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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



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 ( 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
Kimiya Karbasy
, Petra Ariadne
, Stephanie Gaglione
, Michelle Nieuwesteeg
, Khosrow Adeli
CALIPER program, Department of Pediatric Laboratory Medicine, The Hospital for Sick Children,
University of Toronto, Toronto, Ontario, Canada
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
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.
Download Date | 9/25/15 4:32 AM
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-
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 ( 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 (
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
Download Date | 9/25/15 4:32 AM
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
and 97.5
percentiles were
established and the central 95
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
Download Date | 9/25/15 4:32 AM
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
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
Kyriakopoulou et al. (2013)
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
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
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
Download Date | 9/25/15 4:32 AM
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-
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
Download Date | 9/25/15 4:32 AM
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
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 (, 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
Download Date | 9/25/15 4:32 AM
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30 Adeli et al.: Advances in Pediatric Reference Intervals for Biochemical Markers
Received: July 1, 2014
Accepted: July 2, 2014
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Full-text available
Bone turnover markers (BTMs) have been developed many years ago to study, in combination with imaging techniques, bone remodeling in adults. In children and adolescents, bone metabolism differs from adults since it implies both growth and bone remodeling, suggesting an age- and gender-dependent BTM concentration. Therefore, specific studies have evaluated BTMs in not only physiological but also pathological conditions. However, in pediatrics, the use of BTMs in clinical practice is still limited due to these many children-related specificities. This review will discuss about physiological levels of BTMs as well as their modifications under pathological conditions in children and adolescents. A focus is also given on analytical and clinical challenges that restrain BTM usefulness in pediatrics.
Introduction: Patients with suspected adrenal insufficiency undergo screening with a serum morning cortisol level and confirmatory testing with an adrenocorticotropic hormone (ACTH) stimulation test. However, much of the data collected to determine appropriate values for morning cortisol levels are derived from adult populations and may not accurately represent pediatric physiology. The purpose of this study was to evaluate the mean morning cortisol level in the pediatric population based on pubertal status and sex in order to better understand such influences on laboratory evaluation of adrenal insufficiency. Methods: A retrospective chart review was conducted using electronic medical records of patients seen at Children's Mercy Kansas City from 11/01/2007 to 11/01/2017. Patients between 2 and 18 years of age who had pubertal staging assessed by a pediatric endocrinologist and confirmed adrenal sufficiency by high-dose ACTH stimulation testing were included. Two-sample Wilcoxon rank sum (Mann-Whitney) tests or t tests were used to compare morning cortisol levels between females and males - both independent of Tanner stage and by Tanner stage. Multivariable regression models were used to evaluate associations among covariates on two outcomes: morning cortisol levels and peak cortisol values with ACTH stimulation. Results: Morning cortisol levels were greater in females than males independent of Tanner staging (p = 0.0054) and also in Tanner stage 1 (p = 0.0042). No differences in mean morning cortisol levels between Tanner stage 2-5 females and males were found (p = 0.4652). Morning cortisol levels were not significantly different between Tanner 1 patients and Tanner 2-5 patients independent of sex (p = 0.0575). Sex was predictive of serum morning cortisol levels (p = 0.015), and morning cortisol levels were predictive of peak cortisol levels during ACTH stimulation testing (p < 0.001). Conclusions: These data suggest that different normative cortisol values may need to be established for pediatric females and males, and by pubertal status. Larger prospective studies are needed to evaluate the role of sex and pubertal status in identifying adrenal insufficiency in the -pediatric population.
Full-text available
Clinical interpretation of the test results for cortisol based on continuous reference intervals with appropriate partitions improves pediatric diagnosis; however, these values are available only for Caucasians. To develop the pediatric reference intervals for Chinese population, we examined the serum cortisol levels in 1,143 healthy Chinese children aged 4-18 years (566 boys and 577 girls), using an IMMULITE 2000 Immunoassay System (Siemens Healthcare GmbH). Phlebotomy was performed at 7-9 a.m. for 284 boys and 287 girls and at 1-3 p.m. for the others. They were divided into four age groups according to the Clinical and Laboratory Standards Institute guideline EP28-A3c, with the last group further stratified according to sampling time. Separate reference intervals of 49.6-323.7, 70.9-395.3, and 90.1-448.7 nmol/L were established for children aged 4-8, 9-12, and 13-15 years, respectively. Further, reference intervals of 118.2-464.7 and 71.4-446.7 nmol/L were established for morning and afternoon cortisol levels, respectively, in children aged 16-18 years. Further studies are necessary to transfer and validate these reference intervals in other analytical systems and pediatric populations, and to allow for broader applications.
