS].

Symptoms and signs of early infection and sepsis in premature infants are varied; there are no clear criteria for the diagnosis of such conditions in this cohort. The aim of our study was to analyze and identify the risk factors for early infections in premature infants and to develop on their basis the clinical prognostic model with high diagnostic performance. A retrospective cohort study, which included 152 premature infants, was conducted; 121 of them had the signs of early infections, 31 - had no signs of infection. 52 candidates of prognostic variables were considered. According to the results of multiple stepwise logistic regression analysis, the predictive model has been developed. It includes gestational age, visual changes of the placenta, Apgar score at the 1st minute, the level of monocytes more than 6.5%, the history of abortions and premature rupture of membranes. The diagnostic characteristics of the developed model are high: sensitivity - 82.2%, specificity - 93.55%, positive predictive value - 97.98%, negative predictive value - 58%. Therefore, when interpreting the figures of biomarkers, the decision as to the prescription of antibiotics should be based on the presence of maternal risk factors and clinical symptoms of the infection in the newborn.

[Prognostic value of the determination of bone marrow lesion in patients with neuroblastoma based on the gene PHOX2B and TH expression].

Bone marrow (BM) involvement in neuroblastoma patients is commonly detected by cytomorphology and associated with poor outcome. Molecular techniques, flow cytometry and immunocytochemistry were offered to detect low number of tumor cells in BM due to high value of analytical sensitivity, while prognostic significance of results, obtained with these methods is unclear. PHOX2B and/or TH genes expression was selected as molecular marker of BM involvement. It was determined in 411 BM samples obtained from 75 neuroblastoma patients. 263 BM samples were taken at the time of primary diagnosis, 80 during treatment and 68 before autologous stem cells (ASC) apheresis. Prognostic significance of BM involvement was defined using 5-year (in some groups 4-year) overall (OS), event free (EFS) and progression free (PFS) survival. 24 patients (32.0%) were positive for PHOX2B and/or TH expression in the BM at the time of primary diagnosis. They had decreased survival rates: EFS achieved 0.49+/-0.12, OS - 0.57+/-0.12, PFS - 0.54+/-0.12, comparing with 0.75+/-0.07, 0.80+/-0.07 and 0.77+/-0.07, respectively, in patients with negative BM, p=0.014, p=0.029 and p=0.033. The trend to decreased OS and PFS was detected in case of minimal residual disease presence at the end of the induction chemotherapy (OS and PFS both are 0.22+/-0.19 vs. 0.70+/-0.18 and 0.43+/-0.22, correspondingly, p=0.121, p=0.130). Detection of PHOX2B and/or TH genes expression in the BM before ASC harvesting led to significant decreasing of EFS and OS (0.00 vs. 0.59+/-0.14 and 0.75+/-0.13, respectively, p=0.021 and p=0.016).

[Methods of detection and prognostic significance of genetic material increase of the short arm of chromosome 2 and a deletion of the short arm of chromosome 1 in patients with neuroblastoma].

MYCN gene amplification and 1p deletion in neuroblastoma patients are associated with poor prognosis and commonly used for patient's stratification into risk groups. MYCN copy number and 1p deletion status were analyzed with multiplex ligase-dependent probe amplification (MLPA), PCR and FISH. MYCN amplification was revealed in 21 patients (17.2%) simultaneously by MLPA and PCR. In 28 cases (23.0%) 2p gain was detected. 1p deletion was revealed in 28 patients (23.0%) while concordance between PCR and MLPA achieved 95.8%, PCR and FISH - 90.9%. Mean follow-up time achieved 42 months (ranged from 1 month to 13 years). Event-free survival and overall survival in MYCN-amplified patients as well as in patients with 1p deletion were significantly lower comparing with MYCN-negative patients or patients without 1p deletion.

[The TH, ELAVL4 and GD2 gene expression as diagnostic markers of bone marrow lesions in patients with neuroblastoma].

The bone marrow (BM) TH, ELAVL4 and GD2 genes expression was evaluated in 331 samples from 57 different stage neuroblastoma (NB) patients, 26 BM samples from patients without NB and samples from 2 NB cell lines (IMR-32, Kelly) by real-time PCR. BM samples were considered NB-positive if PHOX2B expression was found or tumor cells were detected in BM smears. TH expression was not revealed in normal BM and was significantly lower in NB-negative samples. Expression of PHOX2B, TH and GD2 remained stable throughout NB treatment, while ELAVL4 expression was down-modulated. ROC-analysis revealed similar initial and follow-up values of TH and PHOX2B in NB patients' bone marrow making it possible to be used for disease detection and monitoring. The test prediction value was 0.994 and 0.952, respectively. The additional test for TH didn't increase the test effectiveness in comparison with PHOX2B test. ELAVL4 and GD2 assessment didn't add diagnostic value for BM involvement monitoring in NB patients.

