During storage, red blood cells (RBC) undergo major biochemical and morphological changes.1,2 Progression of this so-called storage lesion varies between donor units,3,4 but key manifestations are a decrease in [2,3-diphosphoglycerate] ([2,3-DPG]), which becomes depleted by 3 weeks,5 and spherical remodeling.6 These changes are predicted to affect gas handling by reducing the amount of O2 off-loaded from RBC and expanding the diffusion path-length across the cytoplasm,7 respectively. Since RBC have only a few seconds to exchange gases in capillaries, impaired O2 handling could compromise the efficacy of transfusions, particularly when an immediate improvement to tissue oxygenation is required, such as with major hemorrhage. Although multiple randomized controlled trials have failed to identify clinical consequences related to the storage lesion,8 several limitations of these trials have been pointed out,9 including the concern that the intervention being tested is storage duration, rather than a functional appraisal of O2 handling by RBC. Consequently, studies that randomize according to storage duration may lack the power to resolve the real effects of storage lesion. It is therefore important to determine how storage affects the gas-handling properties of RBC.
The effect of storage on the kinetics of O2 (un)loading is poorly understood because appropriate measurements are not routinely performed. For example, estimates of O2-binding affinity cannot predict ‘on’ and ‘off’ rates because they relate to the steady-state. A recent study10 found that storage paradoxically hastens gas exchange, despite also increasing O2-affinity, and that normalizing [2,3-DPG] has no effect on O2 release rate. However, these inferences were based on measuring the effect of RBC on extracellular O2 dynamics and are, therefore, indirect. To assess gas exchange more directly, we used single-cell HbO2 saturation imaging,7 which measures the time-constant (t) of O2-unloading and the O2-carrying capacity (k) evoked by anoxia. This technique combines ratiometric fluorescence imaging of HbO2 saturation using CellTracker Green and Deep-Red dyes, with rapid switching between oxygenated and anoxic microstreams delivered to RBC. Whole-blood units from consenting donors were collected in citrate phosphate dextrose, depleted of leukocytes, and manufactured as red cell concentrates (RCC) in saline adenine glucose-mannitol (SAG-M) within 27 h of venipuncture according to National Health Service Blood and Transplant procedures. Reference measurements were made on freshly collected venous blood from six volunteers (CUREC R46540/RE001). Data were fully anonymized.
The first study investigated storage-related changes in RBC O2 handling, and how these vary between different units. RCC from six donors (A-F) were sampled at various points during storage (Figure 1A). Dye-loaded RBC were plated in a superfusion chamber mounted on a Leica LCS confocal system (excitation at 488/633 nm, emission at 500-550/650-700 nm) and O2 unloading was triggered by rapidly changing the microstream bathing cells from an oxygenated to an anoxic solution (N2-bubbled, containing 1 mM of O2 scavenger dithionite). Example time courses of the HbO2 response are shown in Figure 1B. With longer storage, cells from the RCC of donor C released O2 more slowly and in smaller quantities. This trend continued beyond week 3, at which point 2,3-DPG is depleted. In cells from the RCC of donor F, O2-handling reached its nadir already at day 2. Frequency distributions for the six RCC are presented in Figure 1C, ranked by progression of the kinetic decline. After 1 week of storage, some RCC (C, D) maintained fast O2-unloading kinetics, whereas others reached their nadir (F). With storage, the width of t distributions expanded, indicating increasing heterogeneity, but there was no evidence of bimodality. These distributions were transformed mathematically into cumulative frequency diagrams that inform about the fraction of RBC capable of unloading 95% of O2 in a given time-frame, T95 (Online Supplementary Figure S1A). The majority of freshly-collected RBC released their O2 cargo within 1 s, but with longer storage, RBC needed more time to complete gas exchange, reaching levels (T95 >2 s) that can result in diffusion- limited gas exchange at tissues. O2-carrying capacity (k) was also compromised by storage (Figure 1D).
The second study tested whether defective O2 handling is reversible with biochemical rejuvenation, a treatment previously shown to normalize [2,3-DPG].11 To determine the effect of such treatment, ABO-matched RCC units were pooled in order to reduce donor-dependent variation in measured responses. Two RCC pools were rejuvenated at specified time-points under storage, and time-matched controls received no treatment (Figure 1E). Single-cell HbO2 saturation imaging showed that biochemical rejuvenation hastened O2-handling kinetics irrespectively of storage duration (Figure 1F, G). The normalizing effects of rejuvenation on t, k and T95 are summarized in Figure 1G and H and Online Supplementary Figure S1B.
