Future Perspectives in HLA Typing Technologies

A wide range of malignant and nonmalignant diseases require hematopoietic stem cell transplantation (HSCT) as last resort therapeutic approach. Graft versus host disease (GVHD), which is one of the major causes of transplant-related mortality, is minimized whenever increased matching of human leukocyte antigens (HLAs) between donor and recipient is present. Suitable donor selection is determined with the utilization of HLA typing. HLAs are highly polymorphic glycoproteins encoded by a region of genes known as the major histocompatibility complex (MHC). Their biological function is to present antigenic peptides to T lymphocytes. However, they also play important role in HSCT acceptance/rejection. During the previous years, various techniques have been acquired in order to better characterize the HLA profile of transplant donors and recipients. This effort is particularly challenging due to MHC size, but most importantly due to high sequence variability in specific regions of the respective genetic loci, between individuals. Initially, HLA typing was performed using serological typing, hybridization techniques, and restriction fragment length polymorphism (RFLP) approaches. Later on, polymerase chain reaction (PCR) based techniques and direct sequencing (dideoxy-based Sanger sequencing) capillary electrophoretic analyses arose. Nowadays, 2nd and 3rd generation sequencing (NGS) technologies show great potential in effectively identifying these polymorphic regions

Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

BACKGROUND: We have showed that secretory Apolipoprotein J/Clusterin (sCLU) is down-regulated in senescent, stressed or diseased red blood cells (RBCs). It was hypothesized that sCLU loss relates to RBCs vesiculation, a mechanism that removes erythrocyte membrane patches containing defective or potentially harmful components. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this issue we employed a combination of biochemical and microscopical approaches in freshly prepared RBCs or RBCs stored under standard blood bank conditions, an in vitro model system of cellular aging. We found that sCLU is effectively exocytosed in vivo during membrane vesiculation of freshly prepared RBCs. In support, the RBCs' sCLU content was progressively reduced during RBCs ex vivo maturation and senescence under cold storage due to its selective exocytosis in membrane vesicles. A range of typical vesicular components, also involved in RBCs senescence, like Band 3, CD59, hemoglobin and carbonylated membrane proteins were found to physically interact with sCLU. CONCLUSIONS/SIGNIFICANCE: The maturation of RBCs is associated with a progressive loss of sCLU. We propose that sCLU is functionally involved in the disposal of oxidized/defected material through RBCs vesiculation. This process most probably takes place through sCLU interaction with RBCs membrane proteins that are implicit vesicular components. Therefore, sCLU represents a pro-survival factor acting for the postponement of the untimely clearance of RBCs

A swift risk analysis for COVID-19 testing facilities using rapid tests

Introduction. COVID-19 is an infectious disease of International Concern, due to the wide-spread geographic impact and high transmissibility, causing severe illnesses. Many testing facilities were set-up for monitoring the spread of the SARS-CoV-2 virus, at the early days of the coronavirus pandemic. From Biosafety aspect this study investigates a reliable risk assessment method to identify and mitigate the risks of COVID-19 testing facilities using Rapid diagnostic tests (POCT), in order to protect the staff, the people who got tested, the community and the environment. Material and methods. Many techniques have been used so far for performing a risk assessment. In the present study, SWIFT analysis suitable for biosafety facilities and for risks of different magnitude, was used for identifying threats and hazards and to calculate the risks for COVID-19 testing facilities. Results. Our analysis showed several initial and potential risks, which could lead to unwanted exposure or release of the SARS-CoV-2, and/or unwanted infection of staff and patients. With minor adjustments of the testing facility, by creating standard operating procedures and awareness of the potential risks, most of the identified risks could be mitigated. Conclusions. Our study demonstrated that when setting up a COVID-19 testing facility, a proper risk assessment should be part of the process, in order to ensure the safety of staff, patients, and the environment. Additionally, we proposed a number of multiple mitigation measures and recommendations, with the goal to reduce the risks during the rapid testing diagnostic procedure.Introducere. COVID-19 este o boală infecțioasă cu un impact geografic larg răspândit și transmisibilitate ridicată, care poate provoacăboli grave. Încă de la debutul pandemiei de COVID-19 au fost înființate multe stații de testare pentru monitorizarea răspândirii virusu-lui SARS-CoV-2. Din punct de vedere al biosecurității acest studiu investighează o metodă de evaluare a riscurilor în vederea identificării și atenuării riscurilor stațiilor de testare COVID-19, care utilizează teste de diagnosticare rapidă (POCT) pentru a proteja personalul, pacienții, comunitatea și mediul. Material și metode. În prezentul studiu au fost aplicate diferite tehnici pentru realizarea unei evaluări a riscurilor. A fost utilizată analiza SWIFT pentru instalațiile de biosecuritate și pentru riscuri de diferită amploare pentru identificarea amenințărilor și pericolelor, și pentru a calcula riscurile pentru stațiile de testare COVID-19. Rezultate. Analiza noastră a identificat mai multe riscuri inițiale și potențiale, care ar putea duce la expunerea sau eliberarea nedorită a SARS-CoV-2 și/sau la infectarea nedorită a personalului și a pacienților. Cu ajustări minore ale stațiilor de testare, prin crearea de proceduri standard de operare și conștientizarea riscurilor potențiale, majoritatea riscurilor identificate ar putea fi atenuate. Concluzii. Prezentul studiu a demonstrat că atunci când se înființează o unitate de testare COVID-19, o evaluare adecvată a riscurilor ar trebui să facă parte din proces pentru a asigura siguranța personalului, a pacienților și a mediului. În plus, am propus o serie de măsuri și recomandări multiple de atenuare cu scopul de a reduce riscurile în timpul procedurii de diagnosticare a testării rapide

Effect of Irradiation and/or Leucocyte Filtration on RBC Storage Lesions

Red blood cell (RBC) storage lesions have been shown to be associated with some adverse reactions; numerous studies have focused on the lesions caused by storage, and few data on the RBC storage lesions caused by prestorage treatments of leucocyte filtration and irradiation. In this study, we examined the changes related with the RBC storage lesions, including 2,3-diphosphatidylglyceric acid (2,3-DPG), pH, free hemoglobin (Hb), supernatant free K+ and Na+ concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH). Along with the increasing storage time, decreases in 2, 3-DPG levels, pH and Na+ concentration, increases in K+ and free Hb concentrations, and significant morphological changes in RBC in all groups were found. The changes in the groups of irradiation, leucocyte filtration and the combined irradiation and leucocyte filtration were more significant than those in the untreated group. Meanwhile, the MCV levels of the three treated groups were significantly lower than those in the untreated group, while the MCH variations were significantly higher. Our results suggest that irradiation and leucocyte filtration before storage may aggravate blood storage lesions

Reply to Ghirardello et al Letter to the Editor

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The antioxidant capacity of erythrocyte concentrates is increased during the first week of storage and correlated with the uric acid level

Background and Objectives: Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage. Materials and Methods: Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43 days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour. Results: The AOP of total ECs and their extracellular samples attained a maximum after 1 week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors. Conclusion: The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied

The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general “dialogue” was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies

The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set

Accelerated apoptotic death and <i>in vivo</i> turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice

Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking