Vox Sang. 37: 78-83 (1979)
Detection of the H and I Blood Group Antigens in Normal Plasma A Comparison with A and i Antigens P . Rouger, D . Riveau and C. Salmon National Blood Group Reference Laboratory, CNTS, Inserm U76, Paris
Abstract. The levels of A, H, I and i antigens were measured in the plasma of 185 normal subjects by the agglutination-inhibition method. The presence of H in the plasma was only detectable with immune anti-H. The level of H in the plasma was directly correlated with the amount on the red cells, and was affected by the donor secretor status. The plasma of group 0 secretors contained more H than the plasma of donors of other phenotypes. On the other hand, I and i plasma antigens were not related to the other systems studied. Unlike the Ii antigens on the red cell membrane, there was no relationship between the levels of I and i in plasma.
of A and i as a control; (b) the search for the relationships between the levels of these Although there have been many studies antigens; (c) the study of the influence of carried out on the A antigen in plasma [lo- Lewis and secretor systems upon the expres131 and on i antigen in plasma [1-4], there sion of these plasma antigens. have been very few studies on H [7] and I [l,61 in plasma. Materials and Methods The i substance was isolated from plasma as a glycoprotein; the I substance was not In this study, the measurement of H, I, A and found [4].The A substances are present in i antigens in plasma was based on haemagglutinaplasma as glycolipids [13] and glycoproteins tion-inhibition tests. Plasma samples were obtained from 185 healthy [lo]. The site of the synthesis of these plasma substances is unknown; they do not seem men and women, between the ages of 20 and 60 years, and from 6 newborn infants. In haemagto be produced by the red cell precursors. glutination-inhibition tests, 95 samples were tested There are three purpose in this work: with anti-H, 39 samples were tested with anti-I, (a) the levels of H and I antigen in the 64 samples were tested with anti-A, and 33 samplasma of normal subjects, using the levels ples were tested with anti-i.
Introduction
Detection of the H and I Blood Group Antigens in Normal Plasma
79
2% turn settling coil
~
Waste
Saline 0.15M
s0.60$l
0.80
2’/2 turn setting coil Mixer
Waste
,0.60
Air out
1.60
Waste
1.20
4
v Waste ,030
nSaline0.15M v
1.60
v
Fig. 1. Schematic flow diagram of the circuit.
Haemagglutination Inhibition Each plasma sample was tested undiluted, and at dilutions of 1:2, 1:4 and 1:8 in saline. Each dilution of plasma was mixed with a constant antibody concentration. After 2 h at 20 OC, the amount of remaining antibody was measured by haemagglutination in a manifold combined with an autoanalyser. A schematic flow diagram of the circuit is shown in figure 1. Plasma dilutions mixed with antibodies were pumped into a reaction coil. A red cell suspension and PVP were added. This mixture reacted for 30min. The reaction coil was at 10 or 15 OC according to the system used (table I). After double decantation, the residual red cell concentration was determined in a continuous flow counter, and the results were recorded by a pen recorder. From the results obtained, we calculated the agglutination percentage for the chosen dilutions.
The agglutination percentage curve, expressed in terms of the logarithm of tested plasma dilutions, was a sigmoid, which was linearized by ProbitLogarithm transformation [4]. Each tested plasma was referred to a standard inhibitory solution (corresponding to 100 arbitrary inhibition units), and the results are expressed in arbitrary inhibition units (AIU). For these titrations, a very important point is the choice of the most appropriate ‘antibody-antigen system’, which means the lowest antibody concentration that produces the highest efficiency of agglutination; the sensitivity of the method depends upon this choice. Control samples (table I) were used in each set of tests as a check that the inhibition was specific. For each system, all the regressive lines were parallel. The reproducibility of these tests was confirmed by the repeated measurement of the same plasma at the same dilution. The observed variation did not exceed 20%.
