Journal of Immunological Methods, 26 (1979) 193--196
193
© Elsevier/North-Holland Biomedical Press
Short communication ESTERASE STAINING OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
MARCEL C.J.M. DE JONG
Department of Dermatology, State University of Groningen, Groningen, The Netherlands (Received 9 November 1978, accepted 10 December 1978)
Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12--18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.
INTRODUCTION
Recently a simple histochemical staining reaction for 1-naphthyl esterase has been proposed to define mononuclear leucocyte populations for clinical purposes (Ornstein et al., 1976; Horwitz et al., 1977). My own experiences with esterase methods applied to human peripheral blood leucocytes have been disappointing. Although staining of buffy coat smears permitted clear distinction between populations of monocytes and lymphocytes, decreased leucocyte stainability was observed after their separation by the Ficoll-Isopaque method of BSyum (1968). Loss of esterase activity in cytocentrifuge smears of mononuclear cells has also been observed by Horwitz et al. (1977). A slight modification of the esterase method is proposed, which in our hands produces minimal loss of esterase activity in cytocentrifuge smears of human peripheral blood monocytes and lymphocytes. MA TER I ALS AND METHODS
Blood leucocytes. Heparinized venous blood of healthy human donors was used throughout. Leucocyte preparations used for cytocentrifuge smears included (1) buffy coat cells, (2) mononuclear cells isolated according to BSyum (1968), (3) plastic adherent and non-adherent cells, (4) SRBC rosetting and non-rosetting cells (Gmelig, 1977). The final leucocyte suspensions were adjusted to 5 × 10 s cells/ml in RPMI-1640-Hepes (GIBCO) con-
194
taining 1% BSA (medium-BSA), except for buffy coat cells which were contained in autologous plasma. Cytocentrifuge smears. Cells were pelleted onto microscope slides in a Shandon-Elliott cytocentrifuge. The cuvettes with filter paper in place were filled with 0.1 ml medium-BSA and spun for 5 min at 1000 rev/min. After pre-wetting the filters, the cuvettes received another 0.1 ml medium-BSA on to which 50 pl of the respective cell suspension was layered, followed by spinning for 3 min at 750 rev/min. Esterase staining. Fixation and staining were carried out by the methods of Ornstein et al. (1976) and Horwitz et al. (1977), using as substrates 1-naphthyl acetate (Koch-Light) and 1-naphthyl butyrate (Sigma). For optimal staining, the 1-naphthyl butyrate esterase m e t h o d of Ornstein was employed with the following modifications: (1) smears were air-dried in the dark for 12--18 h at room temperature, (2) w i t h o u t further fixation, dried smears were incubated with the substrate mixture for 30--45 min instead of 10 min, (3) after counterstaining for 5 min in 2% m e t h y l green, the smears were rinsed in distilled water, air-dried and m o u n t e d in Entellan-Neu (Merck) or sucrose 85%. The smears were examined by both phase contrast and ordinary transmitted light microscopy. RESULTS AND COMMENTS
Several procedures for cell fixation and esterase staining were compared to establish which was most satisfactory. Best results were obtained when wet b u f f y coat smears were fixed for 9 min in formaldehyde vapour (Ornstein et al., 1976). In general, monocytes developed a diffuse reddish brown cytoplasmic staining pattern as distinct from lymphocytes which showed one or more discrete granules of reaction product. Since the esterase m e t h o d of Ornstein et al. (1976) does not require the use of freshly prepared stock reagents, this m e t h o d was preferred in combination with formaldehyde vapour fixation. When applied, however, to cytocentrifuge smears of Ficoll-Isopaque enriched mononuclear cells, marked loss of staining intensity was observed as compared with the reactive cells in buffy coat smears. Longer incubation with substrate (30 min instead of 10 min) slightly improved the stainability of punctate granules in lymphocytes. In the course of our studies we accidentally f o u n d that air-drying of cytocentrifuge smears for 12--18 h w i t h o u t further fixation resulted in optimal preservation of esterase activity. Control incubations with staining solution deprived of esterase substrate resulted in a complete absence of reaction product. Table 1 illustrates that fixation in comparison with air-drying gives an underestimate of the proportion of esterase positive cells, in particular of granular staining lymphocytes. Air-drying w i t h o u t further fixation appears, therefore, the m e t h o d of choice. A disadvantage of this procedure may be some loss of morphological detail. Furthermore, when E rosette-forming
195 TABLE 1 COMPARISON OF ESTERASE STAINING OF MONONUCLEAR ACETONITRILE-FIXED AND AIR-DRIED SMEARS Cytocentrifuge leucocyte smear
M o n o n u c l e a r cells A d h e r e n t cells E r o s e t t i n g cells b
Positive (%) a Diffuse
L E U C O C Y T E S IN
Negative (%) Granular
Monocytes
Lymphocytes Fixed
Dried
25.3 26.7 20.1
9.8 19.8 7.1
Fixed
Dried
Fixed
Dried
Fixed
12.5 64.0 0
18.5 66.8 0
55.5 5.2 79.9
71.2 12.2 92.1
5.8 4.1 --
Dried
193
© Elsevier/North-Holland Biomedical Press
Short communication ESTERASE STAINING OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
MARCEL C.J.M. DE JONG
Department of Dermatology, State University of Groningen, Groningen, The Netherlands (Received 9 November 1978, accepted 10 December 1978)
Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12--18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.
