Clinical Chemistry 60:3
518–529 (2014)
Pediatric Within-Day Biological Variation and Quality
Specifications for 38 Biochemical Markers in the
CALIPER Cohort
Dana Bailey,1,2,3† Victoria Bevilacqua,1,2† David A. Colantonio,1,2 Maria D. Pasic,1,2,4 Nandita Perumal,5
Man Khun Chan,1 and Khosrow Adeli1,2*
BACKGROUND: Studies of biological variation provide
insight into the physiological changes that occur within
and between study participants. Values obtained from
such investigations are important for patient monitor-
ing and for establishing quality specifications. In this
study we evaluated the short-term biological variation
of 38 chemistry, lipid, enzyme, and protein analytes in
a pediatric population, assessed the effect of age parti-
tions on interindividual variation, and compared the
findings to adult values.
METHODS: Four plasma samples each were obtained
within 8 h from 29 healthy children (45% males), age
4 –18 years. Samples were stored at ⫺80 °C and analyzed
in 3 batches, with samples from 9 –10 study participants
per batch. Within-person and between-person biological
variation values were established using nested ANOVA
after exclusion of outliers by use of the Tukey outlier test.
Analytical quality specifications were established with the
Fraser method.
RESULTS: Biological variation coefficients and analytical
goals were established for 38 analytes. Age partitioning
was required for 6 analytes. Biological variation char-
acteristics of 14 assays (37%) were distinct from adult
values found in the Westgard database on biological
variation. Biological variation characteristics were es-
tablished for 2 previously unreported analytes, uncon-
jugated bilirubin and soluble transferrin receptor.
CONCLUSIONS: This study is the first to examine biolog-
ical variation and to establish analytical quality specifi-
cations on the basis of biological variation for common
assays in a pediatric population. These results provide
1
CALIPER program, Department of Pediatric Laboratory Medicine, The Hospital
for Sick Children, Toronto, Ontario, Canada; 2 Department of Laboratory Med-
icine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 3 cur-
rent address: Gamma-Dynacare Medical Laboratories, London, Ontario, Canada;
4
current address: Department of Laboratory Medicine, St. Joseph’s Health
Centre, Toronto, Ontario, Canada; 5 Department of Pediatrics, The Hospital for
Sick Children, Toronto, Ontario, Canada.
*
Address correspondence to this author at: Clinical Biochemistry, The Hospital for
Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8 Canada. Fax
416-813- 6257; e-mail khosrow.adeli@sickkids.ca.
†
Dana Bailey and Victoria Bevilacqua contributed equally to the work, and both
should be considered as first authors.
518
Pediatric Clinical Chemistry
insight into pediatric physiology, are of use for refer-
ence change value calculations, clarify the appropriate-
ness of reference interval use, and aid in the develop-
ment of quality management strategies specific to
pediatric laboratories.
© 2013 American Association for Clinical Chemistry
Biological variation is an important factor to be con-
sidered when interpreting laboratory test results in a
clinical setting. Studies of biological variation provide
insight into the physiological changes that occur within
and between individuals for a given analyte. Although
several studies have explored biological variation in
adult populations, few studies have examined these at-
tributes in pediatric samples. This information is cru-
cial in result interpretation for 3 key reasons. First,
within- and between-person biological variation can
be used to establish reference change values (RCV),4
which provide the information required to determine if a
change in concentration of a specific analyte qualifies as
clinically significant (1 ). Establishing the usual amounts
of observed analyte fluctuation by use of within- and
between-person biological variation is central to long-
term monitoring and follow-up of children. It is also an
especially important consideration for analytes with cycli-
cal rhythms for which the time of collection may affect the
expected reference interval (2 ).
The second reason why this information is impor-
tant is that data information on biological variation can
be used to establish quality specifications (2 ) such as
bias, precision, and total allowable error (TE). These
Received August 8, 2013; accepted December 3, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.214312
4
Nonstandard abbreviations: RCV, reference change value; TE, total allowable
error; CVI, within-subject/intraindividual biological CV; CVG, between-subject/
interindividual biological CV; CALIPER, Canadian Laboratory Initiative on Pae-
diatric Reference Intervals; A1AT, ␣-1 antitrypsin; AGP, ␣-1 acid glycoprotein;
C3, complement component 3; CRP, C-reactive protein; STfR, soluble transferrin
receptor; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, as-
partate aminotransferase; CK, creatine kinase; GGT, ␥ glutamyl transferase;
HDL-C, HDL cholesterol; LDH, lactate dehydrogenase; CVdd, between-day CV;
CVA, total CV; II, index of individuality.
Pediatric Biological Variation for 38 Biochemical Markers
specifications can have important implications for re-
sult interpretation. For example, in adult populations,
changes in bias can affect how many low-risk individ-
uals are diagnosed with diabetes (3 ) or how many in-
dividuals with cholesterol values within reference in-
tervals are further investigated or sent for treatment
(4 ). Although the same misclassifications are likely
present in pediatric cohorts, data are lacking.
Although there are several ways to establish quality
specifications, a consensus statement states that the use of
biological variation is the preferred method (5 ). Accord-
ing to the hierarchy, a model based on biological variation
is second only to one in which quality specifications are
determined on the basis of the effect of analytical perfor-
mance on specific clinical decision-making. However, the
latter approach can be incredibly time-consuming and
difficult (5 ) and therefore, biological variation represents
both the most rigorous and the most efficient means by
which to establish quality specifications.
Finally, biological variation can provide important
insight into the usefulness of reference intervals for dif-
ferent laboratory tests. Specifically, biological variation
data can be used to generate the index of individuality—
the ratio of within- to between-individual variation for a
given analyte. Although a high index of individuality
(⬎1.4) suggests that the use of reference intervals
would be appropriate, a low index of individuality
(⬍0.6) suggests that other tools such as RCVs should
also be taken into consideration (6 ).
Clearly, the production of data on biological vari-
ation can generate a wealth of information not only
regarding the physiological changes that occur within
and between individuals, but also on what quality spec-
ifications are appropriate for a given test and how and
when to use reference intervals. Although such data are
evidently valuable, there is a clear lack of information on
biological variation in pediatric populations. As such, in
this study we aimed to evaluate the short-term (8 h) bio-
logical variation of 38 chemistry, lipid, enzyme, and pro-
tein analytes in a pediatric population of 29 healthy chil-
dren age 4 –18 years. The within- and between-person
biological variation (CVI and CVG) for these analytes
were estimated and the effects of age-specific partitions on
between-person variation were assessed. Differences in
biological variation between adult and pediatric popula-
tions were also evaluated. RCVs and indices of individu-
ality were generated for each of the 38 analytes and, finally,
the estimates of biological variation were used to generate
analytical goals.
Materials and Methods
STUDY PARTICIPANTS
