Enhancing uniformity and overall quality of red cell concentrate with anaerobic storage

Abstract

Background

Recent research focused on understanding stored red blood cell (RBC) quality has demonstrated high variability in measures of RBC function and health across units. Studies have historically linked this high variability to variations in processing, storage method, and age. More recently, a large number of studies have focused on differences in donor demographics, donor iron sufficiency, and genetic predisposition of the donor to poor storage, particularly through mechanisms of accelerated oxidative damage. A study was undertaken to evaluate a potential additional source of unit to unit variation in stored RBC: the role of variable percent oxygen saturation (%SO₂) levels on blood quality parameters during storage.

Materials and methods

%SO₂ data from 492 LR-RBC/AS-3 units used for internal and external collaborative research was included in the analysis. Whole blood units were processed into red blood cells, AS-3 added, leucocyte reduced, in compliance with American Association of Blood Banks guidelines. LR-RBC/AS-3 products were subsequently analysed for %SO₂ levels within 3–24 hours of phlebotomy using a co-oximeter. Separately, to evaluate the impact of pre-storage as well as increasing levels of %SO₂ during storage, a pool-and-split study was performed. Four units of LR-RBC/AS-3 were split 6 ways; “as is” (control), hyperoxygenated to more than 90%, and four levels of pre-storage %SO₂. The units were periodically sampled up to 42 days and analysed for %SO₂, pCO₂, methaemoglobin, ATP, 2,3-BPG as well as with the metabolomics workflow.

Results

The measured mean %SO₂ in LR-RBC/AS-3 within 24 hours of collection was 45.9±17.5% with (32.7–61.0 IQR). %SO₂ in all products increased to approximately 95–100% in three weeks. Measured blood quality parameters including ATP, % haemolysis, methaemoglobin, oxidised lipids, and GSH/GSSG indicated suppressed cellular metabolism and increased red cell degradation in response to higher %SO₂ levels.

Discussion

The surprisingly high variability in starting %SO₂ levels, coupled with negative impacts of high oxygen saturation on red blood cell quality indicates that oxygen levels may be an important and under-appreciated source of unit-to-unit variability in RBC quality.

Introduction

Blood transfusions save and/or sustain thousands of lives every day in the United States and across the globe. Improvements in pathogen screening, storage solutions, and leucocyte reduction have continued to improve the clinical benefits of blood transfusion and reduce the risks. Nevertheless, the biochemical and biomechanical degradation that occurs over the course of six weeks of storage has been well established. More than 80 million units of red blood cell (RBC) are collected and stored globally. Once RBCs are removed from circulation in donors and separated as red cell concentrate (RCC), they experience progressive damage during refrigerated storage (hypothermic storage lesions). Even when transfused within the current 6-week limit, stored RBC tend to exhibit lower quality (e.g. increased fraction of RBC removed after transfusion, compromised oxygen delivery capacity, reduced deformability) and increased toxicity, often manifested as the clinical sequelae of transfusion therapy^1–15. This view is supported by a large and growing number of articles in the literature^{4,6,7,9–14,16–24}. Oxidative damage is considered the major cause of various storage lesions^25–34. Owing to the high concentration of haemoglobin and molecular oxygen in stored RBC, chemical oxidation of haemoglobin to form methaemoglobin and subsequent denaturation products are the major culprits of oxidative stress in stored RBC³³. Furthermore, oxygen is the substrate for both non-enzymatic oxidation of lipids in the membrane catalysed by the products of oxidised haemoglobin, as well as for the production of biologically active lipid oxidation products during storage. Of two main reactants for methaemoglobin formation during hypothermic storage, haemoglobin and oxygen, the former is regulated by maintaining a minimum donor haemoglobin requirement, while the latter is not specifically controlled or scrutinised. The accumulation of oxidative storage lesions is reduced by storing RBC under hypoxic conditions^35,36, as reducing O₂ during storage removes the primary initiating factor for oxidative stress.

