Erythrocyte Physiology and the Storage Lesion

A mature erythrocyte circulates for about 120 days while carrying oxygen through vessels narrower than its resting diameter. Its biconcave shape, membrane skeleton, and glycolytic metabolism preserve deformability and hemoglobin function. Refrigerated storage slows deterioration while allowing progressive metabolic and structural changes to accumulate.¹

Membrane structure and deformability

The red-cell membrane is a lipid bilayer attached to a protein skeleton. By dry mass, a commonly cited membrane preparation is approximately 52% protein, 40% lipid, and 8% carbohydrate; measured proportions vary with preparation and method. Phosphatidylcholine, sphingomyelin, and glycolipids concentrate in the outer leaflet, whereas phosphatidylserine and phosphatidylethanolamine concentrate in the inner leaflet. Adenosine triphosphate (ATP)-dependent enzymes maintain this lipid asymmetry. External phosphatidylserine marks a damaged or aging cell for clearance and increases during refrigerated storage.^1,2,3

Two membrane-protein groups work together:

The ankyrin complex links spectrin to band 3 and protein 4.2. At junctional complexes, protein 4.1R connects spectrin and short actin filaments to glycophorin C through p55. Adducin stabilizes the spectrin-actin junction. These attachments keep the bilayer and skeleton together as a cell repeatedly bends and recovers its shape.²

The biconcave disc supplies more membrane surface than a sphere of the same volume. This reserve allows the cell to elongate through capillaries and splenic interendothelial slits. Membrane loss lowers the surface-to-volume ratio and promotes a spherical shape. ATP depletion impairs energy-dependent membrane remodeling, while calcium accumulation, oxidation, and weakened protein attachments also reduce deformability. The spleen retains and removes rigid or damaged cells. Membrane loss produces a spherocyte. A bite cell forms when a splenic macrophage removes a Heinz body and the attached membrane from an oxidatively damaged cell.¹

Membrane transport and cell volume

Water moves rapidly through aquaporin 1, and cell volume follows the concentration of dissolved particles inside and outside the cell. Band 3 exchanges chloride for bicarbonate and supports carbon dioxide transport between tissues and lungs. Sodium, potassium, and calcium move more slowly and depend on pumps, channels, and cotransporters to maintain their gradients.

Fresh erythrocyte cytoplasm contains approximately 8 mmol/L sodium and 110 mmol/L potassium, compared with plasma concentrations near 140 mmol/L sodium and 4 mmol/L potassium. These values vary with donor, cell age, temperature, and measurement method. Several hundred sodium-potassium ATPase pumps per cell help maintain the gradients. A calmodulin-activated calcium ATPase keeps cytoplasmic calcium low. Both pumps use ATP from glycolysis. Refrigeration greatly slows pump activity. During liquid storage, potassium leaves the cells, sodium enters, and potassium accumulates in the component supernatant. Calcium entry and falling ATP contribute to membrane rigidity, phospholipid scrambling, and formation of microvesicles, small membrane-bound vesicles shed from the cell. Calcium can also activate the Gardos channel, a calcium-activated potassium channel, promoting potassium and water loss. The net change in cell volume varies with the storage solution and the balance of cation and water movement.^1,4

Erythrocyte metabolism

Loss of nuclei and mitochondria leaves mature erythrocytes dependent on existing proteins and cytosolic glucose metabolism. This metabolism supplies energy, reducing power, and control of hemoglobin oxygen affinity.^1,3

ATP supplies energy for membrane work. Reduced nicotinamide adenine dinucleotide (NADH) supports reduction of methemoglobin, the oxidized form of hemoglobin with ferric (Fe³⁺) heme iron that cannot bind oxygen. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) supports antioxidant defense.