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The aim of this study was to explore oxidative stress status, especially the enzyme myeloperoxidase in children with end-stage renal disease. Also, we investigated possible associations between the atherogenic index of plasma and these parameters. Lipid status parameters, oxidative stress status parameters, and myeloperoxidase concentration were measured in the sera of 20 children in the last stage of chronic renal disease (ESRD) and 35 healthy children of matching age and sex. The Atherogenic Index of Plasma (AIP) was calculated according to the appropriate equation. We did not find any significant differences in myeloperoxidase concentrations between the investigated groups (p=0.394). Oxidative stress parameters were, however, significantly higher in the patient group (p<0.001), as well as the atherogenic index of plasma (p<0.001). Myeloperoxidase concentration and advanced oxidation protein product (AOPP) concentration were independently associated with increased AIP in the patient group (p<0.05). Changes in AIP in children with ERSD are associated with the oxidative stress status and myeloperoxidase concentration.
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Background: In this study, we investigated the relationship of adiponectin with bone marker changes in Egyptian children and adolescents with T1DM and the effect of disease duration on these markers, as well as the possible correlations between adiponectin and bone markers in these patients. Methods: Sixty Egyptian children and adolescent patients with T1DM were studied. Serum adiponectin and collagen breakdown products (cross-linked C-terminal telopeptide of collagen type l »CTX«) were measured and compared to the results of 20 age-matched healthy controls. Results: After adjustment for age, BMI, Tanner stage and gender; (total) adiponectin was significantly higher in all T1DM patients. Serum level of CTX and 25(OH)D showed a marked decrease in diabetics with disease duration > 5 years. Serum level of (total) calcium and inorganic phosphorus (Pi) did not show significant difference from control. CTX was inversely correlated to FBG and T1DM duration. Pi was inversely, while 25(OH)D was directly correlated to FBG. Total calcium showed an inverse correlation with HbA1c. FBG, TC, TAG, LDL-C were independent predictors of CTX in T1DM. Conclusions: Adiponectin showed no correlation with either CTX or bone homeostatic indices. FBG, TC, TAG, LDL-C were independent predictors of CTX in T1DM. We recommend further investigation of adiponectin isoforms in a population-based study, to establish a good age- and sex-related reference.
Full-text available
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 monitoring 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 partitions 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 characteristics of 14 assays (37%) were distinct from adult values found in the Westgard database on biological variation. Biological variation characteristics were established for 2 previously unreported analytes, unconjugated bilirubin and soluble transferrin receptor. Conclusions: This study is the first to examine biological variation and to establish analytical quality specifications on the basis of biological variation for common assays in a pediatric population. These results provide insight into pediatric physiology, are of use for reference change value calculations, clarify the appropriateness of reference interval use, and aid in the development of quality management strategies specific to pediatric laboratories.
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Background: Pediatric endocrinopathies are commonly diagnosed and monitored by measuring hormones of the hypothalamic-pituitary-gonadal axis. Because growth and development can markedly influence normal circulating concentrations of fertility hormones, accurate reference intervals established on the basis of a healthy, nonhospitalized pediatric population and that reflect age-, gender-, and pubertal stage-specific changes are essential for test result interpretation. Methods: Healthy children and adolescents (n = 1234) were recruited from a multiethnic population as part of the CALIPER study. After written informed parental consent was obtained, participants filled out a questionnaire including demographic and pubertal development information (assessed by self-reported Tanner stage) and provided a blood sample. We measured 7 fertility hormones including estradiol, testosterone (second generation), progesterone, sex hormone-binding globulin, prolactin, follicle-stimulating hormone, and luteinizing hormone by use of the Abbott Architect i2000 analyzer. We then used these data to calculate age-, gender-, and Tanner stage-specific reference intervals according to Clinical Laboratory Standards Institute C28-A3 guidelines. Results: We observed a complex pattern of change in each analyte concentration from the neonatal period to adolescence. Consequently, many age and sex partitions were required to cover the changes in most fertility hormones over this period. An exception to this was prolactin, for which no sex partition and only 3 age partitions were necessary. Conclusions: This comprehensive database of pediatric reference intervals for fertility hormones will be of global benefit and should lead to improved diagnosis of pediatric endocrinopathies. The new database will need to be validated in local populations and for other immunoassay platforms as recommended by the Clinical Laboratory Standards Institute.
