Quantitation of immunoglobulins in normal children

CLINICA CHIMICA ACTA

QUANTITATION

JEAN

A. UFFEL~AN,

OF IMMUNOGLOBULINS

WARREN

Departme?zt

of Microbiology, Clinic and Lincoln Medical (Received

November

zest,

E. ENGELHARD

IN NORMAL

AND

CARL

CHILDREN

R. JOLLIFF

University of Nebraska, Lincoln and ~e~ay~~e~~ of Pediatrics, Research Foundation, Lincoln, N&r. (U.S.A.)

Limcoln

1969)

SUMMARY

A modified radial immunodiffusion procedure was used to quantitate normal immunoglobulins IgG, IgA, and IgM in approximately 300 normal healthy agedefined children. The results were recorded as both, the percentage of the adult level and the Student 2 sample t test to indicate significance of difference from the adult level. Both values were used in our comparisons with other investigators’ results. Adult levels of IgM, and IgG were attained at ages of z and 7 years, respectively, while IgA was not realized in our studies at the upper age level used, namely 13 years. Comparative increases and decreases of these 3 immunoglobulins over the r3-year span were also discussed.

INTRODUCTION

Children with low levels of resistance have been identified with deficiencies in one or more of the major immunoglob~ins, IgG, IgA, or IgM. Specifically, patients with upper respiratory infections frequently exhibit normal IgG serum levels with low or absent IgA levels’. Furthermore, IgA deficiencies are associated with increased susceptibility to infections 2. Immunoglobulin abnormalities also occur in the WiscottAldrich syndrome (decreased IgM, increased IgA, with normal .I&); Gideon-Scheideggar syndrome (decreased IgM and IgA with normal IgG) ; and the Telangiectasia ataxia syndrome (decreased IgA, normai IgG and IgM)r. Certainly, deficiencies of all three immunoglobulins are evident in cases of sex-linked congenital agammaglobulinemia. In addition, immunoglobulin excesses occur following neoplastic proliferation of plasma cells. In polyclonal gammopathies, such as liver and collagen diseases an increase of all 3 immunoglobulins is frequently preseut3. Moreover, antibodies detected in allergic children were reported to be associated with immunoglobulin fractions*e5. It cannot be disputed that meaningful interpretations of these anomalies depend upon a consideration of the normal values particularly (in children) in light of the rapidly changing immunoglobulin levels in infancy and early childhood. Furthermore, it is essential to study and compare large groups of normal children to obtain mean values and ranges. For example, relatively few of the published reportsB-12 incorporate a Ctin. Ciim.

Acta, 28 (1970)

185-192

UFFELMAN

186

et a~!.

significant number of children’s serum immunoglobulin patterns from birth to 12 years of age, or moreover, treat their results with basic statistics. This study therefore purports to establish immunoglobulin values of normal children from birth to 12 years of age, to describe the standards, controls and precautions we felt essential for such interpretation, and to evaluate (with our modifications) theaccuracyandreproducibilityof theHylandradialimmunodiffusionprocedure. A statistical comparison of the data with normal adult values is also presented. MATERIALS

AND

METHODS

297 sera were obtained from children seen in the Pediatric Department of the Lincoln Clinic, Lincoln, Nebr. The children were all well babies or those appearing for routine physical examinations. The children ranged in age from birth to 13 years. Complete history and physical examination records were scrutinized to ascertain the possibility of recent or concurrent allergies, infections or immunization procedures. Follow-up studies were completed to insure that infections or allergic episodes had not occurred after the blood samples had been obtained. Blood specimens were collected by either heel puncture or venipuncture. Cord blood was obtained from the clamp to placenta side in each instance before the placenta was delivered. All blood samples were separated from the clot by centrifugation, serum removed and subjected to electrophoresis, immunoelectrophoresis and immunoglobulin quantitation. No samples were allowed to stand for a period of over I day at 4O. All serum specimens were obtained on Caucasians. 62% were males and 387; were females. Electrophoresis of serum proteins was performed on cellulose acetate (Sepraphore III, Gelman*). Immunoelectrophoresis was accomplished using the method of ScheideggarX3 with a modification which allowed the patterns to be placed on a 3.25 inch x 4 inch lantern slide. In this manner the patients’ and the control sera could be reacted with anti-human whole serum as well as anti-human IgG, IgA and IgM sera prepared in goats (Hyland* *). Prior to staining the slides were photographed for reference. Single radial immunodiffusion quantitative studies were performed on “Immunoplates” (Hyland**) of the same lot numbers for IgG, IgA and IgM, respectively. Reference standards for IgG, IgA and IgM were prepared from a pool of IOO serum specimens. Aliquots of this pool were submitted to the National Cancer Institute Immunoglobulin Reference Center (N.C.I.I.R.C.) and to the University of Colorado Medical Center, Department of Allergy and Immunology for quantitation (Table I). Utilizing the values obtained on our pooled reference standard from the N.C.I.I.R.C., z-fold dilutions were made from the pooled serum. Dilutions were prepared with a 49/, ovalbumin, crystallized 3 times (Nutrition Biochemicals***). If values for immunoglobulins were found to be greater than the concentration of the undiluted pooled reference standard the serum was diluted with the 40/bovalbumin. Reproducibility was not achieved when saline or distilled water was used as the diluent. * Gelman Instrument ** Hyland