80
Rouger/Riveau/Salmon
Table I. Reagents and controls used in the detection of A, H, I and i antigens in plasma
Plasma antigens
A
H
I
i
Temperature-inhibition, "C Temperature-agglutination, "C
20
20 10
20 15
20
15
PVP, %
1.5
1
1.5
1
Red blood cells
A,
0
01
Oi (cord)
Inhibition control
A substance from pig and horse stomach
0 salive pool; 0 and A, sera pool
0 sera pool
0 and A, sera pool
Antisera Several sources of anti-H were tested. Of these, only the serum of a B, subject (Cer.), previously transfused with group 0 blood, permitted the detection of H in plasma. Anti-I (Abg.), from a patient with auto-immune haemolytic anaemia, was used for the detection of I in plasma. Each plasma sample was first treated with 2-mercaptoethanol in order to inactivate the naturally occurring agglutinin, which otherwise interferes with the interpretation of inhibition tests [3]. For the detection of A substance in plasma, a pool prepared from the sera of 15 group 0 subjects, immunized by A substance, was used [12]. Anti-i (Pea.), from a patient with haemolytic anaemia, was used for the detection of i in plasma.
15
' i I 20 40 60 80 100 120 140 160 180 200 AIU
Fig. 2. I plasma antigen in normal population. AIU = Arbitrary Inhibition Units.
Results
H Antigen in Plasma
was not proved, because too few Le(a-b-) subjects were tested.
In the 95 samples tested with anti-H, the A Antigen in Plasma inhibition of agglutination was affected by In 64 samples tested with anti-A, the the ABO phenotype of the donor. The level of H was highest in 0 subjects (average, 140 level of A was higher in A, subjects than in units). The level in A, subjects (average, A, subjects (p = 0.001). The highest level of 110 units) was higher than in A, subjects A was observed in the plasma of Le(a-b-) (average, 51 units). The lowest levels were subjects. The lowest A was observed in the in A,B subjects (average, 25 units). Secre- plasma of Le(a+b-) subjects (p = 0.01). Setors had higher levels than non-secretors cretors had higher levels than non-secretors p = 0.01). The influence of the Lewis system (p = 0.05).
Detection of the H and I Blood Group Antigens in Normal Plasma
Fig. 3. i plasma antigen in normal population. AIU = Arbitrary Inhibition Units.
0
1 2oo
Fig. 4. Study of the relationship between I and i plasma activities in 21 normal subjects: there is no correlation between I and i plasma antigens (r = 0.25, p > 0.2). AIU = Arbitrary Inhibition Units.
I Antigen in Plasma In all 39 samples from adults tested with anti-I (Abg.), there was inhibition of agglutination (fig. 2). The level of I was not related to the ABO, Hh, Lewis or secretor phenotype of the donor. The average level of I in the plasma of newborn infants (6 tested) was 25% of the average level in the plasma of adult subjects. i Antigen in PZasma In all the 33 samples from adults tested with anti-i (Pea.), there was inhibition of agglutination (fig. 3); the level of i antigen was not related to ABO, Hh, Lewis or secretor phenotypes of the donor. As shown in
81
20 40 60 80 100 120 140 160 180 200 220 240 260 280 AlU
. .
1
b
100
0 I antigen,
200
300
AlU
figure 4, there was no relation between I and i plasma antigens. The average level of i in the plasma of newborns was similar to the average level in the plasma of I and i adults (2 adults were tested).
Discussion The essential part of this study is the fact that H and T substances were observed in the plasma of all healthy subjects. A and i antigens were studied as a control. The occurrence of I plasma activity was first suggested by Dzierzkowa-Borodej et aE. [ 6 ] and Abbal et al. [l].Dzierzkowa-Boro-
82
dej et al. demonstrated that 11 normal sera
may have a slight inhibitory effect on the anti-I used (anti-I Mrs. W.). De Boissezon et al. [2] were the first to report the presence of i substance in plasma. The agglutination of anti-i was inhibited by all human plasmas (both adult and newborn). Subsequently, the i substance was isolated from the plasma as a glycoprotein with a molecular weight of about 150,000 [4].In the present study, the inhibition of one anti-i (Pea.) was not related to either ABO, Hh, secretor or Lewis phenotype, nor to the Ii phenotype of the donor. These results suggest that the i phenotype of red cells does not depend on the plasma environment. The fact that there was no correlation between I and i antigen in normal plasma favours the theory that there is no biochemical relationship between these two specificities. These data differ from the results obtained with red cell membranes, but these membrane relationships are probably purely topochemical. Until now, only Gouge et al. [7] have described an anti-H formed by an A, patient which detects H in plasma. This antibody reacts preferentially with group 0 cells and is inhibited by the serum of group 0 secretors as well as by the saliva of all secretors, whatever their ABO group. In our study, only immune anti-H (Cer.) permitted the detection of H substance in the plasma. It is possible that the lectins or non-immune 0, serum react only with type 2 H determinants synthesised by red blood cells, and fails to react with type 1 H determinants from the plasma [ 5 ] . Therefore, the serum of B,, (Cer.) probably contains antibodies which react with both H determinants.
