Spotlights on the trends in performance assessment of qualitative in vitro diagnostic medical devices in transfusion medicine.

For reliable clinical decisions in transfusion medicine, assessing the performance of qualitative tests performed in medical laboratories is critical. When false results are reported, these can lead to an adverse reaction to blood components. Good performance assessment practices are essential for this kind of scenario, and they still remain as one of the many unmet high-priority challenges in this area. This paper aims to provide an overview of the current trends in this field. A review of the IFCC-IUPC. qualitative vocabulary was carried out, and a particular focus was given to the evaluation protocols CLSI EP12-A3 and Eurachem AQA, such as the European Union Regulation for class D in vitro diagnostic medical devices. There is a consistency between the current protocols and recognized performance assessment principles, which are mandatory in transfusion service labs. We believe that a revised imprecision interval approach and models based on emerging qualitative test types may prove beneficial in the long run. It is also important to emphasize the uncertainty of proportions to mitigate the risk of misclassification.

Towards personalized and rational use of immunoglobulins amid expanding indications and shortages.

The development of intravenous IgG (IVIG) formulations in the 1970s enabled expanded use for treating primary antibody deficiency syndromes and autoimmune conditions. Recent advancements include the use of IVIG in secondary immune deficiencies related to hematologic malignancies and stem cell transplantation, along with the newly emerging prophylactic applications following chimeric antigen receptor T-cell (CAR-T) therapies. Novel therapeutic areas such as bispecific antibodies (BsAbs) for lymphoma and myeloma have increased the use of IgG, given the associated risks of infections. Today, the concept of a rational personalized clinical use of IgG in the context of evolving clinical indications in high-income countries (HIC) is emerging, as unmet challenges in line with managing shortages due to increasing demands globally. The current work aims to review and link the indications for IgG to their characteristics and formulations, their dose, route and frequency of administrations and duration of therapy to meet the needs of individual patients. It will also explore the means to rationalize and monitor IgG use in HIC in the time of shortage, while explaining pragmatic strategies to improve supply and use in low- and middle-income countries (LMIC).

T-cell lymphocytopenia: An omnipresent predictor of morbidity and mortality in consequence of SARS-CoV disease and influenza A infections.

PURPOSE OF THE RESEARCH: We proposed T-cell lymphocytopenia as a strategic predictor of serious coronavirus and influenza infections. Our preeminent goal was to determine whether a degree of T-cell lymphopenia would identify a distinct threshold cell count to differentiate between severe and non-severe infections. We codified an Index Severity Score to exploit an association between T-cell cytopenia and the grade of disease activity. PRINCIPAL RESULT: A T-cell count of 560 cells/uL or below signified a trend towards advanced disease.

Autoimmune complications of COVID-19 and potential consequences for long-lasting disease syndromes.

The latest WHO report determined the increasing diversity within the CoV-2 omicron and its descendent lineages. Some heavily mutated offshoots of BA.5 and BA.2, such as BA.4.6, BF.7, BQ.1.1, and BA.2.75, are responsible for about 20% of infections and are spreading rapidly in multiple countries. It is a sign that Omicron subvariants are now developing a capacity to be more immune escaping and may contribute to a new wave of COVID-19. Covid-19 infections often induce many alterations in human physiological defense and the natural control systems, with exacerbated activation of the inflammatory and homeostatic response, as for any infectious diseases. Severe activation of the early phase of hemostatic components, often occurs, leading to thrombotic complications and often contributing to a lethal outcome selectively in certain populations. Development of autoimmune complications increases the disease burden and lowers its prognosis. While the true mechanism still remains unclear, it is believed to mainly be related to the host autoimmune responses as demonstrated, only in some patients suffering from the presence of autoantibodies that worsens the disease evolution. In fact in some studies the development of autoantibodies to angiotensin converting enzyme 2 (ACE2) was identified, and in other studies autoantibodies, thought to be targeting interferon or binding to annexin A1, or autoantibodies to phospholipids were seen. Moreover, the occurrence of autoimmune heparin induced thrombocytopenia has also been described in infected patients treated with heparin for controlling thrombogenicity. This commentary focuses on the presence of various autoantibodies reported so far in Covid-19 diseases, exploring their association with the disease course and the durability of some related symptoms. Attempts are also made to further analyze the potential mechanism of actions and link the presence of antibodies with pathological complications.

