Evolution of adverse changes in stored RBCs

Abstract

Recent studies have underscored questions about the balance of risk and benefit of RBC transfusion. A better understanding of the nature and timing of molecular and functional changes in stored RBCs may provide strategies to improve the balance of benefit and risk of RBC transfusion. We analyzed changes occurring during RBC storage focusing on RBC deformability, RBC-dependent vasoregulatory function, and S-nitrosohemoglobin (SNO-Hb), through which hemoglobin (Hb) O(2) desaturation is coupled to regional increases in blood flow in vivo (hypoxic vasodilation). Five hundred ml of blood from each of 15 healthy volunteers was processed into leukofiltered, additive solution 3-exposed RBCs and stored at 1-6 degrees C according to AABB standards. Blood was subjected to 26 assays at 0, 3, 8, 24 and 96 h, and at 1, 2, 3, 4, and 6 weeks. RBC SNO-Hb decreased rapidly (1.2 x 10(-4) at 3 h vs. 6.5 x 10(-4) (fresh) mol S-nitrosothiol (SNO)/mol Hb tetramer (P = 0.032, mercuric-displaced photolysis-chemiluminescence assay), and remained low over the 42-day period. The decline was corroborated by using the carbon monoxide-saturated copper-cysteine assay [3.0 x 10(-5) at 3 h vs. 9.0 x 10(-5) (fresh) mol SNO/mol Hb]. In parallel, vasodilation by stored RBCs was significantly depressed. RBC deformability assayed at a physiological shear stress decreased gradually over the 42-day period (P < 0.001). Time courses vary for several storage-induced defects that might account for recent observations linking blood transfusion with adverse outcomes. Of clinical concern is that SNO levels, and their physiological correlate, RBC-dependent vasodilation, become depressed soon after collection, suggesting that even "fresh" blood may have developed adverse biological characteristics.

Fig. 1.

RBC 2,3-DPG (A), potassium (B), pH (C), lactate (D), pO₂ (E), Hb O₂ saturation (SO₂) (F), cell-free Hb in storage medium (G), and RBC surface phosphatidyl serine (PS) expression (H) as a function of storage time. Data are median with 25th and 75th percentiles. P values represent significance for change over time.

Fig. 2.

SNO-Hb, related NO adducts, and vasoactivity of stored RBCs. (A–C and E) Total Hb-bound NO (A), Hb[Fe]NO (B), SNO-Hb (C, a calculated value equal to total Hb-NO minus Hb[Fe]NO), and RBC membrane SNO (E) were determined by the PC assay. (D) RBC (total) SNO was determined by the 3C assay. (F) Vasoactivity represents the percentage decrease in tension induced by RBCs in the bioassay (percentage of vasorelaxation). Because of the complexity of the membrane SNO assay, samples were assayed only at selected time points. Data are median with 25th and 75th percentiles. Unprocessed samples (open circles) were assayed immediately (0 h) and, for some parameters, after a 3-h delay in addition to assays at the indicated times after processing was begun (filled circles, beginning at 3 h). P values represent comparison between values in RBCs assayed immediately (0 h) vs. 3 h later (in unprocessed samples for A, B, C, and F). No significant change from 3 h to 6 weeks was observed for any of these variables in processed samples.

Fig. 3.

Alternative mediators of RBC-dependent hypoxic vasodilation. (A) RBC-dependent hypoxic vasodilator responses are NOS-independent. Fresh washed human RBCs [0.4% hematocrit (Hct) or ATP (10⁻⁶ M] were added to preconstricted rabbit aortic rings at 1% O₂ [PO₂ 7 mmHg (mmHg = 133 Pa)] in the absence or presence of the NOS inhibitor L-NAME in tissue baths as described, and the percentage of vasorelaxation was measured. *, P < 0.05. (B) Minimal nitrite-induced vasodilation in the absence or presence of RBCs. Nitrite (1 μM) was added to preconstricted rabbit aortic rings at 1% O₂ (PO₂ 7 mmHg) in the absence or presence of RBCs (0.4% Hct). Data are mean ± SD from four experiments each.

Fig. 4.

RBC deformability as elongation index for two representative shear stress levels as a function of storage time. Values at 0 h are from unprocessed RBCs. Data are median with 25th and 75th percentiles. P values represent significance for change over time.