Omics markers of the red cell storage lesion and metabolic linkage

Abstract

The introduction of omics technologies in the field of Transfusion Medicine has significantly advanced our understanding of the red cell storage lesion. While the clinical relevance of such a lesion is still a matter of debate, quantitative and redox proteomics approaches, as well quantitative metabolic flux analysis and metabolic tracing experiments promise to revolutionise our understanding of the role of blood processing strategies, inform the design and testing of novel additives or technologies (such as pathogen reduction), and evaluate the clinical relevance of donor and recipient biological variability with respect to red cell storability and transfusion outcomes. By reviewing existing literature in this rapidly expanding research endeavour, we highlight for the first time a correlation between metabolic markers of the red cell storage age and protein markers of haemolysis. Finally, we introduce the concept of metabolic linkage, i.e. the appreciation of a network of highly correlated small molecule metabolites which results from biochemical constraints of erythrocyte metabolic enzyme activities. For the foreseeable future, red cell studies will advance Transfusion Medicine and haematology by addressing the alteration of metabolic linkage phenotypes in response to stimuli, including, but not limited to, storage additives, enzymopathies (e.g. glucose 6-phosphate dehydrogenase deficiency), hypoxia, sepsis or haemorrhage.

The red blood cell storage lesion(s) and clinical trials

Over the past ten years, concerns have been raised upon the publication of retrospective clinical evidence¹ suggesting the potential negative association between storage “age of blood” and transfusion outcomes. Following these controversial observations, literature has burgeoned around the description of the so-called storage lesion(s), a wide series of biochemical and morphological alterations red blood cells (RBCs)² undergo during storage in the blood bank. Many groups (as extensively reviewed^3–6), including ours^7–20, have contributed to document the energy and oxidative lesions targeting stored RBCs. Compelling biochemical rationale and laboratory evidence^{14–16,21–24} in vitro and in vivo (animal models^25–27) have been produced to support the hypothesis that prolonged storage does not only negatively affect RBC physiology and functionality (e.g. gas transport^18,22,28), but it also influences RBC survival in animal models in vivo²⁹. These observations have led to question whether the storage lesion could thus impair the effectiveness of the transfusion therapy and likely mediate untoward transfusion-related events (e.g. transfusion-related acute lung injury [TRALI], transfusion-related immune modulation [TRIM]^30–33) or aggravate underlying conditions (e.g. sepsis^26,33). However, reassuring evidence from independent randomised clinical trials (RCTs)^34–38 showed no detectable difference between fresh blood and standard of care at the limits of the statistical power of these studies, which prompted the field to conclude (as summarised in the most recent American Association of Blood Banks [AABB] guidelines³⁹) that the general standard of care will not be improved by preferentially issuing fresh blood, at least to the specific categories of recipients enrolled in those RCTs. Many have noted the limitations of the RCTs⁴⁰, as elegantly described in several of the papers appearing in this issue of Blood Transfusion. For example, none of the RCTs performed to date has actually compared the transfusion of fresh blood products vs products close to the end of their shelf-life owing to ethical concerns⁴⁰. However, the general take home message from the RCTs is overall comforting and suggests that, and to quote AABB guidelines, “a restrictive transfusion threshold is safe in most clinical settings and the current blood banking practices of using standard-issue blood should be continued”³⁹. Still, quoting Zimring and Spitalnik in this issue⁴¹, “when approximately 80 million RBC units are transfused annually worldwide, even vanishingly small (transfusion-associated negative) events, if they are real, can affect actual human lives; it then becomes a question of ethics and economics whether it is ‘worthwhile’ to study and attempt to prevent them”.

In the light of these considerations, welcoming the challenge to further advance the field of Transfusion Medicine, in 2016 the National Heart, Lung, Blood Institutes and Food and Drug Administration gathered leading experts in the field to identify current issues associated with blood storage and define an agenda to pursue the amelioration of blood storage strategies^42,43. Discussions in these meetings highlighted the lack of consensus in the definition of parameters of transfusion efficacy, while classic parameters necessary to obtain FDA clearance for new packed RBC storage additives (haemolysis and 24-hour in vivo survival in autologous healthy volunteers) have been unanimously regarded as necessary, but not sufficient, to predict transfusion efficacy in the clinical setting. Several studies suggest that useful, often orthogonal, parameters can be obtained through modern omics technologies.

