Reticulocyte and mature erythrocyte adhesion to laminin (LN) in normoxic and hypoxic microscale flow. (A) Staining of sickle erythrocytes for nucleic acids with acridine orange reveals the reticulocytes. Varying levels of reticulocyte (blue) and mature erythrocyte (red) adhesion under different physiologically relevant oxygenation conditions are shown. The partial oxygen saturation (SpO2) is greater than 95% under (i) normoxia and approximately 83% under (ii) hypoxia. (B) Comparison of adherent sickle erythrocytes under normoxia and hypoxia. Under normoxic conditions, adherent sickle erythrocytes are predominantly reticulocytes. Reticulocyte adhesion to LN under hypoxia was not significantly different from reticulocyte adhesion under normoxia. However, the contribution to overall adhesion by mature erythrocytes increased significantly under hypoxia. (C) Stacked and grouped column bars represent the adherent sickle erythrocyte types individually for patients. Shown above each column is the mature erythrocyte adhesion percentage. Minimal normoxic mature erythrocyte adhesion notably increased because of hypoxia and became the predominant contributor to total erythrocyte adhesion under hypoxia in 12/15 samples. Shown below patient identifiers are the reticulocyte percentages obtained from a clinical laboratory, and clinical reticulocyte percentages are not statistically associated (P=.162, linear regression) (D) Hypoxia enhanced total erythrocyte adhesion is associated with intrapulmonary shunting in subjects with HbSS SCD. Erythrocytes from both subject groups display a significant increase in adhesion under hypoxia compared with normoxia, and normoxic and hypoxic adhesion of erythrocyts are significantly different between the IPS and IPS unknown groups. N_no-IPS=18, N_IPS=23. P values were calculated using the Mann-Whitney test and 1-way ANOVA for non-normal and normal data, respectively. Data are presented as mean ± SEM.