Rouger/Riveau/Salmon
There is a relationship between the H plasma antigen and the ABO phenotype: in the plasma, group 0 and A, subjects have more H than A, and A,B subjects. The same relationship is observed on the red cell membrane and in the saliva. These data confirm the fact that the A substance formed in the plasma is built on an H substrate. The cells synthesising these substances (glycolipids or glycoproteins) are unknown [8, 91. Like many others [5, 10, 11, 131, we found that the amount of A substance is affected by Lewis and secretor status: secretors had more A substance than the nonsecretors; secretors lacking the L e gene had more A substance than secretors possessing the Le gene. Even if the se gene is not completely silent, A and H plasma antigens of sese subjects could be explained by the existence of two kinds of cells able to synthesise these substances: one is affected by secretor status and the other is not. The influence of secretor status seems to be unrelated to molecular type (glycolipids and glycoproteins) [lo, 131.
References Abbal, M.; Smilovic, W.; Boissezon, J. F. de et Ducos, J.: Quelques problkmes posis par les facteurs I et i. C. r. SOC.fr. Himatol. 4: 488 (1972). Boissezon, J. F. de; Marty, Y.; Ducos, J . et Abbal, M.: Prtsence constante d’une substance inhibitrice de l’anticorps anti-i dans le s6rum humain normal. C. r. hebd. S6anc. Acad. Sci., Paris 272: 1448 (1970). Burnie, K.L.: Ii antigens and antibodies. Can. I. med. Technol. 35: 5 (1973). Cooper, A. G . and Brown, M. C.: Serum i antigen: a new human blood group glycoprotein. Biochem. biophys. Res. Commun. 55: 297 (1973). Crookston, M.C.: Antigens common to red blood cells and plasma; in Plenary Session
Detection of the H and I Blood Group Antigens in Normal Plasma
6
7
8
9
10
Book, XV Congr. Int. SOC.Blood Transfusion, pp. 51-61 (Arnette, Paris 1978). Dzierzkowa-Borodej, W.; Seyfried, H.; Nichols, M.; Reid, M., and Marsh, W. L.: The recognition of water soluble I blood group substance. Vox Sang. 18: 222 (1970). Gouge, J. J.; Boyce, F.; Peterson, P., and Marsh, S.: A puzzling problem due to a harmless cold auto-antibody. Unpublished observations, 1976; cited by Mollison, P.L.: Blood transfusion in clinical medicine; 6th ed. (Blackwell, Oxford 1978). Graham, H. A.; Hirsch, H. F., and Davies, 0. M., jr.: Genetic and immunochemical relationships between soluble and cell-bound antigens of the Lewis system; in Human blood groups. Proc. 5th Tnt. Convoc. on Immunology, Buffalo 1976, p. 257 (Karger, Basel 1977). Hirsch, H.F. and Graham, H.A.: Adsorption of plasma antigens onto red blood cells. Commun. Am. Ass. Blood Banks, Atlanta 1977, Abstract S-62. Holburn, A. M. and Masters, C. A.: The radioimmuno-assay of serum and salivary blood group A and Lea glycoproteins. Br. J. Haemat. 28: 157 (1964).
83
11 Hostrup, H.: A and B blood group substances in the serum of normal subjects. Vox Sang. 7: 704 (1962). 12 Renton, P. H. and Hancock, J. A.: Uptake of A and B antigens by transfused group 0 erythrocytes. Vox Sang. 7: 33 (1962). 13 Tilley, C. A.; Crookston, M. C.; Brown, B. L., and Wherrett, J.R.: A and B and Aleb substances in glycosphingolipid fractions of serum. Vox Sang. 28: 25 (1975). 14 Wilkie, M. H. and Becker, E. L.: Quantitative studies in haemagglutination. Effect of certain variables upon the isohaemagglutination assay. Immunology 74: 199 (1955).
