Critical Reviews in Clinical Laboratory Sciences

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Distinguishing reference intervals and clinical

decision limits – A review by the IFCC Committee
on Reference Intervals and Decision Limits

Yesim Ozarda, Ken Sikaris, Thomas Streichert, Joseph Macri & on behalf of
IFCC Committee on Reference intervals and Decision Limits (C-RIDL)

To cite this article: Yesim Ozarda, Ken Sikaris, Thomas Streichert, Joseph Macri & on
behalf of IFCC Committee on Reference intervals and Decision Limits (C-RIDL) (2018):
Distinguishing reference intervals and clinical decision limits – A review by the IFCC Committee
on Reference Intervals and Decision Limits, Critical Reviews in Clinical Laboratory Sciences, DOI:
10.1080/10408363.2018.1482256

To link to this article: https://doi.org/10.1080/10408363.2018.1482256

Published online: 26 Jul 2018.

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CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
https://doi.org/10.1080/10408363.2018.1482256

REVIEW ARTICLE

Distinguishing reference intervals and clinical decision limits – A review by

the IFCC Committee on Reference Intervals and Decision Limits
Yesim Ozardaa, Ken Sikarisb, Thomas Streichertc and Joseph Macrid; on behalf of IFCC Committee on
Reference intervals and Decision Limits (C-RIDL)
a
Department of Medical Biochemistry, Uludag University School of Medicine, Bursa, Turkey; bDepartment of Pathology, Melbourne
University, Parkville, Melbourne, Australia; cInstitute for Laboratory Medicine, Klinikum Bremen-Mitte, Bremen, Germany; dHamilton
General Hospital, Hamilton, Ontario, Canada

ABSTRACT ARTICLE HISTORY

Reference Intervals (RIs) and clinical decision limits (CDLs) are a vital part of the information sup- Received 28 February 2018
plied by laboratories to support the interpretation of numerical clinical pathology results. RIs Revised 3 May 2018
describe the typical distribution of results seen in a healthy reference population while CDLs are Accepted 25 May 2018
associated with a significantly higher risk of adverse clinical outcomes or are diagnostic for the Published online 23 July
2018
presence of a specific disease. However, as the two concepts are sometimes confused, there is a
need to clarify the differences between these terms and to ensure they are easily distinguished, KEYWORDS
especially because CDLs have a clinical association with specific diseases and risks, thereby imply- Reference intervals; clinical
ing that effective clinical interventions are available. It is important to note that, because popula- decision limits; C-RIDL;
tion-based RIs are derived from the range of values expected in a typical community population, clinical laboratories
laboratory results that fall outside a RI do not necessarily indicate a disease but rather that add-
itional medical follow-up and/or treatment may be warranted. In contrast, CDLs are associated
with a risk of specific adverse outcomes, and are commonly used to interpret laboratory test
results, including lipid parameters, glucose, hemoglobin A1c (HbA1c), and tumor markers, to
determine risk of disease, to diagnose or to treat. In recent years, the International Federation of
Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Reference Intervals and Decision
Limits (C-RIDL) has focused primarily on RIs and has performed multicenter studies to obtain
common RIs. However, the broader responsibility of the Committee, from its name, includes
“decision limits”. C-RIDL now aims to emphasize the importance of the correct use of both RIs
and CDLs and to encourage laboratories to specify the appropriate information to clinicians as
needed. This review discusses RIs and CDLs in detail, describes the similarities and the differences
between these two important tools in laboratory medicine, and clearly explains the processes
used to define them. C-RIDL encourages the involvement of laboratory professionals in the estab-
lishment of both RIs and CDLs.

Abbreviations: ADA: American Diabetes Association; ALT: alanine aminotransferase; Apo A: apoli-
poprotein A; Apo B: apolipoprotein B; BMI: body mass index; BNP: B-type natriuretic peptide;
CA125: carbohydrate antigen 125; CDLs: clinical decision limits; CLSI: Clinical Laboratory Standards
Institute; C-RIDL: Committee on Reference Intervals and Decision Limits; CRP: C-reactive protein;
cTn: cardiac troponin; CVD: cardiovascular disease; CVi: within-individual CV; DM: diabetes melli-
tus; EFLM: European Federation of Laboratory Medicine; ERSPC: The European Randomized Study
of Screening for Prostate Cancer; GDM: gestational diabetes mellitus; GGT: gamma glutamyl-trans-
ferase; HAPO: Hyperglycemia and Adverse Pregnancy Outcome; HbA1c: hemoglobin A1c; HDL-C:
high-density lipoprotein cholesterol; IADPSG: International Association of Diabetes and Pregnancy
Study Groups; IFCC: International Federation of Clinical Chemistry and Laboratory Medicine; ISO:
International Organization for Standardization; LDL-C: low-density lipoprotein cholesterol; non-
HDL-C: non-HDL-cholesterol; PLCO: Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial;
PSA: prostate specific antigen; RIs: reference intervals; ROC: receiver operator characteristic

Introduction
diseased?” and “Is the patient worsening?”. The value of
Every laboratory request has a purpose, with specific laboratory test results is in answering these questions
questions such as “Is the patient healthy?” “Is the so that effective interventions can be applied to
patient at risk of developing a disease?” “Is the patient improve clinical outcomes.

