Quantitative Analysis of Howell-Jolly Bodies in Children with Sickle Cell Disease

Objectives Although functional asplenia in sickle cell disease (SCD) begins early in life and has important clinical consequences, quantitative measurement of splenic function is not readily available. A novel high-throughput flow cytometric method for quantitating Howell-Jolly bodies (HJB) has been developed which isolates HJB-containing CD71+ and CD71− erythrocytes. Analysis of these cell populations allows quantitative measurement of splenic filtrative function and possible chromosomal damage. Methods Blood specimens from 147 children with SCD were analyzed using a high-throughput flow cytometric method. Enumeration of the following populations was accomplished: 1) CD71+ reticulocytes among total erythrocytes, identifying the youngest erythroid cell population; 2) HJB-containing CD71+ reticulocytes, which isolate young erythrocytes containing micronuclei as an index of cytogenetic damage; and 3) HJB-containing CD71− erythrocytes, identifying older erythrocytes containing micronuclei, indirectly measuring splenic function. Results Children with HbSC (n = 24) had slightly elevated HJB frequencies, while children with HbSS (n = 125) had highly elevated frequencies within CD71+ cells (0.44% ± 0.40%, normal 0.12% ± 0.06%, p < 0.001) and CD71− cells (2493 ± 2303 per million RBC, normal 20 ± 11, p < 0.001). Using a multiple regression model, the frequency of HbSS CD71+ reticulocytes containing HJB was significantly influenced by hydroxyurea use (p < 0.0001), age (p = 0.0288), and splenectomy (p = 0.0498). Similarly, mature CD71− erythrocytes containing HJB were positively correlated with hydroxyurea (p = 0.0001), age (p < 0.0001), and splenectomy (p = 0.0104). Conclusions HJB quantitation by flow cytometry is a novel assay for measuring splenic function and may be valuable for investigating the efficacy and safety of therapeutic options for children with SCD.