Effects of Aged Stored Autologous Red Blood Cells on Human Endothelial Function

Abstract

Rationale: A major abnormality that characterizes the red cell "storage lesion" is increased hemolysis and reduced red cell lifespan after infusion. Low levels of intravascular hemolysis after transfusion of aged stored red cells disrupt nitric oxide (NO) bioavailabity, via accelerated NO scavenging reaction with cell-free plasma hemoglobin. The degree of intravascular hemolysis post-transfusion and effects on endothelial-dependent vasodilation responses to acetylcholine have not been fully characterized in humans.

Objectives: To evaluate the effects of blood aged to the limits of Food and Drug Administration-approved storage time on the human microcirculation and endothelial function.

Methods: Eighteen healthy individuals donated 1 U of leukopheresed red cells, divided and autologously transfused into the forearm brachial artery 5 and 42 days after blood donation. Blood samples were obtained from stored blood bag supernatants and the antecubital vein of the infusion arm. Forearm blood flow measurements were performed using strain-gauge plethysmography during transfusion, followed by testing of endothelium-dependent blood flow with increasing doses of intraarterial acetylcholine.

Measurements and main results: We demonstrate that aged stored blood has higher levels of arginase-1 and cell-free plasma hemoglobin. Compared with 5-day blood, the transfusion of 42-day packed red cells decreases acetylcholine-dependent forearm blood flows. Intravascular venous levels of arginase-1 and cell-free plasma hemoglobin increase immediately after red cell transfusion, with more significant increases observed after infusion of 42-day-old blood.

Conclusions: We demonstrate that the transfusion of blood at the limits of Food and Drug Administration-approved storage has a significant effect on the forearm circulation and impairs endothelial function. Clinical trial registered with www.clinicaltrials.gov (NCT 01137656).

Trial registration: ClinicalTrials.gov NCT01137656.

Keywords: cell-free plasma hemoglobin; nitric oxide; red cell transfusion; storage lesion; vasoreactivity.

Figures

Figure 1.

Study design Days 5 and 42. Eighteen subjects underwent the same protocol on Days 5 and 42. Subjects received saline, acetylcholine (Ach), stored blood, and then Ach again to assess the effect of blood transfusion on the vascular endothelial response to Ach. At baseline subjects received an infusion of 0.9% normal saline (NS) at 2 ml per minute. This was followed by sequential infusions of Ach at increasing rates of 7.5, 15, and 30 μg per minute, which corresponded with infusion flow doses of 15, 30, and 60 ml per hour. A washout period of 30 minutes was followed by red blood cell infusion and repeated Ach infusion. Blood was transfused for a total duration of 9–12 minutes. The rates of blood infusion were 5 ml per minute for 2 minutes (300 ml/h), 10 ml per minute for 2 minutes (600 ml/h), and then 15 ml per minute until the unit was completed (900 ml/h). Continuous forearm blood flow measurements were performed at each baseline and infusion stage. Venous blood draws were performed before onset of the study and then again after blood infusion.

Figure 2.

(A) Hemolysis in stored blood on storage Days 5 and 42. Scatter dot plots show the median (interquartile range) values for cell-free plasma hemoglobin (CFPHb), arginase-1 (Arg-1), and lactate dehydrogenase (LDH) for subjects. Values for CFPHb (n = 18), Arg-1 (n = 15), and LDH (n = 15) were statistically significant when comparing levels at Day 5 of storage with Day 42 of storage, using Wilcoxon matched-pairs signed rank testing. (B) Correlation of nitric oxide (NO) consumption with CFPHb in stored blood for 5- and 42-day samples. Samples from the stored blood unit were obtained before transfusion for measure of analytes in the supernatant. NO consumption highly correlated with measured levels of CFPHb on Day 5 (n = 18) and Day 42 (n = 18), both of which are statistically significant.

Figure 3.

Forearm blood flow measurements. (A) Days 5 and 42 forearm blood flow acetylcholine (Ach) response postintraarterial autologous red blood cell (RBC) infusion. Days 5 and 42 forearm blood flows are expressed in absolute and % change from baseline forearm blood flows. Compared with Day 5 measurements, the Ach response on Day 42 is reduced by both absolute and % change from baseline values after the transfusion of aged blood. (B) Percent change in forearm blood flow caused by Ach infusion pre- and post-RBC infusion. Infusion of 42-day-old RBCs results in significantly reduced forearm blood flows measured by Ach response when compared with Day 5 pre- and post-RBC infusion and Day 42 pre-RBC infusion. Forearm blood flows are expressed as % change from baseline forearm blood flows. (C) Forearm blood flows during autologous intraarterial RBC infusion. The blood flows, expressed in absolute and % change from baseline forearm blood flows, are plotted against the rate of RBC infusion of Days 5 and 42 aged RBCs. The values for both absolute and % change from baseline values are concordant, with a significantly increased flow of blood after the infusion of older RBCs.

Figure 4.

(A) Viscosities of fresh and aged red blood cells. Viscosity was found by multiplying the efflux time by the shear rate provided with the viscometer. The average viscosities for fresh and older blood were 7.8 ± 0.8 cSt and 10.5 ± 1.8 cSt, respectively (P = 0.015). (B) Effects of saline infusions at different flow rates. The % change from baseline of flows for saline increased as the rate of flow increased at 5, 10, and 15 ml per hour. The % changes from baseline for saline at all infusion rates are lower than that of blood or acetylcholine infusions but do show that higher intraarterial infusion rates produce shear-related increases in blood flow. cSt = centistokes; NS = normal saline.

Figure 5.

Venous samples for hemolysis before and after blood infusion. The median (interquartile range) plasma venous blood values for cell-free plasma hemoglobin (CFPHb) and arginase-1 (Arg-1) obtained on Days 5 and 42. Levels of CFPHb and Arg-1 are increased after red cell infusion. Wilcoxon matched-pairs signed rank testing was performed.

Figure 6.

Mean arterial blood pressure and heart rate. Mean arterial pressures and heart rate values were obtained immediately pre– and post–red blood cell infusion. There is a statistically significant increase in systemic pressures post–blood infusion that was not demonstrated with heart rate, indicating that the mean arterial pressure response was not likely catecholamine driven. Values are expressed as mean (SD).

Figure 7.

Model of the response to acetylcholine (Ach) in the absence and presence of red cell hemolysis. (A) In the absence of hemolysis, intravascular Ach infusion stimulates muscarinic receptors on the surface of endothelial cells to increase the influx of Ca2+. This stimulates nitric oxide synthase (NOS) activity to increase conversion of l-arginine to nitric oxide (NO), increasing NO synthesis and availability, which diffuses to adjacent smooth muscle cells. NO then stimulates guanylate cyclase (GC) activity in smooth muscle to increase conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), resulting in smooth muscle relaxation. (B) In the presence of red cell hemolysis, hemoglobin and arginase-1 (Arg-1) are released from the red cell into the plasma. Cell-free plasma hemoglobin is released from hemolyzed red cells and scavenges available intravascular NO by the dioxygenation reaction. Decreased NO availability results in decreased NO signaling and decreased smooth muscle relaxation. Additionally, the release of Arg-1 from cellular hemolysis metabolizes l-arginine to ornithine, further decreasing availability of NO. In this hemolysis model, infusion of intravascular Ach is relatively ineffective because NO availability and synthesis are reduced, thereby decreasing the ability of smooth muscle to relax, or a relative vasoconstrictive effect. RBC = red blood cell.