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- MATERIALS AND METHODS
Knowledge of what constitute normal serum immunoglobulin (Ig) concentrations are important for the diagnosis of immunologic disorders. Data on normal Ig evaluated by nephelometry are limited in healthy Asian children, none being available for Thai children. One hundred and forty-eight healthy Thai children aged 2–15 years were tested for serum immunoglobulins G, A, M, G1, G2, G3, and G4 (Ig G, A, M, G1, G2, G3, and G4) by nephelometry. Sixty-three percent were girls of median interquartile range age 6.9 (4.8–9.7) years. The geometric means for each Ig were summarized and categorized by age. Statistical analyses were used to compare Igs between sexes and age groups, and to compare IgG in this study with data from other published studies. The average ratios of IgG subclasses/IgG for Ig G1:2:3:4 were 66:22:5:7%. IgG, IgA, IgG2, and IgG3 concentrations showed a gradual increase with increasing age. There were no significant sex differences for any immunoglobulin isotype (P= 0.971). Our mean IgG concentration was lower than that measured by the radial diffusion method in healthy Thai children (P < 0.05). In all age groups, the mean IgG concentration in our study was significantly higher than that reported in Turkish and USA children, evaluated by the nephelometric and radial diffusion techniques, respectively (both P < 0.001). This study provides information about normal Ig concentrations measured by nephelometry in healthy Asian children and illustrates the importance of ascertaining normal Ig values for age- and ethnic-matched controls using the same assay to diagnose immunologic disorders correctly.
Immunoglobulins, also known as antibodies, are glycoproteins that mediate humoral immunity. They function by recognizing and neutralizing foreign antigens such as bacteria and viruses. There are five isotypes: IgG, IgA, IgM, IgD, and IgE (1). Primary and secondary immunodeficiencies can cause abnormal serum Ig concentrations. Immunodeficient patients present with recurrent and/or severe infections or infections by organisms that do not usually cause illnesses in normal hosts (2). It is crucial to determine the normal concentrations of serum Ig in order to correctly diagnose and follow up immunodeficient patients. For example, selective IgA deficiency, which is the commonest primary immune deficiency, occurring in 1 in 333 to 1 in 700 children, is manifested by recurrent respiratory and gastrointestinal tract infections and lower than normal IgA concentrations. Patients with X-linked agammaglobulinemia, a severe primary immune deficiency, present with recurrent and severe infections from 4–6 months of age and all serum Ig concentrations are extremely low, necessitating intravenous immunoglobulin treatment. In addition, serum Igs are abnormal in other immunologic disorders such as HIV and autoimmune diseases (3).
Age, sex, ethnicity, nutritional status, geographic location and type of assay can affect serum concentrations of Ig. McFarlane et al. reported variations in Ig concentrations between dry and wet seasons and also higher concentrations in Nigerians than in Caucasians (4). Stiehm et al. studied Ig concentrations in normal populations from birth to adulthood in 1966 (5). They showed that serum IgG in infants was as high as in adults, because of maternal transmission and subsequently reached its nadir at age 3–4 months. In Thailand, the first study of Ig concentrations in healthy children (age 4–5.5 years) and adults (17–45 years) was performed in 1970 by Sirisinha et al. (6). This study revealed that serum IgM in females was statistically significantly higher than in males in both age groups. However, studies by Thongchareon et al. (7) and Sakulramrung et al. (8) showed no difference between Thai male and female subjects in concentrations of IgG, IgA, and IgM. All these previous studies from Thailand and other countries used the radial immunodiffusion method of Mancini (9). The nephelometric assay has now replaced this method (10), which is automated and has higher precision (11,12). Recently, studies evaluating serum Ig isotypes using the nephelometric assay in various settings have been published (13–15) but none have been performed in healthy Asian children.
We evaluated the serum Ig isotypes in healthy Thai children by nephelometry. The results from this study will allow accurate diagnosis and management of children with immunologic disorders, particularly primary immune deficiencies.
