The Scientific Journal of Iranian Blood Transfusion
Year:2007 | Volume:3 | Issue:4|Papers Count:11

Background and Objectives: Recently, P19c16 has been cloned from P19 cells. This stem cell line has mesodermal traits and can be differentiated into cardiomyocytes without embryoid body formation. The present study was designed to investigate the oxytocin effect on differentiation of P19c16 into cardiomyocytes and production of GFP-labeled cardiomyocytes.Materials and Methods: At first, the P19c16 cells were cultured as monolayer and embryoid body; then oxytocin with 1×10-7 M concentration was added to culture media as the inducer agent. DMSO was used as an inducer agent in positive control under the same conditions. Differentiated cells underwent morphological and immunocytochemical evaluation. The research in its continuation pursued the transfection of pEGFP-C1 plasmid with P19c16 cells by electroporation method; then, stably expressed GFP cells were differentiated into cardiomyocytes by oxytocin.Results: The obtained data indicated that in the experimental group EBs from P19c16 cells could be differentiated into cardiomyocytes whereas monolayer cells could not. In the control group, both EBs and monolayer cells could be differentiated into cardiomyocytes by DMSO. In second part of this study, P19c16 cells were efficiently transfected with GFP, and GFP expressed cells were differentiated into beating cardiomyocytes by oxytocin. The results of morphological and immunocytochemical evaluation confirmed that differentiated cells were cardiomyocytes.Conclusions: Our results showed that the embryoid body formation is necessary for differentiation of P19c16 cells into cardiomyocytes and these cells after stable transfection with GFP could be differentiated into beating cardiomyocytes. It can be concluded that probably P19c16 line is a good stem cell line for cell transplantation in the experimental model of cardiac disease.

Background and Objectives: Hemophilia A is the most common X-linked blood coagulation disorder. The prevalence rate of this disease in various communities is about 1-2/10000 males. The prevalence of hemophilia A is very high in southern Khorasan population, perhaps due to their isolation and high rate of consanguinity. The aim of this research was to detect hemophilia A carriers in two villages of southern Khorasan and set a prenatal diagnosis program for these families.Materials and Methods: Blood samples of 34 patients with hemophilia A out of 51 family members (9 families) from two villages were collected. We were also able to collect samples from 7 mothers out of 9 families. Intragenic polymorphic sites in factor VIII gene including HindIII, Bc1I and AlwNI were used for carrier detection. DNA extraction and PCR-RELP were also performed.Results: The results revealed 3 heterozygote mothers for HindIII, 2 for Bc1I and 1 for AlwNI. We utilized these polymorphic sites for carrier analysis in these families. Ultimately, 4 carrier sisters of affected boys in this population were found; it was then possible to perform prenatal diagnosis procedure for their families.Conclusions: HindIII and Bc1I polymorphisms can be suitable markers for hemophilia prenatal diagnosis in these two villages.

Background and Objectives: Hemophilia B is an inherited recessive X-linked bleeding disorder caused by deficiency or defect of procoagulant factor IX (FIX). The factor IX gene spans 35kb of DNA and comprises of 8 exons. Mutations in the factor IX gene may result in deficient or defective coagulation factor IX causing the bleeding tendency known as hemophilia B. The aim of this study was to identify the causative mutations and genotype-phenotype correlation for mutations in some known patients with hemophilia B in Isfahan province.Materials and Methods: After informed consent was obtained, genomic DNAs of 24 hemophilia B patients referred to Omid hospital were extracted according to standard protocols. PCR amplification and single strand conformation polymorphism (SSCP) on nondenaturing polyacrylamid gel were performed separately on each sample for eight exons and exon-intron boundaries and promoter. The results of SSCP were compared to normal control and sequencing was performed for those with different migration patterns.Results: The sequencing results showed 70.8% missense mutation, 16.7% deletion, 8.3% nonsense mutation, and 4.2% insertion. Many of the mutations had occurred in exon 8; it came out to be similar to haemophilia B mutation database. Malmo polymorphism (Ala 148 Thr) was found in one family. Four novel mutations not previously reported in the database were also found.Conclusions: This study confirms the marked heterogeneity of factor IX mutations in the population. The results could be used to develop a national database and offer genetic counseling to families.

Background and Objectives: Human Immunodeficiency Virus type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS) in man and diagnosis of HIV-1 genome in suspicious specimens is of special importance. Viral transmission through blood and blood products during the window period is considered to be an important concern. In addition, the employment of rapid, sensitive and accurate techniques is highly necessary for diagnosis of HIV-1 prior to antibody production in infants born from infected mothers. In the present study, a sensitive and rapid RT-Nested PCR to detect viral genome has developed.Materials and Methods: In this study, a sensitive RT-Nested PCR technique for detection of a conserved HIV-1 "gag gene" sequence was developed. By using a specific primer pair, the fragment was amplified in two rounds of PCR. In order to confirm positive results, the developed technique was applied to standard Iranian panel as well as to positive specimens from different infection and disease categories in which reactivity was proven by confirmatory HIV-1 tests (ELISA and western blot).Results: Thirty five sera from different stages of the disease as well as 15 standard Iranian panels and 20 negative control sera were collected and tested by the developed technique. In positive cases, a specific band was observed on agarose gel electrophoresis, while no band could be detected for negative control sera.Conclusions: In this study, it was demonstrated that the developed HIV-1 RT-Nested PCR has a high sensitivity and specificity for diagnosis of HIV-1 infection. It has the advantage of viral genome detection prior to seroconversion and can be easily applied to detect HIV-1 infection during the window period.

