The Scientific Journal of Iranian Blood Transfusion
Year:2007 | Volume:4 | Issue:3 (15)|Papers Count:17

Background and Objectives: Serologic methods used in HPA–typing are limited due to restricted access to specific antisera and decreased platelet count in thrombocytopenic patients. Therefore, several DNA-based HPA-genotyping techniques were used to determine the genotype of HPAs. Since nothing is known about the HPA gene frequency in Iran, this study was performed to determine its frequency in some Iranian blood donors.Materials and Methods: DNA was extracted from a 3-ml whole blood sample prepared from donations of 100 Iranian blood donors collected in EDTA-coated blood tubes. Human platelet (PLT) alloantigen (HPA)-1/2/3/4/5 and HPA-15 typing were performed by the Polymerase Chain Reaction – Sequence Specific Primer technique (PCR-SSP), and HPA-1a phenotyping was performed by ELISA method for 40 % of samples.Results: The frequencies of HPA genes were: HPA-1a 98% , HPA-1b 2% , HPA-2a 54% , HPA-2b 46% , HPA-3a 48% , HPA-3b 52% , HPA-4a 100% , HPA-5a 99% , HPA-5b 1% , HPA-15a 47% , and HPA-15b 53%. HPA-4b was not found. The frequencies of HPA phenotypes were determined to be: HPA1a/1a 96% , HPA1a/1b 4% , HPA2a/2a 8% , HPA2a/2b 92% , HPA3a/3a 19% , HPA3a/3b 59% , HPA3b/3b 22%, HPA4a/4a 100% , HPA5a/5a 98% , HPA5b/5b 2% , HPA15a/15a 14% , HPA15a/15b 67% , and HPA15b/15b 19%. 40 HPA-1a phenotyping by ELISA showed 26 positive (OD > 0.5), 4 negative (OD < 0.3), and 10 indeterminate samples (0.3 < OD < 0.5).Conclusions: No HPA-1b/b homozygous genotype similar to other Asian studies was found. Since HPA-1, - 2,-5 frequencies in the population under study differ from the European Caucasian race, it seems that antibody production in our population might be different from other Caucasians. According to HPA frequencies, it seems that HPA-2b, HPA-5b and HPA-15b may induce post transfusion purpura and platelet refractoriness which need further investigation.

Background and Objectives: Serologic methods used in HPA-typing are limited due to restricted access to specific antisera and decreased platelet count in thrombocytopenic patients. Therefore, several DNA-based HPA-genotyping techniques were used to determine the genotype of HPAs. Since nothing is known about the HPA gene frequency in Iran, this study was performed to determine its frequency in some Iranian blood donors. Materials and Methods: DNA was extracted from a 3-ml whole blood sample prepared from donations of 100 Iranian blood donors collected in EDTA-coated blood tubes. Human platelet (PLT) alloantigens (HPA)-1/2/3/4/5 and HPA-I5 typing were performed by the Polymerase Chain Reaction -Sequence Specific Primer technique (PCR-SSP), and HPA-la phenotyping was performed by ELISA method for 40 % of samples. Results: The frequencies of HPA genes were: HPA-1a 98% , HPA-1b 2%, HPA-2a 54%, HPA-2b 46%, HPA-3a 48%, HPA-3b 52%, HPA-4a 100%, HPA-5a 99%, HPA-5b 1%, HPA-15a 47% , and HPA-15b 53%. HPA-4b was not found. The frequencies of HPA phenotypes were determined to be: HPA1a/1a 96%, HPA1a/1b 4%, HPA2a/2a 8%, HPA2a/2b 92%, HPA3a/3a 19%, HPA3a/3b 59%, HPA3b/3b 22%, HPA4a/4a 100%, HPA5a/5a 98%, HPA5b/5b 2%, HPAl5a/l5a 14%, HPA15a/15b 67%, and HPA15b/15b 19%.40 HPA-1a phenotyping by ELISA showed 26 positive (OD> 0.5 ), 4 negative (OD < 0.3 ), and 10 indeterminate samples (0.3 < OD < 0.5). Conclusions: No HPA-I bib homozygous genotype similar to other Asian studies was found. Since HPA-l,- 2,-5 frequencies in the population under study differ from the European Caucasian race, it seems that antibody production in our population might be different from other Caucasians. According to HPA frequencies, it seems that HPA-2b, HPA-5b and HPA-15b may induce post transfusion purpura and platelet retractoriness which need further investigation.

