Vox Sang. 28: 57-61 (1975)
Agglutination Kinetics A Method for Quantitative Red Cell Antigen Assays
M. LOPEZ,B. HABIBI, J . LEMEUD and C . SALMON Centre departemental de Transfusion sanguine et Groupe de Recherches U 76 de I’lnserm sur les Groupes sanguins et I’Immuno-HCmatologie, Paris
Abstract. A simple, sensitive and highly reproducible method is described using a particle counting procedure for the study of agglutination percentage of red cells as a function of time. The method may advantageously be used for the current study of zygosity and other antigenic variations especially in family investigations.
The velocity of agglutination has seldom been exploited in the quantitative assay of antigens rather than that of antibodies [6, 71. Advantage may, however, be taken of the accuracy and reproducibility of electronic particle counting procedures which, applied to the study of agglutination kinetics, may provide information of interest about the red cell antigenic makeup. The present preliminary investigation was designed to compare the agglutination behaviour of various cell types with the same antisera in the hope of presenting further criteria for the definition of qualitative or quantitative red cell antigen variations.
Materials and Methods Washed red cells were suspended in isotonic phosphate buffer pH 7.3 with the following composition: Na,HPO,, 12 H,O: 20 g; KH,PO,: 1.4 g; NaCl: 7.2 g; AB serum: 1 ml; distilled water: 1,OOO ml. Serum dilutions were made in the same diluent immediately before use. Reaction mixtures were prepared in duplicate in 5-ml glass ampoules containing 0.4 ml of serum at appropriate dilution and 0.2 ml of a cell suspension containing 6 x 105 cells/mm3. Control mixtures were similarly prepared containing phosphate buffer
Received: May 29, 1974; accepted: July 7, 1974.
58
LOPEZet al.
in place of antiserum. The ampoules were sealed and placed on a horizontal linear thermostabilized shaker at 60 oscillations per minute. A 4 pl aliquot was removed from each reaction mixture after 20, 40, 60, 90, 120, 180 and 240 min and free cells were counted in a Coulter counter (model B) according to the Bowdler and Swisher ‘aggregate exclusion’ method [i]. The red cell size distribution and the threshold settings were previously determined and the total cell count/mm3 obtained from the phosphate buffer controls; the agglutination percentage was estimated for each period of incubation. Immune anti-A, -B, -C, -c, -D, -E, -e anti-S and -N sera were tested against a variety of red cells of known phenotype as well as cells obtained within the same family. Conventional quantitative antigen studies by serial dilution titration and agglutination percentage measurements at equilibrium were performed in parallel. The cells were generally assayed on at least two separate occasions.
Results ABO antigen assays with selected immune anti-B antibody at 4°C showed clear-cut dynamic differences of agglutination between cells with different weak B phenotypes which otherwise exhibited the same agglutination percentage at equilibrium (fig. 1). Similar curves were obtained in family members indicating that an individual rate of agglutination actually reflects a genetically determined red cell antigenic makeup [3]. N o significant difference was found with respect to the ABO system between homozygous and heterozygous individuals with normal expression of A or B antigens. Dosage effect was, on the contrary, well-demonstrated by the technique with respect to R h , S and N antigens. Out of 2 anti-N, 2 a n t i 4 10 anti-D, 5 anti-C, 2 anti-c, 8 anti-E and 2 anti-e sera, first evaluated against selected cells with known MNS and Rh genotypes, 1 anti-N, 2 anti-S, 1 anti-D, I anti-C, 1 anti-c, 2 anti-E and 2 anti-e sera were found to give a clear effect of zygosity (fig. 1). Among the selected sera, one anti-S, anti-N, anti-c and the 2 anti-e exhibited, though less distinctly, a dose effect in serial dilution titration techniques whereas the others failed by manual procedures to distinguish homozygous from heterozygous cells. Moreover, three cells with silent Rh phenotypes, DcE/D - - ,dce/D - - and DCe/---, were fully distinguished from DcE/DcE, dce/Dce and DCe/DCe cells while this could not be clearly achieved by manual techniques. The agglutination kinetics of the Rh antigens was found to be subject to great variations according to companion antigens and, among cells with the same genotype, according to individuals. These variations were similar to those obtained by other quantitative methods [2, 4, 51. The behaviour of an individual R h antigen was however found to be the same among family
Agglutination Kinetics
59
80
< C
0 40 4
5
-m 3
m
6
0 120
180
240 mln
80
> C'
0 40
c
C ._ c
3
m m
6
0
1
1
80
< C
'J 40
c
r3
c-
-*am m
Anti-6 153
= l o 60
120
180
24Omln
I
60
I
120
180 I
240mtn I
Fig. I . Agglutination kinetics and demonstration of dosage effect using anti-B, -C, -c, -D, -E, e,-Nand -S sera. Figures in shaded areas indicate number of individuals tested representing a full spectrum of common homozygous (upper bands) and heterozygous (lower bands) phenotypes. Anti-B has been assayed on five different weak B samples. A, A =Two related individuals with B, phenotype; X , 0,@=three related individuals with Bx phenotype.