In order to design and develop an optimal commercial disposable device to reduce oxygen content of RCC prior to refrigerated storage, we purchased a large quantity of fresh RCC from several sources over the past two years. All of the units were fresh (less than 24 hours from collection) and key parameters such as complete blood count (CBC), blood gas and haemoglobin oxygen saturation (%SO₂) values were recorded. This relatively large data set allowed us to examine for the first time the oxygen content, as measured by %SO₂, of a large number of leucoreduced RCC shortly after component separation. In this report, we present the %SO₂ distribution from 492 units of post-production RCC and show the kinetics of gain in %SO₂ during 42-day storage. We also describe the effect of oxygen content during six weeks of storage on several oxidative stress markers and discuss the potential implications of uncontrolled %SO₂ distribution on the blood supply.

Materials and methods

Blood products

Blood products, whole blood in CP2D anticoagulant (WB), packed RBC (component-separated RBC without additive solution) and/or leucoreduced red cell concentrate (LR-RCC in AS-3 additive solution) were purchased from collection facilities: BSC (Biological Specialty Corporation, Colmar, PA, USA); ResBC (Research Blood Components, Boston, MA, USA); Bonfils Blood Center, Denver, CO, USA; IBR/MBC (Innovative Blood Resources/Memorial Blood Center, St. Paul, MN, USA); RIBC (Rhode Island Blood Center, Providence, RI, USA). Blood was collected from healthy donors. Blood products from ResBC and BSC are collected from remunerated donors specifically for research use. At the time of donation, all blood donors signed an institutional review board approved informed consent form.

Methods for collection, leucoreduction and shipping of red cell concentrate products from each source

ResBC: WB (CP2D) and pRBC (without AS)

pRBC (no additive solution added) was either placed in 1–6 °C storage or held at ambient temperature until it was transported by courier to the New Health Sciences, Inc. (NHSi) Blood Service Lab (BSL), Cambridge, MA, USA, within 2–6 hours of collection. AS-3 additive solution was added and leucofiltered by Leukotrap SC RC System (Haemonetics, Braintree, MA, USA), 40 units/case (PN: 430-40) at ambient temperature as per the manufacturer's instructions. WB products were collected into Haemonetics CP2D/AS-3 sets and transported by courier at ambient temperature to NHSi BSL. WB units were sampled then further processed by centrifugation followed by the Compomat G5 (Fresenius-Kabi, Bad Homburg, Germany) to express the plasma and add AS-3 additive solution to the pRBC to make RCC in AS-3 additive. The RBC/AS-3 product was leucofiltered as per the manufacturer's instructions and RCC parameters were measured as below.

Whole blood products in CP2D: BSC, IBR/MBC and Bonfils BC

WB products were collected into Haemonetics CP2D/AS-3 set, then shipped overnight on wet ice (IBR/MBC), 1–10 °C (Bonfils) or at ambient temperature (BSC) to the NHSi BSL where it was processed to RCC as described above. Products from BSC were less than 18-hours old upon arrival at the NHSi BSL.

Leucoreduced RCC in AS-3: Bonfils BC

LR-RCC (AS-3) products were prepared from WB (CP2D) or by apheresis, packed at ambient temperature then shipped overnight to NHSi BSL.

RIBC: LR-RCC (AS-3)

LR-RCC (AS-3) products were prepared from WB (CP2D), packed in wet ice and transported via courier for delivery by or before 8:00 am next day to the NHSi BSL.

Extended storage of undisturbed RBC

For the oxygen uptake study, 10 full units of AS-3 LR-RCC in standard PVC RBC storage bag (HAE PN 126-92 or 126-93) were sampled at day 0, then stored upright in blood shoes (acrylic blood unit holders) at 1–6 °C in a blood bank refrigerator. At day 21, the bags were mixed thoroughly and sampled for blood quality testing, then placed back into undisturbed refrigerated storage until the last sampling at day 42.

Preparation of samples for %SO₂ dose response study

Each leucoreduced double red blood cell unit in AS-3 additive solution was split 6 ways: unprocessed control, and five levels of pre-storage %SO₂: hyperoxygenated (90–98%), 20±2%, 10±2%, 5±2% and <3%. Hyperoxygenated unit was made by adding calculated volume of O₂ gas in the bag; for O₂-reduced subunits, RCC was circulated through neonatal membrane oxygenator (Sorin D100, Sorin, Arvada, CO, USA) as per the manufacturer's instruction using N₂ and CO₂ sweep gas adjusted to achieve desired levels of %SO₂ and pCO₂ (25±5mmHg, 37 °C). pCO₂ levels were unaltered for unprocessed control and hyperoxygenated units. Oxygen-reduced units were stored inside gastight canisters filled with N₂, with two packs of oxygen sorbent (SS-200, Mitsubishi Gas Chemical America, New York, NY, USA). Units were sampled bi-weekly for metabolomics workflow and blood gas/co-oximetry. Oxygen-reduced units were sampled inside a glove-box filled with N₂, separated into RBC pellet and supernatant fractions, snap frozen and stored at −86 °C, then shipped in dry ice to University of Colorado, Denver, USA, for metabolomics workflow²⁵. Adenosine triphosphate (ATP), 2,3-bisphosphoglycerate (BPG) and haemolysis were measured at days 1, 21 and 42, as described below. RCC from 4 volunteer subjects were processed and analysed.

Laboratory measurements

Cell counts and RBC indices were measured by a blood cell analyser (Sysmex XE2100-D, Kobe, Japan), blood gas, total haemoglobin concentration and %SO₂ were measured by ABL90 Flex (Radiometer, Copenhagen, Denmark) and supernatant Hb was measured by spectrophotometry (Hemocue PLS LOW HB, Brea, CA, USA) for haemolysis calculation. ATP and 2,3-BPG samples were deproteinised with TCA (DiaSys; Deutschland Vertriebs-GmbH, Holzheim, Germany; cat# G10784) stored frozen at −86 °C until measurement using a CE-marked ATP Hexokinase kit (DiaSysDeutschland Vertriebs-GmbH; reagent cat# 1 6201 99 10 021; standard cat# 1 6200 99 10 065) and the Roche IVD 2,3-BPG kit (Sigma-Aldrich, St. Louis, MO, USA; cat# 10148334001), a standard was prepared from 2,3-BPG pentacyclohexylammonium (Sigma-Aldrich; cat# D9134).

Results

A total of 492 units RCC (including 209 units component-separated from WB received after overnight shipment) from five different sources were examined for oxygen content as represented by %SO₂. The data set is separated according to type of process; handling history and other pertinent characteristics such as duration of RBC exposed to leucocytes/platelets prior to component separation along with mean %SO₂ values are summarised in Table IA and IB.

Table IA.

%SO₂ of leucoreduced red cell concentrate (RCC) obtained as is or manufactured into RCC from various suppliers.

Supplier	N	Mean (95% CI)	SD	Minimum	Median	Maximum	Skewness	Kurtosis	ANOVA
All	492	46.1 (43.5, 47.6)	17.3	11.4	43.8	96.5	0.54	−0.41
Bonfils BC, Denver, CO, USA	22	35.6 (29.9, 41.3)	12.9	18.7	34.4	68.9	1.33	1.99	Note 1
BSC (Biological Specialty Co.), Colmar, PA, USA	83	39.8 (36.1, 43.4)	16.5	11.4	35.6	82.8	0.8	−0.12
Memorial BC, St. Paul, MN, USA	9	41.2 (28.4, 54.0)	16.3	21.5	40.7	74.1	0.79	0.59
Research Blood Components, Boston, MA, USA	136	58 (55.0, 61.0)	17.5	19.1	59.2	94.6	−0.17	−0.53
Rhode Island BC, Providence, RI, USA	243*	42.7 (40.9, 44.5)	14.4	16	39.7	96.5	0.78	0.51
All suppliers except research blood components	367	41.6 (40.0, 43.1)	15	11.4	38.8	96.5	0.77	0.26	Note 2

Note 1: there is a statistically significant effect for %SO₂ by supplier (p<0.001). Note 2: No significant statistical difference between suppliers for %SO₂ if Research Blood Components (ResBC) is excluded (p=0.105).

