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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2017  |  Volume : 60  |  Issue : 3  |  Page : 360-364
Age-related reference intervals for immunoglobulin levels and lymphocyte subsets in Indian children


1 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India
2 PD Hinduja National Hospital and Medical Research Center, Mumbai, Maharashtra, India
3 Department of Pediatric Cardiology, SDM Hospital - Narayana Hrudayalaya, Dharwad, Maharashtra, India
4 Department of Pathology, Army Hospital (R and R), New Delhi, India

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Date of Web Publication22-Sep-2017
 

   Abstract 

Background: In children, innate and adaptive immunity varies with age, disease status, and ethnicity, reflected by lymphocyte subsets and serum immunoglobulin (Ig) levels. The paucity of such data from the Indian subcontinent necessitated this study. Aims: This study aims to determine reference ranges of Ig and lymphocyte subsets in Indian children from birth to 5 years. Settings and Design: Neonates, infants, and children from a tertiary care hospital were selected and categorized into 5 groups from cord blood/newborn to 5 years. Materials and Methods: Samples were taken from cord blood and healthy children up to 5 years of age. Complete blood counts, serum Ig levels (by turbidimetry), and lymphocyte subsets (by flow cytometry) were studied, and reference ranges calculated. Results: Four hundred and three samples were analyzed; 53 from cord blood and 350 from children 1 month to 5 years. High IgG levels were noted at birth, which decreased in the first 6 months followed by a rise thereafter. IgM remained low in infancy and peaked at 13–36 months. IgA levels were very low at birth but increased with age. CD4 counts were high in cord blood till 3 years of age and then declined. CD8 and CD19 counts remained steady till 5 years of age. CD56 increased after the age of 2 years. Conclusions: While our data correlated well with published literature, notable differences were higher IgM levels seen in 1–3 years' age group and higher natural killer cells through all age groups in our study. Our results provide the largest database of its kind from our country.

Keywords: Absolute cell count, flow cytometry, lymphocyte profile, lymphocyte subsets, serum immunoglobulins

How to cite this article:
Narula G, Khodaiji S, Bableshwar A, Bindra MS. Age-related reference intervals for immunoglobulin levels and lymphocyte subsets in Indian children. Indian J Pathol Microbiol 2017;60:360-4

How to cite this URL:
Narula G, Khodaiji S, Bableshwar A, Bindra MS. Age-related reference intervals for immunoglobulin levels and lymphocyte subsets in Indian children. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Dec 10];60:360-4. Available from: http://www.ijpmonline.org/text.asp?2017/60/3/360/215390



   Introduction Top


Pediatric age is fraught with predisposition to infections. The primary host defense against infection involves innate immunity, which responds regardless of previous exposure to the agent and includes complement, interleukins, and cellular components.[1] The cellular component comprises the phagocytes/macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, and natural killer cells (NK cells). These identify and eliminate pathogens by contact and engulfment are nonspecific and do not confer long-lasting immunity.[2]

Acquired (adaptive) immunity is a highly specific response which involves T and B lymphocytes and NK cells.[3] This response is mediated by two primary mechanisms, cellular immunity by T-cells, and humoral immunity with the production of immunoglobulins (Ig) by 5 functionally distinct subtypes of B cells. Synthesis starts in fetal life and proceeds variably according to Ig type, antigen exposure, race, and ethnicity.[4] Abnormalities in the immune system can cause increased susceptibility to infections and other conditions such as allergy, autoimmunity, and malignancy. Estimation of serum Ig levels and absolute counts and percentages of lymphocyte subsets helps in early diagnosis and treatment of such conditions. However, very few age-specific reference ranges are available for these parameters from India. Those available are mostly on Caucasians, or at best include ethnic minorities from this region. The probability of racial differences has been established in at least one large Asian study.[5] It is inappropriate to extrapolate these results to children from the Indian subcontinent who are far more ethnically diverse and grow up in environments and socioeconomic milieu which are vastly different from their Western counterparts. The scarcity of reference ranges in healthy Indian children led to the present study, the aim of which was to determine reference intervals of Ig levels and peripheral blood lymphocyte subsets in cord blood and children from birth to 5 years of age.