INTRODUCTION
Recently a simple histochemical staining reaction for 1-naphthyl esterase has been proposed to define mononuclear leucocyte populations for clinical purposes (Ornstein et al., 1976; Horwitz et al., 1977). My own experiences with esterase methods applied to human peripheral blood leucocytes have been disappointing. Although staining of buffy coat smears permitted clear distinction between populations of monocytes and lymphocytes, decreased leucocyte stainability was observed after their separation by the Ficoll-Isopaque method of BSyum (1968). Loss of esterase activity in cytocentrifuge smears of mononuclear cells has also been observed by Horwitz et al. (1977). A slight modification of the esterase method is proposed, which in our hands produces minimal loss of esterase activity in cytocentrifuge smears of human peripheral blood monocytes and lymphocytes. MA TER I ALS AND METHODS
Blood leucocytes. Heparinized venous blood of healthy human donors was used throughout. Leucocyte preparations used for cytocentrifuge smears included (1) buffy coat cells, (2) mononuclear cells isolated according to BSyum (1968), (3) plastic adherent and non-adherent cells, (4) SRBC rosetting and non-rosetting cells (Gmelig, 1977). The final leucocyte suspensions were adjusted to 5 × 10 s cells/ml in RPMI-1640-Hepes (GIBCO) con-
194
taining 1% BSA (medium-BSA), except for buffy coat cells which were contained in autologous plasma. Cytocentrifuge smears. Cells were pelleted onto microscope slides in a Shandon-Elliott cytocentrifuge. The cuvettes with filter paper in place were filled with 0.1 ml medium-BSA and spun for 5 min at 1000 rev/min. After pre-wetting the filters, the cuvettes received another 0.1 ml medium-BSA on to which 50 pl of the respective cell suspension was layered, followed by spinning for 3 min at 750 rev/min. Esterase staining. Fixation and staining were carried out by the methods of Ornstein et al. (1976) and Horwitz et al. (1977), using as substrates 1-naphthyl acetate (Koch-Light) and 1-naphthyl butyrate (Sigma). For optimal staining, the 1-naphthyl butyrate esterase m e t h o d of Ornstein was employed with the following modifications: (1) smears were air-dried in the dark for 12--18 h at room temperature, (2) w i t h o u t further fixation, dried smears were incubated with the substrate mixture for 30--45 min instead of 10 min, (3) after counterstaining for 5 min in 2% m e t h y l green, the smears were rinsed in distilled water, air-dried and m o u n t e d in Entellan-Neu (Merck) or sucrose 85%. The smears were examined by both phase contrast and ordinary transmitted light microscopy. RESULTS AND COMMENTS
Several procedures for cell fixation and esterase staining were compared to establish which was most satisfactory. Best results were obtained when wet b u f f y coat smears were fixed for 9 min in formaldehyde vapour (Ornstein et al., 1976). In general, monocytes developed a diffuse reddish brown cytoplasmic staining pattern as distinct from lymphocytes which showed one or more discrete granules of reaction product. Since the esterase m e t h o d of Ornstein et al. (1976) does not require the use of freshly prepared stock reagents, this m e t h o d was preferred in combination with formaldehyde vapour fixation. When applied, however, to cytocentrifuge smears of Ficoll-Isopaque enriched mononuclear cells, marked loss of staining intensity was observed as compared with the reactive cells in buffy coat smears. Longer incubation with substrate (30 min instead of 10 min) slightly improved the stainability of punctate granules in lymphocytes. In the course of our studies we accidentally f o u n d that air-drying of cytocentrifuge smears for 12--18 h w i t h o u t further fixation resulted in optimal preservation of esterase activity. Control incubations with staining solution deprived of esterase substrate resulted in a complete absence of reaction product. Table 1 illustrates that fixation in comparison with air-drying gives an underestimate of the proportion of esterase positive cells, in particular of granular staining lymphocytes. Air-drying w i t h o u t further fixation appears, therefore, the m e t h o d of choice. A disadvantage of this procedure may be some loss of morphological detail. Furthermore, when E rosette-forming
195 TABLE 1 COMPARISON OF ESTERASE STAINING OF MONONUCLEAR ACETONITRILE-FIXED AND AIR-DRIED SMEARS Cytocentrifuge leucocyte smear
M o n o n u c l e a r cells A d h e r e n t cells E r o s e t t i n g cells b
Positive (%) a Diffuse
L E U C O C Y T E S IN
Negative (%) Granular
Monocytes
Lymphocytes Fixed
Dried
25.3 26.7 20.1
9.8 19.8 7.1
Fixed
Dried
Fixed
Dried
Fixed
12.5 64.0 0
18.5 66.8 0
55.5 5.2 79.9
71.2 12.2 92.1
5.8 4.1 --
Dried
