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A new micromethod for the evaluation of Fc receptor expression on polymorphonuclear neutrophils
Journal oflmmunologicalMethods, 89 (1986) 49-51
49
Elsevier JIM03874
A new micromethod for the evaluation of Fc receptor expression on polymorphonuclear neutrophils A d a m Szczepanik, R o m a n Klimas and H e n r y k Wysocki * Department of Hernatologv, Institute of Internal Medicine, A('aden~v of Medicine, ul. Szkolna 8/12. 61-833 Poznah. Poland
(Received 10 September1985, accepted 5 December1985)
A simple method for the evaluation of rosetting polymorphonuclear neutrophils is described. It uses small (200 ~1) quantities of heparinized whole blood and is simpler than the micromethod proposed by Buescher and Gallin. The high concentration of neutrophils and paucity of lymphocytes considerably simplify the evaluation of specimens and make the technique particularly useful in granulopenic states. There was a close correlation between results obtained using this technique and those from a standard method requiring the isolation of polymorphonuclear neutrophils. Key words: Polymorphonuelear neutrophils; Leukocyte rosetting," Fc receptor expression; Micromethod
Introduction
The heterogeneity of circulating polymorphonuclear neutrophils (PMN) is well documented. The expression of surface Fc receptors for IgG permits the differentiation of 2 PMN subpopulations, each manifesting distinct functional properties. It has been claimed that in normal conditions ca. 80% of circulating PMN form rosettes using IgG-coated sheep erythrocytes (Klempner and Gallin, 1978). The rosette-forming neutrophils (RFN) have superior adhesiveness, aggregation and chemotaxis to C5a and they are also superior in the phagocytosis and killing of opsonized bacteria (Gallin, 1984). These specific functional properties of RFN probably explain their almost exclusive participation in the formation of abscesses or sterile inflammatory reactions (Wysocki et al., 1981). These cells significantly predominate in the marginal granulocyte pool (Wierusz-Wysocka et al., 1981) and are probably the first to migrate out of the * To whom correspondenceshould be addressed.
microcirculation. Therefore, the evaluation of both RFN and non-RFN in peripheral blood and other biological specimens is of importance. It seems likely that some of the acquired defects of neutrophil function seen in certain inflammatory diseases may be a reflection of differences in the distribution of PMN subpopulations. Until recently the procedures for determining percentages of rosetting PMN in the peripheral blood were complicated by 2 difficulties: the need to purify neutrophils before rosetting and the requirement for relatively large amounts of blood from which to purify the cells. Buescher and Gallin (1983) have recently described a new micromethod for leukocyte rosetting without cell purification. The authors demonstrated that the percentages of rosetting cells in preparations obtained by standard purification methods correlated closely with the percentages obtained by the new method. This paper describes a new technique which is a significant modification of above assay, but retains all its advantages. The reliability of the new technique has been evaluated by comparison with the standard method of Klempner and Gallin (1978).
0022-1759/86/$03.50 ~ 1986 ElsevierSciencePublishers B.V. (Biomedical Division)
50
Estimations were performed on 30 persons, both healthy and suffering from various diseases (diabetes mellitus, Hodgkin's disease, acute myocardial infarction, chronic bronchitis, bacterial pneumonia).
