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Advances in Pediatric Reference Intervals for Biochemical Markers: Establishment of the Caliper Database in Healthy Children and Adolescents/Napredak U Oblasti Pedijatrijskih Referentnih Intervala Za Biohemijske Markere: Izrada Baze Podataka Caliper Kod Zdrave Dece I Adolescenata

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Abstract

Clinical laboratory reference intervals provide valuable information to medical practitioners in their interpretation of quantitative laboratory test results, and therefore are critical in the assessment of patient health and in clinical decisionmaking. The reference interval serves as a health-associated benchmark with which to compare an individual test result. Unfortunately, critical gaps currently exist in accurate and upto-date pediatric reference intervals for accurate interpretation of laboratory tests performed in children and adolescents. These critical gaps in the available laboratory reference intervals have the clear potential of contributing to erroneous diagnosis or misdiagnosis of many diseases. To address these important gaps, several initiatives have begun internationally by a number of bodies including the KiGGS initiative in Germany, the Aussie Normals in Australia, the AACC-National Children Study in USA, the NORICHILD Initiative in Scandinavia, and the CALIPER study in Canada. In the present article, we will review the gaps in pediatric reference intervals, challenges in establishing pediatric norms in healthy children and adolescents, and the major contributions of the CALIPER program to closing the gaps in this crucial area of pediatric laboratory medicine. We will also discuss the recently published CALIPER reference interval database (www.caliperdatabase.com) developed to provide comprehensive age and gender specific pediatric reference intervals for a larger number of biochemical markers, based on a large and diverse healthy children cohort. The CALIPER database is based on a multiethnic population examining the influence of ethnicity on laboratory reference intervals. Thus the database has proved to be of global benefit and is being adopted by hospital laboratories worldwide.
J Med Biochem 2015; 34 (1) DOI: 10.2478/jomb-2014-0063
UDK 577. 1 : 61 ISSN 1452-8258
J Med Biochem 34: 23–30, 2015 Review article
Pregledni ~lanak
ADVANCES IN PEDIATRIC REFERENCE INTERVALS FOR BIOCHEMICAL
MARKERS: ESTABLISHMENT OF THE CALIPER DATABASE IN HEALTHY
CHILDREN AND ADOLESCENTS
NAPREDAK U OBLASTI PEDIJATRIJSKIH REFERENTNIH INTERVALA ZA BIOHEMIJSKE
MARKERE: IZRADA BAZE PODATAKA CALIPER KOD ZDRAVE DECE I ADOLESCENATA
Kimiya Karbasy
1,2
, Petra Ariadne
1
, Stephanie Gaglione
1,2
, Michelle Nieuwesteeg
1
, Khosrow Adeli
1,2
1
CALIPER program, Department of Pediatric Laboratory Medicine, The Hospital for Sick Children,
University of Toronto, Toronto, Ontario, Canada
2
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
Address for correspondence:
Khosrow Adeli
Clinical Biochemistry, The Hospital for Sick Children,
University of Toronto, Toronto, Ontario, M5G 1X8 Canada
List of abbreviations: CALIPER, Canadian Laboratory
Initiative on Pediatric Reference Intervals; RIDL, Reference
Intervals and Decision Limits; IFCC, International Federation
of Clinical Chemistry and Laboratory Medicine; CLSI,
Clinical Laboratory Standards Institute; GGT, Gamma-
Glutamyl Transferase; SHGB, sex hormone-binding globulin;
FSH, follicle-stimulating hormone; LH, luteinizing hormone;
AFP, a-fetoprotein; TSH, thyroid-stimulating hormone; T4,
total thyroxine; T3, total triiodothyronine; CRP, C-reactive
protein.
Summary
Clinical laboratory reference intervals provide valuable infor-
mation to medical practitioners in their interpretation of
quantitative laboratory test results, and therefore are critical
in the assessment of patient health and in clinical decision-
making. The reference interval serves as a health-associated
benchmark with which to compare an individual test result.
Unfortunately, critical gaps currently exist in accurate and up-
to-date pediatric reference intervals for accurate interpreta-
tion of laboratory tests performed in children and adoles-
cents. These critical gaps in the available laboratory
reference intervals have the clear potential of contributing to
erroneous diagnosis or misdiagnosis of many diseases. To
address these important gaps, several initiatives have begun
internationally by a number of bodies including the KiGGS
initiative in Germany, the Aussie Normals in Australia, the
AACC-National Children Study in USA, the NORICHILD
Initiative in Scandinavia, and the CALIPER study in Canada.