^*Includes n=15 with modified saline-adenine-glucose additive in place of AS-3, with %SO₂=44.3±12.5. CI: confidence interval; SD standard deviation.

Table IB.

%SO₂ from various blood product types as received.

Category of blood product	N	Mean (95% CI)	SD	Minimum	Median	Maximum	Skewness	Kurtosis	ANOVA
Received as whole blood	209	34.4 [32.0, 36.8]	17.6	4.8	29	88.2	0.73	−0.31	Note 3
Received whole blood leucoreduced	105	36.5 [33.1, 39.8]	18.5	8.4	32.5	82.0	0.66	−0.4
Received as leucoreduced red cell concentrate#	243	42.7 [40.9, 44.5]	14.4	16.0	39.7	96.5	0.78	0.51

Note 3: there is a statistically significant effect for %SO₂ by product type (p<0.001).

^#Leucoreduced within 8 hours and shipped to NHSi laboratory in Cambridge, MA, USA. CI: confidence interval; SD standard deviation.

Distribution of collected whole blood and processed RCC within 42 hours of blood collection

The %SO₂ of 492 units of RCC, all measured within 24 hours of phlebotomy, is shown in Figure 1. Based on a probability plot to determine normality of the distribution, it was not normal according to the Anderson-Darling test (p<0.005; data not shown). The mean was 46.1% with a standard deviation of 17.3% with a surprisingly large range of 11.4–96.5%. The mean was significantly lower than expected when compared to the normal value for %SvO₂ as measured from a central venous line (approximately 74%)³⁷. In order to further examine the source of the wide pre-storage %SO₂ distribution, the blood sources, handling, and product types of all measured RCC units were further investigated (Table I). From five different suppliers who provided whole blood, leucoreduced whole blood, non-leucoreduced and leucoreduced pRBC, all units were processed to leucoreduced RCC within 24 hours of collection (Table IA). Comparing five suppliers, LR-RCC from ResBC showed 16.4% higher %SO₂ than the other four. Whole blood shipped overnight showed lower oxygen levels compared to leucoreduced RCC made at the collection site and shipped overnight to our laboratory, while leucoreduction processed at our laboratory caused a small increase in %SO₂ (Table IB).

Figure 1.

Histogram of the incoming %SO₂ measured after leucoreduction.

The overall distribution of leucoreduced red cell concentrate failed an Anderson-Darling test for normality when viewed on a probability plot (p<0.005). It was skewed right (skewness 0.54, kurtosis −0.041), though the shape of the distribution is generally normal. Likely the mean SO₂ (46.1% 95% CI [43.5, 47.6]) is a valid measure of central tendency, though median SO₂ (43.8%) is also reported. SO₂: oxygen saturation.

Estimating oxygen absorbed by RCC during storage

Unless stored in an oxygen-free environment or wrapped in an oxygen barrier film, RBC gradually absorb oxygen through the PVC storage bag. We examined this rate by storing 10 whole units of RCC (leucoreduced in AS-3 additive) in a standard PVC bag that was a part of the blood collection kit. Units were sampled on days 0 or 1, 21 and 42. During storage, units were undisturbed, except for mixing to obtain samples at day 21. The results from individual RCC units are shown in Figure 2A. During the 42-day shelf life of these RCC, the rate of %SO₂ can be empirically fitted with a power function.

Figure 2.

Oxygen uptake by red cell concentrate (RCC) stored undisturbed during 6-week storage.