MATERIALS AND METHODS
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- MATERIALS AND METHODS
Healthy Thai children aged 24 months to <16 years were enrolled from the well child clinic of King Chulalongkorn Memorial Hospital, Chulalongkorn University in Bangkok, Thailand as part of the HIV-NAT 108 study. Medical histories were obtained and physical examination performed by pediatricians on the same day as blood was collected. Children were excluded if they had abnormal growth (below the third or above the 97th percentile of the Thai growth chart), a febrile illness, respiratory and other infections at screening, or medical illnesses that might result in abnormal immunity such as HIV infection or exposure and allergic conditions. Children who had used anti-infective agents or corticosteroids in the past month were also excluded. All caregivers consented to the study and healthy children ≥ 7 years old also gave assent. This study was approved by the Institutional Review Boards of Chulalongkorn University and Ramathibodi Hospital.
At enrollment, a 4 mL blood sample was collected, centrifuged for serum, and stored at −70°C at the HIV-NAT laboratory in Bangkok until shipped to the Ramathibodi Hospital laboratory in Bangkok, under controlled conditions, for serum Ig measurement. IgG, IgA, IgM, IgG1, IgG2, IgG3, and IgG4 concentrations were measured by the nephelometric technique using the BN Prospec Nephelometer Analyzer and commercially available kits from Dade Behring, Marburg,Germany (13). The CV of intra- and inter-assays were < 5%.
Data are presented according to median, IQR or frequency percentage distribution according to the types of variables. Immunoglobulin concentrations were summarized according to age or sex groups by using the geometric mean and 95% CI. The children were categorized into the following age groups: 2–4, 4–6, 6–8, 8–10, 10–12, and 12–15 years.
Differences in Ig values according to sex and age groups were compared by Student's t-test or ANOVA techniques as appropriate. The same methods were used for comparing IgG data from this study to published data from Thailand and other countries. Linear regression analysis was used to assess factors associated with increasing IgG, including sex and age. Log transformation was used to improve normalization and anti-logged back to geometric ratio for presentation. A nonparametric test was applied if log transformation did not improve normalization. All variables with significant level less than 0.1 were selected into multivariate model. In all analyses, effect sizes and 95% CI around this effect size are given in addition to P values. All hypothesis tests were two-sided with statistical significance taken at the level of 5%.
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- MATERIALS AND METHODS
The 148 children were enrolled and categorized by age into 2–4, 4–6, 6–8, 8–10, 10–12, and 12–15 years old groups. Sixty-three percent were girls. The median (IQR) age was 6.9 (4.8–9.7) years (Table 1). Median (IQR) weight and height were 21.5 (16.9 – 31.5) kg and 118.5 (107.0 – 133.3) cm, respectively. The concentrations of Ig isotypes IgG, IgA, IgM and IgG1, IgG2, IgG3, and IgG4 classified by age are shown in Table 2.