Healthy community children were recruited to take
part in this 1-day (9:30 –17:30) study via the Canadian
Laboratory Initiative on Paediatric Reference Inter-
vals (CALIPER) outreach program (7 ). The study
was completed at The Hospital for Sick Children in
Toronto, Canada, with institutional ethics board ap-
proval. Before participation, the health of each child
was confirmed via interview and by health and life-
style questionnaires, with specific exclusion criteria as
described (7 ). Children were instructed to fast over-
night for at least 8 h before the study. A total of 30
children ages 4 –18 years were recruited; 1 child was
removed from the study owing to an inability to obtain
sufficient samples at all time points. The demographics
of the children have been previously reported (8 ). For
each participant, blood was drawn at 4 time points,
with a mean time period of approximately 2.5 h be-
tween each collection. Sample collection was per-
formed after an overnight fast, midmorning after
breakfast, within 2 h after lunch, and late afternoon.
After collection into serum separator tubes, all samples
were processed by the clinical chemistry laboratory and
stored at ⫺80 °C until batch testing.
ANALYTICAL PROCESSING
To avoid multiple freeze–thaw cycles and to minimize
analytical variability, all 38 assays were run on all sam-
ples from each child within the same day. Assays were
completed in batches on the Cobas Integra 400 (Roche)
[␣-1 antitrypsin (A1AT), ␣-1 acid glycoprotein (AGP),
complement component 3 (C3), C4, ceruloplasmin,
C-reactive protein (CRP), haptoglobin, IgG, IgA, IgM,
soluble transferrin receptor (STfR), transferrin] and
the VITROS 5,1 FS chemistry systems analyzer (Ortho
Clinical Diagnostics) [albumin, alkaline phosphatase
(ALP), alanine aminotransferase (ALT), amylase, as-
partate aminotransferase (AST), unconjugated biliru-
bin, calcium, cholesterol, creatine kinase (CK), chlo-
ride, CO2, creatinine, iron, ␥ glutamyl transferase
(GGT), glucose, HDL cholesterol (HDL-C), potas-
sium, lactate dehydrogenase (LDH), magnesium, so-
dium, phosphate, total bilirubin, total protein, triglyc-
eride, uric acid, urea] over a 3-day period, with samples
from 9 –10 individuals run per day. All assays were per-
formed according to the manufacturers’ recommenda-
tions. Before use, both analyzers were calibrated using
manufacturer-provided reagents and calibrators. QC
materials and 2 patient samples of known analyte con-
centrations were run with each batch. Analytical spec-
ifications for the 38 analytes tested have already been
published (7 ).
STATISTICAL ANALYSIS
SPSS Statistical Software (version 21, IBM), EP Evalu-
ator (version 9), and GraphPad Prism (version 4.0)
were used for data analysis. Data from males and fe-
males were analyzed together. All data followed a
Clinical Chemistry 60:3 (2014) 519
gaussian distribution (8 ). Outliers were eliminated by
use of the Tukey method, which defines an outlier as
1.5 interquartile ranges below the 25th percentile or
above the 75th percentile (9 ).
Within- and between-person biological variation
were calculated using GraphPad Prism and STATA and
expressed as CVs, CVI and CVG, respectively. CVG was
estimated using a nested ANOVA. CVI was estimated
by averaging the CV for each child, defined as the
square root of the variance in results divided by the
mean concentration for that individual, across all indi-
viduals. In contrast to estimates of CVI, for which the
overall mean is used [e.g., (10 )]. This approach is more
appropriate for situations in which the overall mean is
not representative of the mean for each individual.
Both within- and between-person biological variation
contain components of biological and analytical varia-
tion, which were minimized through experimental de-
sign and removed mathematically by subtracting the
between-day CV (CVdd) or the total CV (CVA), respec-
tively. Analytical CV was calculated using QC material
run concurrently with the collected samples. The index
of individuality (II), RCV, and analytical goals includ-
ing imprecision, bias, and total allowable error, were
calculated as follows:
II ⫽ CVI/CVG (note that the CVA was not included in
calculating II) (11 );
RCV ⫽ z(2)1/2(CVA2 ⫹ CVI2)1/2; z ⫽ 1.96 for P ⬍ 0.05
for a bidirectional change;
Analytical imprecision:
Optimal ⫽ 0.25 ⫻ CVI,
Desirable ⫽ 0.50 ⫻ CVI,
Minimal ⫽ 0.75 ⫻ CVI;
Analytical bias:
Optimal ⫽ 0.125 ⫻ (CVI2 ⫹ CVG 2 1/2
) ,
Desirable ⫽ 0.25 ⫻ (CVI ⫹ CVG) ,
2 2 1/2
Minimal ⫽ 0.375 ⫻ (CVI2 ⫹ CVG 2 1/2
) ;
Total allowable error (TAE) ⫽ (1.65 ⫻ imprecision) ⫹
bias.
When statistically indicated, the population of
children was partitioned according to age on the basis
of previously determined CALIPER reference interval
age partitions (7 ). To determine whether CALIPER-
determined age partitions were appropriate for this
substudy population, a Student t-test and F-test were
performed on analytes containing 2 age partitions (uric
acid, HDL-C, CRP, urea, ALT, haptoglobin, IgG),
whereas a 1-way ANOVA and Bartlett test were per-
formed on analytes containing 3 or more age parti-
tions (total bilirubin, CO2, creatinine, phosphate,
ALP, AST, LDH, albumin, total protein, and IgA) to
520 Clinical Chemistry 60:3 (2014)
identify significantly different group means or vari-
ances, respectively. Reference intervals were not
available for AGP, A1AT, bilirubin (unconjugated),
ceruloplasmin, CK, chloride, glucose, potassium, so-
dium, and STfR (7 ).
Adult values for CVI and CVG were obtained from
the Westgard Biological Variation database (12 ). This
database is an updated compilation of multiple papers
examining biological variation in adult populations
(13 ). The weighted-mean CVG across pediatric age
partitions was used for the pediatric cohort. Pediatric
to adult CVI, CVG, RCV, and total allowable error (TE)
ratios ⬍0.5 or ⬎1.5 were arbitrarily defined as being
significantly different.
Results
A total of 29 children ages 4 –18 years (13 male, 16
female) were recruited to participate in this 1-day (8 h)
study. Before participation, the health of each child was
ensured through an interview as well as health and life-
style questionnaires. Participants were instructed to
fast for at least 8 h before the study. Each child was
sampled at 4 time points with approximately 2.5 h be-
tween each sampling, yielding 116 data points for each
analyte; a total of 38 analytes were examined, yielding
4408 analytical results.
To correctly estimate CVG, it was necessary to par-
tition children by age for 6 of the 38 analytes, specifi-
cally ALP, AST, creatinine, LDH, phosphate, and uric
acid (Fig. 1, panel A). Suitable age partitions for this
cohort were derived from the CALIPER database, as
recently described (14 ). To determine whether age par-
titioning was necessary to minimize CVG, children
were partitioned by age, and the mean concentration
and variance of each age group was compared statisti-
cally, as described in Methods. Partitioning by age did
not significantly affect CVG for HDL-C, CRP, albumin,
total bilirubin, and IgG (Fig. 1B).
Table 1 lists the CVA, between-run CV, and CVdd
CVs, the CVI and CVG, the II, and the RCV for each of
the 38 analytes, as partitioned by age. Only 1 analyte,
AST, had an index of individuality that exceeded 1.4.
The majority of analytes examined (albumin, ALP,
AGP, A1AT, ALT, amylase, AST, C3, C4, ceruloplas-
min, cholesterol, CK, chloride, CRP, iron, GGT, hap-
toglobin, HDL-C, IgA, IgG, IgM, LDH, STfR, total pro-
tein, transferrin, uric acid, and urea) showed marked
individuality, with II values of ⬍0.6.
When comparing the pediatric cohort and adult
populations, CVI and CVG components of 24 of 38
analytes (63%) were found to be consistent with adult
values published in the Westgard database (Table 2)
(12 ). Four analytes showed marked differences, arbi-
trarily defined as a more than 50% reduction or more
Pediatric Biological Variation for 38 Biochemical Markers