Materials and methods
Sheep leukocyte- and platelet-free red blood cells (RBC) were obtained commercially. The rabbit anti-sheep RBC serum (Biomed) was titered against the purified, washed RBC, and the first dilution at which RBC were microscopically unagglutinated was used for RBC sensitization. Equal volumes of purified RBC (at 1 × 109/ml) and diluted antibody were mixed and incubated at 37°C for 30 min. Sensitized RBC were then washed in PBS 3 times and readjusted to 1 x 109 cells/ml in Hanks' balanced salt solution (HBSS). A monolayer of white blood cells was prepared by dripping 0.2 ml of whole heparinized blood (10 U / m l ) onto standard coverslips and allowing the blood to spread out to cover most of the surface. Each of coverslips was supported by a salinemoistened sponge, placed in a petri dish and incubated at 37°C with full humidity for 30 rain. Following incubation, the coverslips were gently washed with HBSS (37°C) and decanted off leaving a barely perceptible film. To each freshly prepared monolayer of adherent cells (mostly polymorphonuclear neutrophils) was added 200/xl of a previously prepared suspension of sensitized sheep RBC. The petri dish was then covered and placed on a turntable rotating at 5 rpm and incubated at 37°C for 20 min. Following incubation t~e coverslips were removed from the petri dishes, washed gently with HBSS (37°C) and stained for 20 30 s with acridine orange (Koch Light) diluted 1 : 10000 in PBS. The excess of dye solution was decanted and the coverslips were then mounted on glass slides, monolayer side down. The coverslips were rimmed, sealed with nail polish and examined under a UV fluorescent microscope using a halogen light source and a 100 x oil immersion objective. By adjusting the amount of incandescent light illuminating the specimen leukocytes located by their fluorescent nuclei were simultaneously scored as rosetting (3
RBC attached) or non-rosetting. The estimations of rosetting PMN using the new and the standard method were always performed separately by 2 laboratory assistants. It was evident that some of the neutrophils were rinsed from the coverslip surface during the procedure. Therefore, in 10 cases we carefully collected all the fluids used in washing the coverslips and performed both total and differential leukocyte counts.
Results
The percentages of rosetting neutrophils in the peripheral blood samples from the various patient and control groups as estimated by both the standard method and the new modification are listed in Table I. Fig. 1 graphically compares percentage rosetting as evaluated by the standard method using purified neutrophils, with neutrophil rosening measured by the new modification. Statistically, the absolute average difference between the 2 A
n,
FIG1
100 /
/
/
90
#
N=30
//
80
/
%
70 60
a?f °
50 40, o,,o
30, 20, 10
oO /
o
cx,//'6
9" I
1; 20 3'o .; 5; ~b ;o 8'0 9; 1;o
,~',of RFN (standard method)
Fig. 1. Comparison of rosette-forming neutrophils (RFN), using whole blood (new method ordinate) and purified PMN (standard method - - abscissa). Each point represents a single comparison. The line represents linear regression (least squares method) through the plotted points.
51 TABLE I PERCENTAGES OF ROSETTE-FORMING NEUTROPHILS (RFN) IN PERIPHERAL BLOOD SAMPLES OF PATIENTS AND HEALTHY INDIVIDUALS (MEAN VALUES, SD, RANGE) ESTIMATED USING THE NEW AND STANDARD METHODS Patients
Controls Diabetes mellitus Hodgkin's disease Acute myocardial infarction Chronic bronchitis Bacterial pneumonia
n
RFN - new method
RFN
2
_+SD
ff
+ SD
8 9 6
61.9 41.7 16.8
6.03 10.70 13.02
60.9 38.6 17.2
6.03 14.19 11.42
3 3 1
40.0 48.3 18
11.78 4.50 -
35.7 42.7 15
14.57 2.30
m e t h o d s was 4.7% a n d the S p e a r m a n r a n k c o r r e l a t i o n c o e f f i c i e n t was s t a t i s t i c a l l y s i g n i f i c a n t ( P < 0.001). T h e e v a l u a t i o n o f cells lost d u r i n g the p r o p o s e d p r o c e d u r e r e v e a l e d that the cells rinsed f r o m the c o v e r s l i p s w e r e m o s t l y m o n o n u c l e a r leukocytes.
standard method
Range New' method
Standard method
max
min
max
irlin
72 58 39
54 29 4
71 60 37
53 18 9
53 53
30 44
51 44
22 40
W e believe that o u r p r o c e d u r e is n o v e l b e c a u s e it c o n s i d e r a b l y r e d u c e s the m a n i p u l a t i o n s used in the s t a n d a r d assay (e.g., c e n t r i f u g a t i o n ) . T h e m e t h o d e n a b l e s the e v a l u a t i o n o f a l m o s t p u r e P M N p o p u l a t i o n s with o n l y a small a d m i x t u r e of m o n o cytes. T h e n e w t e c h n i q u e s u p p l i e s a relative large n u m b e r of cells o n glass slides and s h o u l d be p a r t i c u l a r l y useful in studies of g r a n u l o p e n i c states.
