Group O blood donors in Iran: evaluation of isoagglutinin titers and immunoglobulin G subclasses.

This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non-high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non-high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non-high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5-12 .This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non­high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non­high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non­high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5­12 .

Design and introduction of a rational mechanical eluting system for leukocyte recovery from leukoreduction filters: A cell differential approach.

OBJECTIVES: The use of leukoreduction filters has been highly increased in Iranian Blood Transfusion Centers within the last decade to provide sufficient leukoreduced blood products from healthy repeated donors for alloimmunized or sensitive recipients. Leucoflex LCR5, the dominant brand which procured by the Iranian Blood Transfusion Organization, is the most updated generation of the filters used around the world. MATERIAL AND METHODS: In this study, we recovered trapped leukocytes from these filters using different buffer solutions and optimized elution method. The count of recovered cells assessed by cell counter, and cell viability was detected using trypan blue staining. The percent of leukocyte subpopulations was evaluated using a panel of monoclonal antibodies and flow cytometric analysis. RESULTS: It illustrated that a buffer solution consistent with PBS in pH 7.2 containing 2mM EDTA and 4% (w/w) Dextran 40 was the best buffer for LCR5 filter backflushing. The white cell counted as 4.56×108 Granulocytes, 3.34×108 Lymphocytes, and 0.64×108 Monocytes according to analysis with auto hemoanalysis and flow cytometric methods. CONCLUSION: The study guides and assists blood management systems in arranging a national blood profile database for future cell therapy strategies. Also, the recovered cells could be of significance in stem cell research, cellular interaction studies as well as novel molecular developments in drug discovery.

HLA-DRB1*01:01, but not HLA-DRB1:1503 or HLA-DRB1*11, is associated with decreased inhibitor risk in Iranian hemophilia A patients.

BACKGROUND/OBJECTIVE: Hemophilia A is a genetic disorder through which patients suffer from recurrent bleeding. This can be caused by a defect in human plasma coagulation factor VIII. High incidence of FVIII inhibitors in some severe hemophilia A patients after FVIII therapy is a considerable complication. Determination of good predictive factors can improve the safety of this treatment. HLA-II have been shown as a predictive element for inhibitor development. The goal of this study is to determine the association between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*01:01 alleles and FVIII inhibitors in severe hemophilia A patients in Iran. MATERIALS/METHODS: HLA-DRB1 genotyping was performed using Multiplex sequences Specific Primers (PCR-SSP) in two groups of severe hemophilia A patients comprising 51 and 50 individuals with and without FVIII inhibitors respectively. The levels of inhibitor were determined through Nijmegen-modified Bethesda assay. HLA-DRB1 allele frequencies were compared between groups by using multiple logistic regression models. RESULTS: HLA-DRB1*01:01 allele frequency was significantly higher in patients without inhibitor ORadj: 2.7 (95%CI: 1.08, 6.97; P = 0.034). There wasn't any statistically significant difference in HLA-DRB1*11 allele frequency between groups ORadj: 0.7 (95%CI: 0.27, 1.82; P = 0.47). There was no connection between HLA-DRB1*15:03 and inhibitor development ORadj: 0.94 (95%CI: 0.38, 2.35; P = 0.94). CONCLUSION: An association between HLA-DRB1*01:01 and paucity of FVIII inhibitor showed that this allele has probably a protective effect in severe hemophilia A patients in Iran. Determination of the predictive and protective alleles are beneficial in pre-treatment activities and decrease the risk of unsuccessful therapy with FVIII in each population.

Preparation of albumin from human plasma by heat denaturation method in plasma bag.

AIM: In many countries, there is surplus plasma which is thrown out. Due to the high cost of albumin as a pharmaceutical product, it is not widely available for all patients. Therefore, we considered direct preparation of albumin in the plasma bag. BACKGROUND: Human plasma has many unique features compared with prepared pharmaceutical products because of its human origin. In countries which do not have a plasma fractionation industry, shortages for plasma products are commonplace. METHODS: Albumin stabiliser was added to the plasma in the bag to stabilised albumin molecule and make it resistant to heat. When the plasma bag was heated, other plasma proteins are irreversibly precipitated and can be separated by centrifugation. Finally, albumin is filtered and pasteurized in plasma bag. RESULTS: The purity of prepared albumin is >99% and the yield is 21 g per liter of plasma. Polymer content of pasteurized albumin measuring with High Performance Liquid Chromatography (HPLC) was less than 1%. CONCLUSIONS: Since albumin preparation in plasma bag is a simple and cheap technology, it has the potential for use in the production of a safe plasma derived product, although suitability for human use would require appropriate clinical assessment for safety. Using this method, albumin could be prepared from all types of plasma. This method is suggested as a possible method of albumin production for all countries which do not have a plasma fractionation industry and even for the countries with low plasma supplies.

Preparation, purification and virus inactivation of intravenous immunoglobulin from human plasma.

IVIG can be prepared from fractionation of normal human plasma and it is used as a therapeutic drug for treatment of various diseases. IVIG has been for some time the high-growth product within the plasma derived products, at both a global and a national country level. Fractionation was performed according to Cohn method with some modifications. Fraction II was first produced and then it was used for purification and virus inactivation steps. Two methods of virus inactivation (pasteurization at 60 degrees C for 10 hours and solvent/detergent treatment with TnBP and Tween 80) were used and validated. A chromatography method (cation exchange chromatography on CM Sepharose FF) was also added to obtain high purity. The final product (in liquid and freeze dried formulation) meets European Pharmacopeias requirements. The amount of PKA and aggregates was beyond the acceptance limit. The intactness of the IVIG was also examined by circular dichroism (secondary and tertiary structure). It was stable after 6 months of storage. Since Iran market is completely dependant on importation of plasma derived products, it is important to develop such methods for production of IVIG to obtain regional demands.

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Structural study on immunoglobulin G solution after pasteurization with and without stabilizer.

Pasteurization was investigated as a method of inactivating virus during the preparation of immunoglobulin for intravenous use. The effect of pH, protein concentration and the presence of protein stabilizers on the structure of immunoglobulin G (IgG) molecules during pasteurization was investigated using an immunoglobulin solution derived from a Cohn's fraction II preparation. Changes in the secondary and tertiary structure of IgG molecules as well as the degree of polymerization of protein were investigated using spectrophotometry, circular dichroism and size exclusion chromatography. Only slight changes in secondary and tertiary structure were observed after pasteurization in a 10 g L(-1) immunoglobulin solution at pH 4.5 and 5.5 in the absence of stabilizer and in a 50 g L(-1) immunoglobulin solution at pH 5.5 in the presence of glycine and sucrose or sorbitol. Concentrations of immunoglobulin solution below 20 g L(-1) were not denatured when pasteurized at pH 4.5 in the absence of stabilizers. High concentrations of immunoglobulin solution required stabilizers such as glycine and sorbitol or sucrose to prevent or reduce denaturation during pasteurization.