Aim: Post-bone marrow transplant and during relapse disease, there can be variable amounts of nucleated red blood cells (nRBCs) in the peripheral blood which can introduce gDNA contamination in cell subset enrichment. In this study we assess the (1) clinical accuracy, (2) precision and (3) reportabe range of novel flow cytomety panels which utilize a rapid and simple lyse-no-wash protocol with Syto-16 to identify nRBC contamination when assessing subset enrichment purity.
Methods: Several purity assessment flow cytometry panels were designed. These panels utilize the following markers: CD45 APC-H7 (BD), CD3 PE (Miltenyi), CD19 APC (Miltenyi), CD14 PE-Cy5.5 (BC), CD15 PE (BD), CD34 PE (Miltenyi) and Syto-16 (BD). Residual pateint bone marrow and peripheral blood samples with known concentrations of nRBCs, as calculated by a hematology analyzer (Sysmex), were assessed using the chimerism purity flow panels. Residual patient samples with higher counts of nRBCs were diluted using samples lacking nucleated red blood cells, after which the flow purity assessment panels were utilized to measure nRBCs. Lastley, reproducability studies were performed to assess the precision of the panels at measuring nRBC proportions. All samples were stained by the HLA laboratory at Children's Hospital Los Angeles and aquired on the FACSLyric instrument (BD).
Results: The lyse-no-wash protocol for assessing subset puritty and nRBC contamination can be performed and ready for aquisition on a flow cytometer in about 30 minutes. The correlation anlaysis for measuring the proportion of nucleated red blood cells expected for the CD3, CD19, CD14+15, CD34, and CD15 panels demonstrated R-squared values greater than 0.95. Precision studies for these panels demonstrate excellent reproducability with CV(%) less than 10% on average for assessing nRBC proportions.
Conclusion: These new flow cytometry panels offer a rapid way to assess purity of chimerism subset enrichment with the ability to appropriately measure proportions of contaminating nRBCs which can introduce gDNA contamination when subset enrichment is performed, providing a more comprehensive assessment of cells which contain gDNA. These panels demonstrate excellent correlation with expected values and acceptable precision.