Received: November 2,1978 Accepted: March 12, 1979 Ph. Rouger, Centre National de Transfusion Sanguine, 53, boulevard Diderot, F-75571 Paris CCdex 12 (France)
Detection of the H and I Blood Group Antigens in Normal Plasma A Comparison with A and i Antigens P . Rouger, D . Riveau and C. Salmon National Blood Group Reference Laboratory, CNTS, Inserm U76, Paris
Abstract. The levels of A, H, I and i antigens were measured in the plasma of 185 normal subjects by the agglutination-inhibition method. The presence of H in the plasma was only detectable with immune anti-H. The level of H in the plasma was directly correlated with the amount on the red cells, and was affected by the donor secretor status. The plasma of group 0 secretors contained more H than the plasma of donors of other phenotypes. On the other hand, I and i plasma antigens were not related to the other systems studied. Unlike the Ii antigens on the red cell membrane, there was no relationship between the levels of I and i in plasma.
of A and i as a control; (b) the search for the relationships between the levels of these Although there have been many studies antigens; (c) the study of the influence of carried out on the A antigen in plasma [lo- Lewis and secretor systems upon the expres131 and on i antigen in plasma [1-4], there sion of these plasma antigens. have been very few studies on H [7] and I [l,61 in plasma. Materials and Methods The i substance was isolated from plasma as a glycoprotein; the I substance was not In this study, the measurement of H, I, A and found [4].The A substances are present in i antigens in plasma was based on haemagglutinaplasma as glycolipids [13] and glycoproteins tion-inhibition tests. Plasma samples were obtained from 185 healthy [lo]. The site of the synthesis of these plasma substances is unknown; they do not seem men and women, between the ages of 20 and 60 years, and from 6 newborn infants. In haemagto be produced by the red cell precursors. glutination-inhibition tests, 95 samples were tested There are three purpose in this work: with anti-H, 39 samples were tested with anti-I, (a) the levels of H and I antigen in the 64 samples were tested with anti-A, and 33 samplasma of normal subjects, using the levels ples were tested with anti-i.
Introduction
Detection of the H and I Blood Group Antigens in Normal Plasma
79
2% turn settling coil
~
Waste
Saline 0.15M
s0.60$l
0.80
2’/2 turn setting coil Mixer
Waste
,0.60
Air out
1.60
Waste
1.20
4
v Waste ,030
nSaline0.15M v
1.60
v
Fig. 1. Schematic flow diagram of the circuit.
Haemagglutination Inhibition Each plasma sample was tested undiluted, and at dilutions of 1:2, 1:4 and 1:8 in saline. Each dilution of plasma was mixed with a constant antibody concentration. After 2 h at 20 OC, the amount of remaining antibody was measured by haemagglutination in a manifold combined with an autoanalyser. A schematic flow diagram of the circuit is shown in figure 1. Plasma dilutions mixed with antibodies were pumped into a reaction coil. A red cell suspension and PVP were added. This mixture reacted for 30min. The reaction coil was at 10 or 15 OC according to the system used (table I). After double decantation, the residual red cell concentration was determined in a continuous flow counter, and the results were recorded by a pen recorder. From the results obtained, we calculated the agglutination percentage for the chosen dilutions.
The agglutination percentage curve, expressed in terms of the logarithm of tested plasma dilutions, was a sigmoid, which was linearized by ProbitLogarithm transformation [4]. Each tested plasma was referred to a standard inhibitory solution (corresponding to 100 arbitrary inhibition units), and the results are expressed in arbitrary inhibition units (AIU). For these titrations, a very important point is the choice of the most appropriate ‘antibody-antigen system’, which means the lowest antibody concentration that produces the highest efficiency of agglutination; the sensitivity of the method depends upon this choice. Control samples (table I) were used in each set of tests as a check that the inhibition was specific. For each system, all the regressive lines were parallel. The reproducibility of these tests was confirmed by the repeated measurement of the same plasma at the same dilution. The observed variation did not exceed 20%.