(Left) %SO₂ gain measured in 10 full units during six weeks of storage. Except for mixing and sampling at day 21, units were undisturbed at 4 °C. Lines are empirical fit using power function. (Right) Visualisation of oxygen gain during storage. %SO₂ distribution from Figure 1 was transformed for weeks 2, 4, and 6 based on data from the left panel using an empirical simulation algorithm (equation 1 in the Results section). %SO₂ is shown in X-axis as in Figure 1. Three bars on the right represent frequency of units that were calculated to be above 90%. Numbers above curves and bars represent weeks in storage. %SO₂: percent oxygen saturation.

The oxygen absorption rate depends on the frequency and extent of mixing, as well as how much of the bag surface is exposed to ambient air during storage. To provide a rough estimate of how this oxygen absorption through the PVC bag during RCC storage might affect %SO₂ distribution, Figure 2B was generated by applying an empirical equation obtained from data in Figure 2. For this specific storage configuration, three points in each unit can be empirically fit by a power function with R² more than 0.99:

$$\begin{array}{l}\%{\text{SO}}_{2\hspace{0.17em}(\text{day\hspace{0.17em}}x)}=\%{\text{SO}}_{2\hspace{0.17em}(\text{day\hspace{0.17em}}1)}\times {t_{(x\hspace{0.17em}\text{day})}}^{(k)};\\ k=0.3187-0.00356\times \%{\text{SO}}_{2\hspace{0.17em}(\text{day\hspace{0.17em}}1)}\end{array}$$ (1)

where %SO₂ (day) is simulated %SO₂ at day x with the initial %SO₂ (day 0 or 1) at day t, and the exponent k is a function of the initial value of %SO₂. Using this simulation, the distribution on Figure 1 with a mean %SO₂ increased from 45.9±17.6% (range 11.4–96.5%) at day 0 to 77.1±12.5% (range 32.2–100%) after day 42 of storage.

Effects of oxygen content on the quality of stored RBC

Although evidence for accumulation of oxidative storage lesions is abundant in the literature^24,26,31,38, the relationship between the dose of oxygen and the extent of oxidative damage has not been investigated systematically. We undertook a study to examine this relationship with outcome parameters in standard blood quality as well a full, non-specific “omics” workflow (D'Alessandro et al., 2017; unpublished manuscripts). From each double RBC unit obtained from a single donor, we prepared 6 split units with differing levels of pre-storage %SO₂. In this study, a smaller volume of RCC (initially 100 mL in a 150 mL transfer bag) was mixed and sampled bi-weekly. This bi-weekly mixing and sampling leads to a higher oxygen gain rate compared to the full unit storage (Figure 2), reaching 100% saturation in three weeks for unprocessed control units stored in ambient air. On the other hand, %SO₂ levels slowly decreased in the O₂-depleted units as they were stored in an oxygen-free atmosphere. Instead of rapidly gaining oxygen, these units lost nearly 50% of haemoglobin oxygen saturation during 42 days (20% to 11%, 10% to 5%, 5% to 3% and 3% to less than 1.3%; well below accurate detection capabilities of the the co-oximeter).

Free intracellular methaemoglobin is an unstable molecule that readily denatures into haemichromes and then to globin and haemin during refrigerated storage of RBC. However, it is apparent from Figure 3B that the steady-state concentration of methaemoglobin is %SO₂-dose dependent and it appears to reflect %SO₂ levels at each sampling time.

Figure 3.

Changes in %SO₂ levels and concentration of methaemoglobin.

Parameters are measured weekly using co-oximeter/blood gas analyser. (A) %SO₂ levels during storage. (B) Methaemoglobin levels during storage. Set pre-storage %SO₂ values at day 0: from bottom to top lines; < 3% (. . .); 5% (- - -); 10%, unprocessed (− −); 20% (— — ); unprocessed (thick gray line); and more than 90% (solid line). %SO₂: percent oxygen saturation.