Omics markers of the RBC storage lesion

Over the last ten years⁴⁴, the introduction of omics technologies in the field of Transfusion Medicine has brought about significant advancements in the understanding of the RBC storage lesion^2,4,6,45. We can now perform state-of-the-art quantitative proteomics approaches (see Figure 1A for the QconCAT approach^10,15,46,47) or redox proteomics analyses (e.g. switch-tag redox proteomics^15,18; Figure 1B) to highlight the proteomics storage lesion with unprecedented specificity and sensitivity. In addition, recent advancements in the field of metabolomics enabled us not just to expand on our understanding of the metabolic storage lesion^{2,7,8,11,12,14,17,19,21,45,48–53}, but also to perform ultra-high throughput⁵⁴ quantitative tracing experiments with heavy labelled substrates^14,15,55 to inform biomarker¹⁶ and metabolic flux analyses^14,15,21,55 (Figure 1C). However, we¹⁷ and others²¹ have recently acknowledged that molecular appreciation of the storage lesion is but the first step in defining novel strategies to improve storage quality. Protein^8,10,15,24 and metabolic markers^{13,14,16,21,51} of the RBC storage lesion have been proposed by us and others. The metabolic phenotype of stored RBCs follows a specific 3-stage sequence, as gleaned through multivariate analysis of metabolomics data of SAGM and AS-3 RBCs (Figure 2A)¹⁶. Metabolic reprogramming of stored RBCs has been associated with the necessity to restore reducing equivalents in order to counteract oxidative stress to functional proteins, such as haemoglobins¹⁸ and anti-oxidant enzymes (e.g. peroxiredoxin 2^8,24). Of note, these redox systems appear to be linked, in that irreversible thiol oxidation of cysteine 93 of haemoglobin beta (a residue necessary for haemoglobin S-nitrosylation and thus haemoglobin-mediated nitric oxide signalling) impairs recycling of the oxidised active site cysteine of peroxiredoxin 2, inhibiting catalysis²². Oxidative lesion(s) to stored RBCs also affect redox sensitive amino acid residues in active site pocket of glyceraldheyde 3-phosphate dehydrogenase (GAPDH)^15,23, a biochemical adaptation that promotes RBC metabolic reprogramming from glycolysis to NADPH-generating pentose phosphate pathway (PPP) in response to oxidative stress (Figure 2B). However, these salvage mechanisms appear to be insufficient to fully counteract the oxidative lesion, resulting in the progressive release of irreversibly oxidised/functionally impaired proteins and small molecule metabolites (including oxidised lipids) into packed RBC supernatants^{8,15,18,23,24,31,48}. Of note, protein and metabolite markers of the RBC storage lesion show significant correlations among each other (Figure 2C–G), resulting from the elaboration of data from our previous publications^10,13,16. Importantly, there is a significant correlation between the levels of metabolic markers of the storage age and the absolute concentrations of supernatant haemoglobin (Figure 2D–G), a marker of RBC storage haemolysis/vesiculation, as we recently proposed¹⁰. Though merely correlative, these observations are relevant in that they suggest that monitoring RBC markers of the metabolic age could provide information about the quality of stored RBCs and potentially predict the effectiveness of the transfusion therapy, in addition to guiding the design and development of novel storage strategies/additives to decrease RBC storage haemolysis.

Figure 1.

Advancements in omics technologies for Transfusion Medicine applications.

(A) An overview of the QconCAT approach for quantitative proteomics¹⁰ and (B) switch-tag approach for redox proteomics applications¹⁵,¹⁸. (C) An overview of a tracing quantitative metabolic experiment, a workflow that can be exploited to inform quantitative metabolic flux analysis elaboration with systems biology tools.

Figure 2.

Metabolic markers of the red blood cell (RBC) storage lesion have been identified through statistical analysis, such as Partial least-square discriminant analysis (A) and receiver operating characteristic curves (ROC)¹⁶,²¹.

A combination of redox proteomics, quantitative proteomics and metabolic flux analyses has revealed a role for the oxidative stress-dependent regulation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity in mediating the activation of the pentose phosphate pathway (PPP) to generate reducing equivalents in the attempt to counteract oxidative stress over storage¹⁵,²³. (B). Correlative analysis of metabolic¹³ and protein markers¹⁰ of the storage lesion was here performed and plotted as a heat map (black = R>0.75) (C). Of note, metabolic markers of the RBC storage age correlated with the absolute concentration of supernatant haemoglobin over storage, a marker of RBC vesiculation and haemolysis (D–G). Though only correlative analyses are here provided, it is interesting to note that all the metabolites showing the highest positive correlations with supernatant haemoglobin were part of the purine catabolism/salvage pathway, a pathway that is activated by oxidative lesion to the purine nucleoside pool and is in part counteracted by salvage reactions fueled by aspartate consumption and resulting in fumaratemalate accumulation (H).

