Multiparametric analysis of the specific immune response against SARS-CoV-2.

BACKGROUND: SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. OBJECTIVES: We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. METHODS: Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. RESULTS: We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. CONCLUSION: This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

Multicentric evaluation of sensitivity of eight commercial anti-SARS-CoV-2 antibody assays and their correlation to virus neutralization titers in seropositive subjects.

Diagnosis of SARS-CoV-2 virus is mainly based on direct detection. Determination of specific antibodies has been used mostly for epidemiological reasons. However, select immunoassays showed good correlation to plaque reduction virus neutralization test (PRNT) in smaller patient cohorts, which suggests their potential as predictors of virus neutralization titer. A total of 3,699 samples from Covid-19 patients were included in the multicentric study performed in the Czech Republic. Anti-SARS-CoV-2 antibody levels were evaluated by 8 commercial antibody assays. Simultaneously, PRNT evaluations were performed with the SARS-CoV-2 B.1.258 variant. All immunoassays showed an overall high true positive diagnostic value ranging from 79.17 to 98.04%. Several commercial EIA methods showed highly positive correlation between the assay results and PRNT levels, e.g., Liaison CoV-2 TrimericS IgG DiaSorin (Spearman r = 0.8833; Architect SASRS-CoV-2 IgG Abbott (r = 0.7298); NovaLisa SARS-CoV-2 IgG NovaTec (r = 0.7103) and Anti-SARS-CoV-2 ELISA IgG Euroimmun (r = 0.7094). While this correlation was less positive for other assays, those, conversely, presented higher true positive values. For most immunoassays, the positive percent agreement of the results was ≥ 95% in sera exhibiting PRNT levels of 1:80 and higher. The assays tested have shown variable correlation to PRNT. Those possessing high positive predictive values serve well as qualitative tests, while others can be utilised as quantitative tests highly predictive of neutralization antibody levels.

Effects of pre-freeze pathogen reduction with riboflavin and UV light on red cells stored post-thaw in AS-3 additive solution.

BACKGROUND: Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. STUDY DESIGN AND METHODS: 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. RESULTS: PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. CONCLUSIONS: Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.

Cryopreservation of apheresis platelets treated with riboflavin and UV light.

BACKGROUND: Pathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings. STUDY DESIGN AND METHODS: 16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5-6% DMSO, frozen at - 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score. RESULTS: PRT treated platelet units had lower platelet number (247 vs 278 ×109/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged. CONCLUSIONS: Samples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.

Use of unmanned aerial systems in the health care.

The use of unmanned aerial systems (drones) in the medical care, especially for the distribution of blood, laboratory samples, drugs and medical supplies is the subject of several research and practical pilot projects around the world with a wide perspective of use in civilian and military settings. For the purposes of medical service of troops, this is a suitable and promising solution for strengthening of pre-hospital care in advanced lines and in small combat task forces, which often operate in the rear of the enemy, in conditions of irregular warfare and difficult to access medevac. Deploying drones can effectively enhance the capabilities of mobile medical teams and at the same time life-saving prehospital healthcare concepts such as "remote damage control resuscitation" and "blood far forward". The paper presents a brief overview of the development and use of drones in medical applications for civil and military use.

The EHA Research Roadmap: Transfusion Medicine.

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research 1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1-2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including eleven sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy.

Evaluation of platelet concentrates prepared from whole blood donations with collection times between 12 and 15 min: The BEST Collaborative study.

BACKGROUND AND OBJECTIVES: In many countries, whole blood (WB) donations with collection times between 12 and 15 min are not allowed to be used for platelet concentrates (PC). Since the development of guidelines, many process-related changes have been introduced. We aimed to determine the effect of WB with long collection times on PC quality. MATERIALS AND METHODS: Five participating centres tested buffy coat (BC)-derived PC in platelet additive solution type E prepared from only WB collections lasting <12 min (control) versus similar PC including one BC from a collection lasting >12 min (study group, n = 8). One centre produced platelet-rich plasma (PRP)-derived PC from single donations (<10 or >12 min). All PC were stored at 22 ± 2°C and sampled on Days 1, 6 and 8 post-collection for in vitro quality determination. RESULTS: Average collection time was significantly longer in the study group compared to controls (8.9 ± 2.6 vs. 7.3 ± 1.3 min, p < 0.001). There were no differences in volume, platelet concentration, basal CD62P expression, soluble-CD62P and CCL5 levels, or nucleotide content between the groups. Stimulation with TRAP-6 resulted in comparable levels of cell surface CD62P. On Day 8, all PC fulfilled requirements for pH. The findings from single PRP-derived PC centre were similar. CONCLUSION: PC with one BC and single PRP derived from collections lasting >12 min had equivalent in vitro quality to controls during storage. This study provides evidence that 12-15 min donations should not be excluded for PC preparation and justifies to readdress the guidelines to <15 min instead of <12 min of collection in line with current practice in some countries.

