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CURRENT ISSUE /2-2012

COMPARATIVE DETERMINATION OF GLYCATED HEMOGLOBIN WITH DIRECT PHOTOMETRIC IMMUNOASSAY
AND AFFINITY CHROMATOGRAPHY

E. Vucheva(1), М. Genova(2), Y. Niagolov(3), N. Doncheva(1), G. Peichinova(1) and K. Tsatchev(2)
(1)Department of Clinical Laboratory, Medical Institute - Ministry of Interior, Sofia
(2)Department of Clinical Laboratory and Clinical Immunology, Medical University, Sofia
(3)Department of Physiology, Medical University, Sofia

Summary. Glycated hemoglobin (HbA1c) is the gold standard in long-term assessment of glycaemic control in patients with diabetes mellitus (DM). Liquid Chromatography-Isotope Dilution-Mass Spectrometry is a reference method for HbA1c, but other methods used in practice need standardization and comparison. This study was aimed to perform a comparative evaluation of the results from HbA1c determination by direct immuno-photometric method (A) and affinity chromatography method (B). The study included 50 samples with values from all clinically measurable areas. HbA1c in whole blood was studied using Olympus AU 400 analyzer and NycoCard READER II by, applying method A and method B, respectiovely. The results are compared using the program Method Validator, as for the reference method the chromatographic method was accepted. The imprecision in series for method A has been traced in 10 parallel samples from two patients in border and abnormal area of values. The inaccuracy was evaluated in normal and pathological human control material for 25 consecutive days. The results from correlation and regression analysis show linear regression coefficient r = 0.949, slope = 0.919 and intercept = 0.59. Assessment of imprecision for method A shows CV values of 1.21% and 1.91% for both cases, respectively. Accuracy monitoring of method A with control materials shows d% 4.979 in the normal area, and 5.033 in the pathological area. The results show high positive correlation and comparability between the two methods. Direct immuno-photometric method has good accuracy and precision and is applicable for analyzer Olympus AU 400.
Key words: Glycated haemoglobin, direct immuno-photometric method, affinity chromatography method, NycoCard READER

 

RE-STIMULATION OF T-CELL ANTIGEN RECEPTOR AND REGULATION OF THE TYROSINE
PHOSPHORYLATION IN PERIPHERAL T-CELLS

K. Hristov, J. Lindquist and B. Schraven
Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg,
GERMANY

Summary. TCR stimulation involves ZAP70 tyrosine phosphorylation and activation that is essential for signal initiation and TCR degradation. Here we find that re-stimulation of TCR fails to affect the tyrosine Y319 phosphorylation of ZAP70. Vanadate is a reversible PTP inhibitor, sensitive to the presence of EDTA. By contrast, pervanadate inhibits irreversibly. We find that two successive washes for 10 min at 37oC remove the effects of vanadate on protein tyrosine phosphorylation in T-cells. Hypothermia has been shown to affect the expression of different signalling proteins within 1 hour. We find that incubation on ice does not affect the ability of vanadate to increase the activatory phosphorylation of serine S473 at PKB/AKT, and that the removal of the inhibitory effect of vanadate following two successive washes with culture medium down-regulates to control levels the phosphorylation of S473 at PKB/AKT.
Key words: cellular signalling, system biology, T-cell receptor, re-stimulation, tyrosine phosphorylation, vanadate, pervanadate, PI3K – PKB/AKT signalling pathway

 

A RELATIONSHIP BETWEEN MICRORNA204 AND OCCLUDIN IN PROSTATE CANCER INFLAMMATION SIGNALING
K. Todorova, N. Manolova, D. Zasheva and S. Hayrabedyan
Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences

Summary. For the first time the role of miR-20 in prostate cancerogenesis and metastasis is implicated in the modulation of innate inflammatory signaling in prostate cancer. The modulatory effect of miR-204 exogenous artificial upregulation on human prostate cancer cell lines LNCaP (p53+/+, AR+) and PC3 (p53-/-, AR-), challenged with synthetic analog of common for Gram-negative bacteria peptidoglycan and minimal inducer of the NOD1 innate immunity receptor, was evaluated using flow cytometry (FCS) and immunofluorescent detection of Occludin expression. The peptidoglycan minimal inducer challenge induced Occludin upregulation in both cell lines - an effect modulated by miR-204 mimic towards preventing the Occludin upregulation in LNCaP differentially from its null modulating effect in PC3 cells. Our data suggest that miR-204 modulation is at least p53-dependent and impacts the already altered junctional signaling in prostate cancer cells.
Key words:  prostate cancer, Occludin, microRNA, ie-DAP