CONTACT Yesim Ozarda yesim@uludag.edu.tr Department of Medical Biochemistry, Uludag University School of Medicine, Bursa, Turkey
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
2 Y. OZARDA ET AL.

The first question (“Is the patient healthy or not may be used. The most obvious example is fasting glu-
healthy?”) relates to reference intervals (RIs) that cose, where several decision limits are defined: a level
describe the typical distribution of results seen in an
3.9 mmol/L (
70 mg/dL) for the diagnosis of hypogly-
apparently healthy reference population [1]. In the trad- cemia [
2.2 mmol/L (
40 mg/dL) for life-threatening
itional approach to establishing RIs, termed the “direct” hypoglycemia] [20]; 5.6–6.9 mmol/L (100–124 mg/dL) for
approach, the RI is typically defined as the interval an increased risk of diabetes or prediabetes; and
between the two reference limits (2.5th and 97.5th per- 7.0 mmol/L (125 mg/dL) for diabetes mellitus
centiles) derived from the distribution of results (DM) [21].
obtained from a sample of the reference population [2]. RIs are focused on optimizing specificity (typically to
Historically such ranges have been called “normal 95%) while CDLs are also focused on optimizing sensi-
ranges” [3], but this term has long been considered to tivity for the disease. In other clinical circumstances that
be ambiguous [4] and subject to misuse [5] and is now place importance on both sensitivity and specificity,
formally avoided by the International Federation of optimal limits may be derived from receiver operator
Clinical Chemistry and Laboratory Medicine (IFCC) [2] characteristic (ROC) curves, which balance sensitivity
and International Organization for Standardization (ISO) and specificity [22] (Table 1). Ideally, a Bayesian
[6]. About 30 years ago, the IFCC published a series of approach is also required to balance pretest probability
six articles to clarify this concept [7–12], and over the or prevalence against sensitivity and specificity [23].
last decade, the IFCC’s Committee on Reference “Optimal limits” derived from ROC curves, an intermedi-
Intervals and Decision Limits (C-RIDL) has focused on ate category of threshold, are a compromise between
direct common RIs; it has developed guidelines on con- specificity and sensitivity [24].
ducting such studies and has conducted multicenter This review describes the differences between RIs
studies to derive common RIs at a national level and CDLs, gives definitions and provides examples to
[13–15]. An alternative approach is to perform data- help readers to avoid confusion. We also consider the
mining of results that have been generated as part of preanalytical, analytical, and postanalytical factors influ-
routine clinical pathology testing and then to use encing them and apply the principles of analytical qual-
appropriate statistical techniques to determine the ity specifications to the quality of RIs and CDLs. Finally,
underlying RIs. This is considered the “indirect” we discuss the importance of clearly specifying the use
approach [16]. of either RIs and/or CDLs on reports to improve post-
According to the directive on in vitro diagnostic
analytical interpretation.
medical devices of the European Union, diagnostic
manufacturers are now required to supply their clients
with appropriate RIs for use with their assay platforms RI definitions
and reagents [17], and the ISO 15189 standard for clin- RIs are derived from the reference population value dis-
ical laboratory accreditation states that each laboratory tribution, usually the central 95% interval, and describe
should periodically reevaluate its RIs [6]. Despite these a specific population using a minimum sample size of
efforts and requirements, the process of producing the
120, as recommended by the Clinical Laboratory
RIs is complex and the implementation of RIs in most
Standards Institute (CLSI) guideline EP28-A3c [2]. The
clinical laboratories is still incomplete [18]. Furthermore,
reference individuals form the reference sample group
obtaining RIs for pediatric, geriatric, and pregnant pop-
ulations is particularly challenging because of the tech- Table 1. Comparison of CDLs and RIs.
nical difficulty of enrolling a sufficient number of RIs CDLs
reference individuals [19]. Number of values Statistical limits Clinical threshold
The other questions (“Is the patient at risk of a devel- (two values) (one value)
Derivation A biological characteristic A decision regarding a
oping a disease, or is the patient diseased, or worsen- of the unaffected clinical condition
ing?”) are related to clinical decision limits (CDLs), population
Population General population Clinical population
where values above or below the threshold are associ- Based on 95% central interval of Clinical outcome studies,
ated with a significantly higher risk of adverse clinical the reference guidelines and consen-
outcomes or are defined as diagnostic for the presence distribution sus values, ROC curves,
predictive values
of a specific disease [6]. In contrast to the RIs, where Defined by experts Laboratory experts Clinicians and labora-
there are two limits (upper and lower), there is only one tory experts
Most promin- Laboratory experts Clinicians
CDL, which is usually an upper limit. However, accord- ent experts
ing to the likelihood of various clinical situations or dif- Consensus Well-defined [2] Still to be developed
standard
ferent clinical questions, multiple low and high CDLs
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 3

