Identification by Peroxidase Staining of Monocytes in Surface Immunofluorescence Tests

Abstract Surface immunoglobulins (S.Ig)2 on human lymphocytes have been widely studied in the last few years, mainly with surface immunofluorescence (IF) tests. These studies have led to somewhat contradictory results (reviewed in 1) and most discrepancies are likely explained by methodological reasons since S.Ig detection is exposed to a number of pitfalls (2). Monocytes represent an important source of potential errors. They bind serum IgG by their Fc receptor and therefore stain with conjugated antisera to γ-, κ- and λ-immunoglobulin chains. In addition some monocytes are able to bind rabbit IgG used as reagents and show positivity in IF tests whatever the specificity of the conjugate is. Two approaches may be used in order to avoid erroneous interpretation due to monocytes, either the use of cell suspensions free of monocytes or the identification of monocytic cells. None of the procedures we used to remove monocytes (filtration through glass or nylon wool or through Sephadex G-10 (3), incubation with iron carbonyl followed by Ficoll centrifugation (4)) was satisfactory since they all resulted in a poor lymphocyte recovery and in a selective loss of B cells. Therefore we kept separating blood lymphocytes by Ficoll centrifugation which does not remove monocytes and we tried to identify them on criteria more objective than the mere morphology in phase contrast microscopy. Giemsa staining of IF slides cannot be used due to a strong autofluorescence. We tried, therefore, to combine IF and cytochemical reactions. Esterase staining (5) was first used, but it gave under ultraviolet illumination a diffuse cell staining which made the specific fluorescence barely visible. Moreover, some lymphocytes showed some degree of esterase positivity. By contrast, peroxidase {POX) staining was found to be extremely useful since it made the monocyte identification very easy and took very little time, without grossly affecting the IF patterns.