 

HIGH INCIDENCE OF UNFAVORABLE CYTOGENETIC ABERRATIONS AND LOW REMISSION RATE IN ADULTS OVER
60 WITH ACUTE MYELOID LEUKEMIA

M. Velizarova(1), E. Hadzhiev(2), I. Dimova(3), D. Toncheva(3) and K. Tsatchev(1)
(1)Department of Clinical Laboratory and Clinical Immunology, University Hospital “Alexandrovska”
(2)Clinic of Hematology, University Hospital “Alexandrovska”
(3)Department of Medical Genetics, Medical University – Sofia

Summary. Adults over 60 years of age with acute myeloid leukemia (AML) more frequently have unfavorable molecular-cytogenetic aberrations, poor response to chemotherapy and higher mortality rate than younger patients. The aim of the study was to analyze the clinical, molecular-cytogenetic and induction treatment response features of adult patients over 60 years of age with newly diagnosed AML. Seventy-seven adults (over 18 years of age) with AML were enrolled in our 3-year study. Molecular-cytogenetic aberrations, remission rates, resistant disease and early deaths were compared in the three patient groups: 18-34, 35-59 and 60 years of age or older. Our results confirmed the generally poor outcome of the older AML patients. Patients over 60 had a higher percentage (32%, p = 0.03) of unfavorable cytogenetic abnormalities (i.e., chromosome 5 and 7 abnormalities, t(11q23)/MLL and complex karyotypes) and a lower percentage (3.6%, p = 0.03) of favorable cytogenetic abnormalities (only 1 patient over 60 with t(15; 17)) than younger patients. Out of all AML cases, complete remission following initial induction chemotherapy was achieved in 41 (53.2%). The adults over 60 had the lowest complete remission rate – 35.7% (p < 0.01) and the highest rates of primary chemotherapy-resistant disease (28.5%, p = 0.03) and early deaths (53.6%, p < 0.01), compared to younger patients. We concluded that high frequency of unfavorable cytogenetic aberrations and adverse age were major determinants of complete remission rate in intensively treated acute myeloid leukemia patients older than 60 years. These data confirmed the need of an age-specific management for the disease in the elderly.
Key words: acute myeloid leukemia, cytogenetics, age, treatment


HPV-ASSOCIATED BUSCHKE-LÖWENSTEIN TUMOURS IN A PATIENT WITH DISSEMINATED ANOGENITAL FORM
OF CONDYLOMATA ACUMINATA – A DERMATOSURGICAL PROBLEM

G. Tchernev(1), J. Ananiev(2), M. Gulubova(2), I. Bakardzhiev(3), G. Pehlivanov(4), M. Gonevski(5), L. Zisova(6) and J. C. Cardoso(7)
(1)Polyclinic of Dermatology and Venerology, “Saint Kliment Ohridski University”, University Hospital “Lozenetz”, Sofiа, Bulgaria
(2)Department of General and Clinical Pathology, Medical Faculty, Trakia University, Stara Zagora, Bulgaria
(3)College of Medicine, Medical University of Varna
(4)University Clinic of Dermatology, Sofia
(5)St James’s University Hospital, Leeds Teaching Hospitals, Clinic of Nephrology, UK
(6)Department of Dermatology and Venerology, Medical University, Plovdiv, Bulgaria
(7)Department of Dermatology, University Hospital of Coimbra, Portugal

Summary. Buschke-Löwenstein tumours are a relatively rare clinicopathologi­cal entity. However, due to their malignant potential and frequent association with some types of Human Papillomaviruses (HPV), their diagnosis should be promptly confirmed and appropriate management should be planned (according to the condition). In this article we present a middle-aged patient with long-standing tumoural lesions located in the anogenital area, which were confirmed clinically and histop­athologically as well-differentiated squamous cell carcinomas of Buschke-Löwenstein type, in combination with disseminated anogenital condylomata acuminata. Histologically, large tumour nests of well-differentiated squamous cell carcinoma were observed, as well as a marked mononuclear cell infiltrate and conspicuous koilocytosis. Immunohistochemistry for Ki-67 revealed a relatively high proliferative index, and was strongly positive for HPV with a pan-HPV antibody (AK), confirming the presence of the virus in the tissue. Surgical removal of the various tumoural masses was performed by electrodesiccation, combined with shave-curettage under general anesthesia. Due to the excessive bleeding there was no possibility for complete removal of the perianal tumour formations and the therapeutic strategy was revised. Systemic therapy with interferon was planned with the purpose of reducing the tumour mass before further surgery.
Key words: Buschke-Löwenstein tumour, HPV association, Ki-67, verrucous carcinoma


