An Easy Multiplex PCR-SSP Assay for the Genotyping of KEL1 and KEL2 in Multi-transfused Patients.

Kell blood group system consists of 34 antigens. KEL1 and KEL2 are the most clinically important antigens of this system, causing hemolytic disease of the fetus and newborn (HDFN) and transfusion reaction. A total of 200 samples from blood donors were tested serologically for the presence of KEL1 and KEL2 antigens on erythrocytes. Genomic DNA was analyzed by PCR-SSP method to determine the Kell genotype. A multiplex PCR-SSP assay was designed and tested to genotype KEL1/KEL2 alleles in a single reaction. PCR genotyping revealed samples as; KEL2/KEL2 (93.5%) and KEL1/KEL2 (6.5%), while no sample determined as KEL1/KEL1. A 100% concordance observed between PCR and serological results. Multiplex PCR accurately diagnosed Kell genotype. Kell blood group genotyping by PCR-SSP can be used as an alternative method, especially in multi-transfused patients where serological findings are ambiguous.

miRNA96 expression level within red blood cells is probably associated with RSL indicators during the storage of red blood cell units.

BACKGROUND AND OBJECTIVES: Many biochemical and hematological changes occur during the storage of RBC units. Collectively, these changes are known as RSLs. Previous studies found miRNA96 as non-coding RNA that its expression level changed during RBC storage. However, its correlation with mechanical and biochemical RSL indicators is not yet determined. Therefore, this study aimed to assess possible correlations between miRNA96a and some RSLs indicators to clarify its biomarker capability for evaluating the storage quality of RBC units. MATERIALS AND METHODS: Samples were collected from ten leuko-reduced RBC units on days 0, 14, 28, and 42 of storage. miRNA96 gene expression level and RSLs indicators including hemolysis, mechanical fragility index (MFI), total antioxidant capacity (TAC), lipid peroxidation (TBARs), thiol groups, and RBC indices were measured on the days mentioned above. RESULTS: Significant correlations were found between the changes in miRNA96 expression level and the levels of hemolysis, TAC, TBARs, and MFI indices (p values < 0.05). The donors were classified into the high risk group and low risk group, according to four important characteristics and lifestyle habits (smoking, physical activity, age, and BMI). The high risk group had a significantly lower rate of hemolysis, free hemoglobin, MFI, TAC, and a higher rate of lipid peroxidation compared to low risk group (p values < 0.05). CONCLUSION: The finding suggested that upregulation of miRNA96 could prevent hemolysis of RBCs, despite the accumulation of oxidative injuries in them. The miRNA96 expression level was probably a potential predictor for mechanical and biochemical RSL indicators.

An overview on the investigation of nanomaterials' effect on plasma components: immunoglobulins and coagulation factor VIII, 2010-2020 review.

FVIII and immunoglobulins (Igs) are the most prominent plasma proteins, which play a vital role in plasma hemostasis. These proteins have been implemented frequently in protein therapy. Therefore, their maintenance, durability, and stability are highly essential. Herein, various approaches to improve protein functions have been investigated, such as using recombinant protein replacement. In comparison, advances in nanotechnology have provided adequate context to boost biomaterial utilization. In this regard, the applications of various nanoparticles such as polymeric nanomaterials (PEG and PLGA), metal nanoparticles, dendrimers, and lipid based nanomaterials (liposomes and lipid nanoparticles) in stability and the functional improvement of antibodies and coagulation factor VIII (FVIII) have been reviewed from 2010 to 2020. Reviewing related articles has shown that not only can nanomaterials adequately protect the structure of proteins, but have also improved proteins' functions in some cases. For example, the high rate of FVIII instability has been successfully enhanced by bio-PEGylation. Also, utilizing PEGylated liposomes, using the PEG-lip technique for coating nanostructures, leads to FIIIV half-life prolongation. Hence, PEGylation had most impact on the stability of FVIII. Likewise, PEG-coated liposome nano-carriers also presented such a good effect on stability improvements for FVIII due to their ability to tune the immune system by reducing FVIII immunogenicity. Similarly, Ig PEGylation and conjugation to magnetic nanoparticles resulted in increased half-life and better purification of Igs, respectively, without any loss in structural or functional features. Consequently, metal-organic frameworks and recent hybrid systems have been introduced as promising nanomaterials in biomedical applications. As far as we know, this is the first study in this field, which considers the applications of nanoparticles for improving the storage and stability of antibodies and coagulation FVIII.

Hepatitis C (HCV) Viremic Rate and its Correlation to Demographic Factors among HCV Confirmed Iranian Blood Donors.