80
Rouger/Riveau/Salmon
Table I. Reagents and controls used in the detection of A, H, I and i antigens in plasma
Plasma antigens
A
H
I
i
Temperature-inhibition, "C Temperature-agglutination, "C
20
20 10
20 15
20
15
PVP, %
1.5
1
1.5
1
Red blood cells
A,
0
01
Oi (cord)
Inhibition control
A substance from pig and horse stomach
0 salive pool; 0 and A, sera pool
0 sera pool
0 and A, sera pool
Antisera Several sources of anti-H were tested. Of these, only the serum of a B, subject (Cer.), previously transfused with group 0 blood, permitted the detection of H in plasma. Anti-I (Abg.), from a patient with auto-immune haemolytic anaemia, was used for the detection of I in plasma. Each plasma sample was first treated with 2-mercaptoethanol in order to inactivate the naturally occurring agglutinin, which otherwise interferes with the interpretation of inhibition tests [3]. For the detection of A substance in plasma, a pool prepared from the sera of 15 group 0 subjects, immunized by A substance, was used [12]. Anti-i (Pea.), from a patient with haemolytic anaemia, was used for the detection of i in plasma.
15
' i I 20 40 60 80 100 120 140 160 180 200 AIU
Fig. 2. I plasma antigen in normal population. AIU = Arbitrary Inhibition Units.
Results
H Antigen in Plasma
was not proved, because too few Le(a-b-) subjects were tested.
In the 95 samples tested with anti-H, the A Antigen in Plasma inhibition of agglutination was affected by In 64 samples tested with anti-A, the the ABO phenotype of the donor. The level of H was highest in 0 subjects (average, 140 level of A was higher in A, subjects than in units). The level in A, subjects (average, A, subjects (p = 0.001). The highest level of 110 units) was higher than in A, subjects A was observed in the plasma of Le(a-b-) (average, 51 units). The lowest levels were subjects. The lowest A was observed in the in A,B subjects (average, 25 units). Secre- plasma of Le(a+b-) subjects (p = 0.01). Setors had higher levels than non-secretors cretors had higher levels than non-secretors p = 0.01). The influence of the Lewis system (p = 0.05).
Detection of the H and I Blood Group Antigens in Normal Plasma
Fig. 3. i plasma antigen in normal population. AIU = Arbitrary Inhibition Units.
0
1 2oo
Fig. 4. Study of the relationship between I and i plasma activities in 21 normal subjects: there is no correlation between I and i plasma antigens (r = 0.25, p > 0.2). AIU = Arbitrary Inhibition Units.
I Antigen in Plasma In all 39 samples from adults tested with anti-I (Abg.), there was inhibition of agglutination (fig. 2). The level of I was not related to the ABO, Hh, Lewis or secretor phenotype of the donor. The average level of I in the plasma of newborn infants (6 tested) was 25% of the average level in the plasma of adult subjects. i Antigen in PZasma In all the 33 samples from adults tested with anti-i (Pea.), there was inhibition of agglutination (fig. 3); the level of i antigen was not related to ABO, Hh, Lewis or secretor phenotypes of the donor. As shown in
81
20 40 60 80 100 120 140 160 180 200 220 240 260 280 AlU
. .
1
b
100
0 I antigen,
200
300
AlU
figure 4, there was no relation between I and i plasma antigens. The average level of i in the plasma of newborns was similar to the average level in the plasma of I and i adults (2 adults were tested).
Discussion The essential part of this study is the fact that H and T substances were observed in the plasma of all healthy subjects. A and i antigens were studied as a control. The occurrence of I plasma activity was first suggested by Dzierzkowa-Borodej et aE. [ 6 ] and Abbal et al. [l].Dzierzkowa-Boro-
82
dej et al. demonstrated that 11 normal sera
may have a slight inhibitory effect on the anti-I used (anti-I Mrs. W.). De Boissezon et al. [2] were the first to report the presence of i substance in plasma. The agglutination of anti-i was inhibited by all human plasmas (both adult and newborn). Subsequently, the i substance was isolated from the plasma as a glycoprotein with a molecular weight of about 150,000 [4].In the present study, the inhibition of one anti-i (Pea.) was not related to either ABO, Hh, secretor or Lewis phenotype, nor to the Ii phenotype of the donor. These results suggest that the i phenotype of red cells does not depend on the plasma environment. The fact that there was no correlation between I and i antigen in normal plasma favours the theory that there is no biochemical relationship between these two specificities. These data differ from the results obtained with red cell membranes, but these membrane relationships are probably purely topochemical. Until now, only Gouge et al. [7] have described an anti-H formed by an A, patient which detects H in plasma. This antibody reacts preferentially with group 0 cells and is inhibited by the serum of group 0 secretors as well as by the saliva of all secretors, whatever their ABO group. In our study, only immune anti-H (Cer.) permitted the detection of H substance in the plasma. It is possible that the lectins or non-immune 0, serum react only with type 2 H determinants synthesised by red blood cells, and fails to react with type 1 H determinants from the plasma [ 5 ] . Therefore, the serum of B,, (Cer.) probably contains antibodies which react with both H determinants.