Background and Objectives: GVHD occurs in 20–40% of recipients of HLA-matched sibling donor grafts. A number of studies suggest that polymorphism in cytokine genes influence susceptibility to post-BMT complications. One of the cytokine gene families (IL-1RN) encodes IL-1 receptor antagonist (IL-1Ra), an anti-inflammatory molecule that competes for receptor binding with IL-1a and IL-1b. The overall contribution of IL-1 to the proinflammatory response depends on the balance between these three molecules. In this study, we decided to evaluate the polymorphisms of IL-1Ra gene that might influence the outcome of BMT.Materials and Methods: In this cohort study, patients' (n=175) and donors' (n=175) DNA from a total of 350 human leucocyte antigen (HLA)-identical sibling allogeneic BMTs were analyzed. IL-1Ra gene polymorphism was studied by PCR-VNTR method. PCR products were then visualized by electrophoresis in 2% agarose gel. Then, the correlation between donor and recipient genotype and GVHD grade for their respective transplant was assessed.Results: The frequency of the aGVHD grades was divided to grade 0 (38.3%), grade I (17.7%), grade II (27.4%), grade III (13.7%), grade IV (2.9%). Correlation between donor and recipient genotype and GVHD grade for their respective transplant was assessed. We observed no significant correlation between the IL-1Ra polymorphism and incidence of aGVHD, although possession of the allele 2 in the individual genotype was associated with less severe acute GVHD.Conclusions: The observed allele frequencies were different from those previously reported. None of the polymorphisms showed association with the presence of acute GVHD. However, presence of allele 2 showed association with aGVHD. If the recipient possesed allele 2, the probability of aGVHD was 46% and in the absence of allele 2, it was 62%. Recipient age, donor age, recipient disease particularly thalassemia were the most remarkable risk factors for acute GVHD.

Background and Objectives: GVHD occurs in 20--40% of recipients of HLA-matched sibling donor grafts. A number of studies suggest that polymorphism in cytokine genes influence susceptibility to post-BMT complications. One of the cytokine gene family (IL-IRN) encodes IL-l receptor antagonist (IL-IRa), an anti-inflammatory molecule that competes for receptor binding with IL-la and IL-I~. The overall contribution of IL-1 to the proinflammatory response depends on the balance between these three molecules. In this study, we decided to evaluate the polymorphisms of IL-1Ra gene that might influence the outcome of BMT. Materials and Methods: In this cohort study, patients' (n = 175) and donors' (n = 175) DNA from a total of 350 human leucocyte antigen (HLA)-identical sibling allogeneic BMTs were analysed. IL-1Ra gene polymorphism was studied by PCR-VNTR method. PCR products were then visualized by electrophoresis in 2% agarose gel. Then, the correlation between donor and recipient genotype and GVHD grade for their respective transplant was assessed. Results:  The frequency of the aGVHD grades was divided to grade 0 (38.3%), grade I (17.7%), grade II (27.4%), grade III (13.7%), grade IV (2.9%). Correlation between donor and recipient genotype and GVHD grade for their respective transplant was assessed. We observed no significant correlation between the IL-1Ra polymorphism and incidence of aGVHD, although possession of the allele 2 in the individual genotype was associated with less severe acute GVHD. Conclusions: The observed allele frequencies were different from those previously reported. None of the polymorphisms showed association with the presence of acute GVHD. However, presence of allele 2 showed association with aGVHD. If the recipient possesed allele 2, the probability of aGVHD was 46% and in the absence of allele 2, it was 62%. Recipient age, donor age, recipient disease particularly thalassemia were the most remarkable risk factors for acute GVHD.

Background and Objectives: Thrombotic events are among the most important complications in patients with betathalassemia major. Alterations in natural anticoagulation system have a major contribution to the occurrence of thrombotic events in these patients. This study was performed to evaluate the protein C, protein S and Antithrombin III activity in patients with beta-thalassemia major having referred to Mofid Children Hospital in 1382.Materials and Methods: Thalassemic children older than 2 years of age, with the interval of more than 25 days since last transfusion, and without any evidence of symptomatic liver or heart disease were enrolled in the study. Plasma activity of protein C and protein S was assessed by PTT based tests and the activity of antithrombin III was determined by chromogenic method. The correlation between decreased levels of anticoagulants and other parameters such as splenectomy, liver transaminases and serum ferritin level was analyzed by spss software.T-test and Mann whitney U-test were used to compare the mean values of variables.Results: 20.3% of patients showed a decrease in protein C and 15.3% in protein S. 12 patients had been splenectomized. Mean protein C activity in splenectomized and non-splenectomized patients were 63.4±17.9% and 74.5±15.1%, respectively (p<0.04).Conclusions: There was a decrease in proteins C and S activity in thalassemia major patients. Splenectomy might cause reduction in protein C level. Analytic studies are recommended to evaluate the activity of anticoagulant system in thalassemic patients. In addition, assessment of this system is suggested prior and after splenectomy.