In the present article, we will review the gaps in pediatric ref-
erence intervals, challenges in establishing pediatric norms
in healthy children and adolescents, and the major contribu-
tions of the CALIPER program to closing the gaps in this cru-
cial area of pediatric laboratory medicine. We will also dis-
Kratak sadr`aj
Klini~ki laboratorijski referentni intervali pru`aju lekarima
podatke koji su va`ni za tuma~enje rezultata kvantitativnih
laboratorijskih testova i stoga su od klju~ne vrednosti za pro-
cenu zdravstvenog stanja pacijenta i dono{enje klini~kih
odluka. Referentni interval slu`i kao reper u smislu zdravlja s
kojim }e se porediti rezultat pojedina~nog testa. Na`alost,
trenutno postoje velike razlike u ta~nim i savremenim pedija-
trijskim referentnim intervalima za ta~no tuma~enje labora-
torijskih testova koji se obavljaju kod dece i adolescenata.
Ove velike razlike u dostupnim laboratorijskim referentnim
intervalima o~ito lako mogu dovesti do dijagnosti~ke gre{ke
ili po gre{nog dijagnostikovanja mnogih bolesti. Nekoliko ini-
cijativa pokrenuto je od strane vi{e tela na me|unarodnom
nivou sa ciljem da se re{i problem ovih razlika, me|u njima
inicijativa KiGGS u Nema~koj, Aussie Normals u Australiji,
AACC – Nacionalna de~ija studija u SAD, Inicijativa
NORICHILD u Skandinaviji i studija CALIPER u Kanadi. U
ovom ~lanku da}emo pregled razlika u pedijatrijskim refer-
entnim interva lima, izazova vezanih za utvr|ivanje pedijatri-
jskih normi kod zdrave dece i adolescenata i glavnih dopri-
nosa programa CALIPER u pogledu otklanjanja razlika u ovoj
najva`nijoj oblasti pedijatrijske laboratorijske medicine.
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24 Adeli et al.: Advances in Pediatric Reference Intervals for Biochemical Markers
The Necessity of Reference Intervals
Reference intervals are health-associated
bench marks essential for the interpretation of quanti-
tative laboratory test results by medical practitioners.
An interval is formally defined as a statistically derived
range of values determined from a reference interval
study encompassing the central 95% of values from a
healthy reference population. Biomarker test results
lying outside of the reference interval suggest an
abnormal result and as such, establishing accurate
reference intervals is crucial to informed clinical deci-
sion-making.
Reference Interval Verification
Clinical laboratories are individually responsible
for assuring the validity of reference intervals used to
interpret test results they report. Regulatory, accredi-
tation, and licensing organizations require verification
or establishment of reference intervals for all quanti-
tative test methods offered by the laboratory, with the
exception of tests that utilize decision cut-off limits. If
a laboratory opts not to establish the reference inter-
val, verification must be completed using well-
defined, systematic methods with supporting docu-
mentation demonstrating adherence to international
standards. The process of validating an externally
determined reference interval is referred to as »trans-
ference«, and is required to prevent incorrect classifi-
cation of test results as normal or abnormal. If a lab-
oratory chooses to conduct a reference interval study,
the Clinical Laboratory Standards Institute (CLSI)
guidelines must be followed (C28-A3) (1).
Reference intervals are not necessarily transfer-
able between labs due to differences in the reference
populations of the donor and receiving laboratories,
and differences in analytical methods. In particular,
dissimilarities between reference populations can
include variations in ethnic compositions, geographic
factors, diet preferences, and lifestyle. Analytical
methods can vary in measurement principle, calibra-
tion, reagent formulation, operating environment,
and lot-to-lot variation in reagents and calibrators. To
eliminate the potential for error resulting from these
differences, a transference study can be completed to
assess a reference interval on the demographics of its
reference individuals, pre-analytical and analytical
details, and statistical methods. While transference of
a reference study requires 20 reference individuals, a
de novo reference interval study is far more laborious
and expensive, and is usually reserved for emerging
analytes or new analytical methods.