   Materials and Methods Top


The study population comprised of neonates born in a tertiary care hospital (from whom cord blood was collected) and infants and children visiting well baby clinics or outpatient departments at the same center. They were segregated into 5 age categories:

  1. Newborn-cord blood of term neonates
  2. Early infants (1–6 months)
  3. Late infants (7–12 months)
  4. Toddlers (13–36 months)
  5. Preschool children (37–60 months).


Inclusion criteria were:

  1. Cord blood from normal term deliveries without any antenatal complications or family history of immune deficiencies or malignancies
  2. Normal infants and toddlers attending well baby clinic
  3. Children attending outpatient department for noninfection-related complaints (e.g., fractures) but otherwise in apparent good health determined by routine history and examination
  4. Children on follow-up visits, more than 2 weeks after recovery from routine self-limiting childhood infections such as viral infections of the upper respiratory tract or a solitary episode of a self-limiting viral enteritis
  5. Patients on follow-up visits for surgical disorders that were not related to infections in any way and had healed/recovered completely.


Exclusion criteria were:

  1. Any solitary episode of a systemic infection of the organ system in the past, for example, pneumonia
  2. All cases of infectious disease, such as typhoid, malaria, mumps, measles, varicella, and hepatitis
  3. Children with upper respiratory tract infections (URTIs) - if number of viral self-limiting infections exceeded 10 in the previous 12 months or if they had any associated episode of pyogenic pharyngitis, suppurative otitis, or sinusitis
  4. Children with gastrointestinal tract infections - if number of viral self-limiting infections exceeded 1 in the previous 12 months or if an episode of bacterial, parasitic, or fungal diarrhea occurred
  5. All cases of parasitic or fungal infections
  6. Cases with atopic symptoms or syndromes
  7. Patients with a history of malignancy.


Informed consent and a detailed history were taken from the parents, and a thorough examination of the child was conducted. Cord blood samples were collected from the placenta in the labor room/operation theater immediately after delivery in 2 ethylenediaminetetraacetic acid and 1 sterile vacutainer. Venous samples were collected in the outpatient department. Complete blood counts were performed using Automated Cell Counter, ABX Pentra model no-120. Serum Ig levels were estimated by turbidometric method using automated cuvette-based Quantiamate turbidometer from Tulip Diagnostics Pvt Ltd®. Determination of cell surface markers was done by four-color dual-laser flow cytometry using FACSCalibur™ manufactured by Becton Dickinson Biosciences ®. TruCount Multitest tubes were used to estimate the lymphocyte subsets. Samples which could not be analyzed immediately were stored for up to 24 h at 18 to 22°C before evaluation.


   Results Top


A total of 403 samples were analyzed, of which 53 were cord blood samples and 350 were from children aged 1 month to 5 years, divided into subsets based on age [Table 1]. The male to female ratio was 1.1:1.
Table 1: Demographic profile of subject population

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Serum immunoglobulin levels

Mean Serum IgG levels were high in cord blood (978mg/dL) declining to a nadir of 611.2 mg/dL by late infancy and then increasing to adult levels by the 5th year of life [Table 2] and [Figure 1].
Table 2: Serum IgG, IgM, and IgA levels in mg/dL (n=403)

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Figure 1: Serum immunoglobulin levels at different ages

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Serum IgM levels were low in cord blood (mean 22.4 mg/dL) and gradually increased till 5 years of age [Table 2]. However, in the third year of life, a wide scatter was observed with high values (mean 289.6 mg/dL) [Figure 1].

The mean IgA levels were very low in cord blood (mean 3.35 mg/dl) but rose steadily in a linear fashion till 5 years of age [Table 2] and [Figure 1].