Full-text available
Background: Reference intervals are indispensable in evaluating laboratory test results; however, appropriately partitioned pediatric reference values are not readily available. The Canadian Laboratory Initiative for Pediatric Reference Intervals (CALIPER) program is aimed at establishing the influence of age, sex, ethnicity, and body mass index on biochemical markers and developing a comprehensive database of pediatric reference intervals using an a posteriori approach. Methods: A total of 1482 samples were collected from ethnically diverse healthy children ages 2 days to 18 years and analyzed on the Abbott ARCHITECT i2000. Following the CLSI C28-A3 guidelines, age- and sex-specific partitioning was determined for each analyte. Nonparametric and robust methods were used to establish the 2.5th and 97.5th percentiles for the reference intervals as well as the 90% CIs. Results: New pediatric reference intervals were generated for 14 biomarkers, including α-fetoprotein, cobalamin (vitamin B12), folate, homocysteine, ferritin, cortisol, troponin I, 25(OH)-vitamin D [25(OH)D], intact parathyroid hormone (iPTH), thyroid-stimulating hormone, total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine. The influence of ethnicity on reference values was also examined, and statistically significant differences were found between ethnic groups for FT4, TT3, TT4, cobalamin, ferritin, iPTH, and 25(OH)D. Conclusions: This study establishes comprehensive pediatric reference intervals for several common endocrine and immunochemical biomarkers obtained in a large cohort of healthy children. The new database will be of global benefit, ensuring appropriate interpretation of pediatric disease biomarkers, but will need further validation for specific immunoassay platforms and in local populations as recommended by the CLSI.
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Iron deficiency is the most common nutritional deficiency in the world, but little is known about the iron status of people in Canada, where the last estimates are from 1970-1972. The data are from cycle 2 (2009 to 2011) of the Canadian Health Measures Survey, which collected blood samples from a nationally representative sample of Canadians aged 3 to 79. Descriptive statistics (percentages, arithmetic means, geometric means) were used to estimate hemoglobin and serum ferritin concentrations, and other markers of iron status. Analyses were performed by age/sex group, household income, self-perceived health, diet, and use of iron supplements. World Health Organization reference values (2001) were used to estimate the prevalence of iron sufficiency and anemia. The overall prevalence of anemia was low in the 2009-to-2011 period--97% of Canadians had sufficient hemoglobin levels. Generally, hemoglobin concentration increased compared with 1970-1972; however, at ages 65 to 79, rates of anemia were higher than in 1970-1972. Depleted iron stores were found in 13% of females aged 12 to 19 and 9% of females aged 20 to 49. Lower household income was associated with a lower prevalence of hemoglobin sufficiency, but was not related to lower serum ferritin sufficiency. Self-perceived health and diet were not significantly associated with hemoglobin and serum ferritin levels. The lack of a relationship between iron status and diet may be attributable to the use of questions about food consumption frequency that were not specifically designed to estimate dietary iron intake. Factors other than iron intake might have contributed to the increase in the prevalence of anemia among seniors.
Full-text available
Objectives: To develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. Design and methods: The samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18years were analyzed. The concentrations were measured by using an LC-MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. Results: Reference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. Conclusions: We report a sensitive, accurate and relatively fast LC-MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.