An alternative approach is to establish common
reference intervals using a multicenter study design
(2). Common reference intervals offer significant
advantages including: 1) reduced costs in establish-
ing the reference interval, 2) involvement of multiple
laboratories, 3) implementation of a single universal
reference interval to interpret a test result that does
not require transference, and 4) wide adoption of the
interval by laboratories with similar reference popula-
tions using the same metrologically traceable
method. Nonetheless, several challenges currently
impact the common reference interval approach. The
lack of harmonization of methods by manufacturers
and the limited availability of reference materials and
methods for most analytes reduces the ability of mul-
tiple laboratories to collaborate. Presently, there are
no official guidelines for setting and monitoring the
minimum analytical quality goals that a laboratory
must achieve to contribute to or use reference values,
and reference intervals may not be robust when
applied to ethnically diverse populations.
Challenges in Establishing Pediatric
Reference Intervals
Necessity of Pediatric Reference Intervals
Critical attention is required to close several
accuracy gaps with respect to establishing reference
intervals for use in the pediatric population. The
application of adult reference intervals in a pediatric
setting is inappropriate due to differences in physical
size, organ maturity, immune and hormonal respon-
siveness, nutrition, and metabolism, which collective-
ly influence normal analyte concentrations in chil-
dren. Additionally, unique biomarkers for children and
neonates are often unaccounted for in adult refer-
ence intervals, and partitions (or separate reference
intervals) are necessary for children and neonates of
different age groups, genders, and ethnicities (1).
cuss the recently published CALIPER reference interval data-
base (www.caliperdatabase.com) developed to provide com-
prehensive age and gender specific pediatric reference inter-
vals for a larger number of biochemical markers, based on a
large and diverse healthy children cohort. The CALIPER data-
base is based on a multiethnic population examining the
influence of ethnicity on laboratory reference intervals. Thus
the database has proved to be of global benefit and is being
adopted by hospital laboratories worldwide.
Keywords: biochemical markers; pediatric; reference
intervals; CALIPER; children; adolescents
Tako|e }e biti re~i o nedavno objavljenoj bazi podataka o ref-
erentnim intervalima CALIPER (www.caliperdatabase.com)
koja treba da pru`i sveobuhvatne pedijatrijske referentne
intervale specifi~ne za uzrast i pol za ve}i broj biohemijskih
markera, zasnovane na velikom i raznolikom skupu zdrave
dece. Baza podataka CALIPER zasniva se na multietni~koj
populaciji, zahvaljuju}i ~emu se istra`uje i uticaj etni~ke pri-
padnosti na laboratorijske referentne intervale. Ova baza
podataka pokazala se globalno korisnom, zbog ~ega je usva-
jaju bolni~ke laboratorije {irom sveta.
Klju~ne re~i: biohemijski markeri, pedijatrija, referentni
intervali, CALIPER, deca, adolescenti
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J Med Biochem 2015; 34 (1) 25
Gap Analysis of Various Biomarkers
Several studies exemplified the need for up-to-
date pediatric reference intervals forbiomarkers asso-
ciated with cardiovascular, endocrine, and metabolic
diseases systems (3–6). Although pediatric reference
intervals have previously been calculated for various
biomarkers, many of these were performed prior to
the vasttechnological advancements in recent years.
In addition,most published reference intervals have
been determined in hospitalized inpatients or clinic
outpatients, and in many cases appropriate partition-
ing for key covariates including age, gender, and eth-
nicity have not been made. These deficiencies in
appropriate reference intervals pose a serious risk to
pediatric care, potentially subjecting infants to further
blood collection, pain, anxiety, infection risk, lengthi-
er hospital stays, and unpleasant or invasive diagnos-
tic procedures. The potential for incorrect or delayed
diagnosis and the administration of inappropriate
treatment is unacceptable. Thus, determining age-
and gender-specific pediatric reference intervals is
essential to patient safety (1).
Reference Interval Methodology
Procedures for establishing reference intervals
have been recommended by the CLSI and by the
RIDL (Reference Intervals and Decision Limits)
Committee of the International Federation of Clinical
Chemistry and Laboratory Medicine (IFCC). The most
recent updated guideline, entitled Defining, Estab -
lishing, and Verifying Reference Intervals in the
Clinical Laboratory, Approved Guideline–Third Edition,
Version C28-A3, includes protocols to determine ref-
erence intervals for new or existing analytes, and to
validate existing reference intervals developed in a
different laboratory (1). These guidelines clearly indi-
cate that reference individuals must be recruited from
a healthy population according to a pre-described
definition of healthy. Non-healthy individuals must be
clearly distinguishable and recruitment criteria should
be derived from known sources of biological variation
for the analyte. An a priori approach is also advised to
recruit healthy reference individuals. Indirect sam-
pling may be employed when collecting sufficient
numbers of reference samples is difficult, however,
the CLSI guideline does not generally endorse the sta-
tistical analysis of data derived from a previously sam-
pled population (indirect sampling) (1). Question -
naires must be filled out by reference individuals with
informed consent and, noting that the intention of the
samples is not for medical investigation, ethical
approval for the study must be obtained. A minimum
of 120 samples must be collected for all analytes in
order to accurately calculate a 95% confidence inter-
val. Sourcing a large number of healthy children can
be difficult and requires parental permission.
