Acta path. microbiol. scand. Sect. C, 84: 477-484, 1976
LYMPHOCYTE MARKERS IN NEWBORN INFANTS T. HALLBERG and ANITA HALLBERG Department of Medical Microbiology, Lund and Department of Pediatrics, Malmo General Hospital, University of Lund, Sweden
Hallberg, T. & Hallberg, A. Lymphocyte markers in newborn infants. Acta path. microbiol. scand. Sect. C, 84: 477-484, 1976. Purified peripheral blood lymphocytes from 20 newborn infants and 12 adults have been studied for the presence of surface markers. Adults and infants did not differ in the percentage of sheep RBC-binding cells (means 58-65 per cent) or in Ig-bearing cells measured by the mixed antiglobulin reaction (means 7.2-1 1.6 per cent). However, newborn infants had a significantly lower percentage of lymphocytes binding Fc of IgG (mean 10.4 per cent) as compared to adults (mean 17.2 per cent). No overall correlation between plasma Ig levels and Ig-bearing lymphocytes was found but a single infant with increased plasma IgA also showed the highest level of Ig-bearing lymphocytes among all infants. Key words: Lymphocyte markers ; B-cells, T-cells; newborn infants.
T. Hallberg, Department of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
Received 7.vi.76
Accepted 6.vii.76
Lymphocyte surface markers such as surface immunoglobulin, receptors for immunoglobulin and for complement factors and receptors for sheep erythrocytes are valuable tools in the identification of lymphocyte subpopulations in man (for review see HaEEberg 1975). Various investigators have by now established the normal levels in adults of lymphocytes bearing such markers whereas children and infants have been less extensively studied. In the present investigation, peripheral blood lymphocytes from newborn term and preterm infants were investigated by rosetting techniques for the presence of surface immunoglobulin, binding of Fc of I g G and binding of sheep erythrocytes. The results are compared with those obtained with lymphocytes from normal adult donors. 31
Acta path. microbiol. scand. Sect. C, 84, 6
MATERIAL AND METHODS Newborn Infants Thirteen newborn, preterm infants (gestational age 33-37 weeks) and 7 newborn, term infants (gestational age 38-42 weeks) were investigated. The birth weight of all but one of the infants was appropriate for gestational age (AGA); one term infant was small for gestational age (SGA). None of the infants showed signs of infection or had received blood transfusions before testing. The age of the infants at time of testing is shown in Table 1. All the infants were hospitalized at the Department of Pediatrics, Malmo General Hospital. Among the preterm infants 7 were hospitalized just because of the prematurity whereas 3 infants suffered from hyperbilirubinaemia, 2 infants had asphyxia and one had signs of cerebral damage. In the group of term infants the patients were hospitalized because of hyperbilirubinaemia (2 patients), asphyxia ( Z ) , pulmonary atelectasis ( l ), cleft palate (1) or low birth-weight (1). Before collecting blood from the infants their parents were asked for the allowance to do so.
47 7
TABLE 1. Age Distribution of Newborn Infants Tested for Lymphocyte Markers Total number
Boys
Girls
13
7
5 5
Total number 20
10
Infants Preterm Term
4-7
8-15
16-30
8
5
2
4
3 1
3
2
1
1
10
9
4
4
3
Normal Donors were healthy adult people working in the laboratory. Preparation of Lymphocytes for Testing Approximately 2 ml of blood was collected in heparin from a scalp or cubital vein of the infant. Processing of the blood included sedimentation of red cells with methyl cellulose, magnetic removal of phagocytes after ingestion of iron particles and a final purification of the lymphocytes by centrifugation on Isopaque-Ficoll (for details see Hallberg 1974). For use in the mixed antiglobulin reaction the lymphocytes were treated with formaldehyde as described by Hallberg et al. 1974. Before testing the lymphocytes were made up to 2 x lo6 per ml in diluent (phosphate buffered saline with 0.2 per