Full-text available
Objectives: A comprehensive set of age- and gender-specific pediatric reference intervals is essential for accurate interpretation of laboratory tests in a pediatric setting. Design and methods: 1459 serum/plasma from children attending select outpatient clinics and deemed to be metabolically stable, were collected from five age groups; 0-12 months, 1-5 years, 6-10 years, 11-14 years and 15-20 years. Samples were analyzed for 24 chemistries and 15 immunoassays on ARCHITECT ci8200. Results: Reference intervals were established according to CLSI/IFCC C28-P3 guidelines by the Robust statistical method. The ranges reflect the central 95% confidence intervals for the population tested. Age and gender were partitioned using the Harris-Boyd method. Conclusions: While these intervals are ci8200 method specific, they not only provide robust intervals for users of this system but are also useful for any laboratory requiring pediatric intervals if they can be shown to be transferable and if validated for the local patient population.
To compare pediatric reference intervals calculated using hospital-based patient data with those calculated using samples collected from healthy children in the community as part of the CALIPER study. Hospital-based data for 13 analytes (calcium, phosphate, iron, ALP, cholesterol, triglycerides, creatinine, direct bilirubin, total bilirubin, ALT, AST, albumin and magnesium), measured on the Vitros 5600, collected between 2007 and 2011 were obtained. The data for each analyte were partitioned by age and gender as previously defined by the CALIPER study. Outliers in each partition were removed using the Tukey method. The cumulative distribution function (cdf) was then determined for each analyte value following which, the inverse cdf values of a standard Gaussian distribution were calculated. The analyte values were plotted against the inverse cdf of the standard Gaussian distribution. Piece-wise regression determined the linear portion of the resulting graph using the statistical software R. Linear regression determined an equation for the linear portion in each partition and reference intervals were calculated by extrapolating to identify the 2.5th and 97.5th centiles in each partition based on the inverse cdf values (which would correspond to the values -1.96 and 1.96 of the Gaussian distribution). Using the 90% confidence intervals for the reference intervals defined by CALIPER and the reference change value (RCV) as criteria, these calculated reference intervals were compared to those reported previously by CALIPER. Reference samples were also measured on the Vitros 5600 analyzer in an attempt to validate the calculated reference intervals. In general, the reference intervals calculated from hospital-based data were generally wider than those calculated by CALIPER. None of the reference intervals calculated using the Hoffmann approach fell completely within the 90% confidence intervals calculated by CALIPER. These results suggest that calculating pediatric reference intervals from hospital-based data may be useful, as a guide, in some cases but will likely not replace the need to establish reference intervals in healthy pediatric populations.
Objectives: The CALIPER program recently established a comprehensive database of age- and sex-stratified pediatric reference intervals for 40 biochemical markers. However, this database was only directly applicable for Abbott ARCHITECT assays. We therefore sought to expand the scope of this database to biochemical assays from other major manufacturers, allowing for a much wider application of the CALIPER database. Design and methods: Based on CLSI C28-A3 and EP9-A2 guidelines, CALIPER reference intervals were transferred (using specific statistical criteria) to assays performed on four other commonly used clinical chemistry platforms including Beckman Coulter DxC800, Ortho Vitros 5600, Roche Cobas 6000, and Siemens Vista 1500. The resulting reference intervals were subjected to a thorough validation using 100 reference specimens (healthy community children and adolescents) from the CALIPER bio-bank, and all testing centers participated in an external quality assessment (EQA) evaluation. Results: In general, the transferred pediatric reference intervals were similar to those established in our previous study. However, assay-specific differences in reference limits were observed for many analytes, and in some instances were considerable. The results of the EQA evaluation generally mimicked the similarities and differences in reference limits among the five manufacturers' assays. In addition, the majority of transferred reference intervals were validated through the analysis of CALIPER reference samples. Conclusions: This study greatly extends the utility of the CALIPER reference interval database which is now directly applicable for assays performed on five major analytical platforms in clinical use, and should permit the worldwide application of CALIPER pediatric reference intervals.
The diurnal variation of serum iron found in healthy adults was evaluated in two groups of children of different ages. No variation was found in 12 children aged 2 weeks to 20 months. In contrast, there was a significant variation of the per cent relative change of serum iron from a. m. to p. m. for a group of 12 children from 3 to 14 years of age. These results support the evidence for the late appearance of a biological “clock” in children.