60
LOPEZel al.
CcDee
CcDee
CcDee
.,
NT
Anti-c 4 5 7 2 I
I
I
60
120
180
I
2 4 0 min
80
1 :
/
Ant i - D 326
0 I
I
I
60
120
180
I
240min
60
120
180
240 min
Fig. 2. Agglutination behaviour of cells within the same family.
members having identical genotypes (fig. 2). The rate as well as the final degree of agglutination of D ( + ) cells were found to progress in the wellestablished descending order of D--> DcE> Dce> DCe. N o similar phenomenon was found with respect to C, c, E and e antigens. Incomplete antibodies needing antiglobulin techniques or enzyme-treated cells ( K , Fy, Jk) were not found to be suitable for dosage studies, The reproducibility of the technique was found, irrespective of antibody, to be remarkably high. Assays on multiple samples obtained from the same donors throughout a 2-year period yielded fully identical curves.
Comments
The present method was designed to take advantage of accurate standardization of factors playing a determinant part in quantitation of agglutination : number of cells in the initial reaction mixture, steady continuous shaking and precise counting of free cells throughout the reaction. Two pieces of quantitative information were p;ovided by plotting the
Agglutination Kinetics
61
agglutination percentage as a function of time : the velocity of agglutination and the final extent of equilibrium when a plateau is attained. The precise numerical data obtained in this way proved to be useful for the study of zygosity and other antigenic variations, especially in family investigations, providing saline antibodies are available. The procedure was more sensitive and remarkably more reproducible than the manual method of agglutination scoring. It could therefore advantageously compete with conventional methods for red cell antigen quantitation.
References 1 BOWDLER, A. J. and SWISHER, S. H.: Electronic particle counting applied to the quantitative study of red cell agglutination. Transfusion 4: 153 (1964). 2 GIBBS,M. B. and ROSENFIELD, R. E.: Immunochemical studies of the Rh system.
IV. Hemagglutination assay of antigenic expression regulated by interaction between paired Rh genes. Transfusion 6: 462 (1966). 3 LOPEZ,M.; BOUGUERRA, A.; LEMEUD, J.; BADET,J., and SALMON, C.: Quantitative, kinetic and thermodynamic analysis of weak B 60 erythrocyte phenotypes. Heterogeneity among families, identity within a family. Vox Sang. (in press, 1974). 4 MASOUREDIS, S. P.; DUPUY,M. E., and ELLIOT,M.: Relationship between Rho (D) zygosity and red cell Rho (D) antigen content in family members. J. clin. Invest. 46: 681 (1967). 5 SILBER,R.; GIBBS,M. B.; JAHN,E. F., and AKEYROD, J. H.: Quantitative hemag-
glutination studies in the Rh blood group system. I. The assay of Anti-D (Rho) agglutinin. Blood 17: 282 (1961). 11. A study of D (Rho) agglutinogen. Blood 17: 291 (1961). J. M.; GIBBS,M. B., and BOWDLER, A. J.: Methods in quantitative hemag6 SOLOMON, glutination. Vox Sang. 10: 54 (1965). C., and GOODMAN, H. S.: Rate studies in immune 7 TAU KUANG MING; SEVERSON, hemagglutination. J. Immunol. 93: 576 (1964).
