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Letters to the Editor
Stored blood has compromised oxygen unloading kinetics that can be normalized with rejuvenation and predicted from corpuscular side-scatter
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 respec- tively. Since RBC have only a few seconds to exchange gases in capillaries, impaired O2 handling could compro- mise the efficacy of transfusions, particularly when an immediate improvement to tissue oxygenation is required, such as with major hemorrhage. Although mul- tiple randomized controlled trials have failed to identify clinical consequences related to the storage lesion,8 sev- eral 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 random- ize 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-han- dling 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 con- centrates (RCC) in saline adenine glucose-mannitol (SAG-M) within 27 h of venipuncture according to National Health Service Blood and Transplant proce- dures. 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 vari- ous 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-bub- bled, 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 quanti- ties. 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-col- lected 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 dif- fusion-limited gas exchange at tissues. O2-carrying capac- ity (k) was also compromised by storage (Figure 1D).
The second study tested whether defective O2 han- dling is reversible with biochemical rejuvenation, a treat- ment 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-depen- dent 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 nor- malizing effects of rejuvenation on t, k and T95 are sum- marized in Figure 1G and H and Online Supplementary Figure S1B.
Proxies for O2-handling kinetics were sought by com- paring the variables t and k against standard biochemical ([2,3-DPG], [ATP]) and flow-cytometric parameters (Sysmex XN1000 hematology analyzer), including RET- RBC-Y and RET-RBC-Z channels that correspond to cor- puscular 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 slow- ly, while mean corpuscular volume increased (Figure 2A, Online Supplementary Figure S1C). The efficacy of rejuve- nation 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 relation- ship 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 dys- functional O2 handling. Intriguingly, SSC was found to have good predictive power for t (Online Supplementary Figure S2C), and the power improved further when sam- ple-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 sin- gle-cell resolution. SSC measured conventionally is influ- enced by RBC shape and composition, but upon
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haematologica | 2022; 107(1)