Giuliano Grazzini

Red blood cell storage: the story so far

Red blood cells are still the most widely transfused blood component worldwide and their story is intimately entwined with the history of transfusion medicine and the changes in the collection and storage of blood1,2. At present, the most widely used protocol for the storage of red blood cells (for up to 42 days) is the collection of blood into anticoagulant solutions (typically citrate-dextrose-phosphate); red cell concentrates are prepared by the removal of plasma and, in some cases, also leukoreduction. The product is stored at 4 ± 2° C in a slightly hypertonic additive solution, generally SAGM (sodium, adenine, glucose, mannitol, 376 mOsm/L)1. Despite this, a definitive protocol that reconciles long-term storage on the one hand and safety and efficacy of the transfusion therapy on the other is still the subject of intense debate and discussion. In fact, although the organisation of the blood system, through the achievement of self-sufficiency, currently enables ordinary requests of the transfusion ‘market’ to be met, in the case of a calamity, disaster, or emerging infections3, or in particular periods of the year, local reserves can sometimes reach a minimum. There is still an underlying concern about the real need to store blood components for as long as possible in order to obtain a gradual increase in the interval between the donation and the transfusion, and how much this elastic time span can be prolonged without definitively compromising the quality of the product and, in the final analysis, the recipients’ health2. Indeed, although the transfusion establishment initially pursued both objectives (product quality and prolongation of the storage period), recent retrospective studies (whose results are, therefore, weakened by all the statistical limitations of this type of analysis)5–8 have indicated the apparent irreconcilability of the two aims. These studies seem to suggest that the quality (in terms of safety and efficiency) of red blood cells decreases in proportion to the time the storage period is prolonged. Furthermore, there is extremely convincing molecular evidence9,10 which, together with the results of clinical studies11–33, appears to confirm the preliminary conclusions regarding the likely poorer quality of red blood cells stored for a long time. However, the statistical validity and methodological rigour, in terms of evidence-based medicine, of the clinical studies have recently been challenged, highlighting the need for prospective, double-blind, randomized studies, in like fashion to the one carried out by Walsh et al.34 in 2004, which led the authors to conclude “the data did not support the hypothesis that transfusing red blood cells stored for a long time has detrimental effects on tissue oxygenation in critically ill, anaemic, euvolumaeic patients without active bleeding”. The international scientific community now seems much more convinced of the need of prospective studies, since such studies, on large cohorts of subjects, are currently underway35,36. The key point of the problem is probably the lack of universally accepted standard criteria that closely reflect the dramatic molecular changes that occur during prolonged storage of red blood cells and which, simply put, would enable ‘good’ blood to be distinguished from ‘no longer sufficiently good’ blood. The current standard requirements for patenting new additive solutions in the USA, and also suggested in the recommendations of the European Council37, are essentially based on two parameters: the level of haemolysis (below the threshold of 0.8% at the end of the storage period, following the introduction of the “95/95” rule38) and a survival rate of the transfused cells of more than 75% at 24 hours after transfusion. This latter parameter can be assessed by measuring the half-life of red blood cells labelled with 51 chromium prior to transfusion. These parameters are, however, fairly general and easily affected by the considerable biological variability between donors, given that it is known that blood from some donors resists storage better than that from other donors39. Haemolysis is an easier parameter to monitor. Typically, between 0.2 and 0.4% of red blood cells stored in the presence of standard additive solutions are haemolysed after 5–6 weeks of storage, while pre-storage leukoreduction halves the incidence of this phenomenon40. These widely accepted and well-established parameters do not, however, reflect the profound molecular changes that affect red blood cells during their storage. A brief list of the elements of the so-called “red blood cell storage lesion” includes10: morphological changes, slowed metabolism with a decrease in the concentration of adenosine triphosphate (ATP), acidosis with a decrease in the concentration of 2,3-diphosphoglycerate (2,3-DPG), loss of function (usually transient) of cation pumps and consequent loss of intracellular potassium and accumulation of sodium within the cytoplasm, oxidative damage with changes to the structure of band 341 and lipid peroxidation, apoptotic changes with racemisation of membrane phospholipids and loss of parts of the membrane through vesiculation9. Some of these changes occur within the first few hours of storage, for example, the decrease in pH or the increases in potassium and lactate; others, however, take days or weeks10. Together, these events risk compromising the safety and efficacy of long-stored red blood cells, reducing their capacity to carry and release oxygen, promoting the release of potentially toxic intermediates (for example, free haemoglobin can act as a source of reactive oxygen species) and negatively influencing physiological rheology (through the increased capacity of the red blood cells to adhere to the endothelium42,43 or through their enhanced thrombogenic44 or pro-inflammatory45 potential). These observations at a molecular level were supported by the results of a series of clinical studies (albeit retrospective and not randomised). These studies appeared to show a relationship between the duration of storage and a proportional increase in adverse events in the transfused patients, although the data available are preliminary and the statistically more reliable studies that conform more closely with the gold standard criteria represented by evidence-based medicine are considered necessary by many4 and are, indeed, underway5.

