Simultaneous detection of membrane markers with monoclonal antibodies and peroxidatic activities in leukaemia: ultrastructural analysis using a new method of fixation preserving the platelet peroxidase

Abstract

Simultaneous detection of specific surface markers by immunogold and intracellular peroxidase activity was determined ultrastructurally in [human] normal and leukemic progenitors of platelets, erythrocytes and granulocytes. A new method of fixation was employed to preserve platelet peroxidase activity. Monoclonal antibodies to platelet glycoproteins labeled exclusively platelet peroxidase (PPO) positive cells, i.e., platelets megakaryocytes and promegakaryoblasts (PMKB). In acute megakaryoblastic leukemia, most PMKB possessed both markers, while a few PMKB identified by PPO did not bind monoclonal antibodies. PPO appears earlier in maturation than platelet glycoproteins. Although all glycoproteins (GP) displayed fewer sites in PMKB than platelets, GP Ib was often observed in more mature megakaryocytes. Surface (glycophorin A) and intracytoplasmic markers including ferritin, intra-mitochondrial iron and diffuse peroxidase activity due to Hb of erythroid progenitors, appeared simultaneously. The number of glycophorin A sites increased with maturation. In leukemia involving PMKB and proerythroblasts, the surface markers were coincident with the localization of peroxidase activity; glycophorin A was always absent from blasts which exhibited PPO activity localized in endoplasmic reticulum. Platelet glycoproteins were never expressed in any other cell lineage. The myeloid surface antigen was present on normal late neutrophilic promyelocytes after the cessation of myeloperoxidase synthesis. In some cases of M1 and M2 AML (FAB classification), labeling was identical to normal cells, while in others the antigen appeared earlier than normal. The surface phenotype of blasts from non-lymphoid leukemia and the intracellular peroxidase activity of a given cell type can be simultaneously demonstrated and analyzed by EM.