Request reprints from : M. LOPEZ,Centre Dtpartmental de Transfusion Sanguine, 53, Boulevard Diderot, 75012 Paris (France)
Agglutination Kinetics A Method for Quantitative Red Cell Antigen Assays
M. LOPEZ,B. HABIBI, J . LEMEUD and C . SALMON Centre departemental de Transfusion sanguine et Groupe de Recherches U 76 de I’lnserm sur les Groupes sanguins et I’Immuno-HCmatologie, Paris
Abstract. A simple, sensitive and highly reproducible method is described using a particle counting procedure for the study of agglutination percentage of red cells as a function of time. The method may advantageously be used for the current study of zygosity and other antigenic variations especially in family investigations.
The velocity of agglutination has seldom been exploited in the quantitative assay of antigens rather than that of antibodies [6, 71. Advantage may, however, be taken of the accuracy and reproducibility of electronic particle counting procedures which, applied to the study of agglutination kinetics, may provide information of interest about the red cell antigenic makeup. The present preliminary investigation was designed to compare the agglutination behaviour of various cell types with the same antisera in the hope of presenting further criteria for the definition of qualitative or quantitative red cell antigen variations.
Materials and Methods Washed red cells were suspended in isotonic phosphate buffer pH 7.3 with the following composition: Na,HPO,, 12 H,O: 20 g; KH,PO,: 1.4 g; NaCl: 7.2 g; AB serum: 1 ml; distilled water: 1,OOO ml. Serum dilutions were made in the same diluent immediately before use. Reaction mixtures were prepared in duplicate in 5-ml glass ampoules containing 0.4 ml of serum at appropriate dilution and 0.2 ml of a cell suspension containing 6 x 105 cells/mm3. Control mixtures were similarly prepared containing phosphate buffer
Received: May 29, 1974; accepted: July 7, 1974.
58
LOPEZet al.
in place of antiserum. The ampoules were sealed and placed on a horizontal linear thermostabilized shaker at 60 oscillations per minute. A 4 pl aliquot was removed from each reaction mixture after 20, 40, 60, 90, 120, 180 and 240 min and free cells were counted in a Coulter counter (model B) according to the Bowdler and Swisher ‘aggregate exclusion’ method [i]. The red cell size distribution and the threshold settings were previously determined and the total cell count/mm3 obtained from the phosphate buffer controls; the agglutination percentage was estimated for each period of incubation. Immune anti-A, -B, -C, -c, -D, -E, -e anti-S and -N sera were tested against a variety of red cells of known phenotype as well as cells obtained within the same family. Conventional quantitative antigen studies by serial dilution titration and agglutination percentage measurements at equilibrium were performed in parallel. The cells were generally assayed on at least two separate occasions.
Results ABO antigen assays with selected immune anti-B antibody at 4°C showed clear-cut dynamic differences of agglutination between cells with different weak B phenotypes which otherwise exhibited the same agglutination percentage at equilibrium (fig. 1). Similar curves were obtained in family members indicating that an individual rate of agglutination actually reflects a genetically determined red cell antigenic makeup [3]. N o significant difference was found with respect to the ABO system between homozygous and heterozygous individuals with normal expression of A or B antigens. Dosage effect was, on the contrary, well-demonstrated by the technique with respect to R h , S and N antigens. Out of 2 anti-N, 2 a n t i 4 10 anti-D, 5 anti-C, 2 anti-c, 8 anti-E and 2 anti-e sera, first evaluated against selected cells with known MNS and Rh genotypes, 1 anti-N, 2 anti-S, 1 anti-D, I anti-C, 1 anti-c, 2 anti-E and 2 anti-e sera were found to give a clear effect of zygosity (fig. 1). Among the selected sera, one anti-S, anti-N, anti-c and the 2 anti-e exhibited, though less distinctly, a dose effect in serial dilution titration techniques whereas the others failed by manual procedures to distinguish homozygous from heterozygous cells. Moreover, three cells with silent Rh phenotypes, DcE/D - - ,dce/D - - and DCe/---, were fully distinguished from DcE/DcE, dce/Dce and DCe/DCe cells while this could not be clearly achieved by manual techniques. The agglutination kinetics of the Rh antigens was found to be subject to great variations according to companion antigens and, among cells with the same genotype, according to individuals. These variations were similar to those obtained by other quantitative methods [2, 4, 51. The behaviour of an individual R h antigen was however found to be the same among family
Agglutination Kinetics
59
80
< C
0 40 4
5
-m 3
m
6
0 120
180
240 mln
80
> C'
0 40
c
C ._ c
3
m m
6
0
1
1
80
< C
'J 40
c
r3
c-
-*am m
Anti-6 153
= l o 60
120
180
24Omln
I
60
I
120
180 I
240mtn I
Fig. I . Agglutination kinetics and demonstration of dosage effect using anti-B, -C, -c, -D, -E, e,-Nand -S sera. Figures in shaded areas indicate number of individuals tested representing a full spectrum of common homozygous (upper bands) and heterozygous (lower bands) phenotypes. Anti-B has been assayed on five different weak B samples. A, A =Two related individuals with B, phenotype; X , 0,@=three related individuals with Bx phenotype.
