فصلنامه خون
سال هشتم شماره 2 (پیاپی 31، تابستان 1390)

Background and ObjectivesUmbilical vein mesenchymal stem cells (UVMSC) which are multipotent precursors have been recently isolated. They are capable of differentiating into various cell types including neural and cardiac cells. In this study, the differentiation ability of human UVMSC into hepatocyte cells has investigated with concentration on hepatic genes expression profile. Materials and MethodsThe UVMSC were isolated and cultured in differentiation medium containing hepatocyte growth factor (HGF) and oncostatin M (OSM) using two step protocol. Differentiation of UVMSCs into cells expressing liver-specific genes was investigated by RT-PCR and immunocytochemistry.ResultsThe cells showed the remarkable transition from bipolar fibroblast-like morphology to epithelial and polygonal shapes. The temporal gene expression pattern of hepatocyte-specific genes such as CK8, CK18, HNF3β, c-Met, AFP, TTR, G6P, Transthyretin, and ALB were detected during differentiation. The immunoflourescent analysis also showed that the differentiated cells were stained positively for CK-18 protein. ConclusionsFindings indicate that UVMSC has a potential for differentiation into the hepatic like cells.

Background and ObjectivesTwo factors known to cause thrombophilia in women with unexplained recurrent spontaneous abortion (RSA) are MTHFR polymorphisms including C677T and A1298C. This study aimed to determine the association between RSA and two polymorphisms of MTHFR in Iranian patients.Materials and MethodsIn this case-control study, 30 patients with the background of two or more consecutive unexplained abortions and 10 women with at least two live births without a miscarriage who referred to Baqiyatallah Hospital and Avicenna Infertility Clinic were analyzed for MTHFR C677T and A1298C polymorphisms by the PCR-RFLP method. Results achieved from estimating the genotype of each polymorphism were analyzed by the SPSS16. The Spearman method was also used to evaluate the correlation between the two polymorphisms.ResultsData has shown a significant correlation between MTHFR C677T and MTHFR A1298C polymorphisms. Seventeen women (56.6%) with recurrent spontaneous abortions and 5 women (50%) among the controls were heterozygote for MTHFR C677T polymorphism. T allele frequency in the patient group was more than the control group (28.4% for patients and 25% for controls). Frequency of the MTHFR A1298C polymorphism was 63.3% in patients and 50% in controls. For A1298C polymorphism, 43.3% of patients and 20% of controls were heterozygote. Furthermore, 20% of patients and 30% of controls were homozygote for this polymorphism.ConclusionsThe prevalence of MTHFR C677T and MTHFR A1298C polymorphisms were slightly higher in RSA patients compared to controls. These findings failed to support the relationship between thrombophilia polymorphisms and the increasing risk of RSA in evaluated Iranian women.

Background and ObjectivesTissue factor (TF), a 45-kDa transmembrane glycoprotein, is the major cellular initiator of the coagulation cascade. Prothrombin Time (PT) is the test that evaluates extrinsic pathway of coagulation. Thromboplastin used in this test is mostly prepared of rabbit brain. Thus it causes variation in the PT results. There is an important advantage of using recombinant TF in PT test to get more reproducible results. Materials and MethodsTF mRNA was isolated from human lung fibroblast cells. After preparation of cDNA it has cloned in pcDNA3 plasmid. CHO cells were transfected with recombinant plasmid. Transfected cells were grown in presence of Geneticin. Total proteins were extracted from CHO cells and separated with SDS/PAGE electrophoresis and analyzed by western blotting technique. CHO cells expressing TF were added to citrated plasma in presence of Cacl2 and clotting time was measured. In addition, factor VII activation by recombinant TF in the plasma was assessed by ELISA method. ResultsExtracted proteins from tarnsfected CHO cells separated on SDS/PAGE showed an approximately 40-kDa band that verified with a monoclonal antibody against TF on western blot analysis. Adding transfected cells (106cell/ml) to the citrated plasma in presence of Cacl2 could decrease clotting time from 3 minutes to 21 ± 3 seconds compared with untransfected CHO cells. The level of factor VIIa in citrated plasma was 810 ± 35 ng/ml in presence of transfected cells’ membrane.ConclusionsRecombinant TF expressed in CHO cells was able to clot citrated plasma and to activate factor VII.

Background and ObjectivesThe quantification of fetal cells in the maternal circulation is an important goal to determine the amount of anti-D for prevention of active immunization of a D-negative mother giving birth to a D-positive baby. The aim of this study was to evaluate two flowcytometric staining technics for determination of fetal erythrocytes in maternal blood and to recognize the best.Materials and MethodsIn this experimental study, 34 adult D-negative blood samples were spiked with six serial dilutions of D-positive cord blood (0.125, 0.25, 0.5, 1, 5 and 10%) which was representative of 99.9% of the clinical fetomaternal hemorrhage (FMH); they were stained for flow cytometric analysis. The Fetal Cell Count Kit was used for HbF dual staining and monoclonal anti-D for RhD single staining.ResultsA comparison between RhD and HbF percentages and FMH volume in spiking samples was performed. A significant correlation between two different parameters in flowcytometric percentages was observed (anti-HbF versus anti-D, r= 0.897, p<0.05). FMH volume was calculated and a significant correlation between expected and observed FMHs in RhD and HbF was obtained (p<0.05, r=0.984, r=0.874). The anti-HbF flowcytometric dual staining allowed better distinction between fetal RBCs (HbF+,CA-), F cells (HbF+,CA+), and adult RBCs (HbF-,CA+). Although Rh-D showed better correlation, but higher values with the RhD in suspensions lower than 10% were noted.ConclusionsData showed that anti-HbF labeling is significantly much more accurate than RhD labeling. Quantification of FMH using these two techniques described allows precise dosage of RhD immunoglobulin for protection against anti-D allo-immunization.