Discussion T h e p r o p o s e d p r o c e d u r e for d e t e r m i n i n g perc e n t a g e s of r o s e t t i n g P M N s e e m s to o v e r c o m e the m o s t i m p o r t a n t d r a w b a c k s of the s t a n d a r d assay. T h e d i f f e r e n c e s b e t w e e n the results o b t a i n e d using the n e w a n d s t a n d a r d m e t h o d s are small a n d statistically insignificant. T h e a d d i t i o n a l s t u d i e s p e r f o r m e d to b e t t e r c h a r a c t e r i z e the cells rinsed f r o m the c o v e r s l i p s i n d i c a t e d that g e n t l e w a s h i n g did not s i g n i f i c a n t l y d e t a c h the p o p u l a t i o n of cells h a v i n g d e c r e a s e d adhesive properties {probably non-rosetting PMN).
References Buescher, E.S. and J.l. Gallin, 1983, J. Immunol. Methods 61, 141. Gallin, J.I., 1984, Blood 63, 977. Klempner, M.S. and J.I. Gallin, 1978, Blood 51, 659. Wierusz-Wysocka, B., R. Klimas, H. Siekierka and H. Wysocki, 1981, lmmunol. Pol. 6. 209 Wysocki, H., R. Czarnecki. B. Wierusz-Wysocka, G. Michta, K. Baczyk and K. Wysocki, 1981, in: Advances in Peritoneal Dialysis, eds. G.M. Gahl, M. Kessel, K.D. Nolph (Excerpta Medica, Amsterdam) p. 16.
49
Elsevier JIM03874
A new micromethod for the evaluation of Fc receptor expression on polymorphonuclear neutrophils A d a m Szczepanik, R o m a n Klimas and H e n r y k Wysocki * Department of Hernatologv, Institute of Internal Medicine, A('aden~v of Medicine, ul. Szkolna 8/12. 61-833 Poznah. Poland
(Received 10 September1985, accepted 5 December1985)
A simple method for the evaluation of rosetting polymorphonuclear neutrophils is described. It uses small (200 ~1) quantities of heparinized whole blood and is simpler than the micromethod proposed by Buescher and Gallin. The high concentration of neutrophils and paucity of lymphocytes considerably simplify the evaluation of specimens and make the technique particularly useful in granulopenic states. There was a close correlation between results obtained using this technique and those from a standard method requiring the isolation of polymorphonuclear neutrophils. Key words: Polymorphonuelear neutrophils; Leukocyte rosetting," Fc receptor expression; Micromethod
Introduction
The heterogeneity of circulating polymorphonuclear neutrophils (PMN) is well documented. The expression of surface Fc receptors for IgG permits the differentiation of 2 PMN subpopulations, each manifesting distinct functional properties. It has been claimed that in normal conditions ca. 80% of circulating PMN form rosettes using IgG-coated sheep erythrocytes (Klempner and Gallin, 1978). The rosette-forming neutrophils (RFN) have superior adhesiveness, aggregation and chemotaxis to C5a and they are also superior in the phagocytosis and killing of opsonized bacteria (Gallin, 1984). These specific functional properties of RFN probably explain their almost exclusive participation in the formation of abscesses or sterile inflammatory reactions (Wysocki et al., 1981). These cells significantly predominate in the marginal granulocyte pool (Wierusz-Wysocka et al., 1981) and are probably the first to migrate out of the * To whom correspondenceshould be addressed.
