Detection of terminal deoxynucleotidyl transferase by flow cytometry: a three color method.

Abstract

This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.