Hematological toxicities of immune checkpoint inhibitors and the impact of blood transfusion and its microbiome on therapeutic efficacy and recipient's safety and survival outcome:A systematic narrative appraisal of where we are now!

Classically, patients with solid and hematologic malignancies have been treated with a combination of chemotherapy with or without a holistic targeted strategy using approved conventional therapy. While the evidence-based use of Immunomodulatory drugs and Immune checkpoint inhibitors (ICIs), including those targeting the PD-1, PD-L1 and CTLA-4, have reshaped the treatment paradigm for many malignant tumors and significantly stretched the life expectancy of patients, as for any interventional therapy, the rise in ICI applications, was associated with the observation of more immune-related hematological adverse events. Many of these patients require transfusion support during their treatment in line with precision transfusion. It has been presumed that transfusion-related immunomodulation (TRIM) and the microbiome can pose immunosuppressive effects on the recipients. Looking to the past and beyond and translating available data into practice in the evolving role of pharmaceutical therapy to ICI-receiving patients, we performed a narrative review of the literature on the immune-related hematological adverse events of ICIs, immunosuppressive mechanisms linked to blood product transfusions, as well as the detrimental impact of transfusions and its related microbiome on the sustained efficacy of ICIs and the patients' survival outcomes. Recent reports are pointing to the negative impact of transfusion on ICI response. Studies have concluded that packed RBC [PRBC] transfusions lead to an inferior progression-free and overall survival in patients with advanced cancer receiving ICIs, even after adjustments for other prognostic variables. The attenuation of the effectiveness of immunotherapy likely results from the immunosuppressive effects of PRBC transfusions. It is, therefore, wise to look retrospectively and prospectively at the impact of transfusion on ICI effects and adopt, in the interim, a restrictive transfusion strategy, if applicable, for those patients.

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.

Blood transfusion in autoimmune rheumatic diseases.

Autoimmune rheumatic disorders (ARD) represent a wide spectrum of disorders that affect in priority the joints, bones, muscles, and connective tissues. Examples of ARD include rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, polymyositis, systemic sclerosis, antiphospholipid syndrome and mixed connective tissue disease. Patients with ARD often require transfusion of red cell concentrates (RCC) or other blood-derived components. The presence of an autoimmune background, often complicated by the use of immunosuppressive medications, renders these patients quite vulnerable. Exposing them to RCC, when indicated, can trigger transfusion-related immunomodulation that can be aggravated by the role played by the donor microbiome, and the complement activation and the immune dysregulation induced by iron, leading to an amplification of the immune problems. Furthermore, patients are challenged by the transfused extracellular vesicles which could have a potentially negative role, particularly in patients with antiphospholipid syndrome. Despite the very vigorous screening, transfusion transmissible infections can still represent a risk to these patients, particularly in cytomegalovirus seronegative patients or when dormant pathogens are activated in the immunosuppressed transfusion recipient. The ARD population is also more at risk for transfusion-related reactions. One, therefore, has to consider a restrictive transfusion strategy if possible and, if needed, resort to the numerous blood bank procedures to reduce the immunogenicity of blood products or use safer, more targeted, industrial plasma-derived products subjected to purification and pathogen reduction technologies.

Spotlights on the latest opinions on identification, prevention, and management of newer CoV-2 variants: A roundup appraisal on innovative ideas and designer vaccines for Omicron.

Although mass vaccination combined with some other preventative strategies and lockdown was associated with some early signs that COVID-19 infection might be fading away, the over 35 sites mutated new South African variant, "Omicron", emerged almost globally. Certain predisposed hosts may develop severe inflammatory thrombotic or mild long-Covid conditions due to this variant, which depletes T-cells, neutralizes antibodies circulating in the body, and coincidentally induces hypercoagulability. The surge of Omicron combined with Delta variants may confer unresponsiveness to the currently available vaccines even when the second dose is given up to 90 days. A drop in the antibody levels by 30 % has been identified in omicron-infected individuals, and one in five people is resistant to antibody treatment. This poses major concerns in the transmissibility rate of this new variant, even in a heavy mass vaccinated environment. This heavily mutated Omicron with other spike sites facilitates viral entry into the cells through conformational changes, irrespective of circulating neutralising antibody. Based on this consideration, we believe that speeding up mixed-matched vaccines with higher T-cell stimulation ability may improve the current situation. Moreover, large orders for antiviral drugs and monoclonal antibodies that could tackle Omicron combined with other variants may be valuable. The use of free polyclonal antibody donations and, hopefully, T-cell immunotherapy, may represent further breakthrough therapeutic interventions. However, Omicron infection is relatively milder than the ongoing Delta variant but is extremely contagious, and therefore the development of novel interventions is highly demanding.

