FORSCells: 40-Days Fixed Prepared Reagent for Detection of anti-Forssman Inhumans

In 2012, the FORS system was accepted by the International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is most commonly found on sheep red blood cells (RBC) but rare in human RBC. Anti-Fs antibodies (Ab) are naturally occurring in human sera and are predominantly IgM but they can also be IgG. To this day, the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1-positive RBC available to use for anti-Fs screening in large populations. This study was designed to produce FORS1-positive cells viable for 40 days use in the anti-Fs screening. Three to 5% FORS1-positive cells were produced using sheep\u27s blood and CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated in more than three independent experiments of ABO titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. To demonstrate that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification were carried out in a patient and donor population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good FORS1 Ag preservation was established, and minimal haemolysis was observed. In conclusion, a novel and easy-to-produce reagent has been developed and submitted to a patent with stable FORS1 Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Ab in large populations

FORSCells: 40-days fixed prepared reagent for detection of anti-Forssman in humans

In 2012, the FORS system was accepted by the International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is most commonly found on sheep red blood cells (RBC) but rare in human RBC. Anti-Fs antibodies (Ab) are naturally occurring in human sera and are predominantly IgM but they can also be IgG. To this day, the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1-positive RBC available to use for anti-Fs screening in large populations. This study was designed to produce FORS1-positive cells viable for 40 days use in the anti-Fs screening. Three to 5% FORS1-positive cells were produced using sheep's blood and CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated in more than three independent experiments of ABO titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. To demonstrate that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification were carried out in a patient and donor population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good FORS1 Ag preservation was established, and minimal haemolysis was observed. In conclusion, a novel and easy-to-produce reagent has been developed and submitted to a patent with stable FORS1 Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Ab in large populations.info:eu-repo/semantics/publishedVersio

Apolipoprotein E in degenerative processes in the brain, with focus on Alzheimer's disease

Apolipoprotein E (apoE) is a constitute of several lipoprotein particles with an important role in lipid metabolism in the brain. ApoE has been suggested to have a function in synaptic remodeling after injury. ApoE exist in three major isoforms, apoE2, E3 and E4. An increased frequency of the apoE4 allele has been associated with Alzheimer´s disease (AD).The overall aim of this project was to elucidate the role of apoE and its potential as a biochemical marker for degenerative processes in the brain. To achieve these purposes the following methods have been developed.ELISA methods for measurements of apoE in cerebrospinal fluid (CSF) have been developed. Two different types of ELISAs were used, in paper I a sandwich ELISA and in paper IV and V a direct ELISA. The direct ELISA showed several advantages (lower coefficient of variation and higher specificity) compared with the sandwich ELISA. Possible confounding factors were evaluated with the direct ELISA. It was found that apoE absorbs into test tube walls and this makes handling of CSF samples critical. However, both ELISA methods revealed lower levels of apoE in CSF in AD patients as compared to controls. ApoE was determined in brain tissue (frontal cortex, temporal cortex, hippocampus and cerebellum). The level of apoE was found to be decreased in the frontal cortex and hippocampus in AD patients compared to controls. The apoE level was not related to the apoE isoform, senile plaque or neurofibrillary tangles counts. Also, immunohistochemistry examination revealed generally less staining of apoE in the AD brain compared to controls. The finding of decreased levels of apoE in both CSF and brain tissue in AD, support the suggestion that analysis of apoE in CSF reflects biochemical changes of apoE in the brain. The reduction might have several explanations, apoE might be absorbed into senile plaque and therefore the levels are decreased. However, this explanation is not supported in our studies, since no correlation was found between SP counts and the apoE levels. Another explanation is that the production of apoE is not induced in AD patients and that the apoE regenerative system is defective. To determine the apoE isoform an isoelectric focusing (IEF) method was established. The frequency of the apoE4 allele was increased in the AD patients compared to controls. In the second paper we examined the frequency of the apoE4 allele in a very old population and its relation to cerebrovascular disorders. In the old population the apoE4 allele was only a risk factor for developing dementia, if white matter lesions also occurred. Thus other factors than the apoE4 allele are important for developing dementia in very old age. In the last paper, patients with repeated samples after acute ischemic stroke were examined, in order to evaluate different biochemical markers for AD in CSF. ApoE in CSF exhibited rather stable levels after the stroke. It is possible that there is a difference between different types of damage but this finding might also question the regenerative role of apoE in the human brain.Finally, different procedures to isolate apoE lipoprotein particles from CSF have been evaluated. The purpose was to characterize apoE and to study the presence of described apoE and b-amyloid complexes. Alzheimer´s disease is associated with apoE alterations quantitative as well as qualitative. These findings support an involvement of apoE in the degenerative process in this disease but the mechanism for this involvement is still unclear and needs further studie