*** Nutrition Cl&z. Chim.

Co., P.O. Box 1448, Ann Arbor, Mich. 48106, U.S.A. 450’ CoIorado Boulevard, Los Angeles, Calif. 90039, U.S.A. Riochemicals Corp., Cleveland, Ohio 44128, U.S.A.

Laboratories,

Acta,

28

(1970) 185-192

IMMUNOGLOBULIN ~UANTITATION TABLE

IN NORMAL CHILDREN

=87

I

PooLlED SERUM REFERENCE Refevemc

W (mg/roo

STANDARD

mE#!

N.C.I.I.R.C.’ 2-28-68 1280 -$I 24 University of Colorado Medical Center, Allergy and Immunology Laboratories* * 12-9-66 1080 10-26-67 1130 ~~____ -*Bethesda, Md., U.S.A. ** Denver, Colo., U.S.A. TABLE

II

GIvEX

VALUES

AND

FOUND

VALUES

f&4 (mg/Ioo

IiTM (“g/IO0

ml)

Method ml)

217 + 9

704 sr r

Radial immunodiffusionia

r65 IS2

143 r39

Oudin immunodiffusionls Oudin immunodiffusion

UTILIZING

IgG (mgjroo

ml)

POOLED

SERUM

IgA (mglxoo

REFERENCE

ml)

STANDARD

IgM

Hyland standard Our value

1560 1520

340 325

IO0 roQ

Behringwerke* Our value

760 765

r4o

60 88

standard

* Behring Diagnostics,

160

400 Crossways Drive, Woodbury,

N.Y.,

(mg/100 ml)

.-_______.

11797, U.S.A.

Values obtained by N.C.I.I.R.C. on our pooled reference standard were used as a check on values for immunoglobulins IgG, IgA and IgM in reference standards supplied by Hyland Laboratories and Behringwerke. Comparative results (Table II) indicate that our values were acceptable in so far as we were able to detect known amounts of purified immunoglobulin antigen. Visual observation with a magnifying lens using a superimposed grid was unsatisfactory in our hands for reading the diameter of the precipitin rings on the “Immunoplates”. Measuring the precipitin rings after photographic enlargement of the “Immunoplates”, which a ruler calibrated at r/rooth of an inch, proved to give the desired accuracy and reproducibility as well as a permanent record of the test. A constant amount of serum placed in the antigen wells with the aid of a microsyringe (Hamilton*) adapted with a teflon tip (G. K. Turner**) gave excellent reproducibility. This seemed preferable to filling the antigen wells to the brim, thus disregarding a constant repetitive amount in each well. Immunoelectrophoretic studies were performed using a pool of control serum obtained from the approximate age group of the specimen under study. We felt the use of adult serum as a control allows for misinterpretation of the immunoelectrophoretic pattern in incidences where this is the only test performed along with an electrophoretic pattern in assessing dysgammaglobulinemias. A statistical analysis was performed on the mean levels of IgG, IgA and IgM listed in Table III. The Student two sample t test was employed for the purpose * Hamilton Co., P.O. Box 307, Whittier, Calif., U.S.A. ** G. K. Turner Associates, 2524 Pulgas Ave., Palo Alto, Calif. 94303, U.S.A. Clin. Chim. Acta, 28 (1970)

185-IQZ

III

7

7

4-6 months

7-12

IgG*

81 90



CO.01

(0.05

924 f 234 (640-1420)

I031 * 213 (650-1410)

I000 *

IO40 f 244 (730-1410)

975 + I3I (760-1330) I073 + 181

(730-1350) 1082 + I47 (850-1300)

II43

38

26

I9

18

20

22

I9

26

72

5 years

6 years

7 years

8 years

9 years

IO years

II years

I2-I 3 years

Adult

N.S.