About 90% of glucose normally enters glycolysis and about 10% enters the pentose phosphate pathway, with the proportion changing during oxidant exposure. Pathway defects can produce hemolysis or increased methemoglobin in laboratory testing. Low ATP impairs membrane pumps and deformability. Low NADPH leaves hemoglobin and membrane proteins vulnerable to oxidation. Failure to reduce methemoglobin lowers functional oxygen-carrying capacity.¹

The terms 2,3-bisphosphoglycerate (2,3-BPG) and 2,3-diphosphoglycerate (2,3-DPG) identify the same molecule. The first name shows that its two phosphate groups occupy different carbons. Many blood-bank sources and product labels use 2,3-DPG.^1,5

Hemoglobin oxygen affinity

Hemoglobin shifts between a tense conformation with lower oxygen affinity and a relaxed conformation with higher affinity. Deoxygenation opens the central cleft between the β chains. 2,3-BPG binds in this cleft and stabilizes the tense form, promoting oxygen release. Oxygen binding narrows the cleft and displaces 2,3-BPG. Cooperative binding among the four heme groups produces the sigmoid oxygen dissociation curve.^1,5

P50 is the oxygen partial pressure (PO₂) at which hemoglobin is 50% saturated. Adult hemoglobin has a P50 near 26 to 27 mm Hg at 37 °C, pH 7.4, and a carbon dioxide partial pressure (PCO₂) of 40 mm Hg. A higher P50 means lower affinity and easier oxygen release. A lower P50 means higher affinity and less oxygen release at the same oxygen partial pressure.^1,5

The Bohr effect is the decrease in hemoglobin oxygen affinity caused by increased carbon dioxide and lower pH. At a PO₂ near 100 mm Hg in the lungs, adult hemoglobin is nearly fully saturated. At a typical mixed-venous PO₂ near 40 mm Hg, saturation is about 75%, so resting tissues have extracted about one quarter of the carried oxygen.^1,5

Refrigerated red cells lose 2,3-BPG, which moves their curve to the left. After transfusion, 2,3-BPG synthesis resumes and the curve returns toward the recipient’s physiologic position. The rate of 2,3-BPG recovery varies with the recipient’s acid-base state, phosphate availability, anemia, and severity of illness.^3,5 Large-volume transfusion can temporarily lower 2,3-BPG in the recipient’s circulating red-cell mass; the magnitude depends on unit age, transfused volume, and recipient physiology.^1,3

Changes during refrigerated storage

The red-cell storage lesion is the progressive, partly reversible set of metabolic, structural, and functional changes that develops during storage at 1 to 6 °C. Cold temperature slows metabolism and membrane transport. Glycolysis continues in the closed container, consuming glucose and producing lactate and hydrogen ions. The timing and magnitude of individual changes depend on the storage system and duration: ATP may rise during the first week and then decline, whereas 2,3-BPG generally falls during conventional storage. Falling pH and substrate depletion impair metabolic enzymes and eventually contribute to loss of ATP and 2,3-BPG.^3,4,6

The rate of change differs among donors and processing systems. Anticoagulants, additive solutions, leukocyte reduction, irradiation, oxygen exposure, bag composition, and storage duration all affect the measured lesion. The component’s dating follows the labeling for its collection, processing, and storage system, including the applicable US Food and Drug Administration (FDA) approval or clearance.^1,3,6 The Blood Product Processing, Storage, Components, and Quality Control lists current solutions, temperatures, modifications, and quality-control requirements.