Table 1. Age groups of subjects
|Age groups||N= 148|
|Age, median (IQR), years||6.9 (4.8–9.7)|
|Age group in years, n (%)|| |
| 2–4||25 (16.9)|
| 4–6||27 (18.2)|
| 6–8||37 (25.0)|
| 8–10||25 (16.9)|
| 10–12||21 (14.2)|
| 12–15||13 (8.8) |
Table 2. Immunoglobulin concentrations by age groups
|Age (years)||N||Geometric mean (mg/dl)||SD||Minimum||Maximum||95%CI|
| 2–4||25||1035||226||638||1570||(948, 1129)|
| 4–6||27||1260||217||910||1940||(1181, 1344)|
| 6–8||37||1271||176||840||1570||(1211, 1335)|
| 8–10||25||1329||227||961||1750||(1238, 1427)|
| 10–12||21||1354||179||1080||1760||(1276, 1437)|
| 12–15||13||1326||215||949||1680||(1198, 1466)|
| 2–4||25||73||31||33||170||(62, 86)|
| 4–6||27||124||61||41||344||(104, 148)|
| 6–8||37||126||49||57||257||(112, 142)|
| 8–10||25||161||69||86||396||(138, 187)|
| 10–12||21||177||47||85||267||(155, 201)|
| 12–15||13||217||101||108||473||(170, 277)|
| 2–4||25||130||36||54||211||(116, 147)|
| 4–6||27||137||51||64||279||(120, 157)|
| 6–8||37||136||64||81||394||(121, 153)|
| 8–10||25||135||55||81||280||(117, 156)|
| 10–12||21||137||53||46||242||(113, 167)|
| 12–15||13||155||47||98||275||(131, 183)|
| 2–4||25||789||155||504||1140||(727, 855)|
| 4–6||27||924||157||685||1390||(867, 984)|
| 6–8||37||898||136||598||1160||(852, 946)|
| 8–10||25||902||150||635||1160||(842, 967)|
| 10–12||21||921||115||703||1180||(870, 975)|
| 12–15||13||844||141||653||1100||(766, 929)|
| 2–4||25||186||83||83||467||(160, 217)|
| 4–6||27||247||71||94||394||(218, 280)|
| 6–8||37||273||79||151||489||(250, 299)|
| 8–10||25||321||118||122||646||(277, 372)|
| 10–12||21||338||101||207||608||(298, 384)|
| 12–15||13||382||134||243||685||(312, 468)|
| 2–4||25||50||26||19||114||(41, 62)|
| 4–6||27||56||27||16||131||(47, 68)|
| 6–8||37||60||34||26||220||(52, 70)|
| 8–10||25||64||26||29||120||(55, 75)|
| 10–12||21||72||30||40||155||(61, 85)|
| 12–15||13||81||38||41||147||(61, 106)|
| 2–4||25||33||55||2||211||(21, 53)|
| 4–6||27||63||59||2||203||(42, 95)|
| 6–8||37||63||103||1||459||(42, 92)|
| 8–10||25||84||80||21||407||(63, 112)|
| 10–12||21||60||61||3||249||(37, 96)|
| 12–15||13||49||70||2||241||(20, 119)|
Overall, the geometric mean (SD) IgG concentration was 1251 (227) mg/dL, IgA 131 (72) mg/dL and IgM 137 (53) mg/dL. The mean (SD) IgG1 concentration was 882 (148) mg/dL, IgG2 274 (111) mg/dL, IgG3 61 (31) mg/dL, and IgG4 58 (77) mg/dL. The average ratios of IgG subclasses for IgG1:IgG2:IgG3:IgG4 were 66:22:5:7%. The mean serum IgG concentration was 1252 mg/dL in boys and 1250 mg/dL in girls. There were no statistically significant differences in IgG, IgA, IgM, and IgG subclass concentrations between boys and girls across age groups (P > 0.05).
The values for IgG, IgA, and IgM concentrations by age group are shown in Figure 1a. IgG concentrations increased rapidly between 2–4 and 4–6 years of age, followed by a more gradual increase until age 12–15 years. IgA concentrations rose gradually with increasing age whereas IgM levels were similar in all age groups.
Figure 1. (a) IgG concentrations increased rapidly between 2–6 years then increased gradually. IgA concentrations increased gradually with increasing age. IgM concentrations were similar at all ages. (b) IgG1 concentrations increased rapidly between 2–6 years then increased gradually. IgG2 concentrations increased gradually with increasing age. IgG3 and IgG4 concentrations were similar at all ages.