Lymphocyte profile

Absolute CD4 counts were high in cord blood and remained at the same level till 3 years of age and thereafter started declining. The CD8 counts are low in cord blood but rose at 6 months after which they remained steady till 5 years age. The CD3 counts were high at birth reflecting the elevated CD4 counts and followed the pattern of CD4 counts in all age groups [Table 3]. NK cells could not be enumerated in cord blood however, due to technical issues, but were low at birth and started to increase from the age of 2 years [Table 3]. B-lymphocytes as measured by CD19 were also low in infancy and gradually increased with age [Table 3].
Table 3: Age-wise absolute lymphocyte subset counts in cells/cmm (n=403, for CD16/56, n=350)

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   Discussion Top


Innate and adaptive immunity are required to combat childhood infections caused by various pathogens. Therefore, abnormalities of either the primary or acquired immune function can cause severe morbidity and mortality. Estimation of serum Ig levels and lymphocyte subsets helps in assessing the immune response and also the need for intervention. Studies worldwide have shown that the levels of Ig vary not only with age but also in different geographical regions.[5],[6],[7] Thus, this study was undertaken to determine age-specific reference ranges for these parameters in Indian children. The sample size was adequate for the subgroups analyzed. In our study, we included 138 (39%) children with routine childhood infections which had resolved more than 2 weeks before sample collection in accordance with our inclusion/exclusion criteria.

In the first study of its kind on serum Ig levels, Collins-Williams et al. studied 200 children using Hyland Immunoplate technique and found IgG ranging from 346 mg% in 2–6 months to 645 mg% in 2–5 years age.[6] These were lower than levels of 941 mg% and 1487 mg%, respectively, in our study. This could be due to the higher number of routine childhood infections even in otherwise healthy children in our population. The IgA and IgM levels, however, were comparable. Both the studies show an increase in serum Ig levels with age. However, due to differences in technique, comparisons should be made with caution, especially as the Hyland Immunoplate technique is now obsolete. Another similar study in children and adults by Stoop et al. in 1969 showed gradual increase in serum Ig levels with age; however, the reference ranges differed from other studies of the period, the main reason being lack of an established international reference for Ig determination at that time.[7],[8],[9]

In our study, serum IgG levels were raised in cord blood and in early infancy indicating passive transplacental transfer from mother, which is amply documented.[10] The IgG confers passive immunity from common infections in infancy. In later infancy, IgG levels decreased and reached a nadir at 7–12 months followed by progressively increasing values, a well-noted phenomenon.[11],[12] Our data are comparable with these studies with some notable differences [Table 4].
Table 4: Comparison of age-specific IgG values (mg/dL) with other studies

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In another study, Lockitch et al. clubbed all children below 1 year into one group rendering the subgroup differences incomparable [Table 4]. The upper ranges of IgG in both studies compared are far lower in the 1–5 years' age group than ours, while the lower ranges remain closer to each other [Table 4]. This may be a result of the higher number of recent infections, albeit routine nature and more than 2-week postrecovery, in our patients.

IgM levels were low in cord blood (mean-22 mg/dL) and showed a gradual increase with highest levels in the 13–36 age group (mean-289 mg/dL) similar to known patterns.[8] Low levels in cord blood may reflect the inability of IgM to traverse the placental barrier. Increased levels of IgM in 13–36 months age corresponds with increased incidence of infections in this age group.

IgA levels were also low in cord blood, similar to IgM levels but increased more gradually and maintained a steady level in childhood after 3 years of age. A similar pattern of rise has been noted by others.[7],[8],[9]

Age-specific changes in serum Ig levels in our study with their individual differences are depicted in the consolidated scatter diagram [Figure 1].

