PS1591 FUNCTIONAL PROPERTIES OF THE HEMOGLOBIN FROM STORED RED BLOOD CELLS CONCENTRATES AFTER LEUKOREDUCTION
Authors: Grigoleto M.R. P. , Castro M.L. B. , Costa F.F. , Sonati M.D. F. , Jorge S.E. 
Published: June 2019

Background:

The viability of stored red blood cells (RBCs) after buffy coat reduction presents important implications for hemotherapy. However, the impact of these procedures on the hemoglobin (Hb) is not well established.

Aims:

The aim of the present study was, therefore, to investigate the functional efficiency of the Hb isolated from stored RBC concentrates after leukoreduction processes.

Methods:

Two RBC concentrates (CPD/SAGM), collected from blood donors after consent, were fractioned into 4 subunits (quadruple bags) for: filtration (F), irradiation with γ‐ray (I) and filtration+irradiation (F+I); compared to non‐buffy coat reduced RBCs (controls – C). Hb was extracted after 48 hours, 21 e 42 days of storage and purified by exclusion and ion‐exchange chromatography (Sephadex 25G and Amberlite MB‐3, respectively) in Hepes buffer. O2 affinity of the stripped Hb was evaluated by determining the p50 of Hb [70 M/heme] at pHs of 6.5, 7.0, 7.5 and 8.0 (spectrophotometry‐tonometry method) for Bohr Effect calculations. Heme‐heme cooperativity was determined by Hill coefficient (n) at pH 7.5. The activity of the allosteric effector inositol hexaphosphate (IHP, 0.1 mM) was also tested over pH rage (6.5–8.0). Biochemical parameters such as 2,3‐BPG (2,3‐Diphosphosglycerate Kit, Roche), ATP (ATP Determination Kit, Thermo Fisher), hemolysis percentage (Harboe's method) and pH (by radiometry ‐ Radiometer, ABL 800) were also monitored.

Results:

No functional alterations were observed in 48 hours. However, the Hb‐O2 equilibrium seemed to be affected upon 21 days of storage in leukoreduced samples. The p50 was increased in F+I stripped samples at pH 6.5 [CLog(p50) = 0.68 (SD 0.06); F+ILog(p50) = 0.77 (SD 0.25)] and pH 7.0 [CLog(p50) = 0.52 (SD 0.01); F+ILog(p50) = 0.67 (SD 0.05)], as well as decreased in all leukoreduced samples at pH 8.0 [CLog(p50) = 0.51 (SD 0.30), FLog(p50) = 0.16 (SD 0.09), ILog(p50) = 0.11 (SD 0.01), F+ILog(p50) = 0.17 (SD 0.08)], resulting in altered Bohr Effect. The p50 remained increased in I and F+I samples with 42 days of storage at pH 6.5 [CLog(p50) = 0.60 (SD 0.03); ILog(p50) = 0.77 (SD 0.20), F+ILog(p50) = 0.65 (SD 0.06)] and pH 7.5 [CLog(p50) = 0.32 (SD 0.03); ILog(p50) = 0.48 (SD 0.15), F+ILog(p50) = 0.45 (SD 0.05)]. The presence of the allosteric effector IHP resulted in increased p50 (pH 7.0) in F, I and F+I samples, purified in 48 hours [CLog(p50) = 1.70 (SD 0.04), FLog(p50) = 1.80 (SD 0.02), ILog(p50) = 1.78 (SD 0.02), F+ILog(p50) = 1.77 (SD 0.02)]. Increased p50 was also observed in I samples with 21 days (pH 7.0): [CLog(p50) = 1.65 (SD 0.06); ILog(p50) = 1.80 (SD 0.02)]. As expected, the pH of all blood bags decreased according to storage time [pH 7.03 (48 hours); pH6.64 (21 days) and pH 6.42 (42 days)], higher hemolysis percentage and lower concentrations of 2,3‐BPG and ATP could be found in long‐term samples.

Summary/Conclusion:

Although preliminary, these results suggest that storage time and buffy coat reduction, specially irradiation, could affect the functional efficiency of isolated Hb, mainly in terms of interaction to ions H+ (calculated by Bohr Effect), which could shift the Hb‐O2 equilibrium, conferring less affinity to its ligand. Financial Support: Fapesp, CNPq, Capes, Faepex‐Unicamp.