The Scientific Journal of Iranian Blood Transfusion
Year:2006 | Volume:3 | Issue:1|Papers Count:12

Background and ObjectivesImmunotoxins are comprised of cell targeting and cell killing moieties. Immunotoxins have been proposed as new strategy for cancer treatment. In this study, catalytic domain of Shigatoxin (A1) was fused to human Granulocyte Macrophage-Colony Stimulating Factor (h GM-CSF). The fused gene was already expressed in E.coli and the expressed protein was analysed for its cytotoxic activity on human cancer cell lines expressing GM-CSF receptor (GM-CSFR). Moreover, the impact of GM-CSF receptor on inhibition of cytotoxic effect of Shiga-toxin-GM-CSF recombinant hybrid in cell lines expressing GM-CSF receptor (GMCSFR) was evaluated.Materials and MethodsCell lines were grown in RPMI-1640 medium supplemented with 10-20% heat inactivated fetal bovine serum (Gibco-BRL). Cytotoxic activity was checked on the cell lines and was measured by trypan blue staininig and MTT assay. GM-CSF receptor was blocked by anti-GM-CSF antibody.ResultsCytotoxicity studies revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific due to the presence of killing moiety (A1) that exerts its effect through specific cell targeting domain i.e. GM-CSF, by binding to its receptor present on those cell lines. K562 and LS174T were the most sensitive.Conclusions Our results revealed that the hybrid protein is toxic to the cancer cell lines bearing GM-CSF receptor; this effect could be a potential factor for further consideration of hybrid protein as a therapeutic agent.

Background and ObjectivesIn this study we determined the prevalence of causes for deferral of blood donors in 7 blood centers. These centers are responsible for providing approximately half of the blood units transfused in Iran.Materials and MethodsThis was a cross-sectional study covering 18585 deferred blood donors in Tehran, Fars, Isfahan, Khorasan, East and West Azarbaijan and Ardebil provinces. The simple random sampling method was used. A special form was filled out by the attending physician questioning about demographic characteristics, history of blood donation, and causes of rejection.ResultsThe average age of donors was 32.3±11.3 years. 82.8% of deferred donors were male and 17.8% female. 43% were single, 56.5% married, and 0.5% divorced. 3.9% were illiterate, 38.5% under diploma, 38.3% diploma, and 19.4% university graduates. 38% had no previous history of blood donation, 31.3% were donors with previous history of donation, and 29.2% were regular donors. The most common reason of deferral was unsafe sexual contact (17.8%) followed by drug administration (12.3%), hypotension (8.9%), bloodletting (8%), and polycytemia (5.7%).ConclusionsIn this study the most common reason for deferral was unsafe sexual contact. In comparison to the similar study in 1999 the percent of high-risk behaviors has increased.

Background and ObjectivesKeratinocyte is a suitable model for ex vivo gene expression strategies for systemic release of a protein. In this regard, keratincyte-specific regulatory elements are especially attractive candidates for keratinocyte–mediated gene expression. In the present study, a vector was designed for specific expression of recombinant protein in human epidermal keratinocytes using human keratin 14 gene promoter. The potential of the constructed plasmid was shown by the expression of human FIX-cDNA in human primary keratinocytes.Materials and MethodsA fragment containing human keratin14 gene promoter was inserted in the pcDNA3 plasmid.The human FIX cDNA isolated from a human liver cDNA library was inserted into the recombinant plasmid (phPK 14 H). Human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free medium and transfected with the newly made recombinant plasmid (pK14hFIX) using fugene-6. About 72 hours after transfection, cultured media were collected for expression analysis, and the cells were transferred into selective media containing geneticin. Incubation continued until the appearance of 9 colonies.The expression of human FIX-cDNA was examined by performing one-stage clotting assay as well as RT-PCR.Results The clotting times obtained from the cultured media of different transfection lines were compared to of the negative controls. Indeed the coagulation activity of the expressed rhFIX in the cultured media was detectable both before and after genetic selection of the transected keratinocytes. The expression of hFIX-cDNA in the transected cells was also confirmed by performing RT-PCR.ConclusionOur preliminary results support the idea that human keratinocyte has potential for the production and secretion of biologically active FIX. The expression plasmid constructed in this study, has provided useful means for the expression of different proteins of medical importance in keratinocyte for further biochemical and cellular studies.

