FLOW CYTOMETRIC DETECTION OF INTRACELLULAR ANTIGENS FOR IMMUNOPHENOTYPING OF NORMAL AND MALIGNANT LEUKOCYTES

Intracellular antigens are of major importance for immunophenotyping o f normal leukocytes as well as leukemias and malignant lymphomas. Immu nofluorescence microscopic evaluation of cytocentrifuge preparations h as remained the preferred technique for detection of intracellular ant igens for a long time. Recently, flow cytometric detection of intracel lular antigens has been improved by the development of new permeabiliz ation/fixation solutions. We compared four commercially available solu tions: FAGS Brand Lysing Solution (FAGS Brand; Becton Dickinson, San J ose, CA, USA), Fix & Perm cell permeabilization kit (Fix & Perm; An de r Grub, Vienna, Austria), OptiLyse B lysing solution (OptiLyse B; Immu notech, Marseille, France), and ORTHO PermeaFix(TM)(PermeaFix; Ortho D iagnostic Systems, Raritan, NJ, USA). These solutions were evaluated f or the complexity and duration of the intracellular staining procedure , the effects on light scatter patterns, and the staining results for the intracellular antigens terminal deoxynucleotidyl transferase (TdT) , cytoplasmic CD3 (CyCD3), myeloperoxidase (MPO), and cytoplasmic immu noglobulin light chains (CylgL). The four methods could easily be intr oduced in our laboratory and had only minor effect on the light scatte r patterns of the tested cell samples. Each of the four tested antigen s was detectable with at least one of the four methods. Only the Fix & Perm cell permeabilization kit could be used for reliable detection o f all four intracellular antigens, In a large series of 450 BM and PB samples containing various percentages of TdT(+) cells, the results of flow cytometric TdT staining with FAGS Brand Lysing Solution were hig hly comparable to the results obtained by immunofluorescence microscop y (P = <0.00001). Our comparative study shows that flow cytometric det ection of the intracellular antigens TdT, CyCD3, MPO, and CylgL can no w reliably be performed on a routine basis.