End-Alveolar Carbon Monoxide As a Measure of Erythrocyte Survival and Hemolytic Severity in Sickle Cell Disease
Authors: Minniti Caterina P. , Baird John H. , Xu Dihua , Mendelsohn Laurel , Saiyed Rehan , Jackson Mary , Nichols James , Kim-Shapiro Daniel , Xia Yang , Kato Gregory J. 
Published: October 2019

Abstract
Introduction: In sickle cell disease, polymerization of sickle hemoglobin gives rise to changes in the integrity and viability of the erythrocyte, leading to both extravascular and intravascular hemolysis and shortened life-span. Current gold standard techniques to estimate RBC survival require that a label be placed on the cells that can be followed while the RBCs age in the circulation. These studies are infrequently employed in practice, due to the need for multiple blood draws and an extended period of observation (6 weeks) in order to obtain the necessary time points. Endogenous CO is a technique that has been used in multiple studies to assess RBC survival, based upon the principal that virtually all CO produced in human beings results from cleavage of the α-methene bond of heme and is completely excreted via the lungs. Because RBC destruction accounts for approximately 80% of heme turnover in the body, endogenous CO production can be used as a quantitative indicator of RBC life span. Furne has reported on the development of a simple, rapid, and noninvasive method for determining RBC life span based on gas chromatography measurement of exhaled alveolar CO (EACO) concentration immediately upon awakening corrected for atmospheric CO, as determined with a device that simulates the body's equilibration with CO.
We set up to investigate the use of early morning end-alveolar CO (EACO) concentration as a quantitative measure of RBC life span and correlate it to indirect measurements of hemolytic rate in subjects with sickle cell disease.
Material and methods: EACO was measured within 24 hours of breath collection in 67 SCD adult patients (67 HbSS, 4 HbSC and 1 HbSb-Thalassemia) and 14 HbAA race matched controls on an Ametrek TA 7000 Gas Purity Monitor. Subjects were at steady state, at least 30 days from transfusion or acute exacerbations. Breath samples were collected immediately upon awakening the same day as blood sampling for additional tests. EACO groups were compared using the Mann-Whitney test. EACO values were used to calculate the red blood cell lifespan by the method of Furne (PMID 12878986), using the calculation: RBC survival (days) = 1,380 · [Hb]/EACO. Correlation analyses are reported using Pearson's correlation coefficient. The statistical significance was set at a two-side p < .05. Analyses were performed with R version 2.13.1 (2011-07-08).
Results: EACO was nearly four-fold higher in patients than controls (median 2.25 vs. 0.58 ppm, p<.0001, figure). In patients with SCD, unadjusted EACO values were correlated with %HbS (p<.01), indirect markers of hemolysis: absolute reticulocyte count (p<.02), and percentage of reticulocytes (p<.01), indirect bilirubin (p<.001), and negatively with plasma nitrite (p<.02). EACO-derived red blood cell lifespan correlated negatively with LDH (p<.04), hematocrit (p<.01), hemoglobin (p<.06) indirect bilirubin (p<.01), absolute and percent reticulocyte counts (p<.01), AST (p<.01), and %HbS (p<.01).
Conclusions: Our results confirm the usefulness of exhaled CO measurement as a marker of hemolysis and red cell survival and in substantial, prospective cohort of adult patients with SCD, confirming and considerably extending results of other investigators. EACO-derived RBC survival can be used to update estimates from Crosby in 1955 regarding the proportion of overall hemolysis falling into the extravascular and intravascular categories, the latter related to oxidative stress and scavenging of nitric oxide. This assay may be a useful biomarker in clinical trials of therapeutic interventions aimed at improving red cell life-span in SCD and other hemolytic anemias.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.