SARS-CoV-2 and cancer: the intriguing and informative cross-talk.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has significantly disrupted and burdened the diagnostic workup and delivery of care, including transfusion, to cancer patients across the globe. Furthermore, cancer patients suffering from solid tumors or hematologic malignancies were more prone to the infection and had higher morbidity and mortality than the rest of the population. Major signaling pathways have been identified at the intersection of SARS-CoV-2 and cancer cells, often leading to tumor progression or alteration of the tumor response to therapy. The reactivation of oncogenic viruses has also been alluded to in the context and following COVID-19. Paradoxically, certain tumors responded better following the profound infection-induced immune modulation. Unveiling the mechanisms of the virus-tumor cell interactions will lead to a better understanding of the pathophysiology of both cancer progression and virus propagation. It would be challenging to monitor, through the different cancer registries, retrospectively, the response of patients who have been previously exposed to the virus in contrast to those who have not contracted the infection.

Platelet and extracellular vesicles in COVID-19 infection and its vaccines.

Platelets are at the crossroads between thrombosis and inflammation. When activated, platelets can shed bioactive extracellular vesicles [pEVs] that share the hemostatic potential of their parent cells and act as bioactive shuttles of their granular contents. In a viral infection, platelets are activated, and pEVs are generated with occasional virion integration. Both platelets and pEVs are engaged in a bidirectional interaction with neutrophils and other cells of the immune system and the hemostatic pathways. Severe COVID-19 infection is characterized by a stormy thromboinflammatory response with platelets and their EVs at the center stage of this reaction. This review sheds light on the interactions of platelets, pEVS and SARS-CoV-2 infection and prognostic and potential therapeutic role of pEVs. The review also describes the role of pEVs in the rare adenovirus-based COVID-19 vaccine-induced thrombosis thrombocytopenia.

Polycythaemia vera: molecular genetics, diagnostics and therapeutics.

Polycythaemia vera is one of several classical myeloproliferative neoplasms that may occur in a juvenile onset or late-onset adult forms. It is linked to specific genetic mutations that cause a deleterious elevation in the patient's red cell mass. The discourse on genetics includes an exposé on the molecular biology of the disease and how a shared JAK2 V617F mutation can co-exist among three distinct neoplasms. Concepts of genetics and immunology help define the origin and behaviour of the disease: the tracking of allele burdens of mutations (genetic dosage), the timing or order of acquired mutations, the import of bystander mutations and the onco-inflammatory response; all theories are invoked to explain the progression of disease severity and potential transformational leukaemia. The World Health Organization's diagnostic criteria are accessed to focus on the subtleties of the Hb laboratories and sifting through the challenging listing of differential diagnoses that mimic PV, and our report includes an overview of manual and automated phlebotomy (erythrocytapheresis) procedures, enumerating their clinical indications, significance of temporary phlebotomy resistance and optimizing safety/ efficacy, quality and cost. Stratification of low and high-risk disease distinguishes when to commence chemo-cytoreductive therapy in the high-risk patient to prevent thrombotic complications. Drug resistance is circumvented by artfully switching drugs or using novel drug designs.

The secrets of human stem cell-derived transfusable RBC for targeted large-scale production and clinical applications: A fresh look into what we need most and lessons to be learned.

Blood transfusion, using the safest conventional blood bioproducts, is an irreplaceable part of substitution therapy. It is considered the most essential supportive clinical intervention aimed to restore the health of patients in need. Nevertheless, numerous unresolved problems are still associated with current blood substitution therapy. To alleviate our dependency on blood donors, many investigators have been focusing on the quest for stem cell-derived blood cells in line with major developments in the field of regenerative medicine. The main objective is to provide a safe and highly standardized universal cultured red cell concentrate [CRBC] for all clinical applications, regardless of blood groups. Currently, we are close to overcoming some of the main obstacles in culturing cells. This concise report is a prelude to the immortalized cell lines that are ready for in vivo clinical trials. It is only through the sharing of experimental ideas and knowledge-based strategies that we will be able to achieve such an enormous task and better understand ''the one for all concept'' of CRBCs and their universal usage in all clinical settings.