Potential sources include a campaign in elementary
and secondary schools (with the school board’s sup-
port), and leftover specimens from routine testing of
healthy newborns. Key challenges associated with the
collection of samples from children include small
sample volumes, higher costs associated with the
inability to analyze a large number of analytes from
small samples, and difficulty collecting high volumes
of samples for the many required age partitions.
The CALIPER Initiative to Close the
Gaps in Pediatric Reference Intervals
The Canadian Laboratory Initiative on Pediatric
Reference Intervals (CALIPER) project is a collabora-
tive research initiative spearheaded by The Hospital
for Sick Children in Toronto, Canada in collaboration
with several pediatric clinical laboratories across
Canada. The overall objective is to develop and main-
tain a comprehensive database of reference intervals
for laboratory tests that span the pediatric age range
(birth to 18 years), and include age-, gender-, and
ethnicity-specific partitions. A comprehensive menu
of both traditional and emerging biomarkers have
been considered in developing the database.
CALIPER Pilot Studies
In the preliminary phases of the CALIPER proj-
ect, 14 chemistry and 15 immunoassay analytes were
analyzed using the Abbott ARCHITECT ci8200 sys-
tem (7). Subsequently, an additional14 chemistry
analyteswere tested using the sameplatform (8).
Together, a total of 2809 serum samples from both
studies were collected. Metabolically healthy pediatric
outpatients from five age groups: 0–12 months, 1–5
years, 6–10 years, 11–14 years, and 15–20 years
were deemed appropriate to participate in this study
(7, 8). Following the CLSI/IFCC C28-P3 guidelines,
120 subjects were recruited for each age group, with
the exception of the birth to 1 year age range, as the
sample size proved difficult to achieve (7). After labo-
ratory analysis, the 2.5
th
and 97.5
th
percentiles were
established and the central 95
th
confidence interval
was created using the Robust method (7, 8). Age and
gender partitions were specified using the Harris-
Boyd method. Problems with time of collection for
some biomarkers, such asthyroid hormone, and lack
of sufficient sample size in some age groups, resulted
in weaknesses in the reference intervals for certain
analytes (7).Another pilot study was performed using
the Roche cobas® 6000 analyzer to further enhance
the applicability of CALIPER-derived pediatric refer-
ence intervals (9). In this study, 28 chemistry and
immunoassay analytesthat were previouslytested
using the Abbott ARCHITECT system, were retes -
tedusing 600 new healthy outpatients (9). Speci -
fically, for this study, reference intervals were deter-
mined after eliminating samples found flagged for
hemolysis, icterus, and lipemia, as well as outlier
exclusion using the CLSI recommended Dixon test for
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non-Gaussian distributed populations (9). Gender
was considered amore important variable than age,
and therefore gender partitions were established first,
if necessary (9). A summary of CALIPER pilot stud-
ies can be found in (Table I).
A Priori Studies using a CALIPER
Cohort of Healthy Community Children
Over the past five years, CALIPER has been
recruiting a cohort of healthy children and adoles-
cents across the community at schools, community
centers, daycares, and special community clinics. The
CALIPER promotional campaign has been very suc-
cessful and has resulted in recruitment of over 8500
children and adolescents into the study. Serum sam-
ples collected from this cohort have been used to
establish a CALIPER biobank. The biobank speci-
mens have subsequently been used to complete sev-
eral reference interval studies. Since 2012, CALIPER
has established pediatric reference intervals for over
60 analytes, including 40 biomarkers assessed using
the Abbott ARCHITECT c8000 system (10), 21 using
the Abbott ARCHITECT i2000SR (11, 12), and 8
using the Sciex 4000 QTRAP mass spectrometer (13)
(Table I). These reference intervals have been pub-
lished in scientific journals and can also be accessed
on the CALIPER website, as well as on a recently
developed mobile application.