microcirculation. Therefore, the evaluation of both RFN and non-RFN in peripheral blood and other biological specimens is of importance. It seems likely that some of the acquired defects of neutrophil function seen in certain inflammatory diseases may be a reflection of differences in the distribution of PMN subpopulations. Until recently the procedures for determining percentages of rosetting PMN in the peripheral blood were complicated by 2 difficulties: the need to purify neutrophils before rosetting and the requirement for relatively large amounts of blood from which to purify the cells. Buescher and Gallin (1983) have recently described a new micromethod for leukocyte rosetting without cell purification. The authors demonstrated that the percentages of rosetting cells in preparations obtained by standard purification methods correlated closely with the percentages obtained by the new method. This paper describes a new technique which is a significant modification of above assay, but retains all its advantages. The reliability of the new technique has been evaluated by comparison with the standard method of Klempner and Gallin (1978).
0022-1759/86/$03.50 ~ 1986 ElsevierSciencePublishers B.V. (Biomedical Division)
50
Estimations were performed on 30 persons, both healthy and suffering from various diseases (diabetes mellitus, Hodgkin's disease, acute myocardial infarction, chronic bronchitis, bacterial pneumonia).
Materials and methods
Sheep leukocyte- and platelet-free red blood cells (RBC) were obtained commercially. The rabbit anti-sheep RBC serum (Biomed) was titered against the purified, washed RBC, and the first dilution at which RBC were microscopically unagglutinated was used for RBC sensitization. Equal volumes of purified RBC (at 1 × 109/ml) and diluted antibody were mixed and incubated at 37°C for 30 min. Sensitized RBC were then washed in PBS 3 times and readjusted to 1 x 109 cells/ml in Hanks' balanced salt solution (HBSS). A monolayer of white blood cells was prepared by dripping 0.2 ml of whole heparinized blood (10 U / m l ) onto standard coverslips and allowing the blood to spread out to cover most of the surface. Each of coverslips was supported by a salinemoistened sponge, placed in a petri dish and incubated at 37°C with full humidity for 30 rain. Following incubation, the coverslips were gently washed with HBSS (37°C) and decanted off leaving a barely perceptible film. To each freshly prepared monolayer of adherent cells (mostly polymorphonuclear neutrophils) was added 200/xl of a previously prepared suspension of sensitized sheep RBC. The petri dish was then covered and placed on a turntable rotating at 5 rpm and incubated at 37°C for 20 min. Following incubation t~e coverslips were removed from the petri dishes, washed gently with HBSS (37°C) and stained for 20 30 s with acridine orange (Koch Light) diluted 1 : 10000 in PBS. The excess of dye solution was decanted and the coverslips were then mounted on glass slides, monolayer side down. The coverslips were rimmed, sealed with nail polish and examined under a UV fluorescent microscope using a halogen light source and a 100 x oil immersion objective. By adjusting the amount of incandescent light illuminating the specimen leukocytes located by their fluorescent nuclei were simultaneously scored as rosetting (3
RBC attached) or non-rosetting. The estimations of rosetting PMN using the new and the standard method were always performed separately by 2 laboratory assistants. It was evident that some of the neutrophils were rinsed from the coverslip surface during the procedure. Therefore, in 10 cases we carefully collected all the fluids used in washing the coverslips and performed both total and differential leukocyte counts.
Results
The percentages of rosetting neutrophils in the peripheral blood samples from the various patient and control groups as estimated by both the standard method and the new modification are listed in Table I. Fig. 1 graphically compares percentage rosetting as evaluated by the standard method using purified neutrophils, with neutrophil rosening measured by the new modification. Statistically, the absolute average difference between the 2 A
n,
FIG1
100 /
/
/
90
#
N=30
//
80
/
%
70 60
a?f °
50 40, o,,o
30, 20, 10
oO /
o
cx,//'6
9" I
1; 20 3'o .; 5; ~b ;o 8'0 9; 1;o
,~',of RFN (standard method)
Fig. 1. Comparison of rosette-forming neutrophils (RFN), using whole blood (new method ordinate) and purified PMN (standard method - - abscissa). Each point represents a single comparison. The line represents linear regression (least squares method) through the plotted points.