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For IgG subclasses, IgG1 concentrations followed the pattern of total IgG with a rapid increase between ages 2–4 and 4–6 years, then a plateau. IgG2 concentrations increased gradually, whereas IgG3 and IgG4 were similar across age groups (Fig. 1b). There were statistically significant differences in IgG, IgA, and IgG subclass concentrations between the age groups (P < 0.05). There were no statistically significant differences in IgM concentrations between age groups (P= 0.82)
We compared our IgG concentrations with the results from two studies that have been used as reference values in Thailand, one from Thailand (7) (Table 3) and one from the USA (5) (Table 4). The radial immunodiffusion method was used in both of these studies. In our study, IgG concentrations were significantly lower than those reported in the previous Thai study for all age groups, 2–4, 5–7, and 8–13 years (Table 3). Compared to the same study, the IgM concentrations in our study were higher, but this difference was not statistically significant. The IgA concentrations in our study were lower than in the previous Thai study, this difference being statistically significant in the 5–7 year old age group. In our study, the geometric means of IgG concentrations were higher than those of the USA study for every age group (Table 4). Table 5 shows the comparison of geometric means of IgG concentrations between Thai and Turkish children (15), the only published data available using nephelometry. There is a trend toward higher IgG concentrations in Thai children than in Turkish children for all age groups.
Table 3. Comparison of geometric means of IgG concentrations in Thai children by nephelometry in this study and by the radial diffusion method in a published report (7)
|Age||Thai (HIV-NAT 108) mean (95%CI)||Thai (Thongchareon)* mean (95%CI)||P-value|
|2–4 years||1152 (1068, 1236) N= 25||1494 (1349, 1639) N= 7 || 0.002|
|5–7 years||1269 (1218, 1321) N= 27||1496 (1182, 1810) N= 12|| 0.011|
|8–13 years||1345 (1288, 1402) N= 46||1877 (1739, 2015) N= 40||< 0.001|
Table 4. Comparison of geometric means of IgG concentrations by nephelometry in Thai children in this study and by the radial immunodiffusion method in USA children (5)
|Age||Thai Geometric mean (95%CI)||USA* Geometric mean (95%CI)||P-value|
|2 years||1043 (933, 1166) N= 16 ||685 (424, 1051) N= 50||< 0.001|
|3 years||1020 (896, 1160) N= 9 ||728 (441, 1135) N= 50||< 0.001|
|4–5 years||1260 (1184, 1341) N= 27||780 (463, 1236) N= 50||< 0.001|
|6–8 years||1263 (1212, 1317) N= 52||915 (633, 1200) N= 50||< 0.001|
|9–10 years||1391 (1316, 1471) N= 23||1007 (608, 1572) N= 50 ||< 0.001|
Table 5. Comparison of geometric means of IgG by nephelometry between Thai children in this study and Turkish children (15)
|Age||Thai Geometric mean (95%CI)||Turkish Geometric mean (95%CI)||P-value|
|2–3 years||1043 (934, 1166) n= 16 ||822 (790.4, 906.4) N= 52 || 0.001|
|3–4 years||1020 (896, 1160) n= 9 ||880 (844.1, 944.6) N= 40 || 0.037|
|4–6 years||1266 (1216, 1318) n= 52||986 (958.5, 1058.5) N= 70||< 0.001|
|7–8 years||1255 (1175, 1340) n= 27||1041 (1011.5, 1111.4) N= 66||< 0.001|
|9–10 years||1391 (1316, 1471) n= 23||1063 (1024.9, 1152.7) N= 57||< 0.001|
|11–12 years||1336 (1225, 1458) n= 15||1052 (995.9, 1155.6) N= 34 ||< 0.001|
|13–14 years||1360 (1239, 1492) n= 5 ||1088 (1014.2, 1209.0) N= 25|| 0.028|
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- MATERIALS AND METHODS
This study reports immunoglobulin concentrations evaluated by nephelometry in healthy children in Thailand. Although there is no consensus on the most specific and sensitive method for determining serum immunoglobulin concentrations, the nephelometric assay is easier, less time consuming, and has higher precision and reproducibility than the radial immunodiffusion method (11,12,14).