In a study published in 2012, specimens collect-
ed from 2188 healthy participants (0 to 18 years of
age) from a multiethnic population were analyzed in
order to establish age- and sex- stratified reference
intervals for 40 serum biochemical markers using the
Abbott ARCHITECT c8000 analyzer (10). The estab-
lishment of normative values was in conformity with
CLSI C28-A3 statistical guidelines (1), and the
assessed analytes included 11 serum chemistry
assays, 12 enzymes, 3 lipids or lipoproteins, and 14
protein markers. The study also investigated differ-
ences between the 3 most prevalent ethnic groups in
Canada (Caucasians, East Asians, and South Asians).
Results confirmed that child growth and development
influence the concentration of analytes in healthy
children and adolescents, since all of the assays
(except lipase) required partitioning by age (10).
Partitioning was especially necessary within the 0 to 1
year age range, as levels of many analytes were
remarkably different within the first 14 days, com-
pared to the rest of the first year of life (10). For a
number of biomarkers, sex partitioning within some
age groups was also necessary. Although levels for
seven assays displayed ethnic differences, ethnicity
was not considered a major determinant of normative
values (10). However, further studies assessing the
impact of ethnicity on normal analyte concentrations
in the pediatric population are necessary, since the
number of samples obtained from ethnicities other
than Caucasians in this study was considered small.
Considering that the reference intervals for the
40 biochemical markers assessed in 2012 were only
applicable to hospitals/laboratories using the Abbott
ARCHITECT platform, reference intervals for many
analytes were transferred to other systems in order to
make the CALIPER database more broadly applicable
in Canada and worldwide (14) (Ta b l e I ). The reference
intervals were calculated for the Beckman Coulter
26 Adeli et al.: Advances in Pediatric Reference Intervals for Biochemical Markers
Table I Summary of CALIPER studies published to-date.
Number and type of Analytes Analytical platform Reference
Pilot studies
24 chemistry assays
15 immunoassays
Abbott ARCHITECT ci8200 Chan et al. (2009) (7)
14 chemistry assays Abbott ARCHITECT ci8200 Chan et al. (2008) (8)
11 chemistry assays
17 immunoassays
Roche cobas® 6000 Kulasingam et al. (2010) (9)
Full Reference Interval studies
(in Healthy Community
Children)
11 chemistry assays
12 enzymes
3 lipids/lipoproteins
14 protein markers
Abbott ARCHITECT ci8000 Colantonio et al. (2012) (10)
7 fertility hormones Abbott ARCHITECT ci2000 Konforte et al. (2013) (12)
14 immunoassays Abbott ARCHITECT ci2000 Bailey et al. (2013) (11)
8 steroid hormones
Sciex 4000 QTRAP mass
spectrometer
Kyriakopoulou et al. (2013)
(13)
Transference studies 16–27 chemistry assays,
enzymes, lipids/lipoproteins,
protein markers
Transference from Abbott
ARCHITECT ci8000 Assays
To Assays on Beckman
Coulter DxC800, Ortho Vitros
5600, Roche cobas® 6000,
Siemens Vista 1500
Estey et al. (2013) (14)
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DxC800, Ortho Vitros 5600, Roche Cobas 6000 and
Siemens Vista 1500 systems if deemed transferable.
For a reference interval to be considered transferable,
the relationship between values obtained using the
Abbott and other systems must have met some
required statistical criteria and assumptions. For exam-
ple, only normative values for analytes that displayed
proper correlation between the systems (R
2
value
greater than 0.7) were transferred. In addition, the
residuals distribution was normal and unbiased, and
residuals could not cluster or follow any identifiable
pattern when plotted against the corresponding
Abbott value (14). These assumptions were visually
assessed using normal Q-Q plots, Bland Altman plots,
and residual versus fit plots. After the new reference
intervals were determined, verification of the reference
interval was necessary. In order for a reference interval
to be considered valid, at least 80 to 90% of the levels
obtained from reference individuals (approximately
100 healthy CALIPER participants) should fall within
the lower and upper limits. In this study, many of the
reference intervals previously determined using Abbott
were transferable to the other systems (14). Carbon
dioxide and magnesium were exceptions, since Abbott
reference intervals for these assays were not transfer-
able to any of the other systems due to poor correla-
tion. Also, normative values for phosphate were not
transferable to the Beckman Coulter assay (however, it
could be transferred to all of the other systems exam-
ined) (14). In spite of the strong correlation, Gamma-
Glutamyl Transferase (GGT) reference intervals were
also deemed non-transferable because the assump-
tions in the three graphs assessed were not followed.