51 TABLE I PERCENTAGES OF ROSETTE-FORMING NEUTROPHILS (RFN) IN PERIPHERAL BLOOD SAMPLES OF PATIENTS AND HEALTHY INDIVIDUALS (MEAN VALUES, SD, RANGE) ESTIMATED USING THE NEW AND STANDARD METHODS Patients
Controls Diabetes mellitus Hodgkin's disease Acute myocardial infarction Chronic bronchitis Bacterial pneumonia
n
RFN - new method
RFN
2
_+SD
ff
+ SD
8 9 6
61.9 41.7 16.8
6.03 10.70 13.02
60.9 38.6 17.2
6.03 14.19 11.42
3 3 1
40.0 48.3 18
11.78 4.50 -
35.7 42.7 15
14.57 2.30
m e t h o d s was 4.7% a n d the S p e a r m a n r a n k c o r r e l a t i o n c o e f f i c i e n t was s t a t i s t i c a l l y s i g n i f i c a n t ( P < 0.001). T h e e v a l u a t i o n o f cells lost d u r i n g the p r o p o s e d p r o c e d u r e r e v e a l e d that the cells rinsed f r o m the c o v e r s l i p s w e r e m o s t l y m o n o n u c l e a r leukocytes.
standard method
Range New' method
Standard method
max
min
max
irlin
72 58 39
54 29 4
71 60 37
53 18 9
53 53
30 44
51 44
22 40
W e believe that o u r p r o c e d u r e is n o v e l b e c a u s e it c o n s i d e r a b l y r e d u c e s the m a n i p u l a t i o n s used in the s t a n d a r d assay (e.g., c e n t r i f u g a t i o n ) . T h e m e t h o d e n a b l e s the e v a l u a t i o n o f a l m o s t p u r e P M N p o p u l a t i o n s with o n l y a small a d m i x t u r e of m o n o cytes. T h e n e w t e c h n i q u e s u p p l i e s a relative large n u m b e r of cells o n glass slides and s h o u l d be p a r t i c u l a r l y useful in studies of g r a n u l o p e n i c states.
Discussion T h e p r o p o s e d p r o c e d u r e for d e t e r m i n i n g perc e n t a g e s of r o s e t t i n g P M N s e e m s to o v e r c o m e the m o s t i m p o r t a n t d r a w b a c k s of the s t a n d a r d assay. T h e d i f f e r e n c e s b e t w e e n the results o b t a i n e d using the n e w a n d s t a n d a r d m e t h o d s are small a n d statistically insignificant. T h e a d d i t i o n a l s t u d i e s p e r f o r m e d to b e t t e r c h a r a c t e r i z e the cells rinsed f r o m the c o v e r s l i p s i n d i c a t e d that g e n t l e w a s h i n g did not s i g n i f i c a n t l y d e t a c h the p o p u l a t i o n of cells h a v i n g d e c r e a s e d adhesive properties {probably non-rosetting PMN).
References Buescher, E.S. and J.l. Gallin, 1983, J. Immunol. Methods 61, 141. Gallin, J.I., 1984, Blood 63, 977. Klempner, M.S. and J.I. Gallin, 1978, Blood 51, 659. Wierusz-Wysocka, B., R. Klimas, H. Siekierka and H. Wysocki, 1981, lmmunol. Pol. 6. 209 Wysocki, H., R. Czarnecki. B. Wierusz-Wysocka, G. Michta, K. Baczyk and K. Wysocki, 1981, in: Advances in Peritoneal Dialysis, eds. G.M. Gahl, M. Kessel, K.D. Nolph (Excerpta Medica, Amsterdam) p. 16.