A novel, rapid, multiparametric approach for flow cytometric analysis of intranuclear terminal deoxynucleotidyl...

This report describes a novel, reliable, and simplified approach for determination of intranuclear terminal deoxynucleotidyl transferase (TdT) expression. This approach utilizes standard permeabilization/fixation solutions to reliably detect intracellular antigens with minimal alterations in the light scatter properties of the analyzed cells. In contrast to other described methods, fluorescein isothiocyanate-conjugated anti-TdT antibody is added to previously analyzed and permeabilized cells after a leukemic cell population has been identified using characteristic surface staining patterns. The method eliminates the need for additional sample preparation or cumbersome permeablization steps and can easily be incorporated into any clinical laboratory's existing flow cytometry panels. Sixty-eight cases were analyzed with this method, including 31 acute myelogenous leukemias, 30 acute lymphoblastic leukemias, and 7 chronic lymphoproliferative disorders. To confirm the validity of the method, parallel immunoperoxidase staining and microscopic evaluation of cytocentrifuge test sample preparations were performed. Statistical analysis of the results reveals the method to be highly sensitive and specific, demonstrating exact correlation to the microscopic method. The ease and expeditiousness of this new procedure allows TdT testing to be routinely incorporated into the immunophenotyping repertoire of a busy clinical laboratory. In addition, the method should be readily adaptable to analyze a variety of other clinically relevant intranuclear and intracytoplasmic antigens.