From omics markers of the storage lesion to in vivo survival and haemolysis

Some of the metabolic markers of the storage lesion are not just related to energy metabolism, but also to purine homeostasis and oxidation, such as the adenosine triphosphate catabolites hypoxanthine and xanthine^16,51. Oxidised lipids and purines that accumulate in packed RBC supernatants during refrigerated storage correlate with in vivo performances of transfused cells in animal models²⁹. These observations are suggestive of the likely clinical relevance of the storage lesion, in that it is a safe statement to conclude that, to function, RBCs must at least circulate. Similarly, the metabolic phenotypes (especially in terms of energy and redox homeostasis) correlate with RBC storage haemolysis, both parameters being affected by the genetic phenotype of the donor^56–58. Studies on RBC storage haemolysis and 24-hour in vivo survival suggest that there is room for improvement of the current processing and storage strategies. A large retrospective study of radiolabelled RBC recoveries in autologous healthy volunteers (n=641) by Dumont and Aubuchon reported that end-of-storage RBCs have recoveries of 82.4±6.7%⁵⁹. These numbers indicate that approximately 17% of the RBCs in a transfused unit are lost during storage and transfusion to healthy volunteers⁵⁹. In the light of these data, it is reasonable to assume that heterologous chronically or massively transfused recipients would respond to blood transfusion differently to autologous healthy volunteer recipients owing to their repeated exposure to allogeneic cells or the underlying pro-inflammatory/metabolically-deranged physiology, respectively. Donor and recipient biological variability have often been overlooked in laboratory and clinical studies of the RBC storage lesion, a trend that has been rapidly changing in recent years^60–64. It is still not fully understood whether omics phenotypes of stored RBCs are affected by donor variability and whether that relates to RBC in vivo performances and resistance to the storage lesion, such as in particular haemolysis. Studies such as the REDS III Omics initiative will tackle this important issue in the coming years⁶⁵.

Biological variability, metabolic poise and enzymatic constraints: introducing the concept of metabolic linkage

Despite biological variability, RBC metabolism has evolved to preserve the metabolic poise. To achieve this task, RBC metabolism is tightly regulated by biochemical constraints as a result of evolutionarily conserved enzymatic activities, a phenomenon that can be appreciated through systems biology in silico elaboration of RBC metabolism⁶⁶. Owing to such biochemical constraints, small molecule metabolites do not just show extremely high correlations with storage age¹⁶, but also among each other (Figure 3A–J), a phenomenon we refer to as metabolic linkage. We believe that future advancements in the field of Transfusion Medicine¹⁷, personalised medicine and clinical biochemistry⁶⁷ will be driven by the appreciation of the metabolic linkage and how such linkage is affected by various stimuli; e.g. metabolic responses to hypoxia under physiological (high altitude^68–70) or pathological (haemorrhagic shock⁷¹ or sepsis⁷²) conditions. Similar considerations may also apply with respect to enzymopathies and the role they may play within the framework of the RBC storage lesion. One paradigmatic example is the case of glucose 6-phosphate dehydrogenase deficiency, the most common human enzymopathy that, depending on the variant and thus specific enzymatic mutation, results in different metabolic reprogramming that can affect RBC capacity to cope with stresses upon storage and transfusion into recipients^73,74. Elaboration of data available in the literature reveals that glucose 6-phosphate dehydrogenase deficient donors are characterised by a re-arranged metabolic linkage phenotype (Figure 4A in comparison to Figure 3A), resulting in the loss of significant correlations between some of the metabolites whose level are linked in healthy donors (e.g. lactate and alanine; Figure 4B in comparison to Figure 3I). On the other hand, as a result of a metabolic re-wiring as previously suggested⁷⁴, novel significant correlations emerge (e.g. citrate and 2,3-diphosphoglycerate; Figure 4C), suggesting that recently appreciated metabolic pathways in stored RBCs, such as cytosolic metabolism of citrate in mitochondria-deficient RBCs¹⁴, may play a key role in preserving reducing equivalent homeostasis through alternate pathways in RBCs from glucose 6-phosphate dehydrogenase individuals.

Figure 3.

Metabolic linkage. Metabolite levels in stored red blood cells (RBCs) are significantly correlated, a phenomenon that is explained by enzymatic biochemical constraints and here defined as metabolic linkage (A).

A few examples of metabolites showing significant correlations among each other in 60 packed RBC extracts sampled at random storage time points is shown in (B–J). Figures and panels are the result of the elaborations of metabolomics analyses of samples kindly provided by Dr. Eldad Hod at Columbia University, NY, USA.

Figure 4.

Enzymopathies affect the metabolic linkage.

Determination of the metabolic linkage in stored red blood cells (RBCs) from glucose 6-phosphate dehydrogenase deficient donors reveals a re-wiring of RBC metabolism (A). As a result, significant correlations observed in healthy volunteers are lost (B), while novel ones appear (C). Figures and panels are the result of the elaborations of freely available data from Tzounakas et al.⁷⁴.

Thesis, antithesis and synthesis

As for the three moments of Hegelian philosophy, the transfusion community at first hypothesised that storage age affected the safety and effectiveness of the transfusion therapy (thesis). Despite laboratory and retrospective clinical evidence, reassuring reports from the recent randomised clinical trials have suggested that the current standard of care is not inferior to the transfusion of the freshest units available (antithesis). For the foreseeable future, the combination of omics technologies and clinical evidence, through ambitious studies like the REDS III Omics trial⁶⁵, will enhance our understanding of the effects of handling processes (e.g. leucoreduction, storage additives) and donor/recipient biological variability, and likely reconcile (synthesis) the apparent inconsistencies of the past ten years of Transfusion Medicine research.