BACKGROUND: Hepatitis C virus (HCV) viremia is described as persistent HCV RNA among HCV exposed individuals. HCV viremic rate is defined as the proportion of anti-HCV positive and HCV RNA positive individuals to total anti-HCV positive individuals. Knowledge about HCV viremic rate increases understanding HCV epidemiology and provides the likelihood of HCV viremia infection in a given population. The aim of this study was to evaluate HCV viremic rate and demographic parameter correlations among HCV confirmed Iranian blood donors. METHODS: In this analytical, cross-sectional study, serologically confirmed HCV positive blood donors, who were referred to the Iranian blood transfusion centers around the country from November 2015 to September 2017, were included. HCV RNA RT-PCR was carried out by an in-house qualitative assay. Penalized logistic regression was performed for data analysis. STATA software version 13 was used for statistical analysis. RESULTS: Out of 239 subjects, HCV RNA was amplified in 161 (67.36%, 95% CI 61.21% -73.51%). No statistical associations were found between age, gender, education and marriage status with HCV viremic rate. First time donation was found to be associated with HCV viremia status (adjusted odds ratio [AOR]: 3.26; 95% CI 1.07-9.87). CONCLUSION: The results of this study show the likelihood of active HCV infection occurrence among HCV confirmed Iranian blood donors, as the majority are in the active phase of HCV infection. The viremic rate was associated with first time donation. More effective donor selection process and paying special attention to maintenance of non-infected first time donors as a resource of regular donations are needed to improve blood safety. Follow-up studies on viremic first time blood donors are recommended to clarify impact of factors on the occurrence of HCV viremia.

Reactive Oxygen Species Generated by CD45-Cells Distinct from Leukocyte Population in Platelet Concentrates Is Correlated with the Expression and Release of Platelet Activation Markers during Storage.

BACKGROUND: Platelet stimulation with agonists is accompanied by the generation of reactive oxygen species (ROS) which promotes further platelet activation and aggregation. Considering different cell populations in platelet concentrates (PCs), this study investigates the correlation of ROS generation with the expression and release of platelet activation markers during storage. METHODS: Samples obtained from 6 PCs were subjected to flow cytometry and ELISA to evaluate the expression and shedding of platelet P-selectin or CD40L during storage. Intracellular ROS were detected in either CD45- or CD45+ population by flow cytometry using dihydrorhodamine 123, while ROS production was analyzed in both P-selectin+ or P-selectin- and CD40L+ or CD40L- populations. To further evaluate the correlation between ROS generation and release function, TRAP-stimulated platelets were also subjected to flow cytometry analysis. RESULTS: ROS detected in the CD45-population (leukocyte-free platelets) was significantly increased by fMLP and PMA. P-selectin- or CD40L- platelet did not show significant amount of ROS. Total ROS generation was significantly increased during platelet storage (day 0 vs. day 5; p = 0.0002) while this increasing pattern was directly correlated with the expression of P-selectin (r = 0.72; p = 0.0001) and CD40L (r = 0.69; p = 0.0001). ROS generations were significantly correlated with ectodomain shedding of these pro-inflammatory molecules. CONCLUSION: Our data confirmed increasing levels of intracellular ROS generation in both platelets (CD45-) and platelet-leukocyte aggregates (CD45+) during PC storage. The amount of detected ROS is directly correlated with platelet activation and release in each population while platelet-leukocyte aggregates generate higher levels of ROS than single platelets.

Implementation of Plasma Fractionation in Biological Medicines Production.

CONTEXT: The major motivation for the preparation of the plasma derived biological medicine was the treatment of casualties from the Second World War. Due to the high expenses for preparation of plasma derived products, achievement of self-sufficiency in human plasma biotechnological industry is an important goal for developing countries. EVIDENCE ACQUISITION: The complexity of the blood plasma was first revealed by the Nobel Prize laureate, Arne Tiselius and Theodor Svedberg, which resulted in the identification of thousands of plasma proteins. Among all these proteins, four of which are commercially important for production due to significant need of patients. These four products are: albumin, IgG, factor VIII, and Factor IX. The starting material for the production of biological drugs from plasma is natural which is different from synthetic starting material. So, the quality of plasma as starting material plays an important role in the quality of final product. Introducing new techniques for preparation of the biological drugs from human plasma has resulted in the improvements in purity of products, higher safety, and yield noticeably. Still, the backbone of the modern plasma fractionation technique is mainly based on cold ethanol fractionation of the human plasma that is almost the same as fractionation of crude oil, breaking it down into its components. The demand for IgG for treating immune deficiencies and coagulation factor VIII for hemophilia A determines how to design the plasma fractionation industry in terms of capacity. Nowadays, cold ethanol fractionation has followed by chromatographic methods, since they offer higher purity. In this review, we describe different methods of plasma fractionation such as cold ethanol fractionation, gel filtration, fractionation by salt, and fractionation by polyethylene glycol. There is no doubt that the four main products of human plasma are albumin, IgG, coagulation factor VIII, and IX, which their methods of separation from human plasma have been explained in this paper. CONCLUSIONS: It can be concluded that plasma fractionation with ethanol at low temperature for the preparation of the main human plasma biological components including albumin, IgG, coagulation factors VIII, and IX is still the most widely used method at an industrial scale. Nowadays, this method is being used in combination with different chromatographic techniques in order to achieve a higher quality and the yield.

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography.

BACKGROUND: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. METHODS: PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. RESULTS: Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). RESULTS of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 µg/ml). CONCLUSION: It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII.

Simultaneous purification and polymerization method for bovine serum albumin preparation.

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.

Synthesis and characterization of Pseudomonas aeruginosa alginate-tetanus toxoid conjugate.

Chronic infection with Pseudomonas aeruginosa is the main proven perpetrator of lung function decline and ultimate mortality in cystic fibrosis (CF) patients. Mucoid strains of this bacterium elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of naïve animals elicits opsonic antibodies and leads to clearance of mucoid P. aeruginosa from the lungs. Alginate was isolated from mucoid P. aeruginosa strain 8821M by repeated ethanol precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve immunogenicity, the purified antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoid P. aeruginosa in the CF host.