A similar situation occurred with C-reactive protein,
but only for the Roche Cobas system (therefore, the
reference interval could be transferred to the other
systems) (14). The other analytes were considered
transferable for all of the four systems (14). In brief,
transference from Abbott to the four other major clin-
ical chemistry platforms broadens the scope of the
CALIPER database and allows more children to bene-
fit from the establishment of accurate sex and age par-
titioned pediatric reference intervals. It is important to
note, however, that the comparisons performed
between the Abbott and Beckman assays make no
assumption as to assay accuracy or which is more cor-
rect/accurate, and the differences are largely due to
differences in calibration of assays between the two
platforms.
The project also established pediatric reference
intervals for hormones of the hypothalamus-pituitary-
gonadal axis (fertility hormones) (12) and for steroid
hormones (13). In a study published in 2013, age-,
sex- and Tanner stage-specific normative values for
seven fertility hormones were established on the
Abbott ARCHITECT i2000SR system: estradiol,
testosterone (second generation), progesterone, sex
hormone–binding globulin (SHBG), prolactin, follicle-
stimulating hormone (FSH), and luteinizing hormone
(LH) (12).Complex changes in analyte concentration
according to age were observed for all of the assessed
hormones. There were also statistically significant gen-
der differences for five out of the seven analytes (12).
Partitioning within the first year of life was also obser -
ved for these assays, which further demonstrates the
importance of establishing adequate age partitions for
pediatric reference intervals. Pubertal development
was assessed by self-reported Tanner stage for a sub-
set of participants from 9 to 15 years of age, for whom
data was available (12). All of the assessed fertility hor-
mones displayed Tanner-stage specific differences, and
the patterns were different when comparing boys to
girls. There were modest differences among ethnic
groups for FSH and SHBG, but no differences were
observed for other analytes (12). However, due to the
low number of participants from some ethnicities, eth-
nicity-specific reference intervals for some analytes
could not be calculated. This is a limitation that the
project is currently trying to surpass by recruiting par-
ticipants from specific ethnicities (East Asian and South
Asian) to better represent the Canadian population.
In another study also published in 2013,
referen ce values for 8 steroid hormones (cortisol, cor-
ticosterone, 11-deoxycortisol, androstenedione, 21-
hy dro xyprogesterone, testosterone, 17-hydroxyprog-
esterone, and progesterone) were established after
the development of a mass spectrometric method for
measuring serum steroids (13). The developed assay
was an accurate and sensitive method for simultane-
ously measuring these eight steroid hormones using
the Sciex 4000 QTRAP mass spectrometer and a
small volume of serum (200 mL). This methodology is
important because the ability to accurately and simul-
taneously measure steroid hormones in a small
aliquot of serum is essential in a pediatric setting/fa -
cility due to the challenges and risks associated with
obtaining a large volume of blood from children.
CALIPER samples from 337 participants from 0 to 18
years of age were used to determine the normative
values for the 8 steroid hormones, and age- and sex-
specific reference intervals were established (13). In
the case of cortisol, one extra variable was taken into
account: the time of day when the sample was col-
lected, and therefore reference intervals for this ana-
lyte were set for each period of the day. Age-partition-
ing, especially from birth to 14 days, was necessary
for all analytes (13), which is in accordance with find-
ings from a previous CALIPER study (10). The pattern
for testosterone was especially interesting and worth
noting since it illustrates the importance of partition-
ing pediatric reference intervals by age and sex: in
newborns, serum concentration was significantly
higher in boys than girls. From 1 to 13 years, the lev-
els were very low and similar for both sexes. After 13
years, concentrations increased dramatically (20-fold)
in males, peaking at 15 years. On the other hand, lev-
J Med Biochem 2015; 34 (1) 27
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els were relatively constant for females from 13 to 16
years, after which time there was a modest increase
(but 10-fold less than that observed in males).
Also in 2013, sex- and age- partitioned refer-
ence intervals were determined for an additional 14
analytes using the Abbott ARCHITECT i2000 system.