for measurement of the values from the reference Indirect approach

population. Through statistical analysis of the distribu-
Although the direct approach to establishing RIs has
tion of the obtained values, the reference limits are cal-
been recommended by CLSI guideline EP28-A3c [2], in
culated, and these limits then define the RIs. However,
recent years there has been increasing interest in indir-
at other times, such as for cardiac troponin (cTn) [25,26]
ect methods [35], particularly in situations where it is
and carbohydrate antigen 125 (CA125) [27,28], the 99th
difficult to recruit healthy patients (e.g. pediatrics)
percentile is used as the appropriate upper reference
[36,37]. The rationale for this approach is that routine
limit. The rationale for the use of the 99th centile for
clinical pathology databases, which often contain many
cTn or CA125 is that a false positive rate of 1.0% would
thousands or millions of results from many hundreds or
cause 60% less undue clinical concern compared to a
thousands of patients, also include results from patients
false positive rate of 2.5%.
without disease, and that by using clinical exclusion
While concentrations above the 97.5th (or 99th) per-
[38] and statistical approaches [39,40], the underlying
centiles of the apparently healthy population are associ-
reference distribution can be distinguished. The advan-
ated with an absolute increase in risk for clinical
tages of the indirect approach compared to the direct
outcomes, it could mistakenly be assumed that patients
approach include that it is faster and less expensive and
with results within the RI are healthy and have no risk.
that it more easily overcomes ethical problems such as
However, results within the RI do not all necessarily
obtaining informed consent, issues that relate particu-
imply a minimal risk of adverse clinical outcomes, and
larly to the elderly or children, especially newborns. The
risk still varies within the RI, for example, across the ter-
disadvantage of the approach lies in the statistical and
tiles of C-reactive protein [29], across the quartiles of
clinical assumptions made. However, another assump-
cTn [30], or below and above the median level of pros-
tion is that there is no apparent disease affecting the
tate specific antigen (PSA) [31].
distribution of that analyte, although the observed ref-
It should be noted that RIs have diminishing utility
erence distribution may not be from “healthy” individu-
when the within-individual biological variability is sig-
als. The increased interest in data mining is gradually
nificantly tighter than the between-individual variability,
improving our understanding of the many factors to be
or in other words, when a patient can have a clinically
considered, including preanalytical conditions, analytical
significant change but still remain within the wider
stability, defined mechanisms of outlier removal, statis-
population RI [32].
tical assumptions, minimal sample sizes, partitioning,
and ethical considerations in the use of patient data.
Direct approach The C-RIDL has recently published a review of the
strengths and weaknesses of the indirect approach [41].
With this approach, individuals from a healthy popula-
tion (the reference population) are selected for sam-
pling based on defined criteria. Specimens are then Common RIs (including harmonized RIs)
collected from these individuals and analyzed for the There are two types of common RIs. The first are object-
selected measurand. The designation of good health ive RIs, which have many prerequisites [42] and are
and determination of normalcy for a candidate refer- defined by well-conducted multicenter studies [43–45],
ence individual may involve a variety of examinations and the second are subjective conventional RIs, defined
such as history and physical examination and/or certain by survey(s) and guidance from a group of experts
clinical laboratory tests [2]. Therefore, the identification using the harmonization approach [46–48]. The most
of a healthy status can be self-declared or confirmed by important question is “What is a significant difference in
the absence of disease. Confirmation of the absence of reference limits?” International guidelines indicate that
disease can be by clinical examination that includes if analytical methods and reference populations are the
specific extra testing; examples include conducting same, a subjective approach can be used to adopt com-
semen analysis to confirm testicular health when esti- mon limits [2]. The ISO 15189 quality standard also
mating testosterone RIs in men [33] or conducting B- encourages the use of interpretive comments at a local
type natriuretic peptide (BNP) testing to define healthy or national level [6]. When comparing candidate com-
cardiac function when defining cTn RIs [34]. The CLSI mon RIs, a pragmatic consideration is, what is a clinic-
guideline EP28-A3c [2] also describes the parametric or ally acceptable flag rate variation across analytical
nonparametric statistics that should be applied to the platforms and clinical populations [40]. The flag rate
reference values. represents the balance between false positives, true
4 Y. OZARDA ET AL.