Fig. 1. Relationship of within- and between-person biological variation with age in a pediatric cohort.
Numbers on the y axis refer to the study participant identification numbers, with children sorted by ascending age. The range
of values for each child is indicated by the box-and-whisker plot. (A), Representative analytes for which within- and/or
between-person biological variation change with age in a pediatric cohort. Dark grey shading indicates that the age partitions
determined by prior CALIPER reference interval studies [Colantonio et al. (7 )] significantly affect estimates of CVI and/or CVG.
(B), Representative analytes for which within- and/or between-person biological variation do not change significantly with age
in a pediatric cohort. Light grey shading indicates that the age partitions determined by prior CALIPER reference interval studies
[Colantonio et al. (7 )] did not significantly affect estimates of CVI and/or CVG.

than 150% increase, in both within- and between-
person variation between the pediatric and adult pop-
ulations. Specifically, CRP values in the pediatric co-
hort demonstrated reduced CVI and increased CVG
compared to adult values, GGT showed reduced CVI
and CVG, and ceruloplasmin and glucose showed in-
creased CVI and CVG (Table 2). Additionally, 10 ana-
lytes showed marked differences in either CVI or CVG.
Specifically, AGP, AST, cholesterol, CK, HDL, IgG, and
STfR had reduced CVI, sodium had reduced CVG, and
iron and transferrin had increased CVG. Interestingly,
for iron, the smaller CVI and larger CVG values seen in
the pediatric population resulted in a smaller II (0.38 vs
1.14) (12 ). As a consequence of the differences ob-
served in CVI, the pediatric RCV was lower than that of
adult populations for AGP, AST, CK, CRP, GGT, and
STfR, whereas it was increased for ceruloplasmin and
glucose.
To provide a guide for analytical quality specifica-
tions for pediatric testing based on biological variation,
we calculated optimal, desirable, and minimal analyti-
cal goals for imprecision, bias, and total allowable error
(Table 3) and compared them with adult specifications
(Table 2). The total allowable error based on biological
variation characteristics was reduced by more than half
of that allowed for adult populations for CK (14.3% vs
30.3%, pediatric vs adult, respectively) and STfR (6.8%
vs 17.4%); it was increased to ⬎150% of that allowed
for an adult population for ceruloplasmin (15.1% vs
7.9%) and glucose (13.1% vs 7.2%).