The trend in Ig concentrations across age groups varies between studies. A European study by Gregorek et al. reported that all IgG subclasses except for IgG3 continue to increase with age (16). In contrast, a study in South Africa reported that IgG1 concentrations were high in infants and gradually decreased until adulthood, whereas IgG2, IgG3, and IgG4 concentrations gradually increased with age (17). In the current study, all IgG subclass concentrations except for IgG2 increased rapidly between ages 2–4 years and 4–6 years and increased more slowly thereafter. This is similar to the findings of a Thai study that showed that IgG1 and IgG4 concentrations increase with age, reaching a plateau by the age of 4 years (18). Another Thai study showed IgG1 concentrations increasing to plateau at age 6 years, whereas IgG3 and IgG4 concentrations continued to increase with age until early adolescence (19).
In this study, IgM concentrations were not significantly different among age groups, in agreement with previous studies in Thai children showing that serum IgM concentrations reach adult levels by 12–18 months of age (6–8).
The IgG subclass concentrations are important for diagnosis of IgG subclass deficiency when patients present with recurrent sinopulmonary tract infections (20,21). In contrast to previous studies in Thai children (18,19), IgG4 concentrations were higher than those of IgG3 in most age groups between 4–10 years of age. Atopy, particularly eczema and food allergy (22), and nutritional status (23) are associated with increased IgG4 concentrations, but these were exclusion criteria in our study. The small number of subjects in the 12–15 years age group could account for the increased IgG4 concentrations seen.
Genetic factors, environmental factors such as nutrition, pollution, socioeconomic status, and methodological factors can influence serum immunoglobulin concentrations. A previous study revealed an association between genetic phenotype and the rate of synthesis of the IgG subclass (24). Differences in concentrations of lymphokines and prostaglandin E can result in variable Ig production (25). Compared to Turkish children (15), the Thai children in this study had higher IgG concentrations, which could be due to differences in genetic and environmental factors. The Ig concentrations reported by St. ohm and colleagues in USA children in the 1960s (5) continue to be used by many practitioners in Thailand and perhaps in other countries as reference values for diagnosing primary immunodeficiency. Here we have shown that IgG concentrations in Thai children are higher than those reported by Stiehm et al., which could be due to differences in ethnicity, geography and detection methods. When we compared our results with previous studies in Thai children using radial immunodiffusion assay (6,7), we found that the children in the present study had lower IgG, IgA, and IgM concentrations than in both previous studies. It is possible that the method used and the declining incidence of infections in the community with improvement in health care and sanitation accounts for these differences.
Our study illustrates an important point: over- or under-diagnosis of immunologic disorders can potentially occur if inappropriate normal values are used. It is crucial to have reference values obtained using the same laboratory method from an age-matched population with similar ethnicity and environmental factors. Our study is limited by the small sample size in the different age groups. In addition, we do not have values from children younger than 2 years of age, whom other studies have shown to have markedly different Ig concentrations than older children (5–8). Because many children with primary immunodeficiency present with illnesses before age 2, it is important to have appropriate reference values for these young children.
In conclusion, serum immunoglobulin concentrations can be influenced by age, genetic and environmental factors and the assays used; therefore, reference values from healthy and demographically-matched controls are important for the accurate diagnosis of immunologic abnormalities in children. These include both primary immunodeficiency disorders, such as hypogammaglobulinemia of genetic origin (26) and secondary immunodeficiency due to external factors, such as HIV infection, malnutrition, prematurity, surgery, trauma and immunosuppressive drugs (27).
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We are grateful to the children and their families for participating in this study. This study was supported by the Thailand Research Fund (TRF, grant number BRG5280017) and the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (grant number 53005). We thank Dr Amanda Clarke for proofreading our manuscript. The HIV-NAT 108 Study Group included: from HIV-NAT: Wasana Prasitsuebsai, Oratai Butterworth, Thongsuai Chuanjaroen, Tawan Mengthaisong, Sasiwimol Ubolyam, Apicha Mahanontharit, Phonethipsavahn Nounthong, Channuwat Bouko, Tanyathip Jaimulwong, Bunruan Sopa, Stephen Kerr and from the Pediatric Infectious Diseases Unit, Chulalongkorn University Hospital: Arene Klinklom, Chareeya Thanee, Nichada Naknoi, Napasri Kuljarusiri, Areeratana Piromwong.