Peroxiredoxin-2 as a candidate biomarker to test oxidative stress levels of stored red blood cells under blood bank conditions

BACKGROUND: Several researches on aging red blood cells (RBCs)—performed both in vivo and under blood bank conditions—revealed that RBC membrane proteins undergo a number of irreversible alterations, mainly due to oxidative stress. The individuation of proteins to be used as indicators of irreversible RBC injury and to be proposed as candidate biomarkers of oxidative damage or aging status during blood storage is therefore of great interest.

Zika virus and the never-ending story of emerging pathogens and Transfusion Medicine

In the last few years, the transfusion medicine community has been paying special attention to emerging vector-borne diseases transmitted by arboviruses. Zika virus is the latest of these pathogens and is responsible for major outbreaks in Africa, Asia and, more recently, in previously infection-naïve territories of the Pacific area. Many issues regarding this emerging pathogen remain unclear and require further investigation. National health authorities have adopted different prevention strategies. The aim of this review article is to discuss the currently available, though limited, information and the potential impact of this virus on transfusion medicine.

Deferral of males who had sex with other males

Donor history questionnaires for the determination of blood donor eligibility are a critical layer of blood safety. Early in the course of the AIDS epidemic in North America homosexual men with multiple partners were identified as one of the segments of the population with the highest risk of infection. Voluntary deferral of this group from blood donation led to a dramatic decrease in transfusion-transmitted HIV even before testing was introduced. In the early 1980s blood donors were deferred in England, the US and other nations, if they were ‘homosexual males with multiple partners’. After the implementation of HIV testing in 1985, the majority of the HIV-positive donors identified revealed ‘men having sex with men’ (MSM) behavior, leading the US Food and Drug Administration (FDA) to recommend indefinite deferral of all men who ‘have had sex with men, even once since 1977’; many other regulators and jurisdictions have enacted similar criteria. Three decades later, despite the recognition of other modes of transmission, MSM donors are still among the population segments with the highest prevalence and incidence of HIV in countries around the world. No other donor eligibility criterion has generated as much controversy or public discourse [1,2]. Proponents for change point out that in many countries other key components of blood safety such as donor testing and blood center process control have improved vastly, reducing the contribution of donor questioning to safety. Gay advocates in particular argue that donor selection policies based on MSM are discriminatory against gay and bisexual men in that they amount to a de facto permanent exclusion on the grounds of sexual preference, and are unfair, as other groups with similar risks of HIV infection are allowed to donate blood after shorter time-period deferrals designed to cover the seroconversion window. On the opposite side of the discussion, recipient advocacy groups and regulators are understandably adverse to any change that is not centered on improving safety. Recipient groups argue that they have suffered greatly due to transmission of HIV and HCV by transfusion, and they will be the bearers of any increase in risk that may result from policy changes. Because both MSM and recipients are vulnerable groups that have suffered in the past, the debate over possible changes in criteria has ethical, societal, and emotional dimensions not seen in discussions concerning other donor selection criteria. Of particular concern to blood operators is the prospect that young eligible donors may be dissuaded from donating blood to institutions that are perceived to act in a discriminatory and unfair fashion. This International Forum seeks to describe approaches to this issue and challenges to the status quo, in a snapshot in time. Since it is extremely difficult to obtaindatatoevaluatethepossibleimpactofpolicy changes made to address concerns expressed by advocacy groups, comparison of international practice is particularly valuable, since we may learn from approaches implemented in other jurisdictions. We received responses from 24 respondents representing countries on six continents. In most, but not all, the MSMpolicy isdetermined atthe national level. The following questions were asked of the respondents:

Cord blood banking

Since the first successful haematopoietic stem cell transplantation (HSCT) using cord blood from a sibling in 1988 and the establishment of the first public umbilical cord blood bank in New York Blood Center in 1992, umbilical cord blood banks have been instituted in many countries. Funding was received from regular blood banks, health councils, charity funds or commercial investments. The international medical society is indebted to the New York Blood Center for publishing their procedures and EuroCord for fighting a patent on cord blood processing. Although it is unknown how many cord blood samples are currently banked and have been transplanted worldwide, the figures of the international registration of World Marrow Donor Association (WHDA) show an increase of cord blood use, in addition to other sources of unrelated HSCT (Fig. 1). Cord blood for HSCT is, in addition to national/regional use, exchanged worldwide. Donor counselling, human leucocyte antigen (HLA)-typing, tests for transmittable diseases and product quality control requirements to comply with (inter)national [AABB, Paul-Ehrlich-Institut, NetCord/ Foundation for the Accreditation of Cellular Therapy (FACT)] standards are expensive, making storage of cord blood for unrelated allogeneic haematopoietic transplantation currently a loss-making activity, not particularly attractive for private enterprise. In the last years, hopes have been fueled that other stem cells in cord blood may be used for future repair of metabolic or degenerative diseases. Autologous or personal cord blood banking appeals to individual initiatives and private funding; money is desperately needed by allogeneic cord blood banks, and it would be much better if it could

Reduction of the risk of bacterial contamination of blood components through diversion of the first part of the donation of blood and blood components.

The level of safety of transfusion therapy is now very high thanks to the combination of serological methods and genomic amplification used to screen for transmissible diseases and the meticulous care with which repeat, voluntary, unpaid donors are selected1–4. The main risk of transfusion-related infectious diseases is currently that of bacterial sepsis1,3–5. The risk of bacterial contamination of blood components is, in fact, estimated to be about three orders of magnitude greater than that of post-transfusional HIV and HCV infections5,6; the risk of bacterial sepsis causes by the transfusion of platelets is more than two orders of magnitude greater than the risk of the same viral infections5,6. According to a study published in 20047, the year in which bacteriological screening tests for platelet units were introduced in the USA3,8,9, the average prevalence of bacterial contamination in platelets from whole blood was 33.9/100,000 units, that of platelets from apheresis 51/100,000, while that of red cell concentrates was 2.6/100,000. The overall prevalence of bacterial contamination of units of cellular blood components was, therefore, about 1 in 3,000 donations (33.3/100,000)7,10. Table I reports the data on bacterial contamination of units of platelets and red blood cells derived from studies before 200311–19. More recent estimates indicate that bacterial culture tests on units of platelets are positive, and confirmed as such, in about 1 in 5,000 units (20/100,000)4. The risk of receiving platelet concentrates contaminated by bacteria is, therefore, considerably higher than the risk of post-transfusion infection by HIV, HCV, HBV and HTLV3,4. Table I Prevalence of bacterial contamination of cellular blood components recorded in studies before 2003

