Septimiu Voidăzan, Cosmin Moldovan and Minodora Dobreanu
Adrian Man, Cosmin Moldovan and Minodora Dobreanu
Starting with the first issue of 2013, the Romanian Review of Laboratory Medicine has implemented a new editorial and publishing system. By this editorial, we try to clarify to all the readers and authors the major changes and their outcome in the journal’s evolution. Thus, we present details related to the current internal organization of the editorial board and the editorial workflow of the submitted manuscripts.
Orsolya Benedek, Mihaly Veres and Minodora Dobreanu
Background: Trauma in its early stages leads to an acute inflammatory condition affecting all cellular lines. Neutrophil granulocytes make up the largest population of human white blood cells and are fundamental to the innate immune system. The objective of our pilot study was to evaluate neutrophil death and viability alterations in critically ill trauma patients in correlation with their clinical outcome.
Material and method: Critical ill trauma patients were enrolled in the study. In order to assess alterations in cellular death, blood samples were drawn using EDTA containing tubes and analyzed in the first twenty four hours after admission, then after forty eight and seventy two hours. Annexin V was used as a marker for apoptotic cells and propidium iodide for necrotic cells.
Results: The first two cases exhibited an increase in cellular viability by the second day as shown by a small increase in neutrophil apoptosis and a decrease in neutrophil necrosis. These patients progressed to a positive clinical outcome. The second two cases showed slight modifications in either physiological or pathological cellular death, and increasing levels of cellular necrosis. These patients progressed to a negative clinical outcome.
Conclusions: These cases suggest that neutrophil cell viability and death were associated with the patient’s clinical outcome.
Floredana-Laura Șular and Minodora Dobreanu
Objective: The aim of this study was to verify in our laboratory conditions the performance criteria of a commercial kit (PhagoburstTM, Glycotope Biotechnology) as described by the producers. We have also partially altered the use of the available kit by introducing a non-opsonized Candida albicans stimulus, in addition to the opsonized Escherichia coli stimulus provided by the manufacturer. Material and methods: The peripheral blood samples of 6 clinically healthy adults were tested in triplicate according to the manufacturer recommendations. The intraassay imprecision as well as the ranges of neutrophil and monocyte burst activation triggered by various stimuli were assessed. Results: The activation range of granulocytes and monocytes was similar to the one described by the producer in the presence of E. coli (granulocytes: 78.45-99.43% versus 99.6-99.95%, average %CV of 1.53% versus 0.1%, monocytes: 54.63-92.33% versus 81.80-96.67, average %CV 6.92% versus 1.1%). The leukocyte range of activation in the presence of non-opsonized C. albicans was comparable to the one triggered by the fMLP (N-formyl-methionyl-leucyl-phenylalanine) stimulus. Conclusion: The intra-assay precision obtained in our laboratory conditions, as well as the ranges of activated leukocytes, are comparable to the ones described by the producer when using E. coli as a stimulus. The present study shows that introducing an extra fungal stimulus for burst oxidation assessment could provide additional information regarding the non-specific cellular immune response, particularly in patients at risk for candidemia.
David Remona Eliza and Dobreanu Minodora
Clinical laboratory tests ensure approximately 70% of the medical decisions, so that the time until the release of the results and its accuracy are critical for the diagnosis and the efficiency of the treatment . Risk management involves both the anticipation of what could happen erroneous and the assessment of errors’ frequency as well as the consequences or the severity of the effects caused by it, and finally to decide what can be done in order to reduce the risk to an acceptable clinical level. For this reason, organizations should not see the risk management as a compliance issue, but as an integral part of the decision-making process. EP23-A is a guideline of CLSI that introduces the risk management principles in the clinical laboratory and encourages the laboratories to develop plans of risk management which are addressed to the risks of each laboratory. EP18-A2 proposes 2 techniques for identifying and controlling the errors in the laboratory: FMEA (Failure Mode and Effects Analysis) and FRACAS (Failure Reporting, Analysis and Corrective Action System). The European Committee of Experts and Management of Safety and Quality in Health Care proposed to use the quality indicators to identify the critical stages of each process, thus being possible to assess continuously the medical processes with the aim of identifying the errors when they occur. This review summarizes the principles of the risk management in the clinical laboratory, thus it can achieve its aims to report valid, accurate and reliable test results
Benedek Orsolya, Dobreanu Minodora, Azamfirei Leonard, Veres Mihaly and Copotoiu Sanda-Maria
Trauma affects the activity of the innate immune system. The objective of this case report is to present the case that prompted us to analyse all the peripheral white blood cell lines. A 19 year old male patient was admitted to the Intensive Care Clinic with severe head trauma. The final diagnosis was set to be severe cerebral trauma with subarachnoid hemorrhage, right frontal and temporal cerebral contusions, diffuse cerebral edema, left parietal and temporal fracture, sphenoid hemosinus and right sided lung contusions.