Representative biomarkers reflective of the overall redox status or oxidative damage are shown in Figure 4A-C. Glutathione (GSH)/glutathione disulfide (GSSG) levels are an indicator of the overall redox environment in the RBC cytosol and are inversely dose dependent with %SO₂ levels, especially at the end of storage with the exception of the lowest %SO₂ unit (Figure 4A). In order to examine levels of lipid oxidation during RCC storage, we examined selected oxidation products of polyunsaturated fatty acids using a quantitative lipidomics workflow³⁹ (Figure 4B and C). 16- and 17-hydroxydocosahexaenoic acid (HDoHE) as well 8-, 9-, and 11-hydroxyeicosatetraenoic acid (HETE) are non-enzymatically oxidised products of docosahexaenoic acid and arachidonic acid, respectively. Significant reductions in accumulated oxidation products of polyunsaturated fatty acids were observed with low %SO₂ storage.

Figure 4.

Effects of %SO₂ levels on metabolic and biochemical parameters.

(A) GSH/GSSG ratio during storage. Set pre-storage %SO₂ values at day 0: from bottom to top lines; < 3% (. . .); 5% (- - -); 10%, unprocessed (− −); 20% (— — — ); unprocessed (thick gray line); and more than 90% (solid line). (B) Concentrations of non-enzymatically oxidised PUFA by oxidation of docosahexaenoic acid (16- and 17-HDoHE) at the end of storage. (C) Concentrations of non-enzymatically oxidised PUFA by oxidation of arachidonic acid (8-, 9-, and 11-HETE) at the end of storage. (D) ATP levels at days 1, 21 and 42. (E) 2,3-BPG levels. (F) Haemolysis. Pre-storage %SO₂ levels are indicated in dark to light bars (from left to right). Unprocessed control: more than 90%, 20%, 10%, 5%, and less than 3%. * and #: p<0.05. %SO₂: percent oxygen saturation; GSH: glutathione; GSSG: glutathione disulfide; PUFA: polyunsaturated fatty

The effects of %SO₂ levels on standard parameters of RBC storage quality are shown in Figure 4D-F. ATP levels are reduced in both the control and high %SO₂ levels compared to less than 20% SO₂ after 21 days (Figure 4D). The hyperoxygenated units showed significantly lower ATP levels compared to the unprocessed control. No apparent oxygen dose response was observed below 20%. 2,3-BPG levels are significantly elevated on day 1 and day 21 for all oxygen-reduced units regardless of %SO₂ level compared to units without any O₂ control (Figure 4E). A significant reduction in haemolysis was observed for all O₂-reduced units compared to the non-reduced units. The lowest level of haemolysis was observed with lowest %SO₂ at day 42, reaching significance (p<0.049) compared to the units with 20% SO₂ (Figure 4F).

Discussion

Data shown in Figure 1 resulted from the first detailed study with a large number of units (n=492) on oxygen content of whole blood and prepared RCC. Since more than 95% of the oxygen in a typical unit of blood is bound to haemoglobin, %SO₂ was used as a measure of oxygen content in RCC prepared for blood bank storage.

Measuring %SO₂ using a co-oximeter requires mixing and obtaining a representative sample of RBCs from a unit immediately prior to measurement. To our knowledge, no systematic study to determine freshly collected %SO₂ from blood donors is available. The mean value of 45.9±17.6% appears lower than 61.8±3.7% reported in a reference book for healthy subjects⁴⁰, or 73.5±13.3% reported using central venous line³⁷. The observed distribution of %SO₂ values was surprisingly wider than expected from those published values.