Background and Objectives: The incidence and severity of Graft Versus Host Disease following the use of umbilical cord blood as a source of stem cells for bone marrow reconstitution challenge the scientific findings of the immunocompetence of newborn immune cells. The reports show that self renewal characteristics and the proliferative capacity of primitive hematopoietic progenitors in the preterm cord blood are higher in comparison with term cord blood and bone marrow. In this study, the characteristics of preterm cord blood immune cells were analyzed from a naive point of view especially in comparison with its counterparts in term cord blood.Materials and Methods: Term and pretem MNCs were isolated and cultivated in complete media containing PMA (50 ng/ml) and Ionomycine (1mg/ml) at the presence of monensin; they were then permeabilized with %0.1 saponin. After cell stimulation with PMA and Ionomycine, staining was performed with Moab anti-CD69 antibody to estimate the level of activation and was also conjugated with anti- IL-10, IFN-g, IL-4, IL-2 antibody to evaluate the production of cytokine. Mean percentage frequency of cytokine producing cells and the level of cytokine expression in CD4+/CD8+, CD45RA+/RO+ cells were analyzed by Epics-XL and IMMUNO-4 software. Statistical analysis was carried out using Kolmogrov-Smirnov and Student's t-test.Results: Out of 7700 samples of platelet concentrates studied, 14 (0.18%) were found positive for bacterial contamination. The contamination rate was estimated to be one in every 550 tested platelets (0.18%). Cellular phenotypic analysis showed no significant differences in CD4+CD45RA+, CD8+CD45RO+ and CD4+CD45RO+ cells in term and preterm cord blood (p<0.05). Mean percentage of CD3+, CD4+, CD8+, HLA-DR+CD3+, DR-HLA+CD4+ and CD25+ cells had significant increment in term cord blood. No statistically significant differences in the level of expression and the frequency of cytokine producing cells were observed in distinct gestational ages.Conclusions: Considering the lack of any significant functional differences between term and preterm cord blood immune cells, hematopoietic stem cells of preterm cord blood not only have the same immunological behavior, especially with respect to GVHD, but also have the higher frequency and proliferative capacity. The presence of immature progenitor cells may have priority to term cord blood and be applied in transplantation settings.

Background and Objectives: Major Histocompatibility Complex (MHC) is the most polymorphic system in the genome of different species. In human beings, these genes named HLA are located on the chromosome 6. HLA class I and II undertake genetic control of the immune system. Identification of HLA alleles is useful in transplantation, disease, and anthropological studies. Among different antigens of HLA, DR (DRB1) antigens are the most variable. In this research, due to the importance of DRB1 antigens in bone marrow transplantation, these antigens were studied in normal population. This study was performed on different Iranian races and did not suffice to a specific group.Materials and Methods: DNA was extracted from the whole blood sample of 466 normal individuals after randomized sampling. Some HLA-DRB locus segments were amplified using 23 primer pairs by using PCR-SSP method. Finaly, PCR products were evaluated by electrophoresis in 2% agarose gel.Results: The most prevalent alleles in DRB1 locus in normal poulation of Iran were DRB1*11, DRB1*13, DRB1*15, and DRB1*04 (20%, 11.4%, 11.4%, 10%, respectively). Whereas DRB1* 09 was the least frequent allele.Conclusions: This research showed genetic diversity of HLA DRB1 in the mixed Iranian population. The data suggest that the Iranian population share certain HLA class II genetic components with the populations residing in Russia and Eastern and Southern European countries.

Background and Objectives: Platelets have a major role in haemostasis. Platelet concentrates are used in thrombocytopeny and platelet dysfunction disorders. Due to recent studies reporting loss of in vivo function of platelets in platelet concentrate units, we determined the level of surface expression of PSelectin, conducted platelet aggregation tests and pH measurment to evaluate platelet activation during storage.Materials and Methods: In a cross-sectional study, 100 platelet concentrate units prepared by PRP method were evaluated. 34, 33 and 33 units were taken from the first, second, and third day of storage respectively. For each platelet concentrate unit, pH, CD62P-Selectin and platelet aggregation tests with Arashidonic acid and Ristocetin agonists were conducted.Results: Expression of P-Selectin in third day was significantly higher than first day (p<0.05). Platelet aggregation tests showed significant decrease in third day compared to first day (p<0.05). pH in third day was significantly higher than first day (p<0.05).Conclusions: The present study shows that activated platelets during 3 days of storage have increased PSelectin on their surface. Therefore, the P-Selectin expression can be used as an in vivo marker for platelet activation function.

