Pulmonary Hypertension During Autologous Transfusion of Stored Blood Is Prevented by Nitric Oxide Inhalation in Lambs
Authors: Baron David M. , Yu Binglan , Lei Chong , Bagchi Aranya , Beloiartsev Arkadi , Steinbicker Andrea U. , Stowell Christopher P. , Malhotra Rajeev , Bloch Kenneth D. , Zapol Warren M. 
Published: October 2019

Abstract
Abstract 39

Introduction:
During extended storage, red blood cells (RBCs) undergo biochemical, mechanical, and functional changes. These changes reduce the viability of RBCs, resulting in elevated levels of the potent nitric oxide (NO) scavenger oxyhemoglobin in plasma. Recent studies have shown enhanced systemic vasoconstriction after challenge with tetrameric hemoglobin in murine endothelial dysfunction models. Based on human blood storage techniques, we developed and validated a new model for autologous transfusion of stored RBCs in lambs. We hypothesized that autologous transfusion of leukoreduced ovine RBCs stored for prolonged periods of time would increase plasma hemoglobin levels and induce pulmonary hypertension. We further hypothesized that inhalation of NO would prevent, and endothelial dysfunction would augment the pulmonary vasoconstriction induced by transfusing blood stored for prolonged periods.

Methods:
We studied three- to four-month-old Polypay lambs weighing 32±2 kg. Similar to current blood bank practices, leukoreduced ovine RBCs were stored in Adsol solution (additive solution-1, AS-1) for either 2 days (fresh red blood cells, FRBCs) or 40 days (stored red blood cells, SRBCs). Post-transfusion recovery of circulating biotinylated FRBCs (n=4) and SRBCs (n=4) was determined by flow cytometry.
In separate experiments, 300 ml of autologous FRBCs (n=5) or SRBCs (n=6) were transfused over 30 min into awake lambs, which had been instrumented with carotid artery and pulmonary artery catheters under isoflurane anesthesia. Systemic and pulmonary hemodynamic parameters were measured continuously during and for 4 h after the transfusion. An additional group of animals receiving SRBCs concurrently inhaled 80 parts per million NO (n=4) at FiO2 0.25.
We also studied the effects of transfusing FRBCs (n=4) or SRBCs (n=5) in lambs after acutely inducing endothelial dysfunction by IV injection of 25 mg·kg−1 of NG-nitro-L-arginine methyl-ester (L-NAME). An infusion of 5 mg·kg−1·h−1 L-NAME was continued throughout the experiment.
Plasma hemoglobin and IL-6 levels were determined before and after transfusion. Tissue samples from the lung and liver were harvested 4 h after transfusion. Relative mRNA levels of inflammatory markers (IL-6, TNF-alpha, and myeloperoxidase) were measured by qPCR.
All data are expressed as mean ± SEM.

Results:
Hemoglobin (41±6 vs. 148±8 mg/dl), potassium (3.7±0.4 vs. 7.9±0.9 mmol/l), and lactate levels (1.7±0.2 vs. 5.9±0.9 mmol/l) were higher in the supernatants of SRBCs than in those of FRBCs. Recovery of circulating biotinylated RBCs 24 h after autologous transfusion was 96±2% in FRBCs and 76±3% in SRBCs.
Pulmonary arterial pressure (PAP) transiently increased from 13±0.3 to 18±1 mmHg (p<0.01) and pulmonary vascular resistance index (PVRI) from 108±8 to 156±14 dyne·sec·cm−5·m−2 (p<0.05) during the transfusion of SRBCs, but not FRBCs. This increase of PAP was temporally associated with an increase in plasma concentrations of hemoglobin. Transfusion of SRBCs did not produce systemic vasoconstriction. Concurrent inhalation of NO prevented the pulmonary vasoconstrictor effect induced by transfusing SRBCs, whereas the infusion of L-NAME potentiated the increase in PAP (16±0.3 to 26±2 mmHg, p<0.01) and PVRI (170±15 to 312±38 dyne·sec·cm−5·m−2, p<0.05) associated with transfusion of SRBCs.
Plasma IL-6 levels did not change after transfusion of FRBCs or SRBCs. Lung and liver levels of mRNAs encoding inflammatory markers (IL-6, TNF-alpha, and myeloperoxidase) measured 4 h after transfusion did not differ in lambs receiving FRBCs or SRBCs.

Conclusions:
Ovine RBCs stored for 40 days have many in vitro storage properties and a post-transfusion recovery percentage similar to stored human RBCs. Autologous transfusion of leukoreduced SRBCs induces transient pulmonary hypertension associated with increased cell-free hemoglobin levels. This vasoconstrictor effect is increased in a model of L-NAME-induced endothelial dysfunction. Therefore, patients with disorders associated with pulmonary endothelial dysfunction might be more sensitive to pulmonary vasoconstriction associated with transfusion of SRBCs.

Disclosures:
Yu: Massachusetts General Hospital: Patents & Royalties. Bloch:MGH has received sponsored research grant funding from Ikaria LCC, the producer of NO gas in the US, in support of Dr. Bloch's research program: Research Funding. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.