Transfusion medicine in the era of proteomics

Blood components (BCs) are highly complex mixtures of plasma proteins and cells. At present, BC and blood derivatives (BDs) quality control is mainly focused on standardized quantitative assessment, providing relatively limited information about products. Unfortunately, during the production, inactivation, and storage processes there is the risk of changes in their integrity, especially at the protein level, which could cause negative effects on transfusion. It is therefore a major challenge to identify significant alterations of these products, and, in this context, proteomics can play a potentially relevant role in transfusion medicine (TM) to assess the protein composition of blood-derived therapeutics, particularly for identifying modified proteins. It can provide comprehensive information about changes occurring during processing and storage of BCs and BDs and can be applied to assess or improve them, therefore potentially enabling a global assessment of processing, inactivation and storage methods, as well as of possible contaminants and neoantigens that may influence the immunogenic capacity of blood-derived therapeutics. Thus, proteomics could become a relevant part of quality-control process to verify the identity, purity, safety, and potency of various blood therapeutics. A more detailed understanding of the proteins found in blood and blood products, and the identification of their interactions, may also yield important information for the design of new small molecule therapeutics and also for future improvements in TM. Proteomics, together with genomics in the near future, will presumably have an impact on disease diagnosis and prognosis as well as on further advances in the production, pathogen inactivation and storage processes of blood-based therapeutics.

The role of antenatal immunoprophylaxis in the prevention of maternal-foetal anti-Rh(D) alloimmunisation.

The first description of haemolytic disease of the newborn (HDN) can be traced back to 1609 and was made by a French midwife, Louise Bourgeois, who, from 1600, worked at the royal court of King Henry IV and Queen Marie de Medicis1–4. In the treatise that Bourgeois wrote in 1609 she described the birth of two twins3: the first had hydrops and died immediately, while the second, initially in a better condition, rapidly became jaundiced and, after having developed neurological symptoms (kernicterus), died 3 days after being born. Hydrops foetalis and kernicterus were correctly interpreted as two aspects of the same pathology only in 19325, when Diamond described foetal erythroblastosis secondary to severe haemolysis, although the cause was still unknown. A few years later, in 1938, Ruth Darrow correctly identified the (antibody-related) pathogenesis of HDN6, although erroneously attributing foetal haemoglobin the role of the culprit antigen, which was suggested to have induced a maternal antibody response after crossing the placenta. The true pathogenesis of the disease was definitively clarified in 1940 with the discovery of the Rhesus (Rh) blood group system by Landsteiner and Wiener7 and with the subsequent identification, in 1941, by Levine8, of the Rh(D) antigen. This antigen was, in fact, identified, in D-negative mothers, as being the cause of the immunisation occurring following transplacental passage of foetal D-positive red blood cells. The subsequent passage of maternal anti-D immunoglobulin G (IgG) across the placenta into the foetal circulation was recognised as the final event able to cause the spectrum of clinical events that characterise HDN. It did not take long before the risk of immunisation could be quantified1,3: i) 16% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-compatible foetus; ii) 2% in the case of a Rh(D)-negative mother and a Rh(D)-positive, ABO-incompatible foetus (about 20% of the cases); iii) overall risk of immunisation: 13.2%. Before 1945, about 50% of all foetuses with HDN died of kernicterus or hydrops foetalis. Subsequently, thanks to the progress in treatment, in industrialised countries the mortality decreased to 2–3%; this mortality rate was then very considerably further reduced (100-fold) with the introduction of anti-D immunoprophylaxis to prevent maternal-foetal anti-Rh(D) alloimmunisation 9. At the beginning of the 1960s, Stern demonstrated experimentally that the administration of anti-D IgG could prevent sensitisation to the Rh(D) antigen10; in the same period, other studies clarified the mechanism of Rh iso-immunisation in pregnancy and introduced the clinical practice of passive immunisation with anti-D IgG to protect Rh(D)-negative women from sensitisation against Rh(D)-positive red blood cells11–14. The successes obtained in studies of Rh(D)-negative male volunteers formed the experimental basis for clinical trials in pregnant Rh(D)-negative women15; these trials demonstrated that post-partum immunoprophylaxis decreased the incidence of post-pregnancy anti-Rh(D) immunisation from 12–13% to 1–2%15,16. Subsequently, in 1977, it was shown that 1.8% of Rh(D)-negative women, despite post-natal prophylaxis, continued to develop anti-D antibodies because of small transplacental haemorrhages during pregnancy17,18. One year later, a Canadian study by Bowman et al. showed, in 1,357 Rh(D)-negative primagravida, that the incidence of Rh(D) alloimmunisation could be reduced to 0.1% by prophylaxis with antenatal anti-D IgG, in addition to post-partum prophylaxis19. There is currently sufficient evidence demonstrating that antenatal anti-D prophylaxis also reduces the risk of Rh(D) immunisation in the next pregnancy to below the level of 0.4%. Forty years after Zipursky and Israels first proposed the use of anti-D IgG to reduce the incidence of Rh alloimmunisation in pregnancy14, immunoprophylaxis has drastically reduced the cases of Rh-induced HDN; nevertheless, this pathology continues to be relevant in 0.4 of 1,000 births (0.04%)20, for various reasons21: i) the possible occurrence of anti-D immunisation during the pregnancy (which occurs in about 1% of Rh(D)-negative women carrying a Rh(D)-positive foetus22); ii) the lack of efficacy of immunoprophylaxis because of the administration of an insufficient dose of anti-D IgG that is not congruent with the volume of the foetal-maternal haemorrhage; iii) immunoprophylaxis not administered; iv) possible errors in typing the pregnant or puerperal woman or the neonate; v) possible errors in transfusion therapy in women of child-bearing age.