ULCERATIVE LESION IN THE GENITAL AREA – A RARE CASE OF NON-KERATINIZING POORLY DIFFERENTIATED
METASTASIZING SQUAMOUS CELL CARCINOMA WITH A LETHAL OUTCOME

J. Ananiev(1), G. Tchernev(2) and I. Bakardzhiev(3)
(1)Department of General and Clinical Pathology, Medical Faculty, Trakia University, Stara Zagora, Bulgaria
(2)Polyclinic of Dermatology and Venerology, Saint Kliment Ohridski University, University Hospital Lozenetz, Medical Faculty
(3)College of Medicine, Medical University of Varna

Summary. Introduction: Squamous cell carcinoma (SCC) continues to be one of the most frequent skin neoplasms. A number of factors influence the progression and behaviour of the disease. Assessment of these factors is important in predicting the survival rate of the patients and in defining the best therapeutical options. TNM classification and evaluation of tumour differentiation are important tools in assessing the disease stage in order to define the appropriate management. Case report: We present a case of a patient with an ulcerated lesion in the inguinal area and the proximal portion of the medial left thigh which, after a biopsy, was diagnosed as a non-keratinizing poorly differentiated squamous cell carcinoma. Months after the initial treatment of the primary tumour with simple excision, it relapsed and metastasized, which led to TNM stage upgrade and subsequently showed an aggressive clinical course eventuating into the death of the patient. Conclusion: In this case report we emphasize the morphological features of the tumour, its TNM classification and the implications in the disease course. In conclusion, we want to stress out that a correct tumour staging is of paramount importance in order to select the most appropriate therapeutical approach, although even so there isn’t always a guarantee of success.
Key words: Squamous cell carcinoma, TNM classification, degree of differentiation


ANGIOKERATOMA CIRCUMSCRIPTUM – SUCCESSFUL TREATMENT WITH A NEW TECHNOLOGY
B. Kamoona, J. Kazandjieva, A. Nikolova, E. Bardarov and S. Marina
Department of Dermatology and Venerology, Medical Faculty – Sofia

Summary. The term angiokeratoma means hyperkeratotic, dark-red to purple papules, with dimensions of 1-10 mm in diameter. Histopathologically they are seen as vascular ectasias in the upper dermis surmounted by hyperkeratosis of the epidermis. Several clinical variants of angiokeratomas exist. Some variants are localized on the genitals (Fordyce’s angiokeratoma), on the fingers and toes (Mibelli’s angiokeratoma) or form confluent plaques (Angiokeratoma circumscriptum). We present a 15-year old female patient with Angiokeratoma circumscriptum of one year duration, which expands in size with time. Interestingly, angiokeratoma circumscriptum appeared on the place of an operational scar. Laser treatment is one of the effective options for removal of localized angiokeratoma. The spectrum of treatment options includes ablative lasers, argon laser and IPL. In our case we achieved an excellent and rapid therapeutic result using a laser apparatus with elōs technology.
Key words: Angiokeratoma circumscriptum, laser therapy, elōs technology


OCCUPATIONAL HEALTH MANAGEMENT OF PESTICIDES INTOXICATIONS (A CASE REPORT)
K. Lyubomirova
Department of Occupational Health, Faculty of Public Health, Medical University – Sofia

Summary. Pesticides use is related to risk of occupational intoxication during their production and application. Aim: The aim of the current publication is to present a case of a good occupational health practice in an enterprise involved in field application of pesticides. Methods: The health status of employees applying pesticides was investigated. Serum concentrations of liver enzymes and cholinesterase were determined. Results: The results showed that because of the daily instructions of the employees and adequately chosen personal protective equipment, adverse health effects were not registered among the employees. The serum concentrations of the investigated indices (ASAT, ALAT and cholinesterase) were in the reference range in all samples. Conclusions: Health management of occupational intoxications is based on the good knowledge about the activities and safety use of pesticides, as well as the adequate personal equipment provided by the employers.
Key words: pesticides, health risk, occupational intoxications, health and safety at work, prevention