Clinical Chemistry 60:3 (2014) 521

 Table 1. Biological variation characteristics in a pediatric cohort.

Analytical variation Biological variation

Analyte n Age, years Mean CVr r , % CVdd, % CVA, % CVI, % CVG, % II RCV, %

Albumin, g/dL 29 1 to ⬍19 4.5 1.5 0.2 1.5 2.3 4.7 0.5 7.5
ALP, U/L 5 1 to ⬍10 241.1 1.9 1.0 2.0 5.6 27.2 0.1 16.4
7 10 to ⬍13 219.0 24.0 0.1
3 13 to ⬍15 215.8 27.2 0.1
4 15 to ⬍17 151.4 60.9 0.1
10 17 to ⬍19 86.5 18.7 0.2
AGP, mg/dL 28 1 to ⬍19 80.0 0.9 0.7 1.1 3.7 20.5 0.2 10.8
A1AT, mg/dL 28 1 to ⬍19 120.0 1.9 0.0 1.9 5.0 13.0 0.4 14.7
ALT, U/L 29 1 to ⬍19 16.4 4.8 5.1 7.0 15.6 27.7 0.6 47.4
Amylase, U/L 29 1 to ⬍19 67.0 2.9 1.6 3.3 5.1 24.9 0.2 16.7
AST, U/L 2 1 to ⬍7 35.5 1.7 0.8 1.9 4.7 0.6 8.4 13.9
6 7 to ⬍12 28.5 21.5 0.2
21 12 to ⬍19 22.9 23.9 0.2
Bilirubin (total), mg/dL 29 1 to ⬍19 0.3 1.7 2.0 2.6 28.1 38.2 0.7 78.3
Bilirubin (unconjugated), 28 1 to ⬍19 0.2 1.7 2.6 3.1 51.1 57.0 0.9 141.8
mg/dL
C3, mg/dL 29 1 to ⬍19 120.0 0.9 2.5 2.7 4.8 12.1 0.4 15.2
C4, mg/dL 24 1 to ⬍19 20.0 2.6 2.1 3.4 5.5 28.1 0.2 18.0
Calcium, mg/dL 29 1 to ⬍19 10.0 1.0 0.9 1.3 1.6 2.5 0.7 5.7
Ceruloplasmin, mg/dL 28 1 to ⬍19 19.6 3.8 1.3 4.1 11.3 20.3 0.6 33.4
Chloride, mmol/L 29 1 to ⬍19 105.9 0.2 0.5 0.6 0.8 1.5 0.6 2.8
Cholesterol, mg/dL 29 1 to ⬍19 166.0 0.9 1.1 1.5 2.4 15.7 0.2 7.9
CO2, mmol/L 29 1 to ⬍19 24.6 2.8 2.3 3.7 3.4 5.3 0.6 13.8
CK, U/L 28 1 to ⬍19 89.5 1.7 4.3 4.7 4.1 43.4 0.1 17.2
Creatinine, mg/dL 2 2 to ⬍5 0.3 1.1 0.7 1.3 4.2 4.1 1.1 12.3
5 5 to ⬍12 0.5 23.3 0.2
8 12 to ⬍15 0.6 11.0 0.4
14 15 to ⬍19 0.8 15.7 0.3
CRP, mg/L 27 1 to ⬍19 0.9 1.6 2.9 3.3 19.3 125.4 0.2 54.1
GGT, U/L 28 1 to ⬍19 18.0 0.9 0.5 1.1 2.7 18.7 0.1 8.0
Glucose, mg/dL 29 1 to ⬍19 90.0 1.2 0.0 1.2 11.4 9.1 1.3 31.8
Haptoglobin, mg/dL 29 1 to ⬍19 90.0 1.2 1.3 1.7 10.7 50.8 0.2 30.1
HDL-C, mg/dL 25 1 to ⬍19 54.0 2.2 3.4 4.1 2.9 22.2 0.1 13.9
IgA, mg/dL 29 1 to ⬍19 150.0 0.6 1.3 1.5 4.4 42.1 0.1 12.9
IgG, mg/dL 29 1 to ⬍19 1110.0 2.4 1.1 2.6 1.1 14.7 0.1 7.8
IgM, mg/dL 28 1 to ⬍19 120.0 0.6 1.9 2.0 4.0 37.3 0.1 12.3
Iron, ␮g/dL 29 1 to ⬍19 83.2 2.3 4.2 4.7 14.6 38.9 0.4 42.5
LDH, U/L 5 1 to ⬍10 652.9 2.0 0.9 2.2 4.7 12.5 0.4 14.4
10 10 to ⬍15 528.4 18.1 0.3
14 15 to ⬍19 420.2 13.0 0.4
Magnesium, mg/dL 29 1 to ⬍19 1.9 1.7 0.0 1.7 2.7 4.5 0.6 8.8
Continued on page 523

522 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

Table 1. Biological variation characteristics in a pediatric cohort. (Continued from page 522)

