فصلنامه خون
سال پنجم شماره 2 (پیاپی 19، تابستان 1387)

Background and objectivesThrombophilia is a common and dangerous disease. It may be heritable or acquired. A substitution in the 3' untranslated region of prothrombin gene (PRT-G20210A) has been reported to be related to thrombophilia in Caucasians, but this relationship remains in debate in other populations. The hetrozygote form of PRT G20210A gene variant expresses prothrombin 25% higher than the average level. This study was performed to evaluate the prevalence of G20210A gene mutationas in thrombotic patients. Materials and MethodsIn this descriptive and cross sectional study, 299 patients with venous thromboembolism were selected. The subjects consisted of 116 male and 183 female. Traditional risk factors for venous thrombosis and pulmonary thromboembolism such as proteins C, S, ATIII deficiency and APC-resistance were investigated as well. ResultsWe found that only 1.7% of patients with venous thromboembolism were carrier of prothrombin G20210A mutation which is one of the prevalent point mutations in Caucasians patients (18%). The homozygote form of PRT G20210A was not found. ConclusionsProthrombin G20210A mutations in Iranian population as opposed to western populations are rare (1.7%) with APC-resistance being common in the former.

Background and objectivesHemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia has been found to be associated with a variety of single point mutations encompassing a 40-nucleotide region in factor IX promoter region. Mutations in factor IX gene promoter though relatively rare (about 2% of total) are important because they can give rise to the unique hemophilia B Leyden phenotype. Materials and MethodsOur objective was to study mutations in exon-1 in 4 3 Iranian hemophilia B patients to recognize possible cases of hemophilia B Leyden. Exon-1 of factor IX gene was amplified by PCR; then, conformational sensitive gel electrophoresis (CSGE) was used to distinguish cases having mutations in this region. ResultsTwo cases showed band shifts on CSGE. Exon-1 of the patients was directly sequenced. We found two different mutations in exon-1: the A/T mutation at +6 and the A/G mutation at +13. ConclusionsThe prevalence of hemophilia B leyden in B hemophilia patients (4.6%) in our results shows a higher frequency rate in Iran compared to that of other reported countries.

Background and objectivesIntravenous immunoglobulin (IVIG) preparations are used in several disorders including primary and secondary immunodeficiencies, autoimmune and systemic inflammatory diseases; it is also applied in effective therapy of infectious diseases. IVIG is currently the most widely used plasma component in the world. The addition of multiple steps to the manufacturing process of IVIG lowers the yield of IgG and raises the manufacturing costs. Therefore, manufacturers attempt to employ a cost effective method with respect to safety and quality of the product. In this study we proposed a method which not only reduced the manufacturing steps but also raised product safety by the treatment of pasteurization as a virus inactivation method. Materials and MethodsFor this experimental study, the fraction II paste (rich in IgG) was obtained from fresh frozen plasma (FFP) by the modified cold ethanol fractionation method (Cohn’s method). For further purification, filtration was used to remove impurities. Before performing virus inactivation by pasteurization, protein solution was diafiltered and then the stabilizer was added. Pasteurized solution was diafiltered again and final product was prepared after sterile filtration. ResultsThe quality control results of the finished product obtained by the proposed method revealed that the purity was 100% (determined by cellulose acetate electrophoresis) and the polymer amount was less than 1% (evaluated by HPLC chromatography). The secondary and tertiary structures of IgG molecule were also examined by circular dichroism spectroscopy technique and were then compared to the commercial IVIG products bringing about approximately similar and satisfactory results. The yield calculated for the initial amount of IgG of plasma was 39.1% as measured by nephelometery. ConclusionsThe high purity of the finished product confirmed that the proposed purification process was satisfactory in despite of fewer steps when compared to the current methods. Indeed, the cost- effective preparation together with quatily and safety are taken into account in the proposed method. If the complementary experiments are carried out, the proposed method can escalate at the industry scale.