Lymphocyte subsets analyzed by flow cytometry showed that the levels of absolute CD4 counts were high in cord blood, remained at same levels till 3 years age, and declined thereafter. Two major studies on this aspect have shown a similar trend.[13],[14] [Table 5].
Table 5: Comparison of absolute CD4 counts in cells/cmm with other studies

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In our study, the CD4 counts have declined from mean 2932 cells/μL in infancy to 1977 cells/μL at 3–5 years. It is noteworthy that the values remain comparable with these studies despite the policy of universal BCG immunization at birth, which was in 100% of our study population. This is routinely administered in India and is a known T-cell stimulant.

CD8 absolute cell counts were low in cord blood (mean 836 cells/μL) but rose after birth (mean 1544 cells/μL) after which they remained at a steady level till 5 years of age. Two major studies demonstrated similar trends, though one showed a steady decline in CD8 and total CD3 counts after 3 years.[13],[15] Absolute CD3 counts were high at birth, mirroring the elevated CD4 counts and followed the latter's pattern in all age groups, a pattern well established in the western literature.[14],[15],[16] NK cell (CD16/56) counts were low at birth (mean 15.8 cells/μL) and gradually increased with age (mean 103 and 556 cells/μL at 1–3 and 2–5 years, respectively) comparing well with the scant data available on this cell line.[5],[16],[17] A tendency to higher NK cell counts in our children, as compared to Caucasians, matches results seen by the only other large study in Asian children.[5] Another study from India on T-lymphocyte subsets had 138 children but covered age groups from 3 to 15 years of age and thus was not truly comparable with our data.[18]

CD19 cells showed a peak in early infancy and declined thereafter remaining steady till 5 years of age. Other studies showed a similar pattern till 5 years of age followed by a steady decline till adulthood.[5],[19] The peak in early infancy in all probability relates to the multiple vaccinations stimulating B-cells in this period.

The varying rates of decline in lymphocyte subsets lead to discordance in their percentages and ratios with age as noted in our study. This has been highlighted in a large study consisting of individuals from childhood to adulthood and is particularly relevant to studying the variations in immunodeficiencies including pediatric HIV infection.[20]

In view of the high incidence of recurrent mild viral infection in children in the 1–5 years' age group, we deliberately included children with these conditions but who had recovered 2 weeks before collection of sample so that we could arrive at a more realistic reference range for children. The most common infections included respiratory, followed by gastrointestinal infections. The majority of infections occurred in 13–36 months' age group.

The high IgM levels and IgA levels in 13–36 months' children reflect the immune response due to exposure to infectious agents which is common at this age in our country.

The high absolute lymphocyte counts in infancy in our study are probably due to transplacental passage of T-lymphocytes, which contribute to cell-mediated immunity and T-cell regulated self-recognition in fetal life.

The absolute values obtained in this study are the largest data bank of its kind from India and are indicative of the trends of these parameters in Indian children of different ethnicities and can form a useful reference range for the subcontinent.


   Conclusions Top


  • Age-specific normal serum Ig levels in cord blood and healthy Indian children from birth to 5 years of age as a standard reference were obtained. Immunoglobulin levels of Indian children varied from other populations though trends were comparable
  • Age-specific lymphocyte subsets of normal children were obtained from cord blood till 5 years age. Trends were comparable with other populations though absolute values and range differed in Indian children
  • This study provides the largest and most representative data of its kind in healthy Indian children of wide ethnic diversity
  • So-called healthy children commonly have mild infections URTIs which can alter the Ig levels and lymphocyte counts. We have thus established normalized reference ranges for children which are more realistic than establishing reference intervals on completely healthy children.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Roitt M, Delves PJ, editors. Innate immunity. In: Immunology. 10th ed. Massachusetts: Blackwell; 2001. p. 1-21.  Back to cited text no. 1
    
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Guermonprez P, Valladeau J, Zitvogel L, Théry C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002;20:621-67.  Back to cited text no. 3
    
