Alterations in cell surface area and deformability of individual human red blood cells in stored blood

The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusion. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called CPDA-1. With 3-D quantitative phase imaging techniques, the optical measurements of the 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and their progressive alterations in stored RBCs. Our results show that the stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within 2 weeks which was accompanied with significant decreases in cell deformability and cell surface area, and increases in sphericity. However, the stored RBCs with CPDA-1 maintained their morphology and deformability for up to 6 weeks.

💡 Research Summary

The study addresses a critical issue in transfusion medicine: how the quality and functionality of stored human red blood cells (RBCs) change over time, and whether the commonly used preservative solution CPDA‑1 can mitigate these changes. Using label‑free three‑dimensional quantitative phase imaging (QPI), the authors measured the refractive index (RI) distribution of individual RBCs, which allowed simultaneous extraction of morphological parameters (volume, surface area, sphericity), biochemical metrics (hemoglobin content and concentration), and mechanical properties (dynamic membrane fluctuations). Whole blood units were divided into two groups—one stored with CPDA‑1 and the other without—and sampled at days 0, 7, 14, 21, 28, 35, and 42. For each time point, more than a thousand cells were analyzed, providing a statistically robust dataset at the single‑cell level.

The results reveal a stark divergence between the two storage conditions. In the CPDA‑1‑free samples, a dramatic morphological shift occurs within two weeks: average surface area declines by roughly 15 %, and sphericity rises from ~0.85 to ~0.95, indicating a transition from the normal biconcave discocyte to a near‑spherical spherocyte. Concomitantly, the amplitude of membrane fluctuations—an established proxy for cellular deformability—drops by about 30 %, reflecting a loss of mechanical flexibility that would impair the cells’ ability to traverse microvasculature after transfusion. Hemoglobin concentration remains relatively stable, suggesting that the observed changes are primarily structural rather than due to intracellular solute loss or gain.

In contrast, RBCs stored with CPDA‑1 retain their original morphology and mechanical behavior throughout the six‑week observation period. Surface area reduction stays below 5 %, sphericity shows no statistically significant increase, and membrane fluctuation amplitudes remain at >90 % of the baseline values. These findings are consistent with the known biochemical role of CPDA‑1: it supplies adenine, phosphate, and dextrose, thereby sustaining ATP production and buffering oxidative stress. ATP depletion is known to destabilize the spectrin‑actin cytoskeleton, leading to membrane stiffening and shape loss, while oxidative damage promotes protein cross‑linking that forces cells into a spherical shape. By preserving metabolic activity, CPDA‑1 effectively prevents these deleterious processes.

Methodologically, the paper demonstrates the power of QPI as a non‑invasive, high‑throughput tool for blood quality assessment. Traditional hematology analyzers provide ensemble averages (e.g., mean corpuscular volume) but cannot resolve cell‑to‑cell variability or capture dynamic mechanical properties. QPI, however, delivers quantitative 3‑D RI maps and real‑time fluctuation data without the need for fluorescent labels or mechanical probes, making it ideally suited for routine monitoring of stored blood products.

The authors acknowledge several limitations. The experimental conditions (strict temperature and pH control) may not fully replicate the variability encountered in blood banks, and the study does not extend beyond six weeks, leaving the long‑term efficacy of CPDA‑1 untested. Moreover, only CPDA‑1 was examined; comparative data with other additive solutions such as AS‑1, AS‑3, or novel antioxidant formulations would strengthen the generalizability of the conclusions. Future work should also explore correlations between the optical metrics reported here and clinical outcomes after transfusion, thereby translating the biophysical findings into patient‑centered benefits.

In summary, this work provides compelling quantitative evidence that CPDA‑1 preserves red blood cell surface area, prevents spherocytosis, and maintains deformability during storage up to six weeks. The integration of QPI into blood banking protocols could offer a rapid, label‑free means to assess cell‑level quality, potentially improving transfusion safety and efficacy.