N.S.

2 sample

100

95

94

85


N.S.

9I

87

92

N.S.

* Geometric mean. ** Significance of difference from adult level. Student . mg/roo ml)

II42 i I95 (77o-‘5’0)

(770-15’0)

& 228

(570-1320)

203

(540-1440)

1026 + 208

21

4 years

76


866 f 213 (500-1360)

21

3 years

62


63

33

31

100

705 + IO6 (520-1080)


72’ + 208 (350-1180)

t

y. of adult

level

26



N.S.

p**

556 + III (190-860)

371 f I32 (270-780)

II45 + 252 (75o-‘500)

(mglroo ml + I S.D.)

Levelof

2 years

months

7

20

I-3 months

New born

Number



N.D.,

94

70

6g

72


68

58


63

56

54

40

35

40

28

I4

I

~0.001

106 118

33 N.S.

108 i

(55-210) 121 + zg N.S.

with detectable

(70-150) I02 & 30 (67-208)

(66-155) II4 + 32 N.S.

IIo & 23 N.S.

IgA (22 and 60

106

99 (55-160) Iog + 30 N.S. (80-150)

116

33 N.S. (68-175) IOI + 38 N.S.

118 +

(60-175)

II0

118

9I

112 + 36 N.S. (40-180)

121 & 63 N.S. (52-200)

93 + 37 CO.1 (46-190)

83

75

76 +

(36-104) 85 + 28 co.01 (72-160)

16
69

65

IO

0/Oof adult level

(12-87) 70 f 23
(I I-35) 66 f 34 (0.01

11.8&2.6

Level of IgM * (mg/Ioo ml (& I S.D.)

only 2 samples

0/Oof adult lt?Vt?i


~0.001







N.D.

test. N.S. not significant;

2IO * 44 (‘34-297)

(108-325)

141 + 51 (91-255) 188 + 46

I39 + 34 (70-222)

I44 * 34 (108-200)

I36 + 56 (74-260)

II7 ?c 49 (65-240)

I26 & 39 (83-2’7)

III f 4’ (52-220)

(45-135) 108 + 42 (52-210)

69 k 33 (36-165) 81 & 21

8I i 67 (36-165)

56 * I4 (1096)

(6-58)

28 & 21

N.D.

Level of IgA* P** (mg/100 ml + I S.D.)

L.E”EI,s OF IMMUNE GLOB”LINS IN SERUM OF NORMAL S”B,ECTS AT DIFFERENT AGES

TABLE

k

IMMUNOGLOBULIN

of evaluating

QUANTITATION

IN NORMAL CHILDREN

the age at which the level of the children’s

that of the adult and is no longer significantly the childrens’

mean values f

I SD.

different.

189 immune globulins approaches The method requires that all

must be programmed

according

to age and then

compared with the adult mean & I S.D. The computer determines the t values with a corresponding number of degrees of freedom. The P value indicates whether or not there is a significant difference between the childrens’ mean and the adult mean and is obtained

from a t distribution

table 14.

RESULTS AND DISCUSSION

Certainly, among the other considerations for defining diseases caused by immunological deficiencies is the necessity for documenting the levels in considerable numbers of normal healthy children. This is also essential for accurately evaluating levels during the period of immunological maturation, and as a tool for determining the effectiveness of administering immunoglobulins prophylactically. The immunoglobulin serum levels of zg7 normal healthy children representing age groups from newborn to 13 years are summarized in Table III and Figs. 1-3. 72 adult values are included. IgG. Our measurements of IgG for the first 3 months of age indicated the usual physiological decrease beginning with the high maternal IgG in cord serum of 1145 & 252 mg/roo ml to a low level of 371 & 132 mg/roo ml at 1-3 months. At this point the synthesis of this immunoglobulin protein by the infant is progressing to the extent that by 6 months one-half of the adult level is reached. Other investigators have similarly reported the lowest IgG concentrations occurring at 2-4 month9, 3-6 months’, 4.5 monthW, 6-24 week+‘, and 2-6 months”. Generally, the other quantitative immunoglobulin studies at the end of the first year compare favorably with our findingse+ll. One investigatorlo reported over 75% of the adult level was attained at I year. Considerable variation, however, is apparent concerning the age groups reflecting adult levels of IgG. Concentrations approximating adult levels of IgG were apparent in our study at 7 years. Although a similar observation was reported by Buckley et aL8 and Allensmith et aL6, other investigators have reported adult levels reached

at g-11 years7, 2 years”

and 3 yearP.