Human Parvovirus B19 and blood product safety: a tale of twenty years of improvements

The establishment of systems to ensure a safe and sufficient supply of blood and blood products for all patients requiring transfusion is a core issue of every blood programme. A spectrum of blood infectious agents is transmitted through transfusion of infected blood donated by apparently healthy and asymptomatic blood donors. Recent emerging-infectious-disease threats include West Nile virus1,2, chikungunya3, babesia4, dengue5, hepatitis E virus6, and variant of Creutzfeldt-Jakob disease7. Parvovirus B19 (B19V), long known to be the causative agent of erythema infectiosum (fifth disease), is not a newly emerging agent. However, it deserves discussion because it may be present in blood and in plasma products, can circulate at extraordinarily high titres, can infect recipients, and, in some cases, can cause severe disease8. Its potentially severe pathological effects have become more apparent in the past decade with the widespread use of (pooled) plasma-derived medicinal products and are the main reason for the uneasy relationship between transfusion medicine specialists and B19V9. The aim of this review is to analyse the role played by this virus in compromising safety in transfusion medicine and the progressive measures to reduce the risks associated with the virus.

Hepatitis E: an old infection with new implications.

The availability of safe blood and blood products is an important public health issue. Improvements in donor screening and testing, pathogen inactivation1 and removal methods, the use of serological tests with greater diagnostic efficacy and the introduction of nucleic acid testing (NAT) have resulted in a substantial drop in transfusion-transmitted infections over the last two decades2. Nonetheless, blood supplies remain vulnerable to emerging and re-emerging infections. In recent years, numerous infectious agents found worldwide have been identified or reconsidered as potential threats to blood supplies3–5. Hepatitis E virus (HEV) has long been considered an enterically transmitted virus causing self-limiting acute viral hepatitis. The disease is endemic in many developing countries, but in recent years an increasing number of autochthonous and sporadic HEV infections have been described in developed countries6. This virus usually causes an acute self-limiting hepatitis, but in some cases fulminant hepatic failure resulting in morbidity and mortality may occur, especially in at-risk groups such as the elderly, pregnant women and patients with pre-existing liver disease or those who are immunocompromised. Furthermore, recent seroprevalence studies are questioning the concept of the low circulation of HEV in developed countries7. This narrative review aims at providing a comprehensive view of HEV and its possible “role” in transfusion medicine.