Background and ObjectivesThe most important component of fibrinolytic system is the proenzyme plasminogen that through various activators is converted to its active form plasmin and performs its vital functions that are fibrin clot lysis. The first anti human plasminogen antibody was prepared by Ploplis in 1982 and its effect was studied. Since then many researchers have attempted to prepare and study anti plasminogen antibodies to elucidate important aspects of structure and activation mechanism of plasminogen , physiologic condition of fibrinolysis, etc. In the present study, we studied the probable effects of three antihuman plasminogen monoclonal antibodies A4D10, A5E10 and A2C8 on the activation of the fibrinolytic system.Materials and MethodsAfter separate steps like the culture of antibody-producing hybridoma cells, their injection to mice, extraction of the ascites fluid and purification of antibodies were taken, various methods such as optical evaluation of plasma clot lysis in the presence of antibodies, quantitative measurement of DD/E in D-dimer assay, evaluation with ELIZA assay using S-2251 synthetic substrate lysis and the like were used to study the effects of thses antibodies.ResultsPrimary observations with human pooled plasma showed that in the presence of plasminogen activators (t-PA, u-PA and SK), A5E10 and A4D10 can enhance activation of fibrinolytic system, but A3B2 has no effect on this system. According to D- dimer assay, it was shown that the lytic effects of A5E10 and A4D10 antibodies were dose dependent; the higher the amount of antibodies, the lower their effects. The other test performed with S-2251 synthetic substrate showed plasminogen activation in the presence of Urokinase; therefore, lysis of this substrate enhanced in the presence of A5E10 and A4D10 antibodies. Moreover, in this test ineffectiveness of A3B2 on plasminogen activation was confirmed.ConclusionsIn conclusion, we suggested A4D10 and A5E10 antibodies by modification of structural from of plasminogen facilitate its activation in presence of plasminogen activators.

Background and ObjectivesAlbumin is currently used in greater volume than any other biopharmaceutical solution that is available. The largest- scale production of human albumin is still conducted by cohn cold ethanol fractionation (Cohn's method). In order to save the operating time, labor and material in manufacturing albumin 20%, a two-stage process was carried out based on the cold ethanol fractionation method.Materials and Methods For this experimental study, fresh frozen plasma (FFP) were used as a starting material. In the first stage by addition of supernatant IV to a dissolved fraction V paste, a dense fraction V paste (Vd+I) containing the highest possible albumin concentration and the least impurity (ethanol, α globulins and ionic concentration) was recovered by centrifugation; the recovered paste was then dissolved and filtered through a depth filter in order to remove α globulins as impurity. The filtrate (HPs) was suitable to produce albumin 20%. Two batches were produced and all the methods for the evaluation of the finished product were applied according to the guidelines of the British Pharmacopoeia.ResultsThe quality control of the albumin 20% as a finished product from 2 batches (batches 1 and 2) in the final container produced by the proposed procedure led to satisfactory results.Appearance of the product was clear, purity over 99% , and the molecular size distribution of albumin was revealed for aggregates or polymers less than 2.7 % . The yield of albumin was 24±1 gr/kg of plasma.ConclusionsAdvantages of the proposed process as compared with the rountine process (Cohn & Kistler-Nitschmann methods) during the production of intermediate products (Vd+I, HPs) were remarkable savings in terms of operating time, material and manpower at about 50%.

Background and ObjectivesCH50 method is used for the evaluation of complement system. In this method, sheep red blood cells (SRBCs) are sensitized with specific antibodies. Then, the capability of the complement system in the completion of the hemolytic reaction is assessed. In this study because of need for anti-SRBC antibody in CH50 test, it was produced in rabbit. This antibody is called "Amboceptor".Materials and Methods In order to perform this study, SRBCs or their membrane antigens were injected into rabbits with different methods. Produced antibodies were analyzed in CH50 method using human pooled sera (derived from at least 15 blood donors). Finally the optical densities (ODs) related to the lysis of SRBCs were compared. It is obvious that the amount of OD is correlated with the efficiency of the antibody.ResultsIntravenous or intraperitoneal injections of SRBCs repeatedly result in the formation of high titer antibodies, but the rapid death of the animals due to the anaphylactic shock occurs. While the usage of sonicated or heat-treated antigens of SRBCs in the immunization leads to the formation of antibodies with high specificity and titer; it does not threaten the life of the animals.ConclusionsProduction of anti-SRBC antibodies is applicable using different methods. This study shows that the use of the membrane or heat-stable antigens of SRBCs, which are prepared using sonication and heating (103ºC) respectively, has some advantages. These antigens are safe for rabbits and the titer of the antibody is high enough to be used in CH50 test.