Analytical variation Biological variation

Analyte n Age, years Mean CVr r , % CVdd, % CVA, % CVI, % CVG, % II RCV, %

Phosphate, mg/dL 2 1 to ⬍5 5.3 1.1 0.0 1.1 6.0 7.0 0.9 16.9
9 5 to ⬍13 4.6 8.0 0.8
8 13 to ⬍16 4.6 10.4 0.6
10 16 to ⬍19 4.0 5.1 1.2
Potassium, mmol/L 29 1 to ⬍19 4.4 0.6 1.5 1.6 4.6 5.3 0.9 13.5
Sodium, mmol/L 29 1 to ⬍19 142.7 0.5 0.6 0.8 0.4 0.4 0.9 2.4
STfR, mg/dL 27 1 to ⬍19 350.0 3.2 0.0 3.2 1.4 22.4 0.1 9.6
Total protein, g/dL 29 1 to ⬍19 8.0 1.5 0.8 1.7 1.7 4.6 0.4 6.7
Transferrin, mg/dL 28 1 to ⬍19 2.2 1.9 0.0 1.9 3.0 10.8 0.3 9.8
Triglycerides, mg/dL 29 1 to ⬍19 97.3 1.0 0.4 1.1 27.0 24.8 1.1 74.9
Urea, mg/dL 29 1 to ⬍19 14.6 1.3 0.7 1.5 7.5 22.1 0.3 21.3
Uric acid, mg/dL 7 1 to ⬍12 3.3 1.1 0.7 1.3 6.8 25.1 0.3 19.2
22 12 to ⬍19 4.6 23.0 0.3

Discussion for adults. A reduced CVI was observed for 9 analytes,

Biological variation and its application in serial results
monitoring and analytical goal establishment has been
widely examined in adult populations. However, there
is no literature available exploring biological variation
in a pediatric population. As a consequence, pediatric
laboratories have been obliged to adopt RCV and ana-
lytical quality goals based on adult cohorts. In the ab-
sence of data on biological variation, the clinical pedi-
atric laboratory has been unable to provide a complete
picture of the dynamic changes expected for analytes in
the pediatric population. This is especially crucial given
the many changes associated with childhood and teen-
age development. Previous CALIPER studies have
highlighted the substantial differences between pediat-
ric and adult reference intervals (7, 15, 16 ) and the ef-
fects of fasting and sampling time in a pediatric cohort
(8 ). However, addressing this gap in information rele-
vant to the pediatric population presents particular
challenges, including the acquisition of samples and
the necessary introduction of age partitioning to pro-
vide an accurate estimate of CVG. By examining the
short-term biological variation of 38 chemistry ana-
lytes in a pediatric cohort, we have made the first steps
to fill this gap in pediatric laboratory medicine. The age
partitions established in previous CALIPER studies
were tested in this analysis and it was determined that
age partitioning was required for 6 of the 38 analytes
examined, specifically, ALP, AST, creatinine, LDH,
phosphate, and uric acid (Fig. 1A).
The CVI and CVG estimates in this pediatric pop-
ulation were found to be largely consistent with those
but this may have resulted from the short-term nature
of this study. Previous studies have determined that
measurements made 24 h apart had smaller CVI values
than those taken at intervals of 4 days or longer (17 ).
Additionally, an increased CVG for pediatric vs adult
populations was observed for 5 analytes, which may
indicate a need for further age and/or sex partitioning,
as previously demonstrated by reference interval stud-
ies (18 ). Only 4 analytes, CRP, GGT, ceruloplasmin,
and glucose, showed marked differences in both
within- and between-person variation in the pediatric
vs the adult populations (Table 2).
CRP presented with a reduced CVI (19.3% vs
42.2%) and a larger CVG (125.4% vs 76.3%) in the
pediatric population. This increase in CVG largely re-
sulted from 5 children with median CRP concentra-
tions greater than approximately 1.5 mg/L (Fig. 2). Al-
though the reason for the increase in CRP in these
individuals remains unknown, the findings are consis-
tent with NHANES (National Health and Nutrition
Examination Survey) data for 10- to 15-year-old chil-
dren, in which 15% of children had CRP values be-
tween 0.9 and 2.7 mg/L and 10% had values ⬎2.7 mg/L
(19 ).
GGT demonstrated a reduced CVI and CVG (Fig.
2). Because increases in GGT in adults are known to be
nonspecific, with modest increases noted in conjunc-
tion with various common metabolic risk factors such
as increased blood pressure and decreased HDL-C
(20 ), the tighter biological control of GGT observed in
children may be partially explained by an absence of
these subclinical conditions.

Clinical Chemistry 60:3 (2014) 523

 Table 2. Comparison between pediatric and adult biological variation characteristics.

