Table Of ContentPediatric Reference Intervals
Eight Edition
Edward C.C. Wong, MD, FCAP
Carlo Brugnara, MD
Joely A. Straseski, PhD, DABCC
Mark D. Kellogg, PhD, MT(ASCP), DABCC
Khosrow Adeli, PhD, FCACB, DABCC
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We dedicate this book to our children and grandchildren.
We hope that children worldwide will benefit from this
eighth edition of Pediatric Reference Intervals
(formerly Pediatric Reference Ranges)
Foreword
Thisinvaluablevolumeonpediatriclaboratorymedicineisaveryimportantcontributionto
patient care, now made even more useful by the addition of a greatly expanded chemistry
section and more complete hematology and coagulation reference ranges. Every children’s
hospital, pediatric department, and clinical laboratory should have this well-designed and
easy-to-read manual close at hand. The editors are to be commended for their splendid
addition to the quality of laboratory diagnosis.
David G. Nathan, MD
DanaeFarber Cancer Institute
Boston, Massachusetts
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Preface
The need for pediatric reference intervals is quite evident, as children and adolescents
undergo a series of remarkable changes in growth, organ development, and sexual maturity
from the time they are born to the time they become adults. For example, neonates and
prematureinfantsstartoffwithimmaturehepatic,renal,andpulmonaryfunctionwhichcan
affect the way a wide variety of medications are metabolized. The rapid endocrine changes
evidentduringpubertyareatestamenttotheneedforwell-definedreferenceintervalsinthis
age group. Thus, without appropriate reference intervals, laboratory testing becomes an
inexact endeavor in futility.
fi
How Do We Approach the De nition of Normal Reference
Intervals?
In the ideal situation, samples would be collected directly from a large number of healthy
childrenofallagegroups,thelaboratorywouldtesteachsample,andthoseresultswouldbe
usedtodeterminestatisticallyrelevantreferenceintervalsfortheappropriateagepartitions.
However,weknowthisisnotaneasytask.Childrenareconsideredvulnerablesubjectsfroma
researchperspectiveand,therefore,havelimitationsonthetotalvolumeofbloodthatcanbe
safely collected from them. Strict protocols for consent/assent to participate in research
studies may be challenging to administer to children and require parent or guardian
involvement.Additionally,participantsneedtobecarefullyscreenedfordisordersthatmight
bias any measured results (1). The difficult nature of phlebotomy, particularly in younger
children, can also cause emotional stress on parents who might not want their child to
participate in a potentially painful procedure with no immediate benefit. Because of these
andotherpracticalchallenges,itisoftendifficulttoobtainadequatesamplevolumesfromat
least 120 healthy individuals to achieve a statistically accurate normative reference interval
for an age partition. That challenge is compounded by the need for multiple age and sex
partitions in growing and maturing children. Thus, relatively large studies with “healthy”
volunteers are particularly difficult to perform but provide reassurance in the statistical
determination of reference intervals that typically encompass the 2.5th to 97.5th percentiles
of the reference population data (1).
Statistical Approaches Used to Determine Reference
Intervals
Inlieuofthepreferredapproachofprospectivecollectionoflargesamplenumbersdescribed
above, several investigators have derived or used statistical methods on large databases of
existing laboratory results to retrospectively determine appropriate reference intervals. One
notable approach that has been included in previous editions of this book is the Hoffmann
approximation (2). This approach typically uses either Chauvenet’s or Dixon’s criteria for
removing outliers and involves plotting % cumulative frequency versus the laboratory
xxi
xxii Preface
value (or log of the value, if non-Gaussian distribution). Using this approach, one obtains
a straight linetypically within the central portion of theplotted curve. This straight line can
beextrapolatedtoprovidethe2.5thand97.5thpercentilesforthepopulationbeingstudied.