Background and objectivesEmbryonic stem cells are potential pluripotent stem cells derived from inner cell mass of embryonic blastocyst stage. So far, differentiation of embryonic stem cells to megakaryocyte lineage has resulted from coculture with bone marrow stromal cells with disadvantages such as possibility of stem cell contamination from feeder layer and secretion of unknown factors from this layer ending in unwanted differentiation and genetic changes in embryonic stem cells. In this study, while excluding feeder layer, we differentiated murine bone marrow cells to megakaryocyte lineage. Materials and MethodsIn this experimental study, embryoid bodies resulted from R1 murine embryonic stem cells. The embryoid bodies were then cultured in two groups of test (containing IMDM with FBS, L-glutamin, non-essential aminoacids, beta mercaptoethanol, and TPO and IL-3 as growth factors) and control (the same culture medium used in test group without growth factors). After four days the colony assay test was performed, and after eight days immunohistochemistry staining for CD41 molecule, and finaly transcription test for PF4 gene using RT-PCR to demonstrate differentiation to megakaryocyte lineage. ResultsResults of the colony assay test indicated that the cells considered had the capacity to give rise to colonies. The total number of colonies and banzidine positive colonies were 57 ± 6.1 and 18 ± 3.6 respectively. On the other hand, immunocytochemistry analysis and PCR showed that the CD41 molecule and PF4 gene are expressed in differentiated cells. ConclusionsOur results indicated that murine embryonic stem cells differentiate into megakaryocyte lineage cells without utilizing feeder layer, and embryonic stem cells can be used as an immortal new source for producing megakaryocyte lineage cells on which basic biological research can be conducted.

Background and objectivesThalassemic patients need regular blood transfusion because of chronic nature of disease. Since just major blood groups are controlled in compatibility tests, formation of antibody against minor blood groups (irregular antibodies) can cause transfusion reactions (delayed hemolytic reaction) and may produce complications for patients. In this study, we decided to perform screening and typing of antibodies with gel method and tried to substitute this modern method for routine tests. Materials and MethodsIn a descriptive study 441 thalassemia patients having referred to two thalassemic centers in Tehran and Ghazvin were studied. Data forms about blood transfusion status were filled out for all patients from whom blood samples were then taken. Screening antibody test with tube method was performed for all patients followed by assessment with gel method according to its standard pattern and respective program. Antibody identification test was performed according to the respective program as well. Results of obtained data from forms filled out by patients were assessed with SPSS 11.5 software; chi-square test was done with 95 % confidence interval. Results441 patients consisting of 234 males (53.1%) and 207 females (46.9 %) with the mean age of 22.6 years (SD ± 9.27) participated in the study. Out of the total number of subjects, there are 362 thalassemia major (82.1%) and 79 thalassemia intermedia (17.9%). Screening showed 391 patients (88.7%) without versus 50 (11.3) with alloantibodies. From among identified antibodies, anti-Kell and anti-D were present in 28% and 16% of cases respectively; antibodies to other Rh subgroups including c, C, e, E and Cw appeared in 26% of cases. Comparing results of microtube gel method with tube method for screening shows that 26 patients recognized as positive in gel method were negative in tube method. ConclusionsRegarding the alloimmunization frequency in thalassemic patients, it is suggested that RBC phenotype especially subgroups of Rh, Kell, Kidd, and Duffy be checked (in addition to ABO Rh(D) group) for every newly diagnosed thalassemia patient; it is further recommended that in every crossmatch for blood transfusion. Blood bag be selected based on Rh subgroups and Kell in conformity with patient phenotype; this would decrease alloimmunization by 90%.

Background and objectivesA specific chromosomal abnormality, the Philadelphia chromosome (Ph), is present in more than 90% of CML patients. The aberration results from a reciprocal translocation between chromosome 9 and 22, creating a BCR-ABL fusion gene. Major forms of BCR-ABL fusion are b3a2, b2a2, and e1a2. Less common fusion genes are b3a3 or b2a3 (p203) and el9a2 (p230). The aim of this study was to set up a multiplex RT-PCR assay and determine the frequency of different fusion genes in 75 Iranian patients with CML. Materials and MethodsIn this experimental study, peripheral blood samples from 75 adult Iranian CML patients were analyzed by multiplex RT-PCR to detect different types of BCR-ABL transcripts of the t(9:22). ResultsAll of the examined cases were positive for some type of BCR/ABL rearrangement. The majority of patients (82.6%) expressed one of the P210 BCR-ABL transcripts (b3a2, 62% and b2a2, 20%), while the remaining showed either one of the transcripts of b3a3, b2a3, ela2 or coexpression of b3a2 and b2a2. The rate of coexpression of the b3a2 and b2a2 was 5%. ConclusionsIt is possible to detect rare fusions other than common ones through multiplex method. In Iranian CML patients, b3a2 was three times more frequent than b2a2 shown to be higher as compared with other studies. Probably, the kind of fusion is of clinical importance and helps us in understanding t(9:22) leukemic cells pathogenesis.