Re-introducing whole blood for transfusion: considerations for blood providers.

Whole blood is the original blood preparation but disappeared from the blood bank inventories in the 1980s following the advent of component therapy. In the early 2000s, both military and civilian practice called for changes in the transfusion support for massive haemorrhage. The 'clear fluid' policy was abandoned and replaced by early balanced transfusion of platelets, plasma and red cells. Whole blood is an attractive alternative to multi-component therapy, which offers reduced hemodilution, lower donor exposure and simplified logistics. However, the potential for wider re-introduction of whole blood requires re-evaluation of haemolysins, storage conditions and shelf-life, the need for leucocyte depletion/ pathogen reduction and inventory management for blood providers. This review addresses these questions and calls for research to define the optimal whole blood product and the indications for its use.

NATO and evidence-based military and disaster medicine: case for Vigorous Warrior Live Exercise Series.

The North Atlantic Treaty Organization (NATO) is the premier and only security alliance uniting 30 countries and growing with many partner states in the provision of collective security and against threats posed by conflict and natural disasters. Security of countries and communities is increasingly threatened by a broad spectrum of unconventional types of war and disease threats - from hybrid and asymmetric to multi-domain and peer-to-peer/near-peer conflict. The NATO Centre of Excellence for Military Medicine (MILMED COE) is the centre of gravity for medical best practices and promotion of medical doctrine across the NATO alliance. Disaster medicine is multidisciplinary and in NATO, multinational, requiring best practices that are driven by data and evidence to prevent death on the battlefield and prepare for future conflicts. "Vigorous Warrior" is a live military and disaster medicine exercise series using both civilian and military actors across all sectors of health focused on health security and identifying lessons learned to ready the alliance for future threats. In this brief report, we make the case that the Vigorous Warrior exercise exposes gaps, highlights challenges and generates an evidence base to make NATO military medicine systems more robust, more efficient and in provision of best medical practices. We specifically argue that clinical data capture must be duplicated and continuous across the alliance to ensure evidence-based medicine stays current in NATO military medical doctrine.

Production and use of convalescent plasma in COVID-19 treatment, taking into account the experience in the Central Millitary Hospital Prague.

One of the available treatment alternatives for COVID-19 is the administration of convalescent plasma (CP), blood plasma obtained from people who have undergone the disease. Administration of anti-SARS-CoV-2 antibodies in plasma is a method of passive specific immunization with an expected therapeutic response. CP can also be used for production a specific immunoglobulin. Experience from previous epidemic infections, caused by the coronaviruses SARS-CoV-1 and MERS-CoV, shows that CP contains neutralizing antibodies against the virus, which are probably the main source of its therapeutic potential. However, other immune mechanisms cannot be ruled out, such as antibody-induced cellular cytotoxicity and/or phagocytosis. The use of CP for the treatment of COVID-19 spread during the first half of year 2020 in many countries worldwide and relatively common is also in the Czech Republic, where, at the end of August 2020, about 100 patients were treated with CP. The production and use of CP is governed by the national multidisciplinary guidelines from April 2020 and the recommended therapeutic dose are 2 TU RP (400-450 mL), resp. 4-6 mL/kg. CP is indicated mainly in severe cases of COVID-19, which require oxygen support, ideally within 2-3 days after diagnosis, but our and foreign experience shows a beneficial effect of CP even in moderately severe cases that do not need oxygen treatment.

Emergency Supply Policy of Cryopreserved RBC and PLT: The Czech Republic Concept.

Supply of blood for urgent substitution is a strategic logistical problem for the military medical services across the world. The limited shelf life of blood- derived bioproductsin the liquid state and the need for special transport and use conditions, apart from donor and donations availability are among the causes for concern. To solve these problems many national health-care authorities implemented the national emergency blood crisis policy, to get a large amount of blood at any time at any place in the case of disaster, terrorist attack or war. The civil therapeutic problems in immunohematolgy cases can also be solved by stocks of fresh and cryopreserved homologous or autologous blood for patients with rare RBCs antigens or HLA / HPA platelet refractoriness with no chance to use common blood. The short shelf life of fresh platelets limits their efficient inventory management and availability during a massive transfusion protocol. Building an inventory of frozen blood components can mitigate the risk of insufficient availability. Since the beginning of the century in the Czech Republic, used, like other countries, the use of of cryopreserved blood-derived bioproducts has become the current method used to overcome the shortages of a timely supply. The Military University Hospital, Prague, and its bank of cryopreserved blood have been operating under this policy since 2006. There is currently a stock of frozen RBCs for military reserve, for a national blood crisis and, also, a stock of rare RBC units. For crisis management there are also stored, frozen PLTs, which are used in the treatment of heavily bleeding polytrauma patients. Both the containment and research development mitigation policy programs are in place for civil / military emergency situations. Even pathogen reduced frozen PLTs and frozen RBCs were successfully investigated for clinical use if demands arose. Currently, it is possible to meet operational demand while reducing the number of resupply transports and loss of products due to expiration. A lesson has been learned from the current containment, reseach and mitigation programs of efficient blood supply management with cryopreserved blood and blood derived bioproducts.