Proxies for O2-handling kinetics were sought by comparing the variables t and k against standard biochemical ([2,3-DPG], [ATP]) and flow-cytometric parameters (Sysmex XN1000 hematology analyzer), including RETRBC- Y and RET-RBC-Z channels that correspond to corpuscular forward-scatter and side-scatter (SSC) measured after treating RBC with Cellpack-DFL. [2,3-DPG] became depleted by day 21, ATP, mean corpuscular hemoglobin concentration and SSC decayed more slowly, while mean corpuscular volume increased (Figure 2A, Online Supplementary Figure S1C). The efficacy of rejuvenation was confirmed by the increases in [2,3-DPG], [ATP] and SSC (Figure 2A, Online Supplementary Figure S1D). The most significant correlates to t**********and k (Spearman test) were storage duration, [2,3- DPG], [ATP] and SSC (Figure 2B). The inverse relationship between t and k (Online Supplementary Figure S2A) suggests a common underlying cause of remodeling. A process anticipated to reduce k and raise t is [2,3-DPG] degradation; indeed, an inverse correlation was apparent between [2,3-DPG] and t (Online Supplementary Figure S2B). However, the power of [2,3-DPG] to predict t became compromised beyond day 21, after which t**********continued to increase, despite [2,3-DPG] depletion. An additional factor, such as the expanding path-length in spherically-remodeled RBC, must be contributing to dysfunctional O2 handling. Intriguingly, SSC was found to have good predictive power for t (Online Supplementary Figure S2C), and the power improved further when sample- level variation was factored into the regression (Figure 2C, Online Supplementary Figure S2D). Crucially, SSC was a considerably better predictor of changes in O2 handling than was storage duration. In contrast to [2,3- DPG], SSC continued to fall beyond day-21, is more easily measured, and provides data at single- cell resolution. SSC measured conventionally is influenced by RBC shape and composition, but upon Cellpack-DFL treatment, it also becomes sensitive to membrane permeability, and hence metabolic status. The association between SSC and 2,3-DPG (metabolism) and forward-scatter (volume) was confirmed by a metaanalysis of multiple RCC11 (Online Supplementary Figure S2E, F).
Metabolomic analyses were performed to relate specific metabolites to O2-handling dysfunction4,12 (Figure 3A, Online Supplementary Figure S3A). Established markers of the storage lesion13 were validated, including lactate, hypoxanthine, glucose and reduced glutathione. Donor unit-dependent variation was manifested in the responses of sphingosine 1-phosphate, methionine, S-adenosylmethionine, spermidine, and 20:3 and 20:5 fatty acids (Online Supplementary Figure S3). The metabolic consequences of rejuvenation (Figure 3B, Online Supplementary Figure S3B) provided additional data-points for establishing correlations against t and k (Figure 3C, D). The highest- ranking metabolic correlates to t, equal to or better than [2,3-DPG], are listed in Figure 3E.
Our findings add O2-unloading kinetics to the otherwise static definition of storage lesion. We provide metabolite correlates to O2 handling beyond 2,3-DPG and ATP for future investigations, and show that RBC SSC, a variable influenced by metabolism and corpuscular morphology, is a useful proxy for O2-exchange kinetics. Our assay identified major differences between donor units in the progression of O2-handling dysfunction under storage. The extent to which a gene-function correlation underpins this variation is testable with our method. The normalization of biochemical and flowcytometric parameters with rejuvenation explains how O2 handling is also restored to physiological levels. These observations are significant because of renewed interest in optimizing blood storage regimes for patients receiving large transfusions (in whom cumulative effects of storage lesion can be profound), or patients undergoing procedures in which sub-optimal tissue oxygenation may result in organ injury.
Several high-quality randomized controlled trials14 have shown that transfusion of blood after prolonged storage is not associated with higher rates of adverse clinical outcomes than those after transfusion of blood stored for shorter periods. However, these trials mainly recruited participants with stable anemia and without an imminent threat to organ perfusion, and did not include patients requiring massive transfusion or those with major trauma,14 i.e., cohorts for whom the consequences of impaired gas exchange kinetics are most relevant. In the case of transfusion for major hemorrhage, the clinical imperative is to restore tissue oxygenation immediately. To achieve this, donated RBC must be capable of exchanging gases efficiently at the point of transfusion. This imperative is less relevant in patients with stable anemia because there is evidence that [2,3-DPG] recovers upon re-exposure to a physiological milieu, albeit slowly over up to 3 days.15 A functional assessment of the recovery of O2-handling kinetics in vivo is warranted to determine how the efficacy of transfusion depends on the clinical context of the recipient.
In the aforementioned trials, randomizations were based on storage duration rather than high-quality measurements of RBC physiology. We find that time in storage does not adequately describe the deterioration in O2-handling kinetics, and therefore the use of storage duration for treatment allocation may have compromised the statistical power to detect adverse clinical outcomes related to storage lesion. These considerations may explain the lack of a statistically significant effect of different storage regimes in participants with stable anemia. Given that O2-handling kinetics are mechanistically related to the physiological quality of transfused units, we propose that future investigations should consider RBC function, as determined from O2-unloading kinetics or suitable proxies.