An example for total iron-binding capacity is shown in Fig. 1(3). This approach is simplistic
overallandhasparticularappealinpopulationswithlimiteddataavailablefromfreshsample
collections from volunteers, such as pediatrics. Disadvantages include the need to be very
selective in the population being considered since patients typically have testing performed
duetoaclinicalconcernandmaynotrepresenta“healthy”population.Otherdisadvantages
include the need to carefully select appropriate age interval(s) and the need for a very large
numberofdatapointsinordertoperformthesestatisticalcalculationsforreferenceinterval
derivation.There hasbeen a recentvalidation oftheHoffmann approximation using a large
database from a nationwide chain of clinical reference laboratories in the United States,
without exclusions or filtering (4).
A major criticism of the Hoffman approximation is the observation of narrower than
expected reference intervals (5). There have, therefore, been a number of statistical ap-
proaches that have attempted to improve upon the Hoffman approximation. These include
the Bhattacharya graphical method which involves the identification of Gaussian distribu-
tions within a distribution (6). Another is a computational (nongraphical) strategy using
maximum likelihood through the expectationemaximization algorithm (7). As reviewed by
Holmes and Buhr (5), however, all of these methods should be considered to be “indirect”
estimates of reference intervals in healthy children and should be verified using data from
such individuals.
Another method that has recently been developed is the use of continuous pediatric
reference curves for chemistry analytes (8). The advantages of this approach are that it
providesacontinuumofupperandlowerreferenceintervalsandavoidstheuseofreference
Figure1 Hoffmannplotfortotalironebindingcapacity.ReprintedfromFig.1fromRef.(3)withpermissionfrom
Elsevier.
Preface xxiii
Figure 2 Example of continuous vs. Noncontinuous reference intervals and comparison of continuous versus
partitionedreferenceintervalsforBUN/creationratiosfromages0to19years.Referencevalues,continuous
referenceinterval,andpartitionedreferenceintervalsarecodedineitherpink(females)orblue(males).
Partitionedreferenceintervalsforbothsexesareinblack.Shadedareasrepresent95%confidenceintervals.
ReprintedfromFig.1D(Ref.(9))withpermissionfromElsevier.
intervals that are arbitrarily delineated which may lead to misclassification close to age
“cutoff” points. Current laboratory information systems, however, will need future en-
hancements to incorporate these curves into routine laboratory reports (See Fig. 2) (9).
fi
Method-Speci c Differences
Anotherpotentialcomplicationtoconsiderwhendeterminingpediatricreferenceintervalsis
technology.Giventheplethoraofassaymethodsandinstrumentsavailableforpurchase,itis
extremely unlikely that every laboratory will use the same method or analyzer for measure-
mentofaparticularanalyte.Thecruxofthisconcernisthatthemajorityofanalytesarenot
standardized among methods, resulting in values that cannot be compared well between
laboratories.Agoodexampleisinstrumentationthatquantitatesalkalinephosphataselevels.
Recent proficiency testing data indicate mean values differed among commercial platforms
by as much as 300%.
Therefore, the method or commercial platform used to measure analytes used in
pediatric reference interval determinations should be carefully considered and noted before
intervaluseoradoptionbyalaboratory.Italsospeakstothecriticalneedformethod-specific
reference intervals.
Conclusion
Itisveryclearthatmuchworkandthoughtgoesintothedevelopmentofpediatricreference
intervals including careful consideration and screening of the population studied, the
developmental status of the population, the instrumentation used, and the statistical
xxiv Preface
methods employed. Future advances in laboratory information systems may allow for
reporting of continuous pediatric reference intervals to better provide accurate reference
intervalsforpatients.Referenceintervalsprovidecontexttomeasuredlaboratoryvaluesand
allowforthecomparisonbetweenhealthanddisease.Theirimportance,therefore,cannotbe
overstated.
References
1. AdeliK,HigginsV,TrajcevskiK,White-AlHabeebN.TheCanadianlaboratoryinitiativeonpediatric
referenceintervals:aCALIPERwhitepaper.CritRevClinLabSci2017;54:358e413.