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Lee BW, Yap HK, Chew FT, Quah TC, Prabhakaran K, Chan GS, et al. Age- and sex-related changes in lymphocyte subpopulations of healthy Asian subjects: From birth to adulthood. Cytometry 1996;26:8-15.  Back to cited text no. 5
    
6.
Collins-Williams C, Toft B, Generoso L, Moscarello M. Quantitative immunoglobulin levels (IgG, IgA and IgM) in children, determined by the Hyland immunoplate technique. Can Med Assoc J 1967;96:1510-3.  Back to cited text no. 6
    
7.
Stoop JW, Zegers BJ, Sander PC, Ballieux RE. Serum immunoglobulin levels in healthy children and adults. Clin Exp Immunol 1969;4:101-12.  Back to cited text no. 7
    
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Stiehm ER, Fudenberg HH. Serum levels of immune globulins in health and disease: A survey. Pediatrics 1966;37:715-27.  Back to cited text no. 8
    
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Buckley RH, Dees SC, O'Fallon WM. Serum immunoglobulins. I. Levels in normal children and in uncomplicated childhood allergy. Pediatrics 1968;41:600-11.  Back to cited text no. 9
    
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Nevard CH, Gaunt M, Ockleford CD. The transfer of passive and active immunity. In: Chaouat G, editor. The Immunology of the Fetus. 1st ed. Florida: CRC Press; 1990. p. 193-214.  Back to cited text no. 10
    
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Lockitch G, Halstead AC, Quigley G, MacCallum C. Age- and sex-specific pediatric reference intervals: Study design and methods illustrated by measurement of serum proteins with the Behring LN nephelometer. Clin Chem 1988;34:1618-21.  Back to cited text no. 11
    
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Brugnara C. Reference Values in Infancy and Childhood - Appendix 64, 67, 68. In: Orkin SH, Fisher DE, Look AT, Lux SE, IV, Ginsburg D, Nathan DG, editors. Nathan and Oski's Hematology and Oncology of Infancy and Childhood. 8th ed. Philadelphia: Elsevier Saunders; 2015. p.2527-28.  Back to cited text no. 12
    
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Kotylo PK, Fineberg NS, Freeman KS, Redmond NL, Charland C. Reference ranges for lymphocyte subsets in pediatric patients. Am J Clin Pathol 1993;100:111-5.  Back to cited text no. 13
    
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Denny T, Yogev R, Gelman R, Skuza C, Oleske J, Chadwick E, et al. Lymphocyte subsets in healthy children during the first 5 years of life. JAMA 1992;267:1484-8.  Back to cited text no. 14
    
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Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ, Hop WC, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations. J Pediatr 1997;130:388-93.  Back to cited text no. 15
    
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Spits H, Lanier LL, Phillips JH. Development of human T and natural killer cells. Blood 1995;85:2654-70.  Back to cited text no. 16
    
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Yokoyama WM. Natural killer cell receptors. Curr Opin Immunol 1995;7:110-20.  Back to cited text no. 17
    
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Swaminathan S, Hanna LE, Raja A, Sankaran K, Kumar AN. Age-related changes in blood lymphocyte subsets of South Indian children. Natl Med J India 2003;16:249-52.  Back to cited text no. 18
    
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Hicks MJ, Jones JF, Minnich LL, Weigle KA, Thies AC, Layton JM. Age-related changes in T- and B-lymphocyte subpopulations in the peripheral blood. Arch Pathol Lab Med 1983;107:518-23.  Back to cited text no. 19
    
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Hulstaert F, Hannet I, Deneys V, Munhyeshuli V, Reichert T, De Bruyere M, et al. Age-related changes in human blood lymphocyte subpopulations. II. Varying kinetics of percentage and absolute count measurements. Clin Immunol Immunopathol 1994;70:152-8.  Back to cited text no. 20
    

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Correspondence Address:
Shanaz Khodaiji
PD Hinduja National Hospital and Medical Research Center, Veer Savarkar Marg, Mahim, Mumbai - 400 016, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_542_16

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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