فصلنامه خون
سال پنجم شماره 4 (پیاپی 21، زمستان 1387)

Background and objectivesSeveral studies have shown the positive effects of introns on the expression of heterologous genes in mammalian hosts. In this study, transient expression increment of hFIX in the presence of intron-1 of human beta-glubin was studied. Materials and MethodsThe intron-1 of human beta-glubin (hBG) was introduced into the human factor IX cDNA in donor/acceptor site between exons 1 and 2. The constructed hFIX mini-gene and a native hFIX were inserted separately in two expression vectors next to the CMV promoter. After verification, the two recombinant plasmids with and without intron were used to transfect Chinese Hamster Ovary (CHO) cells. The cultured media taken from the tansfected cells were examined for coagulation activity with a single step clotting test performed on FIX-deficient plasma. Then, to confirm the expression of recombinant hFIX by transfected cells, RT-PCR test was conducted. ResultsThe preliminary data obtained from the expression analysis of the two groups of transfected cells in comparison with the cells with parental plasmid (as negative control) indicate of an increase of about 16 to 62% in coagulation activity of both groups of transfected cells. The same data show an enhanced hFIX coagulation activity of about 1 to 28% in the culture media taken from the cells with intron-containing hFIX-cDNA. Furthermore, RT-PCR confirmed correct splicing process and expression of hFIX from both transfected cells. ConclusionsThe positive function of hBG intron 1 on the expression of hFIX in CHO cells was shown. Besides, the constructed plasmids have provided tools for analysis of the stability of the transfected cells for production of biologically active hFIX in a systematic approach.

Background and objectivesAlthough beta thalassemia is mainly caused by mutations involving single base substitutions and small deletions, there have been reports showing deletions of large regions of beta- globin genes play a similar causing role. The strategy to identify beta thalassemia carriers with known deletions is based on PCR techniques such as Gap PCR. There are however some unknown deletions that can not be detected by the above methods. To overcome this limitation, Real-time PCR and MLPA were developed as two quantitative assays for analysis of beta-globin gene cluster. Materials and MethodsThe subjects were evaluated in a case-control study. Among individuals referred to genetic laboratories of Pasteur Institute of Iran and Kawsar Genetic Research Center, 40 were suspected of having a large deletion in β-globin gene cluster. The including criteria were hematological findings such as low blood indices (MCV <80 fl and MCH <27 pg), normal HbA2 and raised or normal HbF. Genomic DNA was extracted from peripheral blood. A Real-time PCR assay was developed using comparative threshold cycle (Ct) method for analysis of gene copy number. In addition, gene dosage was analyzed using MLPA method. ResultsReal-time PCR results for quantitative analysis of Beta, Delta, G-gamma genes showed the ratio (2-ΔΔCt) of 0.96 ± 0.18 for normal individuals and 0.58 ± 0.04 for carriers of deletions in beta globin gene cluster. MLPA results showed nearly 50% reduction in the height of the peaks corresponding to regions of deletions. ConclusionsMLPA results confirmed the presence of the same deletions detected by Real-time PCR in all of the carrier individuals. It would be ideal to combine these quantitative assays to confirm corresponding results for accurate diagnosis of known and unknown deletions in beta thalassemia carriers.

Background and objectivesSomatic point mutation in JAK2 gene is characterized by G to T transversion at nucleotide 1849 in exon 12 in the JAK2 gene and results in autonomous activation of JAK2 protein. JAK2 mutation leads to independent cytokine signaling and clonal proliferation of hematopoietic cells in MPNs. Due to absence of any reports for JAK2 V617F mutation in Iranian MPNs patients and its important role in diagnosis, we decided to carry out this study. Materials and MethodsIn this experimental study, we evaluated JAK2 mutation in 100 MPNs patients with diagnosis of PV (n=44), PMF (n=31) and ET(n=25) by simple randomized sampling. After extraction of genomic DNA from whole blood buffy coat, detection of mutation was done using allele specific PCR. Agarose gel electrophoresis was used for observation of PCR products. For confirmation of results, PCR-RFLP using BsaXI was applied. ResultsUsing allele specific PCR and PCR-RFLP, frequency of JAK2 V617F mutation was evaluated to be 89%, 61%, and 56% in PV, PMF and ET, respectively. ConclusionsFrequency of JAK2 mutation in our study is compatible with previous reports. According to WHO criteria, allele specific PCR can be applied for detection of JAK2 mutation in Iranian patients with diagnosis of MPNs.

Background and objectivesIn 1997, a novel DNA virus, unrelated to the known hepatitis viruses, was isolated from the serum of a patient with posttransfusion acute hepatitis with elevated alanine aminotransferase of unknown etiology, and it was named TT virus (TTV). In this study, the frequency of TTV and its role in induction of liver damages were evaluated in three groups of healthy, HBV and HCV-infected blood donors in Tabriz. Materials and MethodsIn this descriptive study, sampling was done on 100 healthy donors, 40 HBsAg positive and 40 positive Anti-HCV in East Azarbayjan Blood Transfusion Center. The levels of ALT and AST in the sera were measured; DNA was then extracted and PCR was performed using T801 and T935 consensus primers. One-way ANOVA, Tukey-Kramer, Kruskal-Wallis, Dunn's and Fisher’s Exact tests were finally used for statistical analyses. ResultsTTV was detected in 65%, 70% and 67.5% of the healthy, HBV and HCV-infected blood donors, respectively. In each group of blood donors, the levels of ALT and AST were not significantly different in TTV infected versus non-infected individuals. ConclusionsGiven the high frequency of infection in healthy individuals and considering the level of hepatic enzymes in TTV infected individuals, it seems that the virus or at least certain genotypes present have not been pathogenic for the infected individuals tested.

