Introduction: Hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction, leading to reduced lifespan of RBCs, as they are removed from the circulation. Measuring RBC lifespan is of interest to better evaluate hemolytic anemia severity. To date, the methods used to assess RBC lifespan are invasive and time-consuming, requiring infusion of autologous labeled RBCs and longitudinal follow-up.

Normal RBC aging is characterized by physiological alterations such as cellular dehydration and decrease in enzymes activity. Simultaneously, the concentration of glycated Hb (G-Hb), of which HbA1c form is the most prevalent, increases. Hb glycation is a non-enzymatic reaction whose intensity is modulated by plasma glucose concentration and RBCs longevity. Consequently, in non-diabetic subjects, the level of Hb glycation is directly proportional to RBCs lifespan in a linear-dependent relationship. Moreover, in certain diseases such as sickle cell disease (SCD) or X-linked G6PD deficiency, there can be heterogeneous lifespan that require a cellular approach rather than an average measure.

To circumvent the disadvantages of the currently used methods, we developed a novel assay to measure RBC lifespan based on the assessment of intracellular glycated Hb by flow cytometry. This method is based on technology we have developed to measure fetal and sickle hemoglobins per RBC (Hebert, 2020).

Methods: G-Hb was measured in RBCs from healthy donors (HD) and non-transfused SCD patients, in reticulocytes (RNA-positive cells) and RBCs. Exclusion criteria were diabete or pre-diabete. HD samples were splited into two sub-groups. Group-A (n=50) was used to determine the lifespan of reticulocytes based on the disappearance of RNA staining (time required for a reticulocyte to mature to an erythrocyte). Group-B (n=46) was used to assess accuracy of the measure of the half-life.Single cell G-Hb was assessed by flow cytometry using a fluorescent monoclonal anti-human HbA1c antibody. Glycation of Hb in reticulocytes was assessed by the addition of thiazole orange to stain the RNA. A standard linear equation was calculated as the relationship between thiazole orange and glycated Hb intensity and was used to convert the G-Hb fluorescence into time.

Results: In HD RBCs, glycated Hb fluorescence intensity followed a log-normal distribution in every samples (n=96), indicating homogeneity of the RBC population in aging. G-Hb mean fluorescence intensity (MFI) of every RBC was set as the level of glycation obtained for normal half-life of RBCs (50 days). In HD reticulocytes, G-Hb intensity was negatively correlated to RNA intensity in a Log-linear dependent manner (p<0.001). This relationship was obtained in all HD indicating its consistency. A standard linear curve was calculated as the relationship between decrease in RNA and increase in G-Hb intensity, using mean values measured in HD.

A delta-RNA intensity was calculated as the difference in thiazole orange intensity between the highest and the lowest value. The corresponding delta-glycated Hb intensity is the increase in glycation during the time reticulocytes mature into erythrocytes. Using the standard equation, and by setting glycated Hb MFI equals 50 days, reticulocytes lifespan (time to lose RNA) was 3.6±0.8 days in group-A.

The G-Hb MFI measured in RBCs form HD in group B was converted into time using the standard equation, by setting the time reticulocytes mature into erythrocyte as 3.6±0.8 days. RBCs half-life was 49.5±11.5 days.

In SCD RBCs, GHb intensity was not following a log-normal distribution, indicating intra-individual variability in aging. Using the previously calculated standard equation in HD, the SCD reticulocytes lifespan was 3.3±0.3 days and the mean RBC lifespan was 77±8.5 days (n=16).

Conclusion: Single-cell glycated Hb, as assessed by flow cytometry, enables the precise measurement of RBC lifespan. Normal RBCs half-life we measured using this method aligns with findings from all other studies using reference methods (51 Cr- or biotin-labeled RBCs). The capacity to simultaneously measure the age of different RBC subpopulations makes this method highly valuable in all hemolytic conditions. This novel tool is non-invasive, requires only a single-point measurement, and is easy to implement. It should be considered for evaluating the efficacy of treatments aimed at improving hemolysis, such as pyruvate kinase activators or allosteric Hb modulators.

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2025
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