An Overview of Current Position on Cell Therapy in Transfusion Science and Medicine: From Fictional Promises to Factual and Perspectives from Red Cell Substitution to Stem Cell Therapy.

Stem cell therapy is a relatively novel field of investigation, in which either differentiated cells or stem cells capable of differentiation are transplanted into an individual with the objective of yielding specific cell types in the damaged tissue and consequently restoring its function. The most successful example of cell therapy is hematopoietic stem cell transplantation, leading to regeneration of a patient's blood cells, now a widely established procedure for many oncologic and non-oncologic diseases. Innovative cell-based therapies are being developed to replace, regenerate or repair injured, absent, or diseased tissues and organs. However, cell therapy bioproducts are based on their inherent biological features such as proliferation, migratory, capability, plasticity, and capacity of self-renewal, posing serious challenges during such bioproduct development. The extraordinary promise of stem cells for future treatments of otherwise intractable diseases has raised great hope and expectations in patients, advocates, physicians, and researchers alike. However, despite thousands of scientific publications and research programs, increased efforts need to be put into the identification of the factors involved, biological mechanisms and materials that affect safety/ efficacy, and into the design of cost-effective methods for the harvesting, expansion, manipulation and purification of the cells.

Trends and targets of various types of stem cell derived transfusable RBC substitution therapy: Obstacles that need to be converted to opportunity.

A shortage of blood during the pandemic outbreak of COVID-19 is a typical example in which the maintenance of a safe and adequate blood supply becomes difficult and highly demanding. So far, human RBCs have been produced in vitro using diverse sources: hematopoietic stem cells (SCs), embryonic SCs and induced pluripotent SCs. The existing, even safest core of conventional cellular bioproducts destined for transfusion have some shortcoming in respects to: donor -dependency variability in terms of hematological /immunological and process/ storage period issues. SCs-derived transfusable RBC bioproducts, as one blood group type for all, were highly complex to work out. Moreover, the strategies for their successful production are often dependent upon the right selection of starting source materials and the composition and the stability of the right expansion media and the strict compliance to GMP regulatory processes. In this mini-review we highlight some model studies, which showed that the efficiency and the functionality of RBCs that could be produced by the various types of SCs, in relation to the in-vitro culture procedures are such that they may, potentially, be used at an industrial level. However, all cultured products do not have an unlimited life due to the critical metabolic pathways or the metabolites produced. New bioreactors are needed to remove these shortcomings and the development of a new mouse model is required. Modern clinical trials based on the employment of regenerative medicine approaches in combination with novel large-scale bioengineering tools, could overcome the current obstacles in artificial RBC substitution, possibly allowing an efficient RBC industrial production.

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.

Spotlight on the current perspectives on applications of human blood cell culture and organoids: Introductory remarks.

Culture of blood cells, mainly erythrocytes, at industrial levels complying with cGMP regulations, aim to make them available, at large scale, any time and everywhere, when needed for transfusion, or laboratory applications. Understanding how blood cells differentiate and develop in-vivo, and mechanisms of differentiation and growth factors, has opened newer strategies for in-vitro culture from multipotent stem cells or immortalized lines. This offers interesting perspectives for obtaining such cultured bioproduct cells for medical applications. In addition, many attempts for preparing platelets in-vitro from megakaryocyte culture have been reported. Nevertheless, the quantities of functional viable platelets obtained are still not sufficient to envisage transfusion applications. Other strategic approaches concern culture of organoids, which can synthesize functional blood proteins, but still significant scale-up of yield needs to be addressed. Finally, considerable advances have been made in culturing specific lymphocytes for personalized immunotherapy of some cancer patients with highly promising results in certain applications. This concise mini report focuses on the progress made in these directions, and attempts are made to describe some newer perspectives.

An update on COVID-19 infection control measures, plasma-based therapeutics, corticosteroid pharmacotherapy and vaccine research.

