[Molecular blood genotyping in patients with Thalassemia major in Tehran adult thalassemic clinic]

Blood typing by serologic methods after transfusion has limitations due to presence of donor red cells in recipients. Accurate determination of red blood cells [RBCs] antigens is very important in multitransfused patients including beta-thalassemics and sickle cell anemics. So, the aim of this study was to evaluate DNA- based methods as supplement to the hemagglutination technique to determine the red blood cell [RBC] antigen profile of multitransfused patients with beta- thalassemia. DNA was extracted from peripheral blood of 20 apparently normal people and 44 patients including 35 with beta- thalassemia [out of whom 19 had clinical evidence of delayed hemolytic transfusion reaction], 8 with thalassemia intermedia [out of whom 2 had hemolytic reaction], and one with sickle cell thalassemia. RHD/ RHC/ RHc/ RHE/ RHe/ JKA/ JKB/ FYA/ FYB/ KELL1/ KELL2 alleles were determined by PCR and were then compared with the hemagglutination method. Phenotype and genotype results were the same in all controls. The phenotypes and genotypes of 53 blood antigens of 26 patients were incompatible. Most of the discrepancies [19 cases] occurred in the Rh system, and fifteen in the Duffy and Kidd systems. The results show that screening platelet concentrates for bacterial contamination is necessary for blood transfusion centers and hospital blood banks. Blood typing by serologic method was not accurate in this study but genotyping could determine true blood groups in multitransfused patients and help in selection of RBCs without alloimmunized antigens in future transfusion attempts. Specificity, sensitivity, positive and negative predictive values of hemagglutination method for RhD antigen had good values in comparison to the molecular method. This might be due to pre- transfusion determination of RhD for thalassemic patients so as to receive Rh- matched blood units. It seems pre-transfusion blood typing of Rh and Kell antigens, which are the cause of hemolytic reactions, in comparison to the molecular method could be cost effective. In addition, typing of Rh and Kell antigens in some regular blood donors could be helpdul for selecting antigen-negative RBCs for transfusion dependent patients

Platelet activation and IL-8 production in platelet concentrates prepared by buffy coat and PRP method

The process of platelet concentrate production by plasma rich [PRP] method could activate the platelet and granules secretion of beta thromboglobulin, LDH and CD62P. Platelets activated during the preparation process do not have sufficient efficiency for hemostasis in vivo. It seems that platelet preparation by buffy coat method has an ability less than PRP to activate the platelet. Measuring platelet activation indices, such as CD62P expression and beta thromboglobulin, is a useful means to evaluate the percentage of activated platelet concentrates and compare the two methods of buffy coat and PRP. In this experimental study, 15 concentrates were prepared via PRP method and 15 via BC method; 15 intact blood units were also considered as control group. The percentages of CD62P expression, soluble CD62P concentrates, IL-8 level, and CD14 positive cells were evaluated. Special monocolonal antibodies that conjugated with flourecence dye in flocytometric method were used for CD62P and CD14. ELISA method was used for evaluation of soluble CD62P and IL-8. The average platelet count in both methods showed no significant difference, but WBC contamination rate in PRP-PCs was more than BC-PCs. In PRP-PCs, we found a little decrease in CD62P expression and increase in soluble form and IL-8 level during reservation time. The level of CD14 showed no significant difference in these components. In BC method during the three day reservation, expression of CD62P, its soluble form, and IL-8 concentrates increased and the level of monocyte surface CD14 showed slight decrease ranging from 0.4 to 0.1. It is concluded that there is a close relationship between IL-8 and WBC count in platelet concentrates. In PRP method in contrary to BC method, high speed centrifuge causes adhesion, aggregation and platelet activation

Evaluation of serumic beta-2 microglobulin in HBsAg+ HBV DNA PCR+ and HBsAg+ HBV DNA PCR- subjects: as HBV replication marker

Beta-2 microglobulin [beta2MG] is the light chain of Histocompatibility-Class I human antigen and its normal range is <3mg/ml. beta2MG level in sera of hepatitis B patients increases. In Hepatitis infection the presentation of the viral antigen on the hepatocyte in the presence of Class I HLA antigen plays a major role in the elimination of the virus. In this descriptive study, s beta2MG, HBsAg [by ELISA], and HBV DNA [by PCR] were evaluated in sera of49 patients with hepatitis B and 35 subjects in control group. Our results showed HbsAg was positive in all patients. 29 of patients were HBV-DNA-PCR positive and 20 HBV-DNA-PCR negative.beta2MG in all subjects in control group was in normal range and in 34.7% of patients above normal limit. beta2MG in HBV-DNA-PCR positive patients was higher than HBV DNA PCR negative patients. Such differences were significant [p <0.05]. It seems S beta2MG is a good marker for HBV replication and its absence may exclude HBV replication. The role of beta2MG in monitoring response to therapy needs to be further evaluated

Evaluation of the phagocytosis and killing strength in neutrophils of patients with thalassaemia major