Conventional blood-container systems allow gas exchange; carbon dioxide permeability helps limit acidification during storage. HEMANEXT ONE is an FDA-cleared Class II container system that limits oxygen and carbon dioxide levels in the storage environment. Its current cleared indication specifies the red-cell component, anticoagulant or additive solution, processing conditions, storage temperature, and dating period. Some conventional polyvinyl chloride blood bags contain the plasticizer di(2-ethylhexyl) phthalate (DEHP). In a controlled human study, DEHP-plasticized bags reduced hemolysis and improved 24-hour recovery under the tested storage conditions.^7,8,9

Product quality and posttransfusion recovery

FDA evaluation of a red-cell storage system includes end-of-storage hemolysis and 24-hour in vivo recovery, the percentage of labeled cells remaining in circulation 24 hours after transfusion. In the FDA decision summary for the HEMANEXT ONE system, the hemolysis acceptance plan required a one-sided 95% lower confidence limit showing that at least 95% of units had less than 1% hemolysis at day 42. The 24-hour in vivo recovery criteria included a mean recovery of at least 75%, a standard deviation no greater than 9%, and a lower one-sided 95% confidence limit of at least 70% for the proportion of units with individual 24-hour recovery of at least 75%. Healthy volunteers received small volumes of their own labeled red cells in the autologous recovery study. The complete acceptance plan and labeled conditions are specific to the reviewed system.⁷

After transfusion, membrane-pump activity resumes, and ATP and 2,3-BPG begin to recover. 2,3-BPG synthesis begins within hours and average levels can approach baseline over approximately 36 to 72 hours; ATP recovery has been reported over approximately 24 to 48 hours.^1,3 Cells with irreversible membrane or oxidative injury are cleared, which lowers the recovered circulating fraction. Unit age is an incomplete measure of lesion severity because donor biology and processing alter the rate of change. Release and selection therefore follow the approved system, labeled expiration date, component inspection, quality control, and any recipient-specific procedure.⁶

The clinical importance of temporary 2,3-BPG depletion and the benefit of restoring 2,3-BPG during cardiac surgery or shock remain uncertain.³

Worked interpretation. Relative to a fresh or early-storage sample, a conventionally stored component generally has lower pH and 2,3-BPG and higher supernatant potassium and free hemoglobin; ATP may rise early and decline with prolonged storage. Low ATP during prolonged storage weakens cation control and deformability. Low 2,3-BPG explains the left-shifted oxygen dissociation curve. Increased free hemoglobin indicates greater hemolysis. Release follows the component’s approved quality limits, storage record, inspection findings, expiration date, and laboratory procedure.

References

1. Bloch EM, Campbell-Lee S, McKenna DH Jr, et al, eds. Technical Manual. 22nd ed. AABB; 2026. ISBN 978-1-56395-557-0. Accessed August 27, 2026.
2. Lux SE 4th. Anatomy of the red cell membrane skeleton: unanswered questions. Blood. 2016;127(2):187-199. doi:10.1182/blood-2014-12-512772.
3. Yoshida T, Prudent M, D'Alessandro A. Red blood cell storage lesion: causes and potential clinical consequences. Blood Transfus. 2019;17(1):27-52. doi:10.2450/2019.0217-18.
4. Flatt JF, Bawazir WM, Bruce LJ. The involvement of cation leaks in the storage lesion of red blood cells. Front Physiol. 2014;5:214. doi:10.3389/fphys.2014.00214.
5. Jaafar LS, Kourie CMR, El-Mallah CA, Obeid O. 2,3-Diphosphoglycerate: the forgotten metabolic regulator of oxygen affinity. Br J Nutr. 2025;134(10):803-815. doi:10.1017/S0007114525105345.
6. AABB, American Red Cross, America's Blood Centers, Armed Services Blood Program. Circular of Information for the Use of Human Blood and Blood Components. June 2024. Accessed August 27, 2026.
7. US Food and Drug Administration. De Novo Classification Request for the HEMANEXT ONE System: Decision Summary. BR220665. Published September 15, 2023. Accessed August 27, 2026.
8. US Food and Drug Administration. 510(k) Summary: HEMANEXT ONE. BK251294. Prepared November 13, 2025. Accessed August 27, 2026.
9. AuBuchon JP, Estep TN, Davey RJ. The effect of the plasticizer di-2-ethylhexyl phthalate on the survival of stored RBCs. Blood. 1988;71(2):448-452. PMID: 3337906.