These included a-fetoprotein (AFP), cobalamin (vita-
min B12), folate, total homocysteine, ferritin, cortisol,
troponin I, 25(OH)-vitamin D, intact parathyroid hor-
mone, thyroid-stimulating hormone (TSH), total thy-
roxine (T4), total triiodothyronine (T3), free T4, and
free T3 (11). Samples from 1482 healthy children
were assessed in this study. Age- and sex- partitioning
was necessary for all of the analytes (11). The effect
of ethnicity was also assessed, and statistically signifi-
cant differences in the levels of total T4, total T3, free
T4, cobalamin, ferritin, intact parathyroid hormone,
and 25(OH)-vitamin D across ethnic groups were
observed. In the case of total T3 and free T4, the
effect was small, but total T4 and ferritin were consid-
erably higher in East Asians and lower in South Asians
compared to Caucasians. Cobalamin was noticeably
higher in East Asians, 25(OH)-vitamin D was lower in
East Asians, and intact parathyroid hormone was
higher in South Asians (11).
CALIPER Sub-studies
Parallel to its core studies, which establish and
transfer reference intervals, the CALIPER project has
also published a number of other articles, which
investigate relevant questions and challenges involved
with establishing normative values for a pediatric pop-
ulation.
For example, considering the high costs and
intrinsic challenges associated with obtaining blood
from healthy children in their communities (schools,
day care centers, festivals etc), we compared CALI -
PER reference intervals established using the commu-
nity-based approach with normative intervals
obtained using a modified version of Hoffman’s orig-
inal method (15). The Hoffman method uses hospital
in- and out- patient blood samples (which are easier
and more convenient to obtain) to determine norma-
tive values. Reference intervals for 13 analytes were
determined using the Hoffman method on the Vitros
5600 system (15). The obtained reference intervals
were compared to the corresponding intervals deter-
mined by CALIPER (after transferring the reference
intervals established in 2012 using the Abbott
ARCHI TECT system to the Vitros 5600 system). The
same sex and age partitions established in the previ-
ous CALIPER study were used. In order to compare
the two methods, the 90% confidence interval corre-
sponding to each lower or upper limit previously iden-
tified by CALIPER was used. None of Hoffman limits
fell within the corresponding 90% confidence inter-
vals defined by the CALIPER methodology (15). This
result suggests that using the Hoffman approach
might not be a suitable method for determining pedi-
atric reference intervals, at least not in a tertiary care
hospital such as the Hospital for Sick Children (where
the patient samples were collected), since this
method requires that the majority of assessed individ-
uals be healthy children, which may not be the reali-
ty at an acute care hospital center. The study conclud-
ed that a more feasible approach may be to include
only out-patient data, or use samples from a commu-
nity hospital/health center (15).
Another important CALIPER study performed in
parallel with the main project explored the between-
and within- individual biological variation in levels of
38 analytes in a 1-day study involving 29 healthy par-
ticipants from 4 to 18 years of age (16). In the clinical
setting, it is essential to consider the effect of biologi-
cal variation on analyte levels in order to properly inter-
pret test results. Few studies have investigated the
complex and dynamic biological variation present
within pediatric samples, since most previous studies
have focused on adult populations.Therefore, this
CALIPER study was relevant because it established
pediatric reference change values, which describe the
variation that must be observed before a change in
patient values should be considered clinically im -
portant. Reference change values might be more
appropriate to use than reference intervals in some sit-
uations, particularly when there is significant within-
sub ject variation in the levels of the analyte. In addi-
tion, the effect of time of sample collection on analyte
concentration was also investigated (blood was drawn
from each participant 4 times throughout the day).
Acknowledgement of biological variation is very im -
portant in cases where analytes display cyclic changes
throughout the day. The majority of analytes assessed
in this study displayed significant individuality. In gen-
eral, the within- and between- individual variation was
consistent with data reported for the adult population,
and only four analytes (C-reactive protein (CRP), GGT,
ceruloplasmin, and glucose) showed marked differ-
ences in both within- and between- individual variation
in the pediatric population when compared to adults
(16). Nevertheless, there were essential lessons that
illustrate the importance of specifically studying pedi-
atric samples. For example, the biological variation of
glucose was more pronounced in children than in
adults (16). This is likely due to the fact that children
tend to have lower glucose levels when fasting (17).