Johansson

and Berg8, reporting

on

Fig. I. Mean values f I S.D. of immunoglobulin G concentrations of 297 normal subjects at different ages. The number of subjects represented by each dot is given in Table III. Clin. Chim. Acta,

28

(1970)

185-192

UFFELMAN et 6~1.

Fig. z. Mean values + I S.D. of immonuglobulin A concentrations of 297 normal subjects different ages. The number of subjects represented by each dot is given in Table III.

at

Fig. 3. Mean values i I S.D. of immonuglobulin M concentrations of 297 normal subjects different ages. The number of subjects represented by each dot is given in Table III.

at

the age groups of newborn to 5 years of age, indicated two-thirds of the adult level was reached at 3-5 years of age. Our IgG measurements showed the most marked increase occurred between 3 months and 4 years with a fairly uniform concentration over the r-z-year age periods (Fig. I). After 4 years the rate of increase lessened. In adults the average IgG value was 1140mg/roo ml with a + I S.D. range of 195 mg/roo ml. IgA. With the procedures described above we detected IgA in the cord serum of only z of the 20 samples. The values were zz and 60 mg/roo ml. These results are in agreement with those of Collins-Williams et al.“, Fulginiti et al.lO, one child out of 18; and Johansson and Bergs, one child out of 16. Similarly, West et al.12 could not demonstrate IgA at birth or before the age of 25 days. On the other hand, Stiehm and Fudenberg’, uncovered trace quantities in one-third of 22 subjects and in all children above the age of 3 weeks. Finally, Allensmith et aL6 detected IgA in concenClilz. Chim. Acta, 28 (1970) 185-192

IMMUNOGLOBULIN

trations

QUANTITATION

of 5 mg/roo

IN NORMAL CHILDREN

ml in the cord serum of

IO

newborn

191 infants.

Our IgA values

(Table III, Fig. z) increased slowly during infancy and at the end of 6 months achieved one-fourth of the adult level, and 50% of this value at 4 years. At I year of age Fulginiti et aLlo reported a mean value of one-fifth of the adult level and Allensmith et al.6 ~5~)‘~of the adult value while lower values, namely 13% of the adult level were reported by Johansson and Bergs. Our low values were found at the I-3- and 4--6-month age groups.

In addition

to showing lower values through the entire range of age groups

(1-3 months to II years), a more consistently steady but slower increase was observed than with IgG or IgM. While the rate of increase was generally parallel to that of IgG, the concentration was lower. The most noticeable and marked increase occurred between the 6-month to 4-year and rr-rz-year age groups (Fig. 2). The adult level of IgA was achieved later than was evident for the other immunoglobulins, namely at 12-13 years, 188 & 46 mg/roo ml. IgA concentrations have been reported to reach adult levels by the ninth monthIS, 4-12 years12, 6-7 year$, 5 years?, 12 yearss, and 16 years7. In adults the average IgA value was 200 mg/roo of 44 mg/roo ml.

ml with a _t I SD.