This communication provides a compilation on aspects of COVID-19 infection control measures, describes the potential role of therapeutic plasma exchange to reduce fatality rates, addresses precautions concerning dexamethasone pharmacotherapy and updates the current status on the availability of vaccines. As part of passive immunotherapy, it focuses on various blood derivatives. These include coronavirus neutralising antibodies extracted from different sources to be administered as a pure hyper concentrate intramuscularly or for upgrading and standardising the specific potency of high affinity antibodies. These processes are intended to compose standardised pooled bioproducts of corona convalescent plasma/cryosupernatant that are pathogen inactivated for additional safety by well-established UV technologies. For the best practice of optimising plasma exchange, hyper concentrate NAb should be added to the cryosupernatant, which contains some of the active principles of corona convalescent plasma. The cryosupernatant apart from the high molecular weight viscous part of cold insoluble proteins that are removed, is equivalent to CCP, but makes it safer for general application. Such a bioproduct is often used routinely for substitution therapy of thrombotic thrombocytopaenic purpura. Alternative resources of large-scale specific coronavirus antibodies warrant further exploration such as cadaveric donations. The early uses of therapeutic plasma exchange and low molecular weight heparin, for any clinical trial in development is warranted, in order to interdict the intense inflammatory/kinin driven cascade. Because coronavirus positive patients are highly prone to thrombosis, thromboprophylaxis is necessary, even some time after recovery guided by the laboratory data.

Opportunities for standardization of cold stored, low-titre group O WB products.

Interest in the use of cold-stored low-titre, group O whole blood (LTO-WB) in civilian trauma medicine has motivated regional and national blood services to explore the operational implications of providing this product to their hospital customers. While simpler to produce, store and administer than conventional blood components, LTO-WB is only distributed by a limited number of civilian blood services to date. To improve the availability of LTO-WB, there are still a number of clinical and basic research challenges that need to be addressed including 1. Standardization of the methods and definitions for what constitutes "low-titre" whole blood; 2. Updating regulatory standards for the in vitro quality of cold stored whole blood; 3. Development of standards for the post-storage component separation of red blood cells from cold stored whole blood; and 4. Optimization of the logistics for collection and distribution of cold stored whole blood in regional and national blood systems. The main objective of this concise overview is to highlight the opportunities for future research and product development efforts that will improve the availability of standardised LTO-WB products in emergency cases to the benefit of all concerned.

Assessment of extracellular vesicles using IFC for application in transfusion medicine.

Extracellular vesicles (EVs) have been shown to be involved in various physiological and pathophysiological processes. With respect to Transfusion Medicine, the accumulation of EVs in blood products during hypothermic storage is an indicator of the storage lesion and reportedly correlates with adverse effects after transfusion, including but not limited to immunomodulation, activation of coagulation, endothelial activation, and others. To optimally reduce such an impact on blood product quality degradation and improve post-transfusion outcomes, better methods for detection, enumeration, characterisation by size and phenotype, and functional involvement of EVs in different pathophysiological and physiological processes are required. Currently, Imaging Flow Cytometry (IFC) technology provides the most comprehensive assessment of EV subsets in different body fluids. The unique ability of IFC to detect EVs of 20 nm size by registration of a single pixel of fluorescence signal makes this approach highly promising for comprehensive studies of EVs. In this review, we will focus on the recent breakthrough and advantages of using the ImageStreamX MKII IFC platform for the detection and characterisation of EVs and its future prospects for routine application of IFC in Transfusion Medicine.

Covid-19, induced activation of hemostasis, and immune reactions: Can an auto-immune reaction contribute to the delayed severe complications observed in some patients?

Covid-19 is characterized by weak symptoms in most affected patients whilst severe clinical complications, with frequent fatal issues, occur in others. Disease severity is associated with age and comorbidities. Understanding of viral infectious mechanisms, and antibody immune response, can help to better control disease progression. SARS-CoV-2 has a major impact on the Renin Angiotensin Aldosterone System (RAAS), through its binding to the membrane cellular glycoprotein, Angiotensin Converting Enzyme-2 (ACE-2), then infecting cells for replication. This report hypothesizes the possible implication of an autoimmune response, induced by generation of allo- or autoantibodies to ACE-2, or to its complexes with viral spike protein. This could contribute to some delayed severe complications occurring in affected patients. We also propose a strategy for investigating this eventuality.