 

HEALTH MANAGEMENT OF OCCUPATIONAL DISEASES – A CHALLENGE FOR BULGARIAN ECONOMY AND PUBLIC HEALTH
K. Lyubomirova and N. Tsacheva
Department of Occupational Health, Faculty of Public Health, Medical University – Sofia

Summary. Background: In 2009 more that 400,000 people in Bulgaria have permanent disability and compensations were paid to more that 477,000 people with a disability more that 71%. Many of these people are victims of occupational diseases. Objectives: In 2001 a National Register of Occupational diseases was founded in Bulgaria and a new information system for notification and registration of occupational diseases was introduced into practice. Aim: The aim of the study was to study and analyze the prevalence and type of different occupational diseases and to identify the risky economic branches and work activities. Methodology: Thorough collection of experts’ decisions and registration cards of all patients with occupational diseases in Bulgaria for the investigated period was done. Results: The result showed that during the last 10 years (1998-2008) the prevalence of occupational diseases in Bulgaria increased, especially in some pathological groups. Musculoskeletal diseases and diseases of the peripheral nerves due to occupational factors represent 55% of the cases. Non-specific respiratory diseases (as chronic bronchitis, COPD, bronchiectasis) were 2%, diseases caused by noise and vibrations were 12%. Discussion: The number of people with permanent disability in Bulgaria is extremely high which is unacceptable from social, medical and economic point of view. It is necessary to increase the quality of the occupational health training which will accelerate the early diagnostics and prevention of the occupational diseases and will contribute to improvement of the working conditions in the risky economic sectors.
Key words: occupational diseases, National register, risk factors, economics, public health

 

AN IN VITRO STUDY OF ROOT CARIES OF FRONT TEETH
E. Boteva, M. Marinova and D. Karayasheva
Department of Conservative Dentistry,
Faculty of Dental Medicine, Medical University, Sofia

Summary. Root carieses can occur on root surfaces exposed to bacteria. They are typically diagnosed in patients with periodontal diseases and gingival recessions. Frequencies are higher among elderly patients, although root caries can appear in middle-aged and in young patients. The аim of the present study was to investigate the frequency of root caries of front teeth with sound crowns and high resistance, which has overcome the demineralization and the caries and were extracted due to periodontal reasons. All teeth n = 123 were sound and matured, from the same geographical region and dental practice area. After cleaning and draying, they were observed under magnification x5. The incisors and premolars were separated in six groups. Root caries was observed under magnification x5, diagnosed with probe and registered by two examiners. The data was registered according to the criteria: Margins (2/3-1/2) in dentine and cementum and origin of the lesion on root surface. The size of the lesions observed varied from minimum 1 mm² to massive tissue losses on two to three surfaces of the root surface. The general frequency of root caries was 25%, which was symptomatic for all groups of teeth with generalized elderly and pathological periodontal changes. Early prevention of root caries is essential. Dietary restriction of erosive beverages and sticky sweeteners is an essential part of the general treatment and preventive plan. The usual mouthrinsis, electric toothbrushes, vitamin C, intradental brushes included in standard periodontal treatment must be carefully revised in relation to patients DMFT.
Key words: dental caries, root caries, epidemiology

 

ASSOCIATION OF 13Q14 DELETION WITH CLINICO-LABORATORY PARAMETERS IN B-CELL
CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL)

E. Hadzhiev(1), K. Alexandrova(1), M. Velizarova(2), I. Dimova(3) and D. Toncheva(3)
(1)Department of Internal Medicine, Clinic of Hematology, University Hospital “Alexandrovska”, Sofia, Bulgaria
(2)Department of Clinical Laboratory and Clinical Immunology, University Hospital “Alexandrovska”, Sofia, Bulgaria
(3)Department of Medical Genetics, Medical University – Sofia, Bulgaria