2. HoffmannRG.Statisticsinthepracticeofmedicine.JAMA1963;185:864e873.
3. Soldin OP, Bierbower LH, Choi JJ, Choi JJ, Thompson-Hoffman S, Soldin SJ. Serum iron, ferritin,
transferrin, total iron binding capacity, hs-CRP, LDL cholesterol and magnesium in children; new
referenceintervals usingtheDadeDimensionClinicalChemistrySystem. ClinChimActa2004;342:
211e217.
4. Katayev A, Balciza C, Seccombe DW. Establishing reference intervals for clinical laboratory test re-
sults:isthereabetterway?AmJClinPathol2010;133:180e186.
5. Holmes DT, Buhr KA. Widespread Incorrect Implementation of the Hoffmann Method, the Correct
Approach,andModernAlternatives.AmJClinPathol2019;151:328e336.
6. Bhattacharya C. A simple method of resolution of a distribution into Gaussian components.
Biometrics1967;23:115e135.
7. RednerRA,WalkerHF.Mixturedensities,maximumlikelihoodandtheEMalgorithm.SIAMReview
1984;26:195e239.
8. HoqM,MatthewsS,KarlaftisV,BurgessJ,CowleyJ,DonathS,CarlinJ,YenT,IgnjatovicV,Monagle
P;HAPPIKidsStudyTeam.Referencevaluesfor30commonbiochemistryanalytesacross5different
analyzersinneonatesandchildren30daysto18yearsofage.ClinChem2019;65:1317-1326.
9. BohnMK,HigginsV,AdeliK.CALIPERpaediatricreferenceintervalsfortheureacreatinineratioin
healthychildren&adolescents.ClinBiochem2020;76:31e34.
Introduction
Welcome to the eighth edition of Pediatric Reference Intervals. This edition follows the
publication of its predecessor in 2011, which was the last edition published by AACC Press.
WearehappythatthistextbookhasfounditsnewhomeinAcademicPressandtheElsevier
family.Theeightheditionwitnessedaconsiderablerefreshingoftheeditorialteamwiththe
addition of three new editors:
Joely A. Straseski, Associate Professor (Clinical) in the Department of Pathology at the
University of Utah School of Medicine, and Section Chief of Clinical Chemistry, Medical
Director of Endocrine, and Codirector of Automated Core Laboratories at ARUP
Laboratories. Dr. Straseski is the director of the CHILDx (Children’s Health
Improvement through Laboratory Diagnostics) biorepository of samples from healthy
children.
Mark D. Kellogg, Director for Quality Programs and Associate Director of Chemistry in
the Department of Laboratory Medicine at Boston Children’s Hospital and Assistant
Professor of Pathology at Harvard Medical School.
Khosrow Adeli, Head of Clinical Biochemistry at The Hospital for Sick Children and
Professor at the Departments of Biochemistry, Physiology, and Laboratory Medicine
and Pathobiology at the University of Toronto in Toronto, Canada. He is the principal
investigator of the CALIPER (Canadian Laboratory Initiative on Pediatric Reference
Intervals) project (www.caliperproject.org; www.caliperdatabase.org).
This eighth edition of Pediatric Reference Intervals is a near-complete update of the
analytespublishedintheprioredition.Thisneweditionhasbeensignificantlystrengthened
inhematologywiththeinclusionofseveralnewdatasetsandexpandedincoagulationwith
the inclusion of several additional analytes. In choosing which publications and data to
include in the book, we prioritized published studies that included normal pediatric pop-
ulations and currently used instrumentation. Chemistry analytes have been expanded and
reviewed for clinical relevance. A scoring/metric system was developed to evaluate the
available literature based on the following criteria: age and type of publication, population
size,whetherethnicityandmethodplatformwerereported,andavailability/frequencyofuse
of the method or platform. Scores were used to determine which publications to include
when multiple were available. Where appropriate, the reader is directed to the original
publicationfordetailsthatexceedwhatiscoveredinthisbook.Similartotheseventhedition,
we have kept the same user-friendly format with each analyte having the same layout. We
hope this provides easy access to key information such as sample type, methodology used,
sourcereference,patientpopulation,andthestatisticalbasisonwhichthereferenceintervals
were derived.
xxv