60
LOPEZel al.
CcDee
CcDee
CcDee
.,
NT
Anti-c 4 5 7 2 I
I
I
60
120
180
I
2 4 0 min
80
1 :
/
Ant i - D 326
0 I
I
I
60
120
180
I
240min
60
120
180
240 min
Fig. 2. Agglutination behaviour of cells within the same family.
members having identical genotypes (fig. 2). The rate as well as the final degree of agglutination of D ( + ) cells were found to progress in the wellestablished descending order of D--> DcE> Dce> DCe. N o similar phenomenon was found with respect to C, c, E and e antigens. Incomplete antibodies needing antiglobulin techniques or enzyme-treated cells ( K , Fy, Jk) were not found to be suitable for dosage studies, The reproducibility of the technique was found, irrespective of antibody, to be remarkably high. Assays on multiple samples obtained from the same donors throughout a 2-year period yielded fully identical curves.
Comments
The present method was designed to take advantage of accurate standardization of factors playing a determinant part in quantitation of agglutination : number of cells in the initial reaction mixture, steady continuous shaking and precise counting of free cells throughout the reaction. Two pieces of quantitative information were p;ovided by plotting the
Agglutination Kinetics
61
agglutination percentage as a function of time : the velocity of agglutination and the final extent of equilibrium when a plateau is attained. The precise numerical data obtained in this way proved to be useful for the study of zygosity and other antigenic variations, especially in family investigations, providing saline antibodies are available. The procedure was more sensitive and remarkably more reproducible than the manual method of agglutination scoring. It could therefore advantageously compete with conventional methods for red cell antigen quantitation.
References 1 BOWDLER, A. J. and SWISHER, S. H.: Electronic particle counting applied to the quantitative study of red cell agglutination. Transfusion 4: 153 (1964). 2 GIBBS,M. B. and ROSENFIELD, R. E.: Immunochemical studies of the Rh system.
IV. Hemagglutination assay of antigenic expression regulated by interaction between paired Rh genes. Transfusion 6: 462 (1966). 3 LOPEZ,M.; BOUGUERRA, A.; LEMEUD, J.; BADET,J., and SALMON, C.: Quantitative, kinetic and thermodynamic analysis of weak B 60 erythrocyte phenotypes. Heterogeneity among families, identity within a family. Vox Sang. (in press, 1974). 4 MASOUREDIS, S. P.; DUPUY,M. E., and ELLIOT,M.: Relationship between Rho (D) zygosity and red cell Rho (D) antigen content in family members. J. clin. Invest. 46: 681 (1967). 5 SILBER,R.; GIBBS,M. B.; JAHN,E. F., and AKEYROD, J. H.: Quantitative hemag-
glutination studies in the Rh blood group system. I. The assay of Anti-D (Rho) agglutinin. Blood 17: 282 (1961). 11. A study of D (Rho) agglutinogen. Blood 17: 291 (1961). J. M.; GIBBS,M. B., and BOWDLER, A. J.: Methods in quantitative hemag6 SOLOMON, glutination. Vox Sang. 10: 54 (1965). C., and GOODMAN, H. S.: Rate studies in immune 7 TAU KUANG MING; SEVERSON, hemagglutination. J. Immunol. 93: 576 (1964).
Request reprints from : M. LOPEZ,Centre Dtpartmental de Transfusion Sanguine, 53, Boulevard Diderot, 75012 Paris (France)