cent bovine serum albumin). The final lymphocyte suspension contained more than 95 per cent mononuclear cells, of which almost all excluded trypan blue dye. The suspension also contained some erythrocytes and platelets. Blood samples from normal donors were treated as described above. Rosette Forming Reactions In all tests 1 drop of lymphocytes (2 x 106 per ml) and 1 drop of indicator red cells (0.8 per cent) were spun together in 7 x 508 mm siliconed glass tubes at 200 x G for 5 minutes. After centrifugation the cells were mounted with toluidine blue dye, and the number of rosettes, (i.e. lymphocytes binding at least 4 indicator cells) was counted among 200-300 lymphocytes. Each test was performed in quadruplicate. Sheep Red Cell Rosetting Reaction Lymphocytes and indicator cells were spun at room temperature and kept at + 4" C over night before mounting. Fc-rosetting Reaction Lymphocyte receptors for IgG were demonstrated by a rosetting reaction using antibody-
478
Age (days) at time of testing 1-3
sensitized ox red ceIls as the indicator cells (Hallberg et al. 1973). Ox blood was obtained in Alsever's solution from a slaughter-house. Differences in agglutinability of red cells between individual oxen (Uhlenbruch et al. 1967) may affect the rosetting reaction (Hallberg, unpublished data). However, this difficulty was overcome by treating the red cells with neuraminidase as follows. One ml of a 2 per cent suspension of well washed ox red cells was spun in a glass tube and the supernatant discharged. The packed red cells were mixed with 12.5 IU of neuraminidase (Behringwerke preparation from Vibrio cholerae) in 0.5 ml of saline with 1 mg/ml of CaCI,. The tube was incubated at 37" C for 30 minutes, the cells washed 3 times and resuspended in saline. These neuraminidase-treated ox RBC were sensitized with a pooled rabbit anti-ox red cell antiserum (K78/79) diluted 1:32, i.e. a dose just below the agglutinating capacity of the antiserum (for details see Hallberg et al. 1973). For preparation of rosettes lymphocytes and indicator cells were spun in the cold and kept in ice for 1-2 hours before mounting. Two of the 20 infants could not be tested for Fc-binding lymphocytes because of shortage of lymphocytes.
Mixed Antiglobulin Reaction for Detection of Lymphocyte Surface Immunoglobulin This test was performed on formalinized lymphocytes as described by Hallberg et al. (1974). T h e antiglobulin reagent was a rabbit antiserum (K 8 ) , raised against DEAE-cellulose-purified human IgG, and reactive with both heavy and light chains. Indicator cells were ox red cells sensitized with antibodies from human infectious mononucleosis serum. Controls in the mixed antiglobulin reaction included lymphocytes treated with normal rabbit serum instead of antiglobulin reagent, as well as tests with non-sensitized indicator ox red cells. As a further control of specificity it was checked that all positive reactions could be inhibited by soluble human IgG. Two of the 20 infants could not be tested for Ig-bearing lymphocytes because of shortage of lymphocytes.
TABLE 2. Sheep RBC-binding Lymphocytes in Newborn Infants and Adults
Individuals
N
Preterm infants Term infants All infants Adults
13 7 20 12
Sheep RBC-binding lymphocytes (mean Per cent 62.6 65.3 63.5 58.1
+. 4.2 2 4.2 2 3.1 2 2.3
* SE)
Absolute number per mm8
*
3263 419 4650 2 801 3748 f 407
ND
ND = not done. TABLE 3. Ig-bearing Lymphocytes in Newborn Infants and Adults
Individuals
N
Preterm infants Term infants All infants Adults
11
7 18 12
Ig-bearing lymphocytes (mean i SE) Per cent
*
10.7 1.2 7.2 t 1.6 9.3 i 1.0 11.6 1.6
*
Absolute number per mm3
* *
564 79 519 165 546 F 77
ND
ND = not done. TABLE 4. Fc-receptor-bearing Lymphocytes in Newborn Infants and Adult3 ~~
Individuals
N
Preterm infants Term infants All infants Adults
12 6 18 12
Fc-receptor-bearing lymphocytes (mean f SE) Absolute number per mm3 Per cent
* * *
12.4 1.8 7.1 1.4 10.6 -+ 1.4 17.2 2.4
649 -I- 125 543 -I- 165 613 i 98
ND
ND = not done.