Background and objectivesOrder for a large number of blood and blood components is a very common practice that leads to great reduction in blood supplies, lowers blood unit quality, increases the number of expired units, and imposes heavy cost on blood transfusion centers. To evaluate the most common indications for blood order and blood use, we studied Zahedan teaching hospitals to seek a standard pattern for blood transfusion in Zahedan province. Materials and MethodsThis is a cross sectional study on 2094 blood order forms in Zahedan teaching hospitals including blood banks. The data were collected through questionnairs attached to all blood order forms in Zahedan teaching hospitals from June to September 2007. Data were analyzed in SPSS 14 with the use of Chi square and T test. To calculate C/T ratio, the number of crossmatched units was divided by the number of transfused units. ResultsIn this study, 2094 blood order forms were evaluated. Out of the patients, 57.9% were male with the age average of 27.1. The average number of products ordered for every patient was 2.36 ± 1.89 units and the average number of transfused units was 1.97 ± 1.57. From all blood order forms, 38.3% (CI: 95% = 36.1-40.2) were not transfused. The highest rate of blood orders pertained to surgery emergency (14.2%), ICU (10.3%), general surgery (9.3%), pediatrics (8.6%), orthopedics (8%), and gynecology (6.7%). Packed cell was the product with the highest order rate of 73.4% in Zahedan teaching hospitals; however, out of this rate 44% remained untransfused. ConclusionsThe results of this study showed that the rate of non-transfused ordered blood components in Zahedan teaching hospitals is so high. Thus, we need a regional guideline for blood orders so that we would be able to reduce this rate. We should also reconsider the blood order processing and the blood utilization strategy.

Background and objectivesABO blood group antigens with carbohydrate molecules are found on the surface of uroepithelial cells, which influence human susceptibility to infectious diseases. The present study tries to determine the relation between these antigens and the type of the urinary tract pathogens. Materials and MethodsIn this descriptive study, 250 patients whose urinary tract infection had been confirmed by pyoria and positive urine culture were studied. Sampling method was continued until at least 30 patients from different blood groups were involved. A questionnaire addressing age, sex, blood group type, and type of UTI pathogen was filled out for each patient. Data were analyzed by SPSS 13, chi-sqaure, and fisher test. ResultsThe results showed E.coli as the most prevalent type of the urinary tract pathogen in B and O blood groups with the rates of 72.7% (CI95%= 54.08 - 91.32) and 52.3% (CI95% = 41.54 - 63.06), respectively. Staphylococcus saprophyticus was also the most prevalent pathogen in blood groups A and AB with the rates of 43% (CI95%= 30.1 - 50.9) and 39% (CI95%= 16.48 - 61.52), respectively. The correlation between urinary tract pathogens and blood groups was significant (p<0.05). However, urinary tract pathogens had no significant correlation with sex and age. ConclusionsE.Coli is the most common known cause of UTI; however, it is Staphylococcus saprophyticus being the most common pathogen in blood groups A and AB.

Background and objectivesAlthough physicians are the ones prescribing blood products, they do not receive adequate education about transfusion medicine. As a matter of fact, they are not well aware of preparation, storage, indications, and transfusion reactions of blood components. It is harmful for both blood suppliers and patients. The present study aims to evaluate the knowledge level of recently graduated medical students in this regard. Materials and MethodsIn this descriptive study, 1663 questionnaires were distributed among medical graduates who referred to the Ministry of Health, Treatment, and Medical Education to receive their medical license (2004 to 2005). The questionnaire included 20 multiple choice questions about transfusion medicine, and 2 questions aiming to assess educational needs. The data obtained were finally analyzed with SPSS 11.5. ResultsEach multiple choice question had one score. The mean score was 6.01 ± 2.4 and the mode was five with 93% of the scores being below 10. Out of the respondents, 14 graduates believed that they had not received any education in the field of transfusion medicine. At the same time, 88.9% of respondents expressed their interest to receive training about blood transfusion. ConclusionsKnowledge of medical graduates about transfusion medicine is much lower especially about preparation and storage of blood products than expected. So, it is a necessity for medical students to receive education about blood transfusion in blood transfusion centers.

Background and objectivesThe hemoglobin color scale (HCS) is a simple and inexpensive clinical device for screening of blood donors. This study aimed to assess the validity of the device for anemia screening of blood donors. Materials and MethodsThis cross sectional study was done on 170 randomly selected blood donors. The hemoglobin levels of blood samples taken by both skin puncture and venepuncture were measured by HCS and calibrated automated blood cell counter (standard method), respectively. Sensitivity and specificity as well as false results of the above two measurement methods based on the old and new eligibility criteria of hemoglobin level of >12 g/dl and ≥ 14 g/dl were compared. Data were analysed with SPSS 13.5 by paired t test. ResultsAnalysis of paired results showed that Hb measured by HCS was 0.32 ± 0.65 g/dl lower than the standard method (p<0.001). Sensitivity and specificity of HCS based on previous Standard Operating Procedures (SOPs) were 54.5 and 92.4%, respectively. The frequency rates of false exclusion and inclusion were 7 and 3%, respectively. Sensitivity and specificity of recent SOPs were 85.7% and 87.5%, respectively. based on recent SOPs, 39.4% of blood donors with Hb range of 12-14 g/dl were considered rejected. ConclusionsRecent SOPs have increased the sensitivity of hemoglobin measurement by HCS but almost half of blood donors need to be retested to see if they are eligible as blood donors; this process is however labor intensive and unpleasant for donors. Therefore, further studies are recommended for more appropriate methods of anemia screening in blood donors to be created.