Optimizing the supply of whole blood-derived bioproducts through the combined implementation of cryopreservation and pathogen reduction technologies and practices: An overview.

The essential historical knowledge and expertise developed over the past 5-6 decades on the safety / efficacy of conventional blood components therapy by blood transfusion establishments have guided the development of validated methods which have ensure optimal safety margins for frozen blood and its bioproducts with or even without pathogen reduction. Newer generations of pathogen reduced frozen red blood cell, plasma and platelet products and the standardised and safer pooling of human platelet lysate are now become available for potential clinical use. These types of whole blood-derived bioproducts not only reduce the risk of transmission of range of pathogenic blood-borne pathogen. As cryopreservation can be combined with PRT without significantly compromising in vitro quality characteristics or physiological capabilities, it allows us to maximize the available inventory of these blood products in both civil and military trauma settings. The main objective of this overview is to update readers and scientific / medical communities of the various building blocks needed to optimally grantee the pathogen safety of whole blood-derived bioproducts, with minimal untoward events to the recipients. While this is an emerging area, we are seeing the numerous potential opportunities that cryopreservation and pathogen inactivation can have on the transfused patient outcomes. This manuscript is informed by recent publications on this topic.

Variations in hemoglobin measurement and eligibility criteria across blood donation services are associated with differing low-hemoglobin deferral rates: a BEST Collaborative study.

BACKGROUND: Determination of blood donor hemoglobin (Hb) levels is a pre-requisite to ensure donor safety and blood product quality. We aimed to identify Hb measurement practices across blood donation services and to what extent differences associate with low-Hb deferral rates. METHODS: An online survey was performed among Biomedical Excellence for Safer Transfusion (BEST) Collaborative members, extended with published data. Multivariable negative-binomial regression models were built to estimate adjusted associations of minimum donation intervals, Hb cut-offs (high, ≥13.5 g/dL in men or ≥ 12.5 g/dL in women, vs. lower values), iron monitoring (yes/no), providing or prescribing iron supplementation (yes/no), post-versus pre-donation Hb measurement and geographical location (Asian vs. rest), with low-Hb deferral rates. RESULTS: Data were included from 38 blood services. Low-Hb deferral rates varied from 0.11% to 8.81% among men and 0.84% to 31.85% among women. Services with longer minimum donation intervals had significantly lower deferral rates among both women (rate ratio, RR 0.53, 95%CI 0.33-0.84) and men (RR 0.53, 95%CI 0.31-0.90). In women, iron supplementation was associated with lower Hb deferral rates (RR 0.47, 95%CI 0.23-0.94). Finally, being located in Asia was associated with higher low-Hb deferral rates; RR 9.10 (95%CI 3.89-21.27) for women and 6.76 (95%CI 2.45-18.68) for men. CONCLUSION: Differences in Hb measurement and eligibility criteria, particularly longer donation intervals and iron supplementation in women, are associated with variations in low-Hb deferral rates. These insights could help improve both blood donation service efficiency and donor care.

The use of cryopreserved platelets in the treatment of polytraumatic patients and patients with massive bleeding.