Rouger/Riveau/Salmon
There is a relationship between the H plasma antigen and the ABO phenotype: in the plasma, group 0 and A, subjects have more H than A, and A,B subjects. The same relationship is observed on the red cell membrane and in the saliva. These data confirm the fact that the A substance formed in the plasma is built on an H substrate. The cells synthesising these substances (glycolipids or glycoproteins) are unknown [8, 91. Like many others [5, 10, 11, 131, we found that the amount of A substance is affected by Lewis and secretor status: secretors had more A substance than the nonsecretors; secretors lacking the L e gene had more A substance than secretors possessing the Le gene. Even if the se gene is not completely silent, A and H plasma antigens of sese subjects could be explained by the existence of two kinds of cells able to synthesise these substances: one is affected by secretor status and the other is not. The influence of secretor status seems to be unrelated to molecular type (glycolipids and glycoproteins) [lo, 131.
References Abbal, M.; Smilovic, W.; Boissezon, J. F. de et Ducos, J.: Quelques problkmes posis par les facteurs I et i. C. r. SOC.fr. Himatol. 4: 488 (1972). Boissezon, J. F. de; Marty, Y.; Ducos, J . et Abbal, M.: Prtsence constante d’une substance inhibitrice de l’anticorps anti-i dans le s6rum humain normal. C. r. hebd. S6anc. Acad. Sci., Paris 272: 1448 (1970). Burnie, K.L.: Ii antigens and antibodies. Can. I. med. Technol. 35: 5 (1973). Cooper, A. G . and Brown, M. C.: Serum i antigen: a new human blood group glycoprotein. Biochem. biophys. Res. Commun. 55: 297 (1973). Crookston, M.C.: Antigens common to red blood cells and plasma; in Plenary Session
Detection of the H and I Blood Group Antigens in Normal Plasma
6
7
8
9
10
Book, XV Congr. Int. SOC.Blood Transfusion, pp. 51-61 (Arnette, Paris 1978). Dzierzkowa-Borodej, W.; Seyfried, H.; Nichols, M.; Reid, M., and Marsh, W. L.: The recognition of water soluble I blood group substance. Vox Sang. 18: 222 (1970). Gouge, J. J.; Boyce, F.; Peterson, P., and Marsh, S.: A puzzling problem due to a harmless cold auto-antibody. Unpublished observations, 1976; cited by Mollison, P.L.: Blood transfusion in clinical medicine; 6th ed. (Blackwell, Oxford 1978). Graham, H. A.; Hirsch, H. F., and Davies, 0. M., jr.: Genetic and immunochemical relationships between soluble and cell-bound antigens of the Lewis system; in Human blood groups. Proc. 5th Tnt. Convoc. on Immunology, Buffalo 1976, p. 257 (Karger, Basel 1977). Hirsch, H.F. and Graham, H.A.: Adsorption of plasma antigens onto red blood cells. Commun. Am. Ass. Blood Banks, Atlanta 1977, Abstract S-62. Holburn, A. M. and Masters, C. A.: The radioimmuno-assay of serum and salivary blood group A and Lea glycoproteins. Br. J. Haemat. 28: 157 (1964).
83
11 Hostrup, H.: A and B blood group substances in the serum of normal subjects. Vox Sang. 7: 704 (1962). 12 Renton, P. H. and Hancock, J. A.: Uptake of A and B antigens by transfused group 0 erythrocytes. Vox Sang. 7: 33 (1962). 13 Tilley, C. A.; Crookston, M. C.; Brown, B. L., and Wherrett, J.R.: A and B and Aleb substances in glycosphingolipid fractions of serum. Vox Sang. 28: 25 (1975). 14 Wilkie, M. H. and Becker, E. L.: Quantitative studies in haemagglutination. Effect of certain variables upon the isohaemagglutination assay. Immunology 74: 199 (1955).
Received: November 2,1978 Accepted: March 12, 1979 Ph. Rouger, Centre National de Transfusion Sanguine, 53, boulevard Diderot, F-75571 Paris CCdex 12 (France)
