Application of the avidin‐biotin‐peroxidase complex technique for ultraimmunocytochemical characterization of leukemic cells

Abstract

For simultaneous demonstration of cellular ultrastructure, myeloperoxidase activity, and presence of a membrane-bound antigen in a given blood cell, we examined three different fixatives: periodate-lysine-paraformaldehyde (PLP) and paraformaldehyde and glutaraldehyde for their applicability to preembedding electron microscopic immunocytochemistry using monoclonal antibodies and the avidin-biotin-peroxidase complex (ABC) technique. This procedure was examined in samples from 3 normal volunteers and 29 patients with acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphosarcoma cell leukemia (LSCL), blastic phase of chronic myelogenous leukemia (CML-BC), or other unclassified leukemias. PLP fixation preserved the immunoreactivity of surface glycoproteins as well as immunoglobulins to the most satisfactory extent. Leukemic cells fixed with PLP maintained their fine structural details, so that we could identify their cytoplasmic organelles, although glutaraldehyde produced the best preservation of cellular ultrastructure. In three patients with ALL, our method revealed that a significant portion of blasts possessed both lymphoid surface antigens and peroxidase-positive cytoplasmic granules. Our method was also useful in identifying the lineage of peroxidase-negative leukemic cells, including monoblastic leukemia and megakaryoblastic leukemia cells. Ultraimmunocytochemistry using PLP fixation and the ABC technique may be a promising strategy for determining the nature of blastic cells that remain unclear after a conventional work-up, for characterizing leukemic cells in patients with a relatively low blast cell count in the bone marrow or peripheral blood, and for estimating the presence and frequency of leukemia with multilineage expression.

Use of periodate-lysine-paraformaldehyde for the fixation of multiple antigens in human skin biopsies

“…Nineteen papers are aimed at detecting surface antigens of immune system cells by light or electron microscopy. In these latter studies, PLP has been credited for preservation of several cell surface antigens of the mouse (Parr et al, 1980;van Ewijk et al, 1980;Gendelman et al, 1983;Whiteland et al, 1995), rat (Ruan et al, 1994;Whiteland et al, 1995;Allaerts et al, 1996) and humans (Hancock et al, 1982;Collings et al, 1984;Dienes et al, 1984;van Duinen et al, 1984;Chiba et al, 1986;Holgate et al, 1986;Hall et al, 1987;Pollard et al, 1987;Kadin et al, 1988;Agnarsson and Kadin, 1989;Drach et al, 1989;Yano et al, 1990;Oka et al, 1993;Ahmed et al, 2001), following different preparatory techniques: dissociated cells, cryosections, paraffin sections, pre-embedding immunoelectron microscopy and flow cytometry. It is noteworthy that all but one studies on human cells date back to 1990 or earlier; taking also animal studies into account, only five papers on membrane antigens of immune system cells upon PLP fixation seem to have been published after 1990.…”