[The limited possibility of using a simplified approach to detect minimal residual disease by the flow cytometry technique in children with precursor B-lineage acute lymphoblastic leukemia].

Minimal residue disease (MRD) is a state in which the tumor cells remain in the patient in the amounts unrecognizable with the standard cytological techniques. Flow cytometry is one of the basic methods for evaluation of MRD in precursor B-lineage acute lymphoblastic leukemia (PBLALL). The so-called simplified three-color analysis using the combination of CD19/CD10/CD34 antibodies has been proposed to detect MRD in the midcourse of induction therapy. Four-to-nine-color is presently used to identify MRD. One hundred and thirty-four bone marrow samples taken at different stages of therapy in 55 children with PBLALL were examined to estimate the possibility of using the flow cytometry technique using the 3-color simplified approach to determining MRD. The results of the simplified and standard approaches were compared in the samples stained with 6-8 monoclonal antibodies in the combinations that always included CD19, Cd10 and CD34. The comparison revealed that MRD had been incorrectly identified by the simplified method in 8.0, 17.6, and 75.8% of the patients on therapy days 15, 36, and 85, respectively. In addition, the content of residual tumor cells with respect to the threshold values more frequently proposed to stratify patients was found to be incorrectly calculated in some true positive samples. Thus, when the simplified approach was applied using the results of MRD detection to stratify the patients into risk groups, 16.0, 27.4, and 81.8% of the samples would yield incorrect information on therapy days 15, 36, and 85, respectively. Thus, the simplified approach to identifying MRD is most applicable on day 15 of therapy; however, there may be mistakes in this point of observation. This method used on day 36 more frequently yields incorrect results and is inapplicable on day 85.

[Minimal residual disease monitoring by flow cytometry in children with acute lymphoblastic leukemia].

The cells that have avoided the action of antitumor drugs may be retained after remission achievement during induction therapy and consolidation. A combination of these cells is given the name minimal residual disease (MRD). Multicolor flow cytometry has recently attracted considerable interest as the most promising method for measuring the content of residual tumor blasts. This technique is based on the detection of the so-called leukemia-associated immunophenotype (LAIP), i.e., a tumor-specific combination of the expression of membrane and cytoplasmic markers. Flow cytometry may be successfully used to monitor MRD in 90-95% cases of acute lymphoblastic leukemia (ALL) and in 80-85% of patients with acute myelocytic leukemia. The sensitivity of flow cytometry, which is real for routine flow techniques, is a possibility of identifying one cell among 10(4)-10(5) cells. Multicolor flow cytometry (that involves the simultaneous analysis of the expression of a few markers) is the most reasonable tool for MRD monitoring. The monoclonal antibody panels recommended by different groups of investigators for MRD monitoring in B-lineage ALL include antibodies to the pan-B-cell antigen CD19, markers of different stages of differentiation of B-lineage precursors of CD10, CD34, and CD20 and leukemia-associated markers different for each panel, such as CD22, CD38, CD58, CD45, TdT, CD13, CD33. The hyperexpression of CD10, CD34, CD19, TdT, the decreased expression of CD38, CD45, CD22, CD19, the simultaneous expression of markers of different stages of differentiation of B lymphocytes, such as CD10 and CD20, and the lymphoblast coexpression of myeloid markers of CD13, CD33, CDS15 are the most frequently described immunophenotype aberrations in B-lineage ALL. The selection of combinations of markers for MRD monitoring in children with T-ALL is based on the simultaneous expression of combinations of the antigens characteristic for early stages of differentiation of normal T lymphocytes, namely TdT and cytoplasmic CD3. Some authors consider the use of CD99 versus TdT to be most appropriate. There is recent evidence that MRD-positive patients have a higher cumulative risk for recurrences as compared with those without residual blasts. Moreover, the longer the tumor cells are retained during therapy, the worse the prognosis is. Thus, for choice of the adequate intensity of antitumor therapy, it is necessary to qualitatively and quantitatively assess MRD by multicolor flow cytometry at different stages of therapy.

[Thyroid-stimulating hormone receptor antibodies in the diagnosis of Graves' disease in children].

The informative value of a method for determining total antibodies (Ab) to thyroid-stimulating hormone receptor (TSHR) in the diagnosis of Graves' disease was evaluated in 80 children with various causes of hyperthyroidism. With a RSHR Ab of > 1.6 IU/l, the diagnostic sensitivity and specificity of the test were 100%. Patients with hyperthyroidism and no RSHR Ab require no active therapeutic intervention and need to be followed up.