- Received May 26, 2021
- Accepted September 1, 2021
Disclosures: no conflicts of interest to disclose.
Contributions: KD, AM, FC, KCP, AD'A, PAS and PS performed research; PS, PAS, AD'A, AM, KD and RC designed and supervised the study; KD, PS, FC and AD'A analyzed the data; PS, KD, AM, SS, SM, AD'A and PAS wrote the manuscript.
- Bennett-Guerrero E, Veldman TH, Doctor A. Evolution of adverse changes in stored RBCs. Proc Natl Acad Sci U S A. 2007; 104(43):17063-17068. https://doi.org/10.1073/pnas.0708160104PubMedPubMed CentralGoogle Scholar
- Yoshida T, Prudent M, D'Alessandro A.. Red blood cell storage lesion: causes and potential clinical consequences. Blood Transfus. 2019; 17(1):27-52. Google Scholar
- Tzounakas VL, Georgatzakou HT, Kriebardis AG. Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story. Transfusion. 2016; 56(6):1274-1286. https://doi.org/10.1111/trf.13582PubMedGoogle Scholar
- D'Alessandro A, Fu X, Kanias T. Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity. Haematologica. 2021; 106(5):1290-1302. https://doi.org/10.3324/haematol.2020.246603PubMedPubMed CentralGoogle Scholar
- Hess JR. Measures of stored red blood cell quality. Vox Sang. 2014; 107(1):1-9. https://doi.org/10.1111/vox.12130PubMedGoogle Scholar
- Roussel C, Dussiot M, Marin M. Spherocytic shift of red blood cells during storage provides a quantitative whole cell-based marker of the storage lesion. Transfusion. 2017; 57(4):1007-1018. https://doi.org/10.1111/trf.14015PubMedGoogle Scholar
- Richardson SL, Hulikova A, Proven M. Single-cell O2 exchange imaging shows that cytoplasmic diffusion is a dominant barrier to efficient gas transport in red blood cells. Proc Natl Acad Sci U S A. 2020; 117(18):10067-10078. https://doi.org/10.1073/pnas.1916641117PubMedPubMed CentralGoogle Scholar
- Carson JL, Stanworth SJ, Alexander JH. Clinical trials evaluating red blood cell transfusion thresholds: an updated systematic review and with additional focus on patients with cardiovascular disease. Am Heart J. 2018; 200:96-101. https://doi.org/10.1016/j.ahj.2018.04.007PubMedGoogle Scholar
- Trivella M, Stanworth SJ, Brunskill S, Dutton P, Altman DG. Can we be certain that storage duration of transfused red blood cells does not affect patient outcomes?. BMJ. 2019; 365:l2320. https://doi.org/10.1136/bmj.l2320PubMedGoogle Scholar
- Chng KZ, Ng YC, Namgung B. Assessment of transient changes in oxygen diffusion of single red blood cells using a microfluidic analytical platform. Commun Biol. 2021; 4(1):271. https://doi.org/10.1038/s42003-021-01793-zPubMedPubMed CentralGoogle Scholar
- Smethurst PA, Jolley J, Braund R. Rejuvenation of RBCs: validation of a manufacturing method suitable for clinical use. Transfusion. 2019; 59(9):2952-2963. https://doi.org/10.1111/trf.15426PubMedGoogle Scholar
- Stefanoni D, Shin HKH, Baek JH. Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage. Haematologica. 2020; 105(8):2174-2186. https://doi.org/10.3324/haematol.2019.229930PubMedPubMed CentralGoogle Scholar
- Paglia G, D'Alessandro A, Rolfsson O. Biomarkers defining the metabolic age of red blood cells during cold storage. Blood. 2016; 128(13):e43-50. https://doi.org/10.1182/blood-2016-06-721688PubMedGoogle Scholar
- McQuilten ZK, French CJ, Nichol A, Higgins A, Cooper DJ. Effect of age of red cells for transfusion on patient outcomes: a systematic review and meta-analysis. Transfus Med Rev. 2018; 32(2):77-88. https://doi.org/10.1016/j.tmrv.2018.02.002PubMedGoogle Scholar
- Scott AV, Nagababu E, Johnson DJ. 2,3-Diphosphoglycerate concentrations in autologous salvaged versus stored red blood cells and in srgical patients after transfusion. Anesth Analg. 2016; 122(3):616-623. https://doi.org/10.1213/ANE.0000000000001071PubMedPubMed CentralGoogle Scholar
Figures & Tables
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.