Real-time monitoring of surface-confined platelet activation on TiO2

AbstractFor the development of advanced hemocompatible biomaterial functions, there is an unmet demand for in vitro evaluation techniques addressing platelet-surface interactions. We show that the quartz crystal microbalance with dissipation (QCM-D) monitoring technique, here combined with light microscopy, provides a surface sensitive technique that allows for real-time monitoring of the activation and aggregation of the surface-confined platelets on TiO2. The QCM-D signal monitored during adhesion and activation of platelets on TiO2 coated surfaces was found to be different in platelet-poor and platelet-rich environment although light microscopy images taken for each of the two cases looked essentially the same. Interestingly, aggregation of activated platelets was only observed in a protein-rich environment. Our results show that a layer of plasma proteins between the TiO2 surface and the platelets strongly influences the coupling between the platelets and the underlying substrate, explaining both the observed QCM-D signals and the ability of the platelets to aggregate

Genotyping of Blood Group Systems among Multi-Transfused Thalassemia Patients in Palestine for Safe Blood Transfusion

Study Design This cross-sectional study was conducted in Palestine among multi-transfused thalassemia patients during 2021 (n=100). Demographic and medical data were collected from medical files of patients. In addition, blood samples were collected from patients for DNA extraction, antibody screening and antibody identification, and assessment of the biochemical, hormonal, and hematological parameters of the patients.      Antibody Screening and Identification Three cell antigen panels were used for antibody screening. In the case of positive antibody screening, antibody identification that includes the most common antigens such as D, C, c, E, e, M, N, S, s, K, k, Lea, Leb, Fya, Fyb, Jkb and P was performed by solid phase method using NEO Iris Analyzer (Immucor, USA). Laboratory Assessment of Biochemical, Hormonal, and Hematological Parameters For the assessment of the biochemical, hormonal, and hematological parameters of the patients, the following parameters were tested blood urea nitrogen (BUN), serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Calcium (Ca), serum ferritin (SF), hemoglobin (Hb), 25-hydroxy vitamin D (vit-D), growth hormone (GH), parathyroid hormone (PTH), total triiodothyronine (TT3), free thyroxine (FT4), fasting blood sugar, and thyroid stimulating hormone (TSH).  BUN, serum creatinine, Ca, AST, ALT, and ALP were performed by the photometric enzymatic methods. All measurements were performed using Roche kits and Cobas c 311 analyzer (Roche, Switzerland) following manufacturers’ instructions. TT3, FT4, TSH, and PTH, in addition to Vitamin D and SF, were measured using the Abbott ARCHITECT I1000SR Immunoassay Analyzer (Abbott, USA) following the manufacturer’s instructions using Abbot Kits. In addition, Hb levels were measured using the automatic blood cell counter MEK-6510 (Nihon Kohden, Japan). GH was measured using Maglumi 800 (Snibe Co. Ltd., China). DNA Extraction Genomic DNA was obtained from peripheral venous blood samples collected in EDTA blood tubes. The DNA was extracted using Promega Wizard Genomic DNA Purification Kit (Promega, USA) according to manufacturer instructions. The DNA concentration and purity of each sample were determined by the measurement of optical density For each sample, a total of 9 μL of 10–30 ng/μL DNA were sent to the laboratory at the University of Gothenburg for genotyping.  Molecular Genotyping ERY Q Kits (BAG Diagnostics, Germany) were used for the genotyping of blood groups. These kits are intended for second-line determination of blood group characteristics. The molecular genetic typing was carried out using the sequence-specific primers (SSP)-PCR technique and real-time PCR (RT-PCR). In the first stage, DNA samples were tested using three kits: ERY Q® RH (REF 728405), ERY Q® KKD/MNS (REF 728407), and ERY Q® Rare (REF 728408). The combination of primers and probes chosen for the particular ERY Q® Kit allows the detection of clinically relevant alleles in the targeted blood groups which include Rhesus, Duffy, Kell, Kidd, Colton, Knops, Lewis, Luth, Dombrock, MNS, Diego, Cartwright (Yt), and Vel. Five samples did not show clear results for the RHD using the ERY Q® RH (REF 728405) and therefore were tested using the ERY Q® Partial D (REF 728403). Test procedures and evaluation of data were performed according to the described procedures in the Instructions Manual. In addition, the specificities of the primers and probes chosen for each particular ERY Q® Kit are also described in the Manual. </p

Genotyping of Blood Group Systems among Multi-Transfused Thalassemia Patients in Palestine for Safe Blood Transfusion

