Factors Associated with Heterocellular Aggregate Formation In Sickle Cell Disease

Abstract 2669

White blood cells (WBCs) have emerged as key contributors to the pathophysiology of sickle cell disease (SCD), in part because they become activated and can form heterocellular aggregates with sickle red blood cells (SS RBCs) and platelets. These heterocellular aggregates are potentially detrimental to individuals with SCD because they may obstruct blood vessels and be themselves both pro-inflammatory and pro-coagulant. In non-SCD individuals, these aggregates are almost undetectable. In order to detect heterocellular aggregates, we collected blood into EDTA, and 10 μl of the each individual's whole blood was added to 190 μl of Dulbecco's phosphate buffered saline with Ca⁺⁺, Mg⁺⁺, and 2% (w/v) albumin in each tube. A 4-color flow cytometric assay was used to detect heterocellular aggregates as events positive for both erythrocyte and leukocyte markers, as well as to detect component cells within those aggregates. RBCs were detected using a fluorescein isothiocyanate (FITC)-conjugated antibody against the RBC-specific protein glycophorin A (CD235a). WBCs were detected using an allophycocyanin (APC)-conjugated antibody against the common leukocyte antigen (CD45). Platelets were detected using a phycoerythrin (PE)-conjugated antibody against the platelet-specific antigen GPIb (CD41a). To quantify leukocyte subsets, monocytes were detected using a PE-conjugated antibody against the lipopolysaccharide receptor antigen CD14, and lymphocytes were detected using a peridinin chlorophyll protein (PerCP)-conjugated antibody against the pan-lymphocyte marker CD3. We found that the mean percent of all events that contained both WBCs and RBCs was 0.40% for 33 SCD individuals in steady state and only 0.05% for 9 non-SCD individuals. In order to obtain a comprehensive picture of the factors associated with an increase in the number of aggregates in the blood of SCD individuals, we examined the association of reticulocyte count, WBC count, monocyte count, neutrophil count, lymphocyte count, platelet count, hydroxyurea treatment, and RBC exposure of phosphatidylserine with aggregate formation. Information regarding patients' cell counts was obtained from hospital records. The percent of WBCs in heterocellular aggregates was calculated by dividing the number of aggregates by the total WBC count. In our study of 33 patients, we found the strongest associations between percent events representing aggregates and total WBC count, monocyte count, and neutrophil count. In general, the higher the total WBC count, the greater was the percent of heterocellular aggregates. For the 8 patients in the quartile with the highest WBC count, the mean percent of heterocellular aggregates was 0.51% ± 0.06%, while the mean percent of heterocellular aggregates for the 8 patients with the lowest WBC count was 0.31% ± 0.06% (p = 0.028). Similar statistically significant associations were seen with both monocyte and neutrophil counts. Platelet counts were associated with the percent of leukocytes in heterocellular aggregates (p = 0.037); the higher the platelet count, the greater was the percent of WBCs in aggregates. The mean percent of WBCs in aggregates was 78.9% ± 3.5% and 62.2% ± 6.3%, for the uppermost and lowermost quartiles of platelet counts, respectively. On average, 61.8% of aggregates included platelets. There was no association between levels of annexin V binding or reticulocyte count and aggregate counts. We also examined the effect of hydroxyurea (HU) treatment on aggregate formation. Hydroxyurea treatment increases the production of hemoglobin F, reduces hemolysis and reticulocyte counts, and reduces white blood cell and platelet counts (Platt 2008). Hydroxyurea has also been associated with reduced RBC adhesivity. In our study, individuals not on hydroxyurea treatment had a higher percentage of aggregates than those on hydroxyurea (0.46 ± 0.05 without HU vs 0.37 ± 0.05 on HU, n=33), but this difference was not statistically significant. In summary, heterocellular aggregate formation in SCD involves many different cell types, but the factors promoting their participation in aggregate formation in SCD remain to be completely elucidated. Hydroxyurea may downregulate heterocellular aggregate formation, but this remains to be confirmed.