Additional Tests I n all infants total as well as differential counts of white blood cells were performed. T h e level of plasma IgG, IgA and IgM was determined by electroimmuno assay (LaurelE 1972). RESULTS
The outcome of the rosetting tests in infants and adults is shown in Tables 2-4. In Table 2 the results in the sheep RBC rosetting test ( T cell marker) are shown. Adults and infants had 58-65 per cent sheep RBC-binding lymphocytes. T h e observed differences between the groups were not signifi31,
cant as judged by Student’s t-test (p>0.05). Nor was there any significant difference in absolute number of rosetting lymphocytes between preterm and term infants. T h e mixed antiglobulin reaction, visualizing Ig-bearing lymphocytes, is accounted for in Table 3. Adults and infants had 11.6-7.2 per cent Ig-bearing lymphocytes, the lowest values being found in the group of term infants. Again, no significant differences were seen between the groups. As shown in Table 4, all infants had a mean of 10.6 per cent Fc-rosetting lymphocytes, thereby differing significantly ( p
LYMPHOCYTE MARKERS IN NEWBORN INFANTS T. HALLBERG and ANITA HALLBERG Department of Medical Microbiology, Lund and Department of Pediatrics, Malmo General Hospital, University of Lund, Sweden
Hallberg, T. & Hallberg, A. Lymphocyte markers in newborn infants. Acta path. microbiol. scand. Sect. C, 84: 477-484, 1976. Purified peripheral blood lymphocytes from 20 newborn infants and 12 adults have been studied for the presence of surface markers. Adults and infants did not differ in the percentage of sheep RBC-binding cells (means 58-65 per cent) or in Ig-bearing cells measured by the mixed antiglobulin reaction (means 7.2-1 1.6 per cent). However, newborn infants had a significantly lower percentage of lymphocytes binding Fc of IgG (mean 10.4 per cent) as compared to adults (mean 17.2 per cent). No overall correlation between plasma Ig levels and Ig-bearing lymphocytes was found but a single infant with increased plasma IgA also showed the highest level of Ig-bearing lymphocytes among all infants. Key words: Lymphocyte markers ; B-cells, T-cells; newborn infants.
T. Hallberg, Department of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
Received 7.vi.76
Accepted 6.vii.76
Lymphocyte surface markers such as surface immunoglobulin, receptors for immunoglobulin and for complement factors and receptors for sheep erythrocytes are valuable tools in the identification of lymphocyte subpopulations in man (for review see HaEEberg 1975). Various investigators have by now established the normal levels in adults of lymphocytes bearing such markers whereas children and infants have been less extensively studied. In the present investigation, peripheral blood lymphocytes from newborn term and preterm infants were investigated by rosetting techniques for the presence of surface immunoglobulin, binding of Fc of I g G and binding of sheep erythrocytes. The results are compared with those obtained with lymphocytes from normal adult donors. 31
Acta path. microbiol. scand. Sect. C, 84, 6
MATERIAL AND METHODS Newborn Infants Thirteen newborn, preterm infants (gestational age 33-37 weeks) and 7 newborn, term infants (gestational age 38-42 weeks) were investigated. The birth weight of all but one of the infants was appropriate for gestational age (AGA); one term infant was small for gestational age (SGA). None of the infants showed signs of infection or had received blood transfusions before testing. The age of the infants at time of testing is shown in Table 1. All the infants were hospitalized at the Department of Pediatrics, Malmo General Hospital. Among the preterm infants 7 were hospitalized just because of the prematurity whereas 3 infants suffered from hyperbilirubinaemia, 2 infants had asphyxia and one had signs of cerebral damage. In the group of term infants the patients were hospitalized because of hyperbilirubinaemia (2 patients), asphyxia ( Z ) , pulmonary atelectasis ( l ), cleft palate (1) or low birth-weight (1). Before collecting blood from the infants their parents were asked for the allowance to do so.
47 7
TABLE 1. Age Distribution of Newborn Infants Tested for Lymphocyte Markers Total number
Boys
Girls
13
7
5 5
Total number 20
10
Infants Preterm Term
4-7
8-15
16-30
8
5
2
4
3 1
3
2
1
1
10
9
4
4
3