Background and Objectives: Iron deficiency anemia (IDA) and beta thalassemia minor (BTM) are the most common causes of hypochromic microcytic anemia. Many indices have been defined for rapid differentiation of these diseases via red blood cell indices. The objectives of this study were to introduce a new index, evaluate its validity, and compare it with other indices.Materials and Methods: In this descriptive observational study, new patients with hypochromic microcytic anemia referred to hematology clinic were studied. 130 patients with IDA and 154 with BTM with the mean age of 24.2 years were assessed. Inclusion criteria were anemia [Hemoglobin (Hb) <mean ± 2 SD for age and sex] and Mean corpuscular volume (MCV) < 80 fl in patients over 6 years and MCV < [70 + Age (year)] fl for patients under 6 years of age. Exclusion criteria were pregnancy, anemia due to multifactorial causes, other hemoglobinopathies and chronic diseases. For all patients, serum iron, serum iron binding capacity, serum ferritin, and HbA2 were measured. BTM and IDA diagnoses were confirmed with HbA2>3.5% and ferritin<12 ng/ml or anemia responding to iron respectively. All patients were assessed according to red blood cell (RBC), Mentzer index, England index, Srivastava index and red cell distribution width, and our new index: [MCV-(10.RBC)]. Sensitivity, specificity, as well as Youden's index have been calculated for all indices.Results: Only Mentzer index and the newly introduced index had the sensitivity and specificity of over 90%. Mentzer Index and the newly introduced index had the highest Youden's index.Conclusions: Compared to other well-known indices, the newly introduced index is straightforward and has a good sensitivity and specificity to discriminate IDA and BTM. Its calculation is easy and does not need calculator.

Background and Objectives: Platelets prepared by random donor method were studied to measure metabolic parameters during 5 days of storage.Materials and Methods: The random donor platelets were obtained by two-phase centrifugation; during the first phase the derived component was platelet-rich plasma from which platelet concentrate was obtained. They were then stored at 22oC on an agitator. Four pooled platelets (each pool containing 5platelet concentrates from random blood donors) were examined. Platelet count, LDH (Lactate Dehydrogenize), changes in pH value, PO2, PCO2, O2 sat, O2:CO2, glucose and lactate concentrations were compared on the first and fifth day.Results: The average of pooled- platelet count in fresh units (day 1) was 2.7´1011 and in stored units (day 5) 2.1´1011. In stored units, platelet counts were lower as compared with fresh units. This indicates of platelet elimination during platelet storage. The pH values for all random pooled platelets were almost stable and no significant changes were observed. Changes between pH values of samples from the first day of collection and those of the fifth day of collection were very small. In fact, all pH values fell within the range of 7.4. Reduction in PO2 level in comparison to PCO2 level was sharply lower. In average, PO2 reduction in random pooled platelets on the fifth day was 12.4 ml Hg and it was 16.8 ml Hg for PCO2. The average O2/CO2 ratio on the first day was 2.7 and this ratio on the fifth day was 3.6 which show an increase of 1.33% in the O2/CO2 ratio on the fifth day. In fact, PO2 level was higher than PCO2 thus contributing to pH stability. The concentration of Lactate dehydrogenizers (LDH) and Lactate considerably increased on the fifth day. The level of glucose decreased gradually from 24.8 to 21.5 m mol/l.Conclusions: The results indicate that the selection of suitable bags for platelets collection and storage can keep O2/CO2 ratio significantly higher even after the fifth day of collection; it can contribute to the preservation and storage of platelets with acceptable quality.

Background and Objectives: Thalassemia is a congenital anemia which is endemic in our region. In our country the national prevention program is followed for the couples who want to get married to be screened via CBC test; then, according to the results, complementry tests are conducted. Through this program the carriers will be diagnosed and new cases might be avoided by prenatal diagnosis.Case: We had a 2.5 year old patient with anemia who came to our thalassemia clinic for exact diagnosis. In her history, her mother had minor beta thalassemia and her father had Hb D or G in Hb electrophoresis pattern. Their hematology consultation before showed that their infant will not have any chance of being affected with major thalassemia. CBC, Hb electrophoresis (Cellulose Acetate pH:8.4) and molecular diagnosis of beta genes were performed for parents and the child. In hematological study, the patient had low Hb level, high reticoulocyte count (10%) and severe morphologic changes in smear. Hb electrophoresis pattern showed 15.9% HBA, 79 % HbF and 4.1% HbD or G. Genetic study showed IVS II-I/N for mother and Hb Lepore/N, Hb D negative for father. The patient was Hp Lepore/IVS II-I which is the combined form of Hb Lepore and b thalassemia mutation. The diagnosis indicated of hetrozygote b thalassemia and Lepore hemoglobin which is the same as intermedia thalassemia phenotype.Conclusions: Hemoglobin Lepore is a variant of thalassemia syndrome with the crossing over of chromosome 11. In Hb electrophoresis, hemoglobin Lepore band is overlapped with Hb D or G and can not be differentiated by these laboratory methods. In our country, in screening program for prevention of thalassemia, we have many cases with thalassemia minor. Since Hb Lepore electrophoresis band overlaps with Hb D or G, in Hb D or G patients who want to get married with other hemoglobinopathy patients, the possibility for Hb Lepore to go undetected should be considered.