Establishing Blood Group Genotyping to Resolve ABO Discrepancies in Iran.

ABO discrepancies are recognized when the reactions obtained in the forward type do not "match" the reactions obtained in the reverse type. Discrepant results are often caused by a variant ABO gene. Molecular analysis is required to confirm the type of subgroups and discrepancy. In this study ABO genotyping was performed on a series of blood donors and patients to determine their definite blood groups. We examined 100 samples with ABO discrepancies from blood donors and patients referred to Tehran Blood Transfusion Center between October 2015 and August 2016. ABO genotyping was performed on all samples with allele specific PCR for differentiation of A, B and O alleles. Exon 6 and 7 of ABO gene were sequenced to confirm the results. The genotyping of donor/patients samples with discrepant results of ABO blood typing consisted of 61 cases of A2 and A2B, 3 cases of B 302 and 4 cases of Aw06. Genotyping of 6 samples that had extra antibody in their serum (AB blood group) confirmed the cell type reaction results. 6 samples that had shown a very weak reaction with anti-AB (similar to O blood group) and had no anti-A in their serum were genotyped as O 1 O 2. Blood group genotyping laboratory provides an efficient service for evaluation of ABO discrepancies and resolve the problems encountered in serology reactions.

Expression analysis of microRNA-125 in patients with polycythemia vera and essential thrombocythemia and correlation with JAK2 allele burden and laboratory findings.

INTRODUCTION: The JAK2V617F mutation has emerged in recent years as a diagnostic as well as a treatment target in patients with polycythemia vera (PV) and essential thrombocythemia (ET). The disease phenotype is also influenced by other factors such as microRNA (miRNA) deregulation. The aim of this study was to investigate miR-125 expression level in these patients with those obtained from healthy control subjects and its correlation with JAK2 allele burden and laboratory findings. METHODS: In total, forty patients with a clinical diagnosis of PV and ET were examined at the time of diagnosis. Ten healthy subjects were checked as controls. We performed JAK2 V617F allele burdens measurement and expression analysis of miR-125b-5p, miR-125b-3p, miR-125a-5p, and miR-125a-3p in leukocytes isolated from peripheral blood by quantitative real-time polymerase chain reaction. RESULTS: MiR-125b-5p and miR-125a-5p were upregulated in both patients with PV (P = 0.00 and P = 0.003, respectively) and ET (P = 0.02 and P = 0.002, respectively). In PV group, a significant correlation was observed between miR-125a-5p and platelet counts (P = 0.01, r = 0.531). The correlation between miRNA and JAk2 allele burden was not significant. CONCLUSION: In conclusion, our data indicate that other factors such as aberrant miR-125 expression may influence on the disease phenotype in patients with PV and ET.

Expression pattern of key microRNAs in patients with newly diagnosed chronic myeloid leukemia in chronic phase.

INTRODUCTION: Chronic myeloid leukemia (CML) is caused by reciprocal translocation in hematopoietic stem cells (HSCs). This translocation forms the BCR-ABL1 oncogene, which alters several signaling pathways that control malignancy. CML has three phases: chronic, accelerated, and blast crisis. The microRNAs (miRNAs or miRs) are noncoding RNAs that downregulate their target gene by targeting 3' UTR of mRNA or through translational inhibition. It has been shown that miRNAs regulate many biological processes, and dysregulation of these regulatory RNAs is involved in disease development, particularly in cancer. The important role of miRNAs as therapeutic agents and biomarkers has been demonstrated in CML patients at different phases of the disease. METHODS: Stem-loop reverse transcription polymerase chain reaction was used to characterize differentially expressed miRNAs of leukocytes in the peripheral blood of 50 newly diagnosed CML patients in chronic phase. RESULTS: Some onco-miRNAs were found to be downregulated (miR-155 and miR-106), and some tumor suppressor miRs (miR-16-1, miR-15a, miR-101, miR-568) were upregulated. CONCLUSION: These results show that very few miRNAs alone would be good candidates for CML diagnosis independently of conflicting results, but together could be an additional tool for CML diagnosis. Moreover, miRNAs might be good candidates for prognosis prediction and CML therapy.

A comparison between the efficacy of Bio-Oss, hydroxyapatite tricalcium phosphate and combination of mesenchymal stem cells in inducing bone regeneration.