The immunity system of thalassemics cause of different reasons faces dysfunction. So they are susceptible to recurrent infections. One of these reasons pertains to deficiency in phagocytosis, chemotaxis, and bacterial killing ability neutrophils. As there are several reports about decline in neutrophilic functions in such patients, we aimed at studying phagocytosis and candida killing in major thalassemics.In this study, we analyzed blood samples drawn randomly from 30 patients with thalassemia major [16 splenectomized], and 30 healthy subjects as control group without any familial background of the disease. We evaluated phagocytosis and candida killing in these groups.Our findings showed that there is no relationship between sex and age with neutrophil function in the patients.There is a significant difference in phagocytosis [p<0.1] and killing ability between the patient group and control group [p<0.0001].Since phagocytosis and candida killing are of lowest activity in patients than in the control group, there may be relative deficiency in these patients' neutrophils function

Flowcytometric evaluation of antibodies against histocompatibility antigens and platelet-specific antigens in patients with hematological disorders following the transfusion of platelets concentrates

Blood transfusion may lead to the manifestation of anti-HLA and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. It appears that the study of antibodies against HLA-Class I and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. The aim of this study was to detect anti-HLA and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders [including Acute Leukemia, Aplastic Anemia] and patients with ITP. In this descriptive study, anti-HLA and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different hematological disorders who showed a poor response to platelet transfusion and 20 from patients with ITP. The results of anti-HLA antibodies were then compared by Panel Reactive Antibodies [PRA]. Our results showed 44 [53.7%] out of 82 patients had anti-HLA Class-I antibodies in their sera. The frequency of each antibody isotype was found to be as follows: IgM [51.2%], IgG [32.9%] and IgA [1.2%]. 36 [43.9%] out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: IgM [40.2%], IgG [30.5%] and IgA [12.2%]. 27 [31.7%] out of 82 patients had both antibodies. No difference was found between the two groups in platelet specific antibodies. Despite significant correlation between flowcytometry and PRA methods, PRA can only detect antibodies which react with complement. With increase in the number of platelet transfusion, immunization to HLA antigens occures; moreover, immunization against platelet specific antigens may also occure during autoimmunity. The presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. Conducting similar studies with higher number of samples, platelet cross-match, and the use of HLA- matched platelets for these patients are recommended

Comparison of prevalence of anti-CMV antibodies [IgM and IgG] and CMV Ag in renal transplant recipients

Human cytomegalovirus [HCMV] is a DNA virus, approximately 200nm in diameter, belonging to the herpes virus family. CMV infection in immunocompromised patients including organ transplantation recipients, patients with AIDS patients under immunosuppressive therapy, and in developing fetus may result in either localized or disseminated diseases. Patients are at both primary CMV infection and reactivation of latent infection CMV can be transmitted through blood transfusion and organ transplantation. In this descriptive study, 62 recipients of kidney transplant [26 females, 41.9% and 36 male, 58.1%] ranging from 2-58 years of age [mean 34 +/- 15] were analysed to detect CMV antibodies by ELISA technique; CMV antigen was also evaluated by Indirect Immunofluorescence Technique. All patients were CMV IgG positive, 10 [16.1%] were CMV IgM positive and 7 [11.4%] were at borderline. 23 [37.1%] of recipients were CMV Ag positive. Statistical analysis showed no relation between CMV Ab and CMV Ag. In spite of the presence of anti-CMV IgG and IgM antigenemia appears in several patients. There is not a strong correlation between antibodies against cytomegalovirus and the detection of CMV antigen in patients with acute infections

[Flow cytometric survey of RBC chimerism after bone marrow transplantation]

The aim of the present study was to evaluate red blood cell chimerism after bone marrow transplantation by flow cytometry. In order to perform this assay, FITC labeled antibodies against blood groups ABH, Rh, Kell, Duffy, Kidd, MNS were used.14 hematologic patients under BMT were selected for this study. The required sample was 5 ml peripheral blood that is collected in tubes containing EDTA. At first, donor and recipients red cells phenotypes were identified with the use of both agglutination and flow cytometry methods; then, on post-transplantation days of 15, 30 and 60, only blood samples of the recipients were analyzed by flow cytometry for the antigens differing from donors to recipients. Antibody screening test and titration of ABH Isohemagglutinins were performed on recipients' plasma samples and then repeated on post-transplantation day of 60. After BMT, red cell chimerism was detected in all 14 patients [in 9 patients on post-transplantation day of 15 and in 5 patients on day of 30]. Antibodies against minor blood groups and Rh blood group were not detected at all. The occurrence of chimerism was not inhibited by ABO incompatibility of donors and recipients but in patients who were ABH incompatible with their donors, ABH isohemagglutinins titer following transplantation decreased. Although the presence of isohemagglutinins did not prevent chimerism but it seems these antibodies by attaching to their related antigens on chimeric red cells membrane prevented corresponding antigen detection. Now by using flow cytometry, red cell phenotyping is applicable and reticulocyte analysis is much easier to perform so that chimerism can be detected in patients who have recently experienced blood transfusion. Moreover, through further evaluation of red cell chimerism and detection of recipient autologous red cells, disease relapse can be predicted