Optimized detection of cytoplasmic immunoglobulin and CD3 in benign and malignant lymphoid cells: enhanced sensitivity combined with differential staining of endogenous peroxidases and light microscopic morphology.

Abstract

A novel immunocytochemical approach to the detection of cytoplasmic antigens, exemplified by immunoglobulin and CD3, was evaluated using benign and 155 malignant lymphohematopoietic cell specimens and conventional techniques for detailed comparison. It relies on (a) electrostatic cell attachment to poly-L-lysine-coated multispot slides, (b) sequential use of glutaraldehyde fixation and detergent permeabilization, and (c) staining of endogenous peroxidases and antigens in contrasting colors. Differential staining instead of inactivation of endogenous peroxidases and avoidance of artifacts incurred in conventional cytocentrifugation, dehydration, and alcohol or acetone fixation uniquely afforded improved precision in cell identification, clear distinction of cytoplasmic from surface antigenic staining, and greatly enhanced sensitivity in antigen detection, all combined with increased performance efficiency achieved by the use of multispot slides. With this method, cytoplasmic immunoglobulin and CD3 were found more widely distributed than has been previously recognized. Thus, almost all malignant cells in all cases of B-cell lymphoma/leukemia (encompassing the major subtypes) and T-ALL, as well as substantial proportions of benign mature B- and T-lymphocytes, stained strongly positive for cIg and cCD3, respectively, whereas myeloid cells were consistently negative. High sensitivity combined with excellent cytomorphology and differential myeloperoxidase staining permitted unequivocal differentiation of minute fractions of malignant cells against a heterogeneous background of benign cells.