Material and Method: Whole blood was immediatly analyzed by flow cytometry for leukocytes. Apoptosis was detected with Annexin V, necrotic cells were stained with propidium iodide. Samples were drawn three consecutive days.
Results: Lymphocytes, monocytes and granulocytes all showed marked increase in viability and decrease in necrosis during the biological monitoring in correlation with a positive clinical outcome. The most important changes were noted in the monocyte population.
Discussion: Although we started out monitoring neutrophil viability and death, this particular case prompted us not to overlook other leucocyte populations.
Conclusion: The apparent positive relationship between this patient’s positive clinical outcome and cellular viability and death changes is promising but they warrant further study.
Adrian Man, Claudia Bănescu, Minodora Dobreanu and Cornel Fraefel
Successful experiments in molecular biology require good knowledge about various methods and protocols. In molecular biology, nucleic acid manipulation is the essence, starting with the quality of extraction and ending with several analysis assays (PCR, RT-PCR, qPCR, PCR arrays, molecular cloning, etc). Though many of these are so called “standardized”, in practice there are many variables that can influence the outcome of the experiment. Due to the importance of optimal primer design in PCR assays, we will focus on primer designing and checking software, but we also present other useful free tools that can help researchers in the molecular biology field
Voidăzan Septimiu, Morariu Silviu-Horia, Căpâlnă Mihai, Mărginean Claudiu and Dobreanu Minodora
Background. Cervical cancer (CC) is a major public health problem worldwide. Knowledge of human papillomavirus (HPV) genotype prevalence and distribution is important for the introduction of an effective vaccination program and the corresponding epidemiological monitoring. The aim of this study was to identify and analyze the distribution of high-risk HPV genotypes.
Methods. Data were collected from 136 patients for the detection of circulating HPV genotypes, where Pap test results revealed the presence of koilocytes or high risk (HR) dysplastic lesions, elements that raise the suspicion of HPV infection.
Results. HPV infection was identified in 72 (55.4%) of the patients tested, 34 (47.3%) with single infection, and 38 (52.7%) with multiple infections. Twenty-two different types of HPV were identified: 14 high risk HPV types, 7 low risk HPV types, 1 probable high risk HPV type. HPV 16 was the most frequently detected (55.6%) one, it was involved in single (15 cases) and multiple (25 cases) infections, primarily associated with type 18 (12 cases), and type 52 (11 cases). The presence of HPV 18 (29.2%) and HPV 52 (23.6%) was identified after HPV type 16.
Conclusions. Oncogenic HPV genotypes 16, 18, and 52 were most frequently associated in women with dysplastic lesions, which require the use of polyvalent HPV vaccines when assessing cross-protective effects of specific immunoprophylaxis programs.
Georgiana Mihaela Şerban, Ion Bogdan Mănescu, Doina Ramona Manu and Minodora Dobreanu
Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation.
Methods: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol.
Results: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes.
Conclusions: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.
Simona Cernea, Adina Huţanu, Ligia Coroş and Minodora Dobreanu
Objectives: The primary aim of this study was to assess residual beta cell function at diagnosis of type 2 diabetes and identify accessible laboratory markers that best estimate it. The secondary objective was to evaluate the change in beta cell function 6 months after starting different therapeutical regimens. Materials and methods: Forty seven subjects were included in the study and each performed a 75-g oral glucose tolerance test (OGTT) at baseline and after 6 months. Metabolic and immunologic parameters were determined from fasting samples. According to the degree of metabolic decompensation, specific therapy was started: metformin, metformin plus gliclazide or insulin therapy (with/out metformin). Early and total beta cell function was evaluated by the disposition index (DI) calculated for 30 minutes and 120 minutes, respectively. Results: At diagnosis, fasting blood glucose (BG) and HbA1c varied largely (129-521 mg/dl and 5.5-14%, respectively). The DI30 and DI120 decreased with more severe glycemic decompensation. For both DI30 and DI120 significant negative correlations were found for glycemic markers (HbA1c, 2-hour BG and maximal BG amplitude) and positive correlation for 2- hour C peptide (p<0.0001 for all). HbA1c value of 7% discriminated an important decrease of DI30 and DI120. Insulin and combined therapy significantly improved DI120 at 6 months (p: 0.0062 and 0.01, respectively), while DI30 was improved only with insulin therapy (p: 0.0326). Conclusions: Beta cell function at onset correlated with HbA1c, 2-hour BG and C peptide during OGTT. Thus OGTT and HbA1c are pivotal for evaluation of beta cell function. Insulin therapy improved early and total insulin secretion at 6 months.