BACKGROUND: The short shelf-life of fresh platelets limits their efficient inventory management and availability during a massive transfusion protocol. Risk of insufficient availability can be mitigated by building an inventory of cryopreserved platelets (CPs). METHODS: A comparative study of fresh apheresis platelets (FAPs) and CPs was performed. Type-O CPs were processed with DMSO frozen at -80°C and reconstituted in thawed AB plasma. All patients enrolled in the study had the following parameters evaluated on admission: vital signs (body temperature, heart rate, mean arterial pressure), blood count, prothrombin time, activated partial thromboplastin time, fibrinogen level, and, in trauma patients, international severity score. Several outcomes were evaluated: 30-day survival, adverse events, quantity of administered blood products, fibrinogen concentrate and thromboxane (TXA), and laboratory parameters after transfusion (blood count, prothrombin time, activated partial thromboplastin time, fibrinogen level). RESULTS: Twenty-five (25) patients in the study group received transfusions totaling 81 units of CPs. Twenty-one (21) patients in the control group received a total of 67 units of FAPs. There were no significant differences in patient characteristics (p > 0.05) between groups. Both groups were comparable in clinical outcomes (30-day survival, administered blood products, fibrinogen concentrate, TXA, and adverse events). Among posttransfusion laboratory parameters, platelet count was higher in the group transfused with FAPs (97.0 ×109 /L) than in the group transfused with CPs (41.5 ×109 /L), p = 0.02025. Other parameters were comparable in both groups. CONCLUSION: The study suggests that CPs are tolerable and a feasible alternative to FAPs. However, larger randomized studies are needed to draw definitive conclusions.

Comparison of titer results obtained using immediate spin one-dilution techniques to a reference method.

BACKGROUND: Many transfusion services determine the titer of potentially incompatible plasma-containing products by performing a one-dilution titer at their selected titer threshold. This study compared the results of immediate spin (IS) one-dilution titers determined by three methods with a reference standard method. METHODS: Plasma-containing products from group A and O donors were titered using the participant's routine IS one-dilution titer method. No time or temperature incubations were performed, and antihuman globulin reagent was not used. The samples were then tested using a reference method, which was a saline tube test with a 1-hour room temperature incubation; antihuman globulin was not used in the reference method. The results of the one-dilution titer were then compared to that obtained in the reference method. RESULTS: Nine centers participated in this study. There were 698 antibodies from 374 units tested by the manual IS tube one-dilution titer method; sensitivity was 0.88 (95% confidence interval [CI], 0.83-0.92), and specificity was 1.00 (95% CI, 0.98-1.00). There were 412 antibodies from 206 units tested by the manual and automated IS buffered gel card one-dilution titer method; sensitivity was 0.95 (95% CI, 0.91-0.98), and specificity was 0.87 (95% CI, 0.81-0.91). There were 98 antibodies from 49 units tested by an automated microplate IS one-dilution titer method; sensitivity was 0.76 (95% CI, 0.71-0.93), and specificity was 0.96 (95% CI, 0.92-0.99). All three methods had an accuracy rate of 90% or greater. CONCLUSION: The manual and automated one-dilution titer methods are suitable for screening plasma-containing units, although more evaluation of the automated microplate method might be required.

NATO Blood Panel perspectives on changes to military prehospital resuscitation policies: current and future practice.

The North Atlantic Treaty Organization (NATO) Blood Panel exists to promote interoperability of transfusion practice between NATO partners. However, it has served as an important forum for the development of prehospital transfusion and transfusion in the austere environment. There are synergies with the trauma hemostasis and oxygen research community especially in the areas of innovation and research. Four presentations are summarized together with a review of some scientific principles. The past decade has already seen significant changes in early transfusion support. Sometimes practice has preceded the evidence and has stretched regulatory and logistic constraints. Ethical and philosophical issues are also important and require us to question "should we" and not just "could we." The challenge for the combined communities is to continue to optimize transfusion support underpinned by evidence-based excellence.

FY*A silencing by the GATA-motif variant FY*A(-69C) in a Caucasian family.

BACKGROUND: The c.1-67C variant polymorphism in a GATA motif of the FY promoter is known to result in erythroid-specific FY silencing, that is, in Fy(a-) and Fy(b-) phenotypes. A Caucasian donor presented with the very rare Fy(a-b-) phenotype and was further investigated. STUDY DESIGN AND METHODS: Genomic DNA was analyzed by sequencing to identify the cause of the Fy(a-b-) phenotype. Samples were collected from some of his relatives to establish a correlation between the serology and genotyping results. Red blood cells were analyzed by gel column agglutination and flow cytometry. Genomic DNA was analyzed on genotyping microarrays, by DNA sequencing and by allele-specific PCR. RESULTS: In the donor, a single-nucleotide polymorphism T>C within the GATA motif was found at Position c.1-69 of the FY promoter and shown to occur in the FY*A allele. His genotype was found to be FY*A(-69C), FY*BW.01. In six FY*A/FY*B heterozygous members of the family, a perfect correlation was found between the presence vs. absence of the FY*A(-69C) variant allele and a Fy(a-) vs. Fy(a+) phenotype. CONCLUSION: The location of the c.1-69C polymorphism in a GATA motif whose disruption is known to result in a Fy null phenotype, together with the perfect correlation between the presence of the FY*A(-69C) allele and the Fy(a-) phenotype support a cause-effect relationship between the two.