The biological variation of iron in children was also
interesting: there was less within-individual variation
than in adult populations (16), which is in accordance
with the observation that infants and young children
lack diurnal variation of serum iron due to the absence
of a sustained period of sleep (18). On the other hand,
there was higher between-individual variation (16),
which may be explained by the fact that iron deficien-
cy is common in pediatric populations, especially in
girls from 12 to 19 years of age (19).
28 Adeli et al.: Advances in Pediatric Reference Intervals for Biochemical Markers
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Current CALIPER Studies
The CALIPER project has been continuing to
establish accurate and accessible pediatric reference
intervals by analyzing common biomarkers across var-
ious platforms, as demonstrated by a number of stud-
ies recently presented at the Canadian Society of
Clinical Chemists Annual Meeting in Prince Edward
Island (June 2014), and the American Association of
Clinical Chemistry in Chicago, IL (July 2014).
Usingthe Abbott ARCHITECT ci4100 platform, 14
special chemistry and endocrine markers (including
free testosterone indexes) were tested using 400-700
samples from healthy children aged birth to 18 years.
To date, there has been a lack of pediatric refer-
ence intervals for biochemical markers that are key
for prognosis and diagnosis of various childhood can-
cers. The CALIPER project aimed to establish new
pediatric reference value distributions for tumor mark-
ers and determine the effects of important covariates
like age, gender, and ethnic background. Using the
Abbott ARCHITECT ci4100 system, 11 analytes were
tested illustrating significant fluctuations in circulating
levels for 10 of the 11 cancer biomarkers.
Additionally, testing for 29 immunoassays has
been recently performed on the Beckman Coulter DxI
Immunoassay system to ensure broader application of
the CALIPER database. The CLSI EP28-A3c guide-
lines were followed when statistically analyzing all
samples. It was noted that concentrations for some
assays were higher on the Beckman Coulter platform
compared to the Abbott platform necessitating instru-
ment-specific reference intervals.
To ensure a wider application of the CALIPER
database, reference intervals for an additional 34 ana-
lytes were studied for potential transference from the
Abbott ARCHITECT to the Beckman Coulter Synchron
Unicel DxC800 platform. The methodology was essen-
tially the same as described for the previous trans -
ference study, and included an assessment of the qual-
ity of the correlation between the two systems for each
analyte, establishment of the transference equation,
assessment of the distribution of residuals (which must
be unbiased and normal), and verification of the trans-
ferred reference interval. Reference intervals for most
Beckman assays were transferable and considered ver-
ified following analysis of healthy CALIPER specimens.
Concluding Remarks & Future
Endeavors
Since the beginning of the project, CALIPER has
taken huge steps toward providing up-to-date and
comprehensive pediatric reference intervals that are
accessible to health institutions in Canada and glob-
ally. The CALIPER outreach and recruitment efforts
have led to collection of over 8500 blood specimens
from healthy community children and adolescents,
which has enabled the creation of a CALIPER
biobank where specimens are stored at –80 °C for
later use. Over 70 common chemical biomarkers
have been studied using the Abbott platforms and
many of these have been successfully transferred to
the four other analytical platforms used in clinical
laboratories including Beckman Coulter DxC, Ortho
Vitros 5,1, Roche Cobas, and Siemens Vista.
The long-term objective of the CALIPER pro-
gram is to ensure that every child presenting at any
clinic or hospital in Canada (and around the world)
benefits from the CALIPER program and the availabil-
ity of up-to-date and accurate pediatric reference
intervals. In the short-term, current and future proj-
ects are focused on establishing a comprehensive
pediatric reference interval database on all major clin-
ical chemistry and immunoassay platforms. This is
being achieved by both transference studies and de
novo reference interval studies of biochemical and
immunochemical assays.
A key objective of the CALIPER program has
been to ensure extensive knowledge translation
through peer-reviewed publications, development of
an online and easy-to-use database listing reference
intervals for over 70 analytes, accessible through the
CALIPER website (www.caliperdatabase.ca), and
most recently, a smartphone application for Apple
and Android smartphones and tablets. Once down-
loaded, the new CALIPER app allows any physician or
laboratorian worldwide to access the CALIPER data-
base without the need for a network connection.
Efforts are currently underway to continue full knowl-
edge translation through new peer-reviewed publica-
tions, additions to the online database, and updates
to the CALIPER application available through the
Apple Store and Google Play.
Conflict of interest statement
The authors stated that they have no conflicts of
interest regarding the publication of this article.
J Med Biochem 2015; 34 (1) 29
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Received: July 1, 2014
Accepted: July 2, 2014
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