range

1gM. IgM was found in all cord serum with an average concentration of approximately 10% of the adult level, 11.8 t_ 2.6 mg/roo ml. This is consistent with the observations of Stiehm and Fudenberg’, Fulginiti et al.‘O, Buckley et aL8 and Johansson and Bergs whose reports indicated cord serum values of II%, slightly less than IO%, 12% and IO%, respectively. Over 50% of the adult level was reached by the end of 3 months. Allensmith et ad8 observed the same percentage by 4 months of age. In two of our cord serum samples from pre-natally infected infants (Table III) an IgM value of over 150 mg/roo ml was found. Obviously, screening of umbilical cord serum for increased amounts of IgM and IgA can be of value in the detection of clinically inapparent and atypical types of congenitally acquired infections. Our studies indicated that when compared to the IgG and IgA values the IgM levels revealed an earlier synthesis of this immunoglobulin. The early synthesis makes this immunoglobulin particularly interesting in the light of analyzing immunological deficiencies suspected in the newborn, among them, agammaglobinemias. Our IgM level at the end of 3 months was more than one-half the adult level. While these results are in agreement with Collins-Williams et al.‘l, essentially the same results were reported by otherss-8,10. Our marked increases were observed between the early ages of 3-6 months (Fig. 3). While some investigators observed a decline in IgM concentration after age 3 yearsI and 3, 7, and 13 years?, we noted a decrease after 4 and 8 years. Larger numbers of samples must be analyzed before significance can be given to these observations. The earlier synthesis of IgM as demonstrated by increases within 5-10 days after births and the realization in early childhood of adult levels are most apparent with this immunoglobulin. Our results indicate that adult levels were achieved at z years of age. The results are in agreement with Allensmith et aLs. An earlier age, I year, was reported by Buckley et aL8 and Collins-Willams et a1.l’ and g months by West et a1.12 and Hitzig’s. In analyses of IgM of Johansson and Berg8 and Fulginiti et al.‘O in the newborn to 5 years age group and newborn to 52 weeks study, respectively, 70% of the adult level was reached by I year. In the former study, however, the adult level was shown to be present only after 5 years. 80% of the adult level was attained in an older age group of g-11 year olds by Stiehm and Fudenberg’. In adults Clin. Chin?. Acta,

28 (1970)

185--Igz

192

UFFELMAN

et d.

the average IgM value was IOO mg/roo ml with a $- I SD. range of 30 mg/xoo ml. Our comparative observations with other investigators’ studies of the normal immunoglobulin values in children of various age groups underscores the invalidity of defining our normal values using all sera taken collectively. Most assuredly, methodology, preparation of standards, serum reference pools, technique and perhaps geography are factors responsible for variation. In the absence of a world-wide or regional reference center, and in order to achieve a reasonable degree of significance regarding abnormal values, each laboratory should develop their own normal standards for children. A respectable number of samples must be tested using current proven methodology, preferably serum pools of a similar age group used as standards and checked by at least one or preferably two outstanding reference laboratories. Fortunately, further advances in our knowledge are being made with identification of subgroups. This may open new vistas in treatment where specific subgroup deficiences exist17’. REFERENCES I 2 3 4 5 6 7

8 9

10 II

R. SMITH. Pediatrics, 17 (1966) 822. F. Bus& R. BuTI.ER-&dk,‘M. DUPAN, J. Pediat., 72 (1968) 29. Lab Synapsis, 2nd Ed., Hoechst Pharmaceutical Co., Kansas City, MO., 1967. P. FIREMAN, W. C. VANNIER AND H. C. GOODMAN,J. Exptl. Med., 117 (1963) 603. R. T. REID, P. MINDEN AND R. S. FARR, J. Exptl. Med., 123 (1966) 845. M. ALLENSMITH, B. H. MCCLELLAN, M. BUTTERWORTH AND J. R. MALONEY, J. Pediat., 72 (1968) 276. R. E. STIEHM AND H. H. FUDENBERG, J. Pediat., 37 (1966) 715. R. H. BUCKLEY, S. C. DEES, AND W. i?iI.O’FALLON, .I. Pediat., 4r (1968) 600. G. S. JOHANSSON AND T. BERG, Acta Paediat. &and.; 56 (1967) j72. V. A. FUZGINITI, 0. F. SIEBER, H. N. CLAMAN AND D. MERRILL, J. Pediat., 68 (1966) 723. C, COLLINS-WILLIAMS, B. TOFT, L. GENERO~O AND M. MOSCARELLO,Can. Med. Assoc. J.,

96 (1967) X.510. 12 C. C. WEST, R. HONG AND N. H. HOLLAND, .J. C&z. Invest., 41 (1962) 2054. 13 J. L. SCHEIDEGGAR,Intern. Arch. Allergy, 7 (1955) 103. 14 B. OSTLE, Statistics in Research, Iowa State College Press, Ames, Iowa 1954, Chapter 5. 15 ‘CV.H. HITZIG, Springer Verlag, Berlin, 1963. 16 C. C. HUNTLEY AND A. D. LYERLY, Am. J. Diseases Children, 106 (1963) 545. 17 M. E. MILLER, Hosp. Pratt., 4 (1969) 38. 18 J. L. FAHEY AND E. M. MCKELVEY, J. Immunol., 94 (1965) 84. 19 H. N. CLAnsAN AND D. MERRILL, J. Lab. Clin. Med., 64 (1964) 685. Clin. Chim. Acta, 28 (1970) 185-r92