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

Children with sickle cell disease (SCD) have repeated vaso-occlusion within the spleen, which leads to early loss of splenic filtration function and an increased risk of sepsis from encapsulated bacteria. Functional asplenia in SCD is believed to be a gradual and cumulative process that occurs during the first few years of life, although quantitative analyses of splenic function have not been readily available. Litron has recently developed a novel high-throughput flow cytometric method of quantitating Howell-Jolly bodies (HJB, also known as micronuclei), standardized using venous blood collected in heparin and fixed within 2 hours of collection. This novel technique may be useful for understanding the normal physiological decline in splenic function that occurs in SCD. Since erythrocytes that contain HJB are normally cleared by the healthy spleen, the frequency of HJB inversely reflects splenic filtration function. To investigate the utility of this novel assay for children with SCD, a tiny aliquot (10 μL) of leftover discarded blood specimens was obtained from unselected children receiving routine clinical care in the Duke Pediatric Sickle Cell Program, fixed in ultracold methanol, stored at −80C, and then shipped to Litron for flow cytometric analysis. Erythrocytes containing HJB were identified by flow cytometry: mature CD71⁻ erythrocytes that contain HJB are an accepted surrogate marker of splenic filtration, while HJB within the youngest CD71⁺ erythrocytes may reflect genotoxic damage associated with increased chromosome breaks. Initial studies determined that blood collected in EDTA was acceptable for analysis; no differences were noted with EDTA collection compared to heparin (R²=.994, n=5, p<.001). Samples fixed after 24 hours of refrigeration also showed excellent correlation with samples fixed within 2 hours (R²=.996, n-16, p<.001); blood stored for 48 hours also gave reliable results. Samples analyzed in duplicate or triplicate demonstrated high intrasample reproducibility (R²=.998, n=8, p<.001). To investigate the clinical utility of this assay, de-identified blood specimens were analyzed using EDTA-anticoagulated blood samples fixed 24-hours after collection. Children with HbSS (n=126) had increased numbers of HJB within mature CD71⁻ erythrocytes (2504±2311 per million RBC versus 28±42 in 50 healthy controls, p<.001). The frequency of HJB was more prevalent with increased age (R²=0.30, p<.001). For HbSS children without splenectomy or hydroxyurea (HU) exposure, 89% over age 1 year, and 100% over age 3 years had >300 HJB per million RBC. Children with HbSC (n=20) had a lower frequency of HJB (131±228 per million RBC), and only 10% with >300 HJB per million RBC. Increased HJB in HbSS patients were also identified in the young CD71⁺ erythrocytes (0.43±0.40%, n=126, normal <0.08%). Children with HbSS and HU exposure had significantly higher numbers of HJB in CD71⁺ erythrocytes (0.67±0.50%, n=46) compared to children without HU exposure (0.22±0.13%, n=59, p<.001). Serial measurements in one young child on HU therapy showed decreased HJB frequency, suggesting recovery of splenic function. Quantitation of HJB by flow cytometry is a rapid, robust, and reproducible assay that will be valuable in studies of organ damage in children with SCD. Serial measurements are needed to assess preservation or recovery of splenic filtration function, as well as genotoxic damage associated with HU therapy.