Peripheral blood %SvO₂ is likely determined from a small volume of blood obtained from the antecubital vein pressurised with a tourniquet prior to venipuncture. On the other hand, the higher values measured in central venous lines were from patients in the intensive care unit. Although it is generally assumed that %SvO₂ of free-flowing blood collected in the bag is similar to reference values in text books, this newly collected data may offer new insights into oxygenation status of healthy individuals. The high end of observed values cannot be explained by trapped air inside the blood collection bag, O₂ dissolved in an anticoagulant solution, or O₂ added with leucofitration and additive solution as evidenced by lack of a large increase in %SO₂ between WB before and after filtration, as well as the final resultant LR-RCC shown in Table IB. Respiring leucocytes and platelets may have caused a decrease in %SO₂ levels from whole blood units held overnight at room temperature as evidenced by approximately 7% higher oxygen saturation of LR-RCC observed produced shortly after phlebotomy at the collection facility and shipped overnight. However, it cannot explain a large number of units with low %SO₂ values that were not held overnight as whole blood. These data might suggest that %SvO₂ values in healthy subjects are not as tightly controlled as blood pH, and that it has a wide range among the diverse donor population, donor age, sex, fitness status, body mass index, as well as genetic predisposition represented by this data. When mean %SO₂ from different suppliers was compared, ResBC showed significantly higher %SO₂ than the other suppliers. It is noteworthy that ResBC is a “for profit” supplier of blood components for research purposes that relies on remunerated blood donors, located near several college campuses. Their donor profile is probably different from typical non-remunerated volunteer blood donors. Data shown here warrant further study to identify various donor characteristics affecting the observed wide distribution of %SO₂ of collected blood.

For leucoreduced red cells, oxygen content increases gradually as the PVC film of the storage bag is permeable to O₂. This phenomenon was previously described with a small study of non-leucoreduced blood⁴¹, but no study has reported on leucoreduced RCC stored undisturbed for a 3-week interval. Because of the relatively low solubility of oxygen in an aqueous salt solution (RBC additive solution), coupled with the moderate permeability of PVC, the rate of %SO₂ increase during storage is dependent on mixing, as well the storage bag surface area-to-RCC volume ratio. Increased surface area-to-volume ratio and mixing substantially increase O₂ uptake rate as compared to the full volume that was mixed only once (Figure 3 vs Figure 2A). On the other hand, %SO₂ gain rate was slower than in Figure 3 when full units were undisturbed for the entire 42 days and then measured (data not shown).

There is a general consensus among the researchers that oxidative damage is one of the major drivers of RBC storage lesion development^24,26,31,38, and the critical role played by oxidised haemoglobin had been recognised more than two decades ago^29,31,33. Since oxygen is a substrate for haemoglobin oxidation, as well as a sustaining substrate for lipid peroxidation cycle, the rate of oxidised product formation is expected to be positively correlated with concentration of free oxygen in the cytosol.

Methaemoglobin is unstable and readily denatures to haemichromes, haemin and globin at cold temperature⁴², and thus its concentration does not increase significantly during RCC storage. However, steady-state concentration of methaemoglobin is highly dose dependent on oxygen (Figure 3B). The ratio of reduced to oxidised glutathione indicates an extent of oxidative stress in stored RCC, and such higher ratios were observed with lower %SO₂. An exception was found in the lowest %SO₂ unit, which measured less than 1.5% beyond day 28, well below the limit of detection of 3% by co-oximetry. At such an extremely reduced intracellular environment, there might be some unknown chemical reactions that can cause GSH depletion.

In order to examine the effect on oxygen depletion on oxidative stress to membrane lipids, quantitative metabolomics of selected samples was undertaken to quantify concentrations of non-enzymatically oxidised polyunsaturated fatty acids in supernatant. The results confirm a significant reduction in specifically non-enzymatic polyunsaturated fatty acids (PUFA) oxidation products arising from docosahexanoic acid as well as arachidonic acid (Figure 4B and C).

Dependence of %SO₂ levels on standard RBC quality parameters, ATP, 2,3-BPG and haemolysis were generally better with oxygen reduction but not dose dependent as long as oxygen was reduced to at least 20%. Day 21 and 42 ATP levels of hyperoxygenated units were significantly lower than the control, while %SO₂-reduced units were higher than control. Higher ATP and 2,3-BPG levels achieved by O₂-reduced units is in agreement with enhanced glycolytic flux through metabolic modulation caused by increasing the ratio of deoxy/oxyhaemoglobin after %SO₂ reduction. For control and hyperoxygenated units, by diverting the glycolytic flux away to the pentose phosphate pathway reducing ATP production, it allows NADPH replenishment and subsequent GSH synthesis⁴³.

