The Detection of Intracellular Antigens in Human Leucocytes by Immunoperoxidase Staining

Abstract

An immunoperoxidase procedure is described for staining intracellular leucocyte antigens in peripheral blood and bone marrow smears. Brief exposure of cell smears to a buffered formol acetone mixture was found to give optimal fixation, combining good cellular morphology with preservation of antigenic reactivity. The immunoperoxidase method is superior to immunofluorescence in that it provides a permanent preparation which can be counterstained with orthodox reagents and viewed by conventional light microscopy. In addition the technique is considerably more sensitive than immunofluorescence procedures. Immunoglobulin was demonstrated in plasma cells, Türk cells and a minority of peripheral blood lymphocytes. Lysozyme was found in cells of the neutrophil series from promyelocytes to mature granulocytes. Monocytes stained for lysozyme but the reaction was less intense than in neutrophils and some monocytes were devoid of activity. Lactoferrin stained strongly in mature neutrophil polymorphs and metamyelocytes, but was weak or absent in earlier myeloid cells. These reaction patterns are in keeping with previous reports on the distribution of these antigens in human leucocytes. In the case of immunoglobulin and lysozyme it was possible to abolish leucocyte staining by incubation of the specific antisera with the appropriate purified antigen, providing additional proof of the specificity of the reactions. Anti-ferritin antisera stained granulocytes and myeloid precursors strongly, and reached weakly with a minority of monocytes. These latter observations are not entirely in accordance with published data on the leucocyte distribution of ferritin and may be attributed to antibody activity of unknown specificity in the anti-ferritin antiserum.

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

“…Multiple samples, -150,000 cells each, were centrifuged onto glass slides, fixed in acetone, and then stained by utilizing the biotin-avidin-horseradish peroxidase technique as described above. Immunophenotyping of tissue sections and cytocentrifuge preparations after fixation reveals intracellular as well as surface antigen expression (25)(26)(27)(28) 10 Mg, and goat anti-mouse IgG (EY Labs, San Mateo, CA) at 4°C over 6 h while slowly rotating. Specific immunoprecipitation, using 10 ,g of specific, purified, anti-Leu-4 antibody and goat antimouse IgG at equivalence, was performed in a similar fashion over 18 h. Precipitates were collected by centrifugation and washed six times in NaCl-EDTA-Tris buffers containing NaCl 0.15 M, EDTA 5 mM, Tris-HCI 50 mM, pH 7.4, Na azide 0.02%, NP-40 decreasing from I to 0%, and additional NaCI to 0.5 M in the first four washes.…”

Membrane-bound lactoferrin alters the surface properties of polymorphonuclear leukocytes.

“…After the addition of the lactoferrin antibody, swine antirabbit immunoglobulin (Dako Accurate Chemical Co.) at a 1:50 dilution was added for 30 min and removed by washing three times. A rabbit anti-horseradish peroxidase:horseradish peroxidase complex was added for 30 min and the complex was developed by washing in Tris-buffered saline containing 0.5 mg/ml diaminobenzidine (Polysciences, Inc., Warrington, PA) and 0.01% hydrogen peroxide for 60 s (14). The (16,17), purple membranes (18), and mitochondria inner membranes (15,19) (20), the membrane order parameters of intact PMN in suspension treated with 60 Mg/ml lactoferrin, 60 ,ug/ml transferrin, or buffer were 0.653, 0.655, and 0.654, respectively.…”

Cytochemical Demonstration of Transferrin in the Mitochondria of Immature Human Erythroid Cells

“…Neither the treatment with ethanol containing 0.3% hydrogen peroxide for 30 min, as described by Mason et al [9], nor the treatment with 0.005 M periodic acid for 15 min, previously employed to block the en dogenous peroxidase of intestinal tissue [7], was directly applicable to bone marrow cells which contain high endogenous peroxi dase activity ( fig. 1).…”