[Significance of determination of antibodies to thyroid-stimulating hormone after discontinuation of antithyroid therapy for Graves disease in children].

Determination of thyroid-stimulating receptor antibodies (TSRA) a promising criterion for discontinuation of antithyroid drug therapy; however, such studies are extremely limited in childhood. The purpose of this investigation was to establish the prognostic value of TSRA on discontinuing antithyroid drug therapy in children with Craves' disease (CD). At the moment of therapy discontinuation, the level of TSRA was higher than the reference range in 15 of 31 children; there were very high values in 2 cases of hypothyroidism that preserved after discontinuation of antithyroid drug treatment. After omitting these cases while making an analysis, the authors ascertained that a relapse developed in 12 of the 13 children and following 3 months CD remission occurred in 1 case. Recurrent thyrothoxicosis occurred in 7 of the 16 patients with normal values of TSRA and 9 children were at remission. Analysis of a ROC-curve revealed that in children having a TSRA level of > 1.95 IU/l at the discontinuation of antithyroid drug therapy, the risk for recurrent thyrotoxicosis is 100% within the first year.

[Genetic polymorphism of glutathione-S-transferase M1 and T1 in children with acute lymphoblastic leukemia].

Children with acute lymphoblastic leukemia (ALL) and deletions of glutathione-S-transferase M1 (GSTMI) and glutathione-S-transferase T1 (GSTT1) have better event-free survival and lower rates of relapses than those with GSTM1 and/or GSTT1. It is concluded that deletions of GSTM1 and/or GSTT1, double-null genotype are closely associated with the good prognosis of childhood ALL treated according to the ALL-BFM 90 and ALL-MB 91 protocols.

[Comparison of albumin analysis by fluorescent method with other clinical laboratory parameters in purulent peritonitis]. / Sravnitel'nyi analiz al'bumina, issledovannogo fliuorestsentnym metodom, i drugikh kliniko-laboratornykh pokazatelei pri gnoinom peritonite.

The concentration and properties of the blood-albumin linkage centers were examined by the method fluorescent probes, besides, the general clinical-and-laboratory indices in the blood serum of patients with diffusive purulent peritonitis were determined. The parameters as obtained by the method of fluorescent probes revealed a valuable information on a patients' severity condition and on a forecasted disease evolution; finally, they surpass, in a majority of cases, the generally accepted clinical-and-laboratory tests applicable to similar conditions.

[Effect of fluvastain and its combination with aspirin and trental on hemostasis and microcirculation in atherosclerosis]. / Vliianie fliuvastatina i ego sochetanii s aspirinom i trentalom na gemostaz i mikrotsirkuliatsiiu pri ateroskleroze.

AIM: To assess effects of fluvastatin monotherapy and combined therapy with aspirin and trental on hemostasis and microcirculation in atherosclerosis. MATERIAL AND METHODS: Hemostasis and microcirculation were studied in 68 patients with coronary atherosclerosis and aortic atherosclerosis on fluvastatin monotherapy and on combined therapy fluvastatin + aspirin + trental. RESULTS: Hypolipidemic treatment with fluvastatin reduced thrombogenic blood potential due to enhancement of plasmic fibrinolytic activity [the time of XII-a dependent fibrinolysis decreased by 33% (p < 0.05)], decreased thrombinemia [blood level of soluble fibrin-monomeric complexes fell by 44% (p < 0.05)] and reduced stimulated platelet aggregation: by 18.2% in response to ADP (p < 0.05) and by 11% in response to adrenalin (p < 0.05). CONCLUSION: Correction of lipid metabolism and blood hypercoagulation improved microrheology. Combination of fluvastatin with aspirin made platelet aggregation reduction in response to both inductors faster and more stable, while combination of fluvastatin with trental contributed to better results in microcirculation improvement.

[Fluorescent method of studying the binding sites of serum albumin for assessing status of patients with purulent-septic diseases]. / Fliuorestsentnyi metod issledovaniia sviazyvaiushchikh tsentrov syvorotochnogo al'bumina dlia otsenki sostoianiia bol'nykh gnoino-septicheskimi zabolevaniiami.

The concentration and properties of the binding centers of the albumin molecule in blood serum of patients with pyo-septic diseases (peritonitis, pancreatonecrosis and sepsis) were investigated. The patients of this category were shown to have a considerably decreased and greatly variable indices of the general concentration of albumin and of effective concentration of albumin in their blood serum. The fluorescent indices were established to correlate with the severity of the disease, efficiency of treatment, prognosis of the development of the disease and its outcome.