A consequence of uncontrolled %SO₂ levels in collected blood at the time of storage is the contribution this variable could make to the inconsistency of red blood cells at the time of use. This study has shown that the starting %SO₂ levels vary widely at the beginning of storage and change at variable rates during storage. The effect oxygen has on various in vitro parameters of blood quality has also been shown, including dose-dependent relationships, e.g. methaemoglobin levels. By choosing to not control oxygen at the time of and throughout storage, this variability becomes part of the characteristics of the blood supply. In contrast, applying a strategy to manage oxygen levels below a certain range (ex. 15% in Figure 5) and maintaining this range during storage, two benefits are possible: 1) the improvement of the many parameters measured that are affected by %SO₂; and 2) significantly reduced inconsistency with regard to %SO₂, and by extension the parameters that change based on oxygen saturation. This strategy would be consistent with best practices in biopharmaceutical manufacturing where building quality into a product means establishing identity, strength, purity, and other quality characteristics designed to ensure the required levels of safety and effectiveness, and by designing the product and manufacturing process in such a way as to consistently produce a quality product (e.g., Code of Federal Regulations Part 211, USA). The improvement of in vitro characterisation tools has allowed the degradation of red blood cells during storage to be sufficiently elucidated such that the root causes of these changes can be identified and dealt with. Management and control of oxygen levels during storage of RBC is a process that can provide increased quality and consistency of this critical therapy.

Figure 5.

Controlling %SO₂ levels throughout storage using a disposable device.

Pre-storage %SO₂ distribution (from Figure 1) is shown as a gray curve in centre. Right curve and a bar graph beyond 90% represent %SO₂ gain after 42 days of storage (from Figure 2). The left curve is generated based on transforming the %SO₂ distribution days of storage (from Figure 2). %SO₂: percent oxygen saturation.

Limitations

Quality of RCC varies depending on specific procedures used for blood collection, transportation and component processing that include time-course, temperature history and component separation/ leucoreduction methods^44,45. %SO₂ distribution reported in Figure 1 is for whole blood or RCC that underwent specific processing conditions as shown in Table I, and thus may not be applicable for other processes, such as overnight room temperature hold, buffy coat method as well as immediate cool down (on ice pack) followed by component processing/ filtration within 72 hours. The same limitations apply to the kinetics of oxygen gain by stored RCC during 6-week shelf life. Several factors affect the rate of %SO₂ gain independent of factors attributed to donor and blood collection (haemoglobin content, volume, and haematocrit). Such examples include placement during storage (surface area of the bag exposed to air) and transportation, as well as mixing. Ten units used for this study were mixed and sampled only once and were otherwise undisturbed during 6-week storage, which may cause %SO₂ increase rate to be underestimated at blood banks.

Conclusions

Oxidative damage is often cited as an important source of damage to red blood cells during storage and is implicated as a source of adverse events or reduced efficacy of transfusions. In order to improve the understanding of the impact that oxidative damage has on both individual units of blood and the blood supply as a whole, this study evaluated %SO₂ in red blood cell units, including the variability of %SO₂ in fresh blood at the beginning of storage, the changes in the %SO₂ level during storage, and the %SO₂ dose dependence of a subset of salient measures of blood quality. The study reveals that the degradation of important blood quality parameters is clearly %SO₂ dose dependent. Coupled with the findings that %SO₂ levels vary widely at the start of storage and inevitably increase towards full O₂ saturation, these data strongly indicate that %SO₂ levels in blood is an important and underappreciated source of both reduced quality for a given unit of blood during storage and inconsistency across all units of blood in storage. Thus, control of %SO₂ may lead to a reduction of adverse events associated with RBC transfusion while increasing its efficacy.