Background and ObjectivesABO typing discrepancies in blood donors are not properly considered in Iran, and the only solution to this problem is to exclude the blood unit without any reasonable explanation to blood donor. In this study we examined 75066 blood units of Isfahan Blood Transfusion Center in 15 months for ABO discrepancies in order to evaluate the prevalence and relevant factors of this complication.Materials and Methods Cell and back typing were conducted in a descriptive study on blood donors’ red clots and sera.Special serological tests were done while observing ABO discrepancies to identify the correct blood group. Relevant factors were evaluated using existing data and information obtained from blood donors’ consultation sessions. Data analysis was performed by utilizing Chi-square test.ResultsWe found 41 cases of ABO discrepancies (0.054%) that were more prevalent among men of 41 to 50 years of age with O blood group (p<0.5, p<0.1, p<0.1). The most important reason for these discrepancies was low titer of anti B in serum samples (41.46%) that made confused results in ABO back typing. Low levels of antigen concentration on red cell membrane (34.16% including 50% for A antigen and 50% for B antigen), presence of naturally occurring cold agglutinins in blood sera (12.19%), and errors made by technicians in detecting agglutinations (12.19%) were other reasons in this regard. In autologous controls, we identified 6 cases of warm autoantibody in sera. Autoantibodies did not interfere with ABO typing: one case was drug induced and others were idiopathic; nevertheless, such blood donors were referred to hematological clinicians. 83.3% of cases had additional cold agglutinins and one was low-titered natural ABO antibody.Conclusions35 saved blood units in this study indicate the importance of considering ABO discrepancies in blood transfusion centers which is necessary for both blood donors and recipients. The presence of autoantibodies in healthy blood donors is recommended to be further studied.

Background and ObjectivesBeta-2 microglobulin (ß2MG) is a low weight molecular protein (mol wt: 11800) synthesized by all nucleated cells and originally isolated from human urine in patients with renal failure.Increased ß2MG levels have been reported in rejection of transplantation, in patients with lymphocytic leukemia, hematological malignant disease and especially in patients with multiple myeloma. The aim of this study is to evaluate serum ß2MG and some related factors in patients with monoclonal and polyclonal gammapathy.Materials and MethodsThis study is a cross - sectional study and we select patients by non - random method. We studied 8 patients with gammapathy for 3 months. They were 10-26 years old. Serum ß2MG concentration was measured by ELISA method and serum CRP was done by qualitative latex method. Serum creatinine level was measured according to colotimetric Jaffe reaction. The separation of serum globulins was conducted by cellulose acetate protein electrophoresis.Immunoglobulin levels were measured by Radial Immunodiffusion (RID) method. We use SPSS statistic program and spearmen's index (Rho) to estimate correlation between parameters in this study.ResultsSerum ß2MG levels increased in 87.5% of patients with monoclonal gammapathy. Serum CRP levels also increased in patients with monoclonal gammapathy. According to statistical results, there was a high correlation between increased CRP and ß2MG levels in patients (Rho=0.693, p<0.05). However, serum ß2MG and creatinine levels both had been increased only in 40% of patients. In evaluation of serum protein electrophoresis, in one patient with renal failure, there was a sharp peak in gamma globulin region.ConclusionsThis study indicated that quantitative measurement of serum creatinine, serum ßMG, serum CRP and evaluation of monoclonal band in serum protein electrophoresis can be strong factors for staging the disease and predicting the survival and prognosis in patients with monoclonal gammapathy. So evaluation of these factors is recommended for follow up of patients in relapse and remission stages. It is better to further evaluate and study the main role of these factors in predicting and diagnosing multiple myeloma as well.

Background and ObjectivesIn chronic blood transfusion, antibody production against minor blood groups may occur and sometimes it could be clinically significant lowering RBC life span. This is a case report of all immunization against M-antigen in a 15-month baby with neuroblastoama. This antibody was IgG type and produced some trouble for blood transfusion in this patient.CaseA 15-month baby with neuroblastoma was referred to our center. Neuroblastoma is one of the most solid tomors in children and chemotherapy must be done for the patient suffering from it.During the treatment course the patients may need blood transfusion because of the suppressive effect of BM by chemotherapeutic drugs. This patient had history of blood transfusion one year ago, but this time he faced a severe blood transfusion reaction and was then referred to our center to be considered for alloimmunization and receive compatible blood.ConclusionsOur tests (Ab screening, pannel case) showed this patient has anti-M antibody IgG type. This finding was very important because antibody production against minor or rare blood groups produced significant problems. M-antibodies are mostly of IgM type and not clinically important but in some cases as in our patients it brought about adverse reactions. After preparing and administering compatible blood, the patient manifested no severe reactions or any other problems.