BACKGROUND: Recently, tissue engineering has been introduced as a regenerative treatment for bone defects. There is some evidence showing bone regeneration from mesenchymal stem cells (MSC) loaded on hydroxyapatite ß-tricalcium phosphate (HA/TCP) as a scaffold in large defects. This study aimed to compare the quality and quantity of regenerated bone using Bio-Oss, HA/TCP and MSC loaded HA/TCP scaffolds. METHODS: Mesenchymal stem cells were aspirated from iliac crest bone marrow after extracting the first, second and third premolars and the first molar in five mature hybrid dogs. The cells were cultured and their osteogenic differentiation potential was evaluated after the third cell passage using Alizarin red staining in experimental conditions. The HA/TCP scaffold (3 × 3 × 3 mm) was loaded with undifferentiated mesenchymal stem cells. Bilateral bone defects were then prepared in the jaws using trephine burs. The defects were randomly filled with HA/TCP, Bio-Oss, or HA/TCP + MSCs. One defect served as a control and was left as an empty cavity. All defects except the control defect were covered with an absorbable membrane. Histological and histomorphometric evaluations were conducted after 6 weeks and data were subjected to analysis of variance (ANOVA) (p < 0.05). RESULTS: The empty cavity demonstrated more bone formation (60.80%) than the HA/TCP (44.93%) and Bio-Oss (40.60%) (p < 0.05) groups. However, the difference from the HA/TCP + MSCs group was not significant (46.38%) (p > 0.05). CONCLUSION: An MSC-loaded HA/TCP scaffold is a more effective alternative than Bio-OSS or HA/TCP in inducing bone regeneration.

Production and characterization of liquid-stored and lyophilized reconstituted human infusible platelet membranes.

INTRODUCTION: During recent years, the need for platelet concentrate (PC) has increased. Infusible platelet membranes (IPM) have been developed as an alternative to standard PCs, with the additional advantage of long shelf-life and increased viral safety. In this study, IPM construction and the morphological and biological features of these microvesicles were surveyed to determine their binding capacities in vitro. METHODS: Thirty-five PC units prepared by the Iranian Blood Transfusion Organization were used to produce IPM. Platelets were lysed by repeated cycles of freezing and thawing, virally inactivated with wet heat in the presence of different concentrations of sodium octanoate as a heat stabilizer, and then sonicated. IPM were separated, kept at 4°C or lyophilized, and examined for binding to collagen and von Willebrand factor (VWF). RESULTS: IPM retained the binding capacity for collagen and VWF, and the extent of VWF binding was dependent on the concentration of the heat stabilizer. Additionally, a higher binding capacity was demonstrated for liquid-stored compared with lyophilized IPM. CONCLUSION: This study revealed the potential of IPM microvesicles to mimic the binding features of platelets in vitro.

Functional hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel 3-dimensional biocompatible nanofibrous scaffold.

AIM: To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. METHODS: After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. RESULTS: Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and alfa-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05). CONCLUSION: Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like cells.

OBJECTIVES: The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes. METHODS: Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments. RESULTS: Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10(9) platelets/ml) was about threefold greater than that of FBS (P < 0.001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and alpha-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS (P < 0.001). CONCLUSION: Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.

The beta-thalassemia mutation spectrum in the Iranian population.

Beta-thalassemia is the most common hereditary disease in Iran. More than two million carriers of beta-thalassemia live in Iran. Since the Iranian population is a mixture of different ethnic groups, it is necessary to determine the frequency and distribution of mutations in the different parts of the country. For this purpose, we divided Iran in to eight different regions according to the geographic and ethnic distribution of the population. Over a 10-year period 1,217 beta-thalassemia chromosomes of 164 affected patients and 889 unrelated carriers were studied using the amplification refractory mutation system-polymerase chain reaction technique. We detected 81% beta-thalassemia mutations in the studied chromosomes. IVS-II-I (G --> A) was the predominant mutation found in our study (34%). Its relative frequency in the north was much higher than other regions, and it lessened toward the south, where the IVS-I-5 (G --> C) mutation was more common. IVS-I-5 (G --> C) (7.55%), codons 8/9 (+ G) (4.76%), and IVS-I-110 (G --> A) (4.76%) were the other most common mutations. The results presented here can be used as a basis of prenatal diagnosis of beta-thalassemia in different regions of Iran.