Pediatric Adulta

Analyte N Age, years CVI, % CVG, %b RCV, % TE, %c CVI, % CVG, % RCV, % TE, %c

Albumin 29 1 to ⬍19 2.3 4.7 7.5 3.2 3.1 4.2 9.5 3.9
ALP 29 1 to ⬍19 5.6 28.1 16.4 11.8 6.4 24.8 18.2 11.7
AGP 28 1 to ⬍19 3.7d 20.5 10.8d 8.3 11.3 24.9 31.5 16.2
A1AT 28 1 to ⬍19 5.0 13.0 14.7 7.6 5.9 16.3 16.8 9.2
ALT 29 1 to ⬍19 15.6 27.7 47.4 20.8 18.0 42.0 53.5 26.3
Amylase 29 1 to ⬍19 5.1 24.9 16.7 10.6 8.7 28.3 25.8 14.6
AST 29 1 to ⬍19 4.7d 21.8 13.9d 9.5 11.9 17.9 33.4 15.2
Bilirubin (total) 29 1 to ⬍19 28.1 38.2 78.3 35.0 23.8 39.0 66.4 31.1
Bilirubin (unconjugated) 28 1 to ⬍19 51.1 57.0 141.8 61.3
C3 29 1 to ⬍19 4.8 12.1 15.2 7.2 5.2 15.6 16.2 8.4
C4 24 1 to ⬍19 5.5 28.1 18.0 11.7 8.9 33.4 26.4 16.0
Calcium 29 1 to ⬍19 1.6 2.5 5.7 2.1 1.9 2.8 6.4 2.4
Ceruloplasmin 28 1 to ⬍19 11.3e 20.3e 33.4e 15.1e 5.8 11.1 19.7 7.9
Chloride 29 1 to ⬍19 0.8 1.5 2.8 1.1 1.2 1.5 3.7 1.5
Cholesterol 29 1 to ⬍19 2.4d 15.7 7.9 6.0 5.4 15.2 15.5 8.5
CO2 29 1 to ⬍19 3.4 5.3 13.8 4.4 4.8 5.3 16.8 5.7
CK 28 1 to ⬍19 4.1d 43.4 17.2d 14.3d 22.8 40.0 64.5 30.3
Creatinine 29 2 to ⬍19 4.2 14.9 12.3 7.3 6.0 14.7 17.0 8.9
CRP 27 1 to ⬍19 19.3d 125.4e 54.1d 47.6 42.2 76.3 117.3 56.6
GGT 28 1 to ⬍19 2.7d 18.7d 8.0d 7.0d 13.8 41.0 38.4 22.2
Glucose 29 1 to ⬍19 11.4e 9.1e 31.8e 13.1e 6.1 6.1 17.1 7.2
Haptoglobin 29 1 to ⬍19 10.7 50.8 30.1 21.8 20.4 36.4 56.7 27.3
HDL-C 25 1 to ⬍19 2.9d 22.2 13.9 8.0 7.1 19.7 22.7 11.1
IgA 29 1 to ⬍19 4.4 42.1 12.9 14.2 5.4 35.9 15.5 13.5
IgG 29 1 to ⬍19 1.1e 14.7 7.8 4.6 4.5 16.5 14.4 8.0
IgM 28 1 to ⬍19 4.0 37.3 12.3 12.7 5.9 47.3 17.3 16.8
Iron 29 1 to ⬍19 14.6 38.9e 42.5 22.4 26.5 23.2 74.6 30.7
LDH 29 1 to ⬍19 4.7 14.7 14.4 7.7 8.6 14.7 24.6 11.4
Magnesium 29 1 to ⬍19 2.7 4.5 8.8 3.5 3.6 6.4 10.7 4.8
Phosphate 29 1 to ⬍19 6.0 7.6 16.9 7.4 8.5 9.4 23.7 10.2
Potassium 29 1 to ⬍19 4.6 5.3 13.5 5.5 4.8 5.6 14.0 5.8
Sodium 29 1 to ⬍19 0.4 0.4d 2.4 0.5 0.7 1.0 2.9 0.9
STfR 27 1 to ⬍19 1.4d 22.4 9.6d 6.8d 13.6 20.8 38.4 17.4
Total protein 29 1 to ⬍19 1.7 4.6 6.7 2.6 2.7 4.0 8.8 3.4
Transferrin 28 1 to ⬍19 3.0 10.8e 9.8 5.3 3.0 4.3 9.1 3.8
Triglycerides 29 1 to ⬍19 27.0 24.8 74.9 31.4 20.9 37.2 58.0 27.9
Urea 29 1 to ⬍19 7.5 22.1 21.3 12.0 12.3 18.3 34.3 15.7
Uric acid 29 1 to ⬍19 6.8 23.5 19.2 11.7 9.0 17.6 25.2 12.4
a
Westgard website available: http://www.westgard.com/biodatabase1.htm.
b
Weighted CVG.
c
Desirable TE ⫽ 1.65 (0.5 ⫻ CVI) ⫹ [0.25 ⫻ ⻫(CVI2 ⫹ CVG2)].
d
Reduced variation compared to adult cohorts (pediatric CV/adult CV ⬍0.5).
e
Increased variation compared to adult cohorts (pediatric CV/adult CV ⬎1.5).

524 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

Table 3. Analytical goals for pediatric testing based on short-term biological variation.

Analytical goals

Imprecision, CV, % Bias, % TE, %

Analyte Age, Years Optimal Desirable Minimal Optimal Desirable Minimal Optimal Desirable Minimal