Background and Objectives: The complications of blood donation are the first reasons why donors do not return for further blood donation attempts. This study was designed to determine the frequency of these complications and their associated risk factors among blood donors in Tehran. It also aimed to provide suitable methods to decrease the frequency of adverse reactions of blood donation, thus eliminating the most important causes of nonreturn, while ensuring the health of donors.Materials and Methods: This analytical descriptive cross-sectional study was performed on 554 blood donors who had donated blood from February 2004 through September 2005 in four fixed blood donation bases and four mobile blood collection buses. Each base was considered as a stratum, and a stratified random sampling proportional to size was done to select the donors.Results: Reported results showed donor reaction rate of 26%, with ecchymosis (22.7%), pain (8.5%), tenderness (5.6%), and hematoma (5.1%) as the most common. The frequency of donor complications has a significant statistical correlation with manner of needle entrance in vein, lack of change in needle position under skin, prolonged phlebotomy, incomplete phlebotomy, and hard work with hand within 12 hours after donation.Conclusions: Regarding the frequency values derived from different complications, it can be concluded that attention to these complications and their control can help encourage donors to become repeat donors preventing their lack of return for further blood donation.

Background and Objectives: For a successful prevention program, globin chain synthesis, as a complementary test beside DNA analysis, is necessary. So, it is important that each laboratory establishes its own reference range for the classification of thalassemia syndromes by globin chain synthesis. Globin chain synthesis is a relatively complex test introduced in the study of thalassemia syndromes as a reference method. The technique is also useful for variant chain identification. This study aims to stablish the method of globin chain synthesis to determine a/b chain ratio in healthy individuals.Materials and Methods: In this study globin chain analysis was performed on 30 healthy laboratory personnel's with normal HbA2 and normal hematological indices. In this method a reticulocyte-rich sample is incubated with a mixture of amino acids, one of which (leucine) radioactively labeled. After washing the excess radioactivity and precipitating the globin, different chains are separated by cation exchange chromatography.Results: The mean a/b ratio was 1.045±0.12 (mean±1SD) in healthy subjects. Our findings were in agreement with those of the other investigators in the world.Conclusions: In any screening program, diagnostic problems will arise that can not be solved without biosynthetic studies. The Clegg and Weather all method has been proved to be very reliable and reproducible, but time-consuming (requiring four days). New methods like reverse phase HPLC are now available for chain separation. Therefore, according to the procedures each laboratory should determine its own reference range.

Background and Objectives: In-vitro differentiation of embryonic stem cells to different cell types provides a valuable tool to study the interaction between these cells and growth factor. Recent investigation showed that recognition of the factors and their influence on the hematopoiesis is important. The aim of this research was to study the effect of different dose of EPO and BMP4 on the differentiation of embryonic stem cells; the number and size of total colonies and benzidine positive colonies were also evaluated.Materials and Methods: 4-day-old EBs were trypsinized and dissociated to single cells. These cells were cultured on the semisolid medium in two groups with different doses of EPO (10, 20, 40, 80, 160ng/ml) and BMP4 (5, 10, 20, 40, 80, 160ng/ml). The colonies were evaluated after 10 days and stained with benzidine and giemsa.Results: The size of colonies in all concentrations of EPO and BMP4 faced an increase showing a significant difference with the control group (PI 0.05). Total number of colonies had a negative correlation with the dose of BMP4 but there was not any significant difference between benzidine positive colonies with the control group. The number and percentage of benzidine positive colonies had significant difference with the control group in all doses of EPO. 20ng/ml of EPO contrary to other concentrations of EPO caused increase in benzidine positive colonies.Conclusions: Our results showed that EPO had better effect on ES cells elytroid colony differentiation than BMP4. It also indicated that 20ng/ml of EPO was the best concentration.

Background and Objectives: Umbilical cord blood (UCB) contains a high number of primitive progenitor cells for transplantation. However, the rate of UCB CD34+ stem cell engraftment is low. In this study we examined the effect of human MSCs (mesenchymal stem cell) on engraftment of human UCB-derived CD34+ cells in irradiated Balb/c mice.Materials and Methods: Human UCB CD34+ cells were obtained from full-term normal deliveries by immunomagnetic techniques and MSCs were isolated by a standard methodology from bone marrow aspirates. Isolated MSCs were evaluated for cell-surface antigens of CD 166 and CD 105 by flowcytometry. The absolute count of CD34+ UCB stem cells was also determined by flowcytometry. Irradiated (7Gy) Balb/c mice were transplanted intravenously with 0.2 to 1.0×106 human UCB CD34+ cells in the presence or absence of 0.25 to 1×106 human bone marrow-derived MSCs. The mice in every group on day 11 after transplantation were killed and their spleen dissected. In every group, colony assay and H and E staining were performed. To make sure of the presence of human stem cells in spleen colonies, UCB CD34+ cells were labeled with super paramagnetic iron oxide (SPIO) before transplantation. The colonies in spleen then underwent Prussian blue staining. In the statistical analyis, Kruskal-Wallis test using SPSS software was employed to determine the number of colonies.Results; Flowcytometry assay showed that after purification, 90% of the cells were CD34+ and 96% marker positive for MSCs. Viablity of cells was 100%. Cotransplantation of low doses of UCB CD34+ cells (0.2 and 0.3×106) and MSC (0.5 and 1×106) resulted in a significant increase of the number of colony forming units spleen, in comparison with the engraftment of UCB CD34+ stem cells without MSC after 11 days (p<0.01). Prussian blue staining showed the Fe+2 granules in UCB stem cells. This indicated that cells in the colonies were engrafted UCB CD34+ stem cells.Conclusions: The results showed that cotransplantation of MSC with UCB CD34+ cells promote engraftment of UCB CD34+ cells.