Summary. A deletion of chromosome band 13q14 (13q-) detected by fluorescence in situ hybridization (FISH) analysis is the single most common cytogenetic abnormality in patients with chronic lymphocytic leukemia (CLL). In our study we disccused the incidence of molecular-cytogenetic deletion of 13q14 marker in B-CLL in order to determine the association of this aberration with clinical manifestation, laboratory parameters and poor disease prognosis. Patients with 13q14 aberrations were significantly older. They had ZAP (+) status, rapid disease progression and therapy dependency. A determination of 13q14 genetic abnormality should, therefore, be included in the investigations of the prognostic factors of B-cell chronic lymphocytic leukemia.
Key words: 13q14 deletion, chronic lymphocytic leukemia, Fluorescence in situ hybridization, ZAP70

 

BCR/ABL MOLECULAR TESTING BY FLUORESCENCE IN SITU HYBRIDIZATION IN UNTREATED ADULT ACUTE LEUKEMIA
M. Velizarova(1), I. Dimova(2), E. Hadzhiev(3) and K. Tsatchev(1)
(1)Department of Clinical Laboratory and Clinical Immunology, University Hospital “Alexandrovska”, Sofia, Bulgaria
(2)Department of Medical Genetics, Medical University – Sofia, Bulgaria
(3)Department of Internal Medicine, Clinic of Hematology, University Hospital “Alexandrovska”, Sofia, Bulgaria

Summary. The BCR/ABL detection influences diagnosis, prognosis and treatment decisions in patients with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). We studied the feasibility and the sensitivity of LSI BCR/ABL dual-colour, dual-fusion probe as a usual assay for detection BCR-ABL gene fusion in acute leukemia. A total of 3229 successfully hybridized interphase nuclei from 34 newly diagnosed patients with acute leukemias (12 AML and 22 ALL cases) were analyzed. We used interphase fluorescence in situ hybridization (FISH) method and LSI BCR/ABL dual-colour, dual-fusion probe (Vysis) for detection of BCR-ABL gene that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR-ABL fusion in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). Fusion BCR-ABL gene was observed in 9 (28.13%) of the cases with successful hybridization and in 6 (18.75%) of them the results had clinical reliability. The aberrant cell lines were of different sizes – 53%-96% of cells were positive for BCR-ABL gene. Fusion signals for BCR-ABL were reported in 2 (16.67%) of AML and in 4 (18.18%) of ALL cases. In conclusion, we proved that LSI BCR-ABL dual-colour, dual-fusion probe (Visys) is characterized with high specificity and sensibility.
Key words: Interphase FISH, BCR-ABL gene, adult acute leukemia

 

IMPORTANCE OF SOME CLINICAL FACTORS (EPILEPSY DURATION, CLUSTERS OF SEIZURES AND/OR  
EPILEPTIC STATUS, TYPE OF EPILEPSY, TYPE  OF SEIZURES, MONO-/ POLYTHERAPY) FOR THE QUALITY
OF LIFE OF PATIENTS WITH REFRACTORY EPILEPSY

E. Viteva
Department of Neurology, Medical University of Plovdiv

Summary. To assess the impact of the duration of epilepsy, the clusters of seizures and/or epileptic status, the type of epilepsy and seizures, and the mono-/ polytherapy on the quality of life (QOL) of Bulgarian patients with refractory epilepsy (RE). Seventy patients with RE were studied using a purposeful interview on the patients’ disease course, as well as an exam of the patients’ medical documentation. All study participants completed QOLIE-89. The greater duration of epilepsy has a negative impact on the assessments of the change in health during the last year. Seizure clusters and/or epileptic status in the disease course are associated with lower assessments of the sexual relations. The type of epilepsy has an impact on: 1. Seizure worries – participants with generalized epilepsy have less worries; 2. The overall score of QOLIE-89 – the patients with partial epilepsy make lower assessments. An impact of the seizure type on the subscale “Overall health” has been demonstrated. The lowest scores are given by the patients having partial seizures with secondary generalization. The highest scores are given by the participants having generalized tonic-clonic seizures, generalized myoclonic seizures, and polymorphic seizures. The prescribed treatment as mono-/polytherapy has an impact on the assessments of the overall health - the scores were higher in cases with polytherapy. The clinical factors duration of epilepsy, clusters of seizures and/or epileptic status, type of epilepsy, type of seizures, mono-/polytherapy have an impact on single aspects of the QOL of the patients with RE.
Key words: quality of life, refractory epilepsy, duration, mono-/polytherapy, seizure clusters, epileptic status

 
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