Albumin 1 to ⬍19 0.6 1.1 1.7 0.6 1.3 1.9 1.6 3.2 4.8
ALP 1 to ⬍10 1.4 2.8 4.2 3.5 6.9 10.4 5.8 11.5 17.3
10 to ⬍13 3.1 6.2 9.2
13 to ⬍15 3.5 7.0 10.4
15 to ⬍17 7.6 15.3 22.9
17 to ⬍19 2.4 4.9 7.3
AGP 1 to ⬍19 0.9 1.9 2.8 2.6 5.2 7.8 4.1 8.3 12.4
A1AT 1 to ⬍19 1.2 2.5 3.7 1.7 3.5 5.2 3.8 7.6 11.3
ALT 1 to ⬍19 3.9 7.8 11.7 4.0 8.0 11.9 10.4 20.8 31.2
Amylase 1 to ⬍19 1.3 2.5 3.8 3.2 6.3 9.5 5.3 10.5 15.8
AST 1 to ⬍7 1.2 2.3 3.5 0.6 1.2 1.8 2.5 5.0 7.5
7 to ⬍12 2.7 5.5 8.2
12 to ⬍19 3.0 6.1 9.1
Bilirubin (total) 1 to ⬍19 7.0 14.1 21.1 5.9 11.9 17.8 17.5 35.1 52.6
Bilirubin (unconjugated) 1 to ⬍19 12.8 25.6 38.3 9.6 19.1 28.7 30.7 61.3 92.0
C3 1 to ⬍19 1.2 2.4 3.6 1.6 3.2 4.9 3.6 7.2 10.8
C4 1 to ⬍19 1.4 2.8 4.2 3.6 7.2 10.8 5.9 11.7 17.6
Calcium 1 to ⬍19 0.4 0.8 1.2 0.4 0.7 1.1 1.0 2.1 3.1
Ceruloplasmin 1 to ⬍19 2.8 5.7 8.5 2.9 5.8 8.7 7.6 15.2 22.8
Chloride 1 to ⬍19 0.2 0.4 0.6 0.2 0.4 0.6 0.5 1.1 1.6
Cholesterol 1 to ⬍19 0.6 1.2 1.8 2.0 4.0 6.0 3.0 6.0 8.9
CO2 1 to ⬍19 0.8 1.7 2.5 0.8 1.6 2.3 2.2 4.3 6.5
CK 1 to ⬍19 1.0 2.0 3.0 5.4 10.9 16.3 7.1 14.2 21.4
Creatinine 2 to ⬍5 1.1 2.1 3.2 0.7 1.5 2.2 2.5 5.0 7.5
5 to ⬍12 3.0 5.9 8.9
12 to ⬍15 1.5 3.0 4.4
15 to ⬍19 2.0 4.1 6.1
CRP 1 to ⬍19 4.8 9.6 14.4 15.9 31.7 47.6 23.8 47.6 71.4
GGT 1 to ⬍19 0.7 1.3 2.0 2.4 4.7 7.1 3.5 6.9 10.4
Glucose 1 to ⬍19 2.9 5.7 8.6 1.8 3.7 5.5 6.5 13.1 19.6
Haptoglobin 1 to ⬍19 2.7 5.4 8.0 6.5 13.0 19.5 10.9 21.8 32.7
HDL-C 1 to ⬍19 0.7 1.4 2.2 2.8 5.6 8.4 4.0 8.0 11.9
IgA 1 to ⬍19 1.1 2.2 3.3 5.3 10.6 15.9 7.1 14.2 21.3
IgG 1 to ⬍19 0.3 0.6 0.8 1.8 3.7 5.5 2.3 4.6 6.9
IgM 1 to ⬍19 1.0 2.0 3 4.7 9.4 14.1 6.3 12.7 19.0
Iron 1 to ⬍19 3.6 7.3 10.9 5.2 10.4 15.6 11.2 22.4 33.7
LDH 1 to ⬍10 1.2 2.4 3.5 1.7 3.3 5.0 3.6 7.2 10.8
10 to ⬍15 2.3 4.7 7.0
15 to ⬍19 1.7 3.5 5.2
Magnesium 1 to ⬍19 0.7 1.3 2.0 0.7 1.3 2.0 1.8 3.5 5.3
Continued on page 526

Clinical Chemistry 60:3 (2014) 525

 Table 3. Analytical goals for pediatric testing based on short-term biological variation. (Continued from
page 525)

Analytical goals

Imprecision, CV, % Bias, % TE, %

Analyte Age, Years Optimal Desirable Minimal Optimal Desirable Minimal Optimal Desirable Minimal

Phosphate 1 to ⬍19 1.5 3.0 4.5 1.1 2.3 3.4 3.6 7.2 10.9
5 to ⬍13 1.3 2.5 3.8
13 to ⬍16 1.5 3.0 4.5
16 to ⬍19 1.0 2.0 2.9
Potassium 1 to ⬍19 1.1 2.3 3.4 0.9 1.7 2.6 2.8 5.5 8.3
Sodium 1 to ⬍19 0.1 0.2 0.3 0.1 0.1 0.2 0.2 0.4 0.7
STfR 1 to ⬍19 0.3 0.7 1.0 2.8 5.6 8.4 3.4 6.7 10.1
Total protein 1 to ⬍19 0.4 0.9 1.3 0.6 1.2 1.8 1.3 2.6 4.0
Transferrin 1 to ⬍19 0.7 1.5 2.2 1.4 2.8 4.2 2.6 5.3 7.9
Triglycerides 1 to ⬍19 6.8 13.5 20.3 4.6 9.2 13.7 15.7 31.5 47.2
Urea, 1 to ⬍19 1.9 3.8 5.6 2.9 5.8 8.7 6.0 12.0 18.1
Uric acid 1 to ⬍12 1.7 3.4 5.1 3.2 6.5 9.7 6.1 12.1 18.2
12 to ⬍19 3.0 6.0 9.0