Background and ObjectivesUncontrolled bleeding is the first leading cause of preventable death in the battlefield and the second cause of mortality in civil accidents. Incompressible hemorrhage control is among the interventions that drastically increase the survival rate of the wounded individuals. Materials and MethodsThree healthy hybrid dogs were selected and after induction of anesthesia with Ether, either arterial puncture by a needle or arteriotomy was performed on both groin regions of the dogs. For control arteries, only a 4 min pressure using a sterile gauze was performed, while for the femoral arteries of the opposite side, Cool Clot hemostatic agent was topically used before the pressure. To assess the Coagulation Time (CT), blood samples were collected from 10 volunteer students. CT was measured by a chronometer according to standard Lee -White method.ResultsIn dog animal model, the Bleeding Time (BT) in control arteries (no hemostatic agent) was more than 15 minutes, while in case of the opposite sides which were treated with Cool Clot hemostatic agent, the BT was only up to 5 minutes. In the human phase of the study, the mean coagulation time in control blood samples was 253.4 ± 4.1 sec, while for blood samples treated with bentonite, zeolite and Cool Clot the mean times were 149.5 ± 50.0, 162.3 ± 74.5 and 143.4 ± 114.6 sec, respectively.ConclusionsThe results obtained in this study indicate the significant efficacy of Cool Clot in shortening the BT in dog model or CT in human blood samples.

Background and ObjectivesThe concerns about blood transfusion have given prominence to the role knowledge improvement can play as a strategy to improve health care quality and reduce health cost. This study is aimed at defining the learning opportunities available in blood centers for medical students. Materials and MethodsIn this quasi-experimental research study, fourteen medical students participated in a five day course designed to introduce learning opportunities in blood centers. At the end of the course, participants’ knowledge was measured through a test; they were also retested after six months. Students’ marks were then compared by means of Wilcoxon “T” test. SPSS18 was used for data analysis.ResultsThe mean of students’ marks in the first exam was 15.7 +2 (out of 20) but in the second exam it declined to 12.8+2 (p< 0.001). However, 85.7% of participants recommended this course for other medical students and 71.4% believed they were competent enough in selecting appropriate blood products. ConclusionsTransfusion medicine should be educated through a multi-disciplinary manner and current clinical fields seem inadequate for this purpose. Iranian Blood Transfusion Organization can be considered as an effective center to provide learning opportunities for medical students.

Background and ObjectivesIn recent years we have been faced with more precise, multi-stage, and highly stringent blood donor screening criteria. The present study aimed at evaluating these criteria through comparing the rate of infection in blood donations with deferral rate for high-risk behaviors.Materials and MethodsThis descriptive analytic study was carried out in Esfahan Blood Transfusion Organization (EBTO) with census method through 2004 to 2009. Annual deferral rate due to high-risk behaviors and major viral markers were analyzed separately by using Pearson correlation. The data were obtained by Negareh software. ResultsThe viral markers frequencies were 54, 45, 34, 25, 22 and 22 percent in volunteers that referred to EBTO from 2004 to 2009. Overall deferral rates due to high-risk behaviors were 3.69, 4.71, 5.29, 5.19, 3.93, and 4.04 percent, respectively. It had a slightly negative correlation with HBV (r=0.148; p=0.78) and HCV (r=0.165; p=0.75) markers.ConclusionsData showed that recent procedures of Iranian Blood Transfusion Organization declined viral markers considerably. However, slightly negative correlation of deferral rate due to TTDs with hepatitis B and C markers makes it necessary for donor selection criteria to be reviewed. More appropriate counselling procedures and post-donation counselling can help improve blood safety.

Background and Objectives Although recent screening policies have markedly decreased the incidence of HCV infection in transfusion dependent thalassemia paitients, this problem is still one of the most important causes of mortality and morbidity in thalassemic patients.Materials and Methods A retrospective cross-sectional study was conducted on 206 thalassemia patients referred to the Shahid Bahonar Hospital during 2009-2010. Serum specimens were tested with anti –HCV assay and RIBA –II technique to asses HCV infection. Statistical analysis was done with SPSS18 (χ2 and T-test). Results The overall prevalence rate of anti-HCV was 15%)11 male, 20 female). Moreover 29 anti-HCV positive patients were RIBA –II positive(11 male, 18 female). All HCV affected patients but one had received their first transfusion before 1996 when HCV screening had became mandatory. Conclusions HCV infection rate compared with the general population is higher in multi transfused thalassemia patients. Out of HCV infected thalassemias, 80% are at risk of developing chronic liver diseases and fibrosis; thus preventive measures such as blood donor screening for anti-HCV and stringent infection control procedures are crucial steps to be implemented for HCV prevention.

Background and ObjectivesCerebral vein thrombosis is a relatively rare but important complication during treatment of Acute Lymphoblastic Leukemia(ALL) in children.CaseA 12 year old boy with pre-B ALL during the consolidation phase of treatment with BFM protocol was admitted with severe headache; imaging study showed cerebral venous thrombosis in left sigmoid and lateral sinuses. Diagnostic evaluation revealed heterozygot mutation in G20210A prothombin gene. ConclusionsSome hereditary hypercoagulability states such as prothrombin G20210A heterogeneity, play an important role in childhood thrombotic events during treatment of hematologic malignancy with medications such as L-asparginase. So venous thrombosis prophylaxis in this situation should be considered.