Study DesignThis cross-sectional study was conducted in Palestine among multi-transfused thalassemia patients during 2021 (n=100). Demographic and medical data were collected from medical files of patients. In addition, blood samples were collected from patients for DNA extraction, antibody screening and antibody identification, and assessment of the biochemical, hormonal, and hematological parameters of the patients.Antibody Screening and IdentificationThree cell antigen panels were used for antibody screening. In the case of positive antibody screening, antibody identification that includes the most common antigens such as D, C, c, E, e, M, N, S, s, K, k, Lea, Leb, Fya, Fyb, Jkb and P was performed by solid phase method using NEO Iris Analyzer (Immucor, USA).Laboratory Assessment of Biochemical, Hormonal, and Hematological ParametersFor the assessment of the biochemical, hormonal, and hematological parameters of the patients, the following parameters were tested blood urea nitrogen (BUN), serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Calcium (Ca), serum ferritin (SF), hemoglobin (Hb), 25-hydroxy vitamin D (vit-D), growth hormone (GH), parathyroid hormone (PTH), total triiodothyronine (TT3), free thyroxine (FT4), fasting blood sugar, and thyroid stimulating hormone (TSH).BUN, serum creatinine, Ca, AST, ALT, and ALP were performed by the photometric enzymatic methods. All measurements were performed using Roche kits and Cobas c 311 analyzer (Roche, Switzerland) following manufacturers’ instructions. TT3, FT4, TSH, and PTH, in addition to Vitamin D and SF, were measured using the Abbott ARCHITECT I1000SR Immunoassay Analyzer (Abbott, USA) following the manufacturer’s instructions using Abbot Kits. In addition, Hb levels were measured using the automatic blood cell counter MEK-6510 (Nihon Kohden, Japan). GH was measured using Maglumi 800 (Snibe Co. Ltd., China).DNA ExtractionGenomic DNA was obtained from peripheral venous blood samples collected in EDTA blood tubes. The DNA was extracted using Promega Wizard Genomic DNA Purification Kit (Promega, USA) according to manufacturer instructions. The DNA concentration and purity of each sample were determined by the measurement of optical density For each sample, a total of 9 μL of 10–30 ng/μL DNA were sent to the laboratory at the University of Gothenburg for genotyping.Molecular GenotypingERY Q Kits (BAG Diagnostics, Germany) were used for the genotyping of blood groups. These kits are intended for second-line determination of blood group characteristics. The molecular genetic typing was carried out using the sequence-specific primers (SSP)-PCR technique and real-time PCR (RT-PCR).In the first stage, DNA samples were tested using three kits: ERY Q® RH (REF 728405), ERY Q® KKD/MNS (REF 728407), and ERY Q® Rare (REF 728408). The combination of primers and probes chosen for the particular ERY Q® Kit allows the detection of clinically relevant alleles in the targeted blood groups which include Rhesus, Duffy, Kell, Kidd, Colton, Knops, Lewis, Luth, Dombrock, MNS, Diego, Cartwright (Yt), and Vel. Five samples did not show clear results for the RHD using the ERY Q® RH (REF 728405) and therefore were tested using the ERY Q® Partial D (REF 728403). Test procedures and evaluation of data were performed according to the described procedures in the Instructions Manual. In addition, the specificities of the primers and probes chosen for each particular ERY Q® Kit are also described in the Manual.</p

Adrenaline enhances in vitro platelet activation and aggregation in blood samples from ticagrelor-treated patients

Temporarily improved platelet reactivity may reduce the bleeding in patients on antiplatelet therapy who have ongoing bleeding or who are in need of acute surgery. Adrenaline can bind to adrenergic a2A-receptors on platelets and potentially enhance platelet reactivity

The apolipoprotein E epsilon4 allele frequency is normal in fronto-temporal dementia, but correlates with age at onset of disease

The apolipoprotein (apoE) epsilon4 allele was studied in fronto-temporal dementia (FTD), a diagnostic category including the specific disorders Pick's disease and frontal lobe degeneration of non-Alzheimer type (FLD). These dementing diseases have neuronal and synaptic degeneration in common with Alzheimer's disease (AD), for which the presence of the apoE epsilon4 allele is a known risk factor, and lowers the age of onset of disease. Previous studies on the apoE epsilon4 allele frequency in FTD have been inconclusive. The structural hallmarks of AD, allegedly linked to apoE presentation, neuritic plaques (NP), primarily composed of aggregates of beta-amyloid, and neurofibrillary tangles (NFT), primarily composed of hyperphosphorylated tau, are lacking in FTD. However, tau-positive cytoskeletal pathology is found in Pick's disease, but not in FLD. Resolving whether the epsilon4 frequency is increased in FTD or not may thus give clues to the pathogenetic mechanism of apoE in AD. We therefore studied apoE alleles in a well characterized material of FTD patients. The epsilon4 allele frequency was similar in 25 patients with FTD (14.0%) as compared with 26 healthy controls (13.5%). A post-mortem neuropathological examination was performed in 10 cases (nine had FLD and one Pick's disease). Our finding of a normal epsilon4 allele frequency in our group of FTD, principally consisting of FLD cases, support hypotheses involving differential binding of apoE to beta-amyloid and/or tau, in the development of beta-amyloid deposition and NP formation and/or tau hyperphosphorylation and NFT formation, for the pathogenetic role of apoE in AD. The age at onset was significantly lower (P < 0.01) in FTD patients possessing the epsilon4 allele (48.7 +/- 8.0 years) than in patients not possessing this allele (58.9 +/- 7.6 years). We conclude that, although the apoE epsilon4 allele frequency is not increase in FTD, the epsilon4 allele is not an etiological factor, but may rather be an accelerating factor in the degenerative process of FTD, thereby resulting in an earlier presentation of the disorder in individuals predisposed to develop FTD