Interestingly, glucose presented with increases in
both CVI and CVG relative to adult populations (Fig.
2). These increases are believed to be due to the more
prominent effect of fasting on glucose homeostasis in
children. In comparison to adults, children are more
prone to decreases in plasma glucose and increases in
ketone body production [reviewed in (21 )], thereby
widening the range of observed glucose concentra-
tions. Indeed, 7 pediatric study participants had fasting
glucose concentrations ⱕ4.0 mmol/L or 72 mg/dL,
with a nonfasting upper limit of 8.0 mmol/L or 144
mg/dL.
Due to a decrease in CVI (14.6% vs 26.5%) and a
modest increase in CVG (38.9% vs 23.2%), the II of
iron was reduced from 1.14 to 0.38 (adult vs pediatric)
(Fig. 2). Consistent with the reduction in CVI, it has
previously been shown that infants and young children
lack the diurnal variation of serum iron due to the ab-
sence of a sustained period of sleep (22 ). Furthermore,
the discrepancy in CVG may be explained, in part, by
the fact that iron deficiency is common in the pediatric
population, with females aged 12–19 years at especially
high risk for anemia (23 ). The observation that the
CVG for a related analyte, transferrin, was also mark-
edly increased in the pediatric population (10.8% vs
4.3%) suggests substantial variations in pediatric indi-
viduals with respect to iron status. Future long-term
studies will be needed to explore whether or not this
observation persists when the duration of the sampling
period is increased.
The need for properly established analytical goals,
and the consequences that result from a lack thereof,
have been well documented (3, 4, 6, 24 ). As such, it
was necessary to determine whether any differences ex-
ist in the laboratory test quality specifications required
in the pediatric population compared with those in the
adult population. Our analysis revealed that 4 analytes
showed discernible differences in TE between the adult
and pediatric populations: CK and STfR in the pediat-
ric population had a decreased TE compared with the
adult population, whereas ceruloplasmin and glucose
had an increased TE (Table 2). It should be noted that
interpretation of these results should take into consid-
eration the clinical context and the downstream effect
of clinical misclassification. For example, as a conse-
quence of the increase in both components of biologi-
cal variation, the calculated TE for glucose increased
from 7.2% to 13.1%. However, we argue that the ob-
servation of increased susceptibility to hypoglycemia in
pediatric individuals suggests the need for accurate and
precise glucose measurements in this population, par-
ticularly at low concentrations.
In terms of the II, it has been argued that analytes
with II values of ⬍0.6 show a high degree of individu-
ality and, therefore, the RCV as opposed to a reference
interval should be used to assess the patient. On the
other hand, analytes with II values of ⬎1.4 show very
little individuality and, therefore, the use of reference
intervals is deemed to be appropriate (6 ). Analysis of
the samples obtained from this pediatric population

526 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

Fig. 2. Within- and between-person biological variation characteristics unique to pediatric samples.
Estimates of CVG for glucose, GGT, CRP, and iron are indicated by the top horizontal line and vertical dashed lines. Numbers
on the y axis refer to study participant identification numbers, with individuals sorted by ascending age. The range of values
for each child is indicated by the box-and-whisker plot.

revealed that only 1 analyte, AST, had an II value that
exceeded 1.4, whereas 27 of the 38 analytes examined
had an II value below 0.6. Of particular interest, the
reduction of II for iron to 0.38 suggests further inves-
tigation of the utility of an iron reference interval for
clinical decision-making in a pediatric population.
Although it may be appropriate to further examine
the usefulness of population-based reference intervals
in cases in which the II falls below 0.6, it is also impor-
tant to consider the clinical context in which the ana-
lyte is likely to be used (25 ). In many cases, a patholog-
ical state would result in a dramatic increase in analyte
concentration that would clearly fall outside of the es-
tablished reference interval. For example, bilirubin has
an II value of 0.74, which falls only slightly above the
0.6 cutoff, showing a fair degree of individuality. Ref-
erence intervals derived from a sample of healthy chil-
dren indicate that total bilirubin for children of ages
birth to 14 days should fall between 0.19 and 16.60
mg/dL, and for children ages 15 days to 1 year, total
bilirubin should fall between 0.05 and 0.68 mg/dL (7 ).
Cases of kernicterus have rarely been reported in neo-
nates with bilirubin concentrations of ⬍25 mg/dL and
are not reported in neonates with bilirubin peak con-

Clinical Chemistry 60:3 (2014) 527

centrations of ⬍20 mg/dL (26 ). These clinically rele-
vant values far exceed the reference intervals estab-
lished, and it is unlikely that, despite the small II value
for bilirubin, a clinical diagnosis based on bilirubin
concentrations would be adversely affected by compar-
ison with a population-based reference interval. There-
fore, it is important that measures like II, as well as
clinical context, be taken into account when interpret-
ing results and determining a course of action for the
patient in question.
Finally, it is important to note certain limitations
of our study. First, previous CALIPER studies have de-
termined that, for the creation of reference intervals,
certain analytes (e.g., creatinine, albumin, and ALT)
show sex differences and, therefore, must be parti-
tioned accordingly (7, 16, 27 ). However, due to sample
size limitations, we were unable to assess the appropri-
ateness of partitioning CVI and CVG by sex. Larger-
scale studies will be needed to determine the role of sex
in biological variation for these analytes. Second, ow-
ing to the sample size of our study and the need to run
samples in batches over several days, our protocol in-
troduced components of between-run analytical im-
precision into estimates of CVG which were removed
mathematically. Third, owing to the challenges associ-
ated with obtaining pediatric samples, this study was
restricted to a single day. Therefore, calculations such
as analytical quality values or RCV might be relevant
only for repeat samples collected within the same time-
frame. Fourth, in comparing the pediatric estimates of
CVI to those of adult populations, it was assumed that
the within-person biological variation in a single day
would be representative of long-term variation. Al-
though we attempted to account for some effects of
diurnal variation and fed/fasting-related variation by
sampling in a fasting and postprandial state, the esti-
mates of CVI are likely smaller than what would be
obtained from a study of longer duration. Lastly, given
the short-term nature of this study, preanalytical sam-
pling variation may contribute significantly to the vari-
ation observed. Future long-term studies will be
needed to confirm the findings described herein.
In conclusion, this is the first study to analyze the
components of biological variation in a healthy pediat-
ric cohort. We have derived estimates of RCVs and
quality indicators for precision, bias, and total allow-
able error. Future studies are needed to refine our esti-
mates of the components of biological variation in pe-
diatric populations, to extend our analysis of short-
term biological variation to longer time periods, and to
increase the sample size investigated.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: K. Adeli, CIHR.
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled participants, review and interpre-
tation of data, or preparation or approval of manuscript.

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