Normal Donors were healthy adult people working in the laboratory. Preparation of Lymphocytes for Testing Approximately 2 ml of blood was collected in heparin from a scalp or cubital vein of the infant. Processing of the blood included sedimentation of red cells with methyl cellulose, magnetic removal of phagocytes after ingestion of iron particles and a final purification of the lymphocytes by centrifugation on Isopaque-Ficoll (for details see Hallberg 1974). For use in the mixed antiglobulin reaction the lymphocytes were treated with formaldehyde as described by Hallberg et al. 1974. Before testing the lymphocytes were made up to 2 x lo6 per ml in diluent (phosphate buffered saline with 0.2 per cent bovine serum albumin). The final lymphocyte suspension contained more than 95 per cent mononuclear cells, of which almost all excluded trypan blue dye. The suspension also contained some erythrocytes and platelets. Blood samples from normal donors were treated as described above. Rosette Forming Reactions In all tests 1 drop of lymphocytes (2 x 106 per ml) and 1 drop of indicator red cells (0.8 per cent) were spun together in 7 x 508 mm siliconed glass tubes at 200 x G for 5 minutes. After centrifugation the cells were mounted with toluidine blue dye, and the number of rosettes, (i.e. lymphocytes binding at least 4 indicator cells) was counted among 200-300 lymphocytes. Each test was performed in quadruplicate. Sheep Red Cell Rosetting Reaction Lymphocytes and indicator cells were spun at room temperature and kept at + 4" C over night before mounting. Fc-rosetting Reaction Lymphocyte receptors for IgG were demonstrated by a rosetting reaction using antibody-
478
Age (days) at time of testing 1-3
sensitized ox red ceIls as the indicator cells (Hallberg et al. 1973). Ox blood was obtained in Alsever's solution from a slaughter-house. Differences in agglutinability of red cells between individual oxen (Uhlenbruch et al. 1967) may affect the rosetting reaction (Hallberg, unpublished data). However, this difficulty was overcome by treating the red cells with neuraminidase as follows. One ml of a 2 per cent suspension of well washed ox red cells was spun in a glass tube and the supernatant discharged. The packed red cells were mixed with 12.5 IU of neuraminidase (Behringwerke preparation from Vibrio cholerae) in 0.5 ml of saline with 1 mg/ml of CaCI,. The tube was incubated at 37" C for 30 minutes, the cells washed 3 times and resuspended in saline. These neuraminidase-treated ox RBC were sensitized with a pooled rabbit anti-ox red cell antiserum (K78/79) diluted 1:32, i.e. a dose just below the agglutinating capacity of the antiserum (for details see Hallberg et al. 1973). For preparation of rosettes lymphocytes and indicator cells were spun in the cold and kept in ice for 1-2 hours before mounting. Two of the 20 infants could not be tested for Fc-binding lymphocytes because of shortage of lymphocytes.
Mixed Antiglobulin Reaction for Detection of Lymphocyte Surface Immunoglobulin This test was performed on formalinized lymphocytes as described by Hallberg et al. (1974). T h e antiglobulin reagent was a rabbit antiserum (K 8 ) , raised against DEAE-cellulose-purified human IgG, and reactive with both heavy and light chains. Indicator cells were ox red cells sensitized with antibodies from human infectious mononucleosis serum. Controls in the mixed antiglobulin reaction included lymphocytes treated with normal rabbit serum instead of antiglobulin reagent, as well as tests with non-sensitized indicator ox red cells. As a further control of specificity it was checked that all positive reactions could be inhibited by soluble human IgG. Two of the 20 infants could not be tested for Ig-bearing lymphocytes because of shortage of lymphocytes.
TABLE 2. Sheep RBC-binding Lymphocytes in Newborn Infants and Adults
Individuals
N
Preterm infants Term infants All infants Adults
13 7 20 12
Sheep RBC-binding lymphocytes (mean Per cent 62.6 65.3 63.5 58.1
+. 4.2 2 4.2 2 3.1 2 2.3
* SE)
Absolute number per mm8
*
3263 419 4650 2 801 3748 f 407
ND
ND = not done. TABLE 3. Ig-bearing Lymphocytes in Newborn Infants and Adults
Individuals
N
Preterm infants Term infants All infants Adults
11
7 18 12
Ig-bearing lymphocytes (mean i SE) Per cent
*
10.7 1.2 7.2 t 1.6 9.3 i 1.0 11.6 1.6
*
Absolute number per mm3
* *
564 79 519 165 546 F 77
ND
ND = not done. TABLE 4. Fc-receptor-bearing Lymphocytes in Newborn Infants and Adult3 ~~
Individuals
N
Preterm infants Term infants All infants Adults
12 6 18 12
Fc-receptor-bearing lymphocytes (mean f SE) Absolute number per mm3 Per cent
* * *
12.4 1.8 7.1 1.4 10.6 -+ 1.4 17.2 2.4
649 -I- 125 543 -I- 165 613 i 98
ND
ND = not done.
Additional Tests I n all infants total as well as differential counts of white blood cells were performed. T h e level of plasma IgG, IgA and IgM was determined by electroimmuno assay (LaurelE 1972). RESULTS
The outcome of the rosetting tests in infants and adults is shown in Tables 2-4. In Table 2 the results in the sheep RBC rosetting test ( T cell marker) are shown. Adults and infants had 58-65 per cent sheep RBC-binding lymphocytes. T h e observed differences between the groups were not signifi31,
cant as judged by Student’s t-test (p>0.05). Nor was there any significant difference in absolute number of rosetting lymphocytes between preterm and term infants. T h e mixed antiglobulin reaction, visualizing Ig-bearing lymphocytes, is accounted for in Table 3. Adults and infants had 11.6-7.2 per cent Ig-bearing lymphocytes, the lowest values being found in the group of term infants. Again, no significant differences were seen between the groups. As shown in Table 4, all infants had a mean of 10.6 per cent Fc-rosetting lymphocytes, thereby differing significantly ( p
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