Background and objectivesAcute lymphoblastic leukemia though the most frequent malignancy in children has well responded to medical treatment as compared with other types of malignancies in recent years. In addition to age, sex and number of white blood cells, genetic survey has been one of the prognostic determinants. This research has been carried out on children diagnosed after being referred to Mofid Children Hospital during a period of three years. This study aims at determining the factors effective on prognosis of lymphoblastic leukemia with an emphasis on patient acute karyotype. Materials and MethodsOut of 102 patients with the diagnosis of acute lymphoblastic leukemia being referred to this hospital between 1990-2002, 74 (within the age groups varying from 6 months to 13 years old) underwent cytogenetic test; out of the latter 28 were excluded. These 74 were classified according to their WBC count and age into high (42 cases) and low risk groups (32 cases). Then, the results were analyzed and compared according to the subfactors such as flowcytometry, age, sex, and blood cell count (WBC, hemoglobin and platelet) both in high and low risk groups. ResultsFrom 74 cases under study, flowcytometry showed that 64 (86.5%) suffered from leukemia type pre B cell; out of the latter 38 (59.4%) based on the number of white cells were placed in the low-risk group. In cytogenetic survey, 34 (45.9%) patients had normal karyotype, 16 (21.6%) had abnormality of chromosome number, and 14 (19%) abnormality of chromosomal structure. No conclusion could be drawn from cytogenetic test conducted on the remaining 10 cases. Comparison of the two groups of high and low risk showed no significant differences. From gender point of view, number of boys was considerably higher than girls in the low risk group (61.9% vs. 39.1%). But as far as karyotype was concerned, there were no differences between the two groups. ConclusionsRegarding the research findings, there was no significant difference between the two groups from genetic disorders point of view. Accordingly, we can conclude that age and number of WBC by themselves are not suitable preliminary factors for prognosis and genetic determination in genes domain. So, it seems necessary for a similar research at a larger scale to be conducted in which prognostic determinants are complied with disease trend.

Background and objectivesTesting for hemoglobin is important in detection and prevention of anemia before and after blood donation respectively. Thus, hemoglobin screening test should have high sensitivity and accuracy for selection of blood donors.The present study aimed at comparative evaluation in measurement of hemoglobin by hemoglobin color scale (HCS) and reference method. Materials and MethodsIn this descriptive study, 144 female donors (the mean age of 33 ± 11) tested by Hb strips were found to be eligible for donation. In addition to the samples taken for Hb test, the second series of blood samples were also drawn from donors and were added to EDTA-coated tubes for measurement of hemoglobin by automated cell blood counter (Sysmex K-800) as a complementary measurement. The results obtained by the two methods were entered in SPSS statistical program (version 11.5) and analyzed statistically using Wilcoxon and Spearman correlation index. ResultsStatistically remarkable differences were observed between the mean of Hb results by cell blood counter (12.8 gr/dl) and the mean obtained by Hb strip color scale (13.2 gr/dl) in female blood donors. So the median rates of Hb tested by Hb color scale and cell blood counter were 14 gr/dl and 12.8gr/dl, respectively. Pearson correlation index showed no significant correlation between the two methods of measurement. ConclusionsResults showed that there was a remarkable difference in hemoglobin levels as measured by the two methods. Therefore, the use of Hb color scale for blood donor screening may fail to detect anemic donors or those with potentiality for post-donation anemia. It is then recommended that donors with Hb>13 gr/dl in Hb color scale be retested by more precise and sensitive methods including Hemo Cue or cell blood counter.

Background and objectivesStorage, distribution, and appropriate use of blood and blood components require a great deal of supervision and control. Higher than needed blood orders not only adversely affect blood quality but also impose extra expenses on treatment center and patients. The aim of this study was to study the rates of blood order and use in different wards of Yahyanejad Hospital. Materials and MethodsThis was a descriptive study. Blood orders issued within 3 months for 1042 patients hospitalized in 14 wards of Yahyanedjad Hospital were reviewed. The data were then analyzed by the use of SPSS 11.5. ResultsOut of 1042 patients whose respective blood orders were reviewed, 599 (57.5%) were female and 243 (42.55%) male. Out of 724 patients experiencing blood administration, 361 (49.9%) were female and 363 (50.1%) male. The overall ratio of C/T and TI index were 2.01 and 0.86 respectively that are considered to be optimal as compared with the standard figures of C/T<2.5 and TI ³ 0.5. ConclusionsThe present findings show that adherence to appropriate principles for blood order can considerably reduce the rate of redundant orders, blood inadequacy, pressure upon blood centers, length of blood storage period, blood loss, and transmission of transfusion transmitted infections.