positives and the understanding of the expected preva- 3. The physiopathological approach involves the use
lence of disease in that particular population. of “critical values” that represent a pathophysio-
logical state with such variance from normal as to
be life-threatening unless prompt action is taken.
Significant figures used in RIs
While many clinical endpoints can be difficult to
The number of significant figures in the reported value define, the endpoint of mortality is clear and,
is defined largely by the analytical confidence [49]. The because it defines the risk of dying or of major
number of significant figures used in a reference limit patient harm (e.g. neonatal hypoglycemia), it
should also be agreed upon when defining common RIs defines a particular set of high risk CDLs often
[50]. For example, is a serum albumin value of 45.4 g/L called “critical values” [55]. Prognostic CDLs for
greater than an upper reference limit of 45 g/L? tumor markers may also belong to this category.
Conversely, if a research study has defined the 97.5th
percentile for the serum albumin reference sample to Regardless of the approach used and the clinical
be 45.4 g/L, should this be rounded down to 45 g/L endpoints defined, CDLs are defined according to an
(slightly increasing the false positive rate) or should it arbitrary risk level.
be rounded up to 46 g/L (ensuring the false positive
rate does not rise above 2.5%)? Diagnostic and staging cutoff values
A diagnostic cutoff is defined by a question such as,
CDLs definitions “When does a patient have diabetes or prediabetes?”
As previously mentioned, RIs relate to studies based on For hemoglobin A1c (HbA1c), the definitions of the
apparently healthy individuals while CDLs are based American Diabetes Association are <39 mmol/mol
mainly on clinical outcome studies (e.g. prospective (<5.7%) for healthy, 39–46 mmol/mol (5.7–6.4%) for
cohort studies, meta-analysis), guidelines and consensus prediabetic, and 48 mmol/mol (6.5%) for diabetic
status [56]. Prediabetes, defined as impaired glucose
values. It is important that these two approaches are
tolerance, impaired fasting glucose, or intermediate
clearly distinguished [51,52].
HbA1c, is of particular importance as a diagnostic cat-
The approaches to identifying CDLs can be catego-
egory because it has been associated with an increased
rized [1]:
risk of cardiovascular disease (CVD) [57], and cardiovas-
cular risk has also been shown to be increased in peo-
1. The Bayesian approach is probably the most evi-
ple with a fasting glucose concentration as low as
dence-based approach to modifying the manage-
5.6 mmol/L or HbA1c of 39 mmol/mol [58].
ment of the patient. Following these criteria, a
While risk may begin at a certain threshold, the def-
value resulting from a diagnostic test that serves
inition of what constitutes a significantly increased risk
to distinguish between two clinical subgroups is
is usually defined arbitrarily by an expert consensus
based on stated assumptions regarding (i) the clin-
group. An example is the definition of gestational dia-
ical sensitivity of the diagnostic test; (ii) the clinical
betes mellitus (GDM) from the International Association
specificity of the diagnostic test; (iii) the relative of Diabetes and Pregnancy Study Groups (IADPSG) [59],
distribution of individuals between the two sub- which was established using the results of the
groups; and (iv) the clinical costs of misclassifica- Hyperglycemia and Adverse Pregnancy Outcome
tion [4]. This concept has been applied to (HAPO) study [60] and which used an odds ratio of
troponins [23,34], where the balance among these 1.75 in selecting the likelihood of complications such
factors had to be considered to maintain the spe- as macrosomia. The prevalence of GDM would have
cificity and sensitivity required for patients pre- been altered if an odds ratio of 2.0 had been chosen
senting with chest pain. instead [61].
2. The epidemiological approach for defining CDLs is Tumor markers represent another group where cut-
based on clinical outcome derived from popula- off values are used instead of RIs. These markers are
tion-based studies and is typically applied to lipid often present in nondiseased people at concentrations
parameters (total cholesterol, low density lipopro- that overlap significantly with those in people with
tein cholesterol [LDL-C], high density lipoprotein malignancies. This typically impedes the general use
cholesterol [HDL-C], etc.) [53] as well as to inter- of tumor markers as a screening tool. For staging
vention-based studies such as diabetes interven- purposes, the International Germ Cell Consensus
tion [54]. Classification represents a tumor marker-based staging
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 5

system in which three biomarkers contribute to the values/delta change). Reference change values,
staging of men suffering from nonseminomatous germ which can be defined by absolute (± delta) or relative
cell tumors; they are classified into three prognostic (± delta%) means, can help in the interpretation of the
groups, depending on the levels of the markers: good results of serial measurements. The example of absolute
(alpha fetoprotein [AFP] < 1000 ng/mL, human chorionic and relative kinetic changes of cTn in patients with
gonadotrophin [(hCG] < 5000 IU/L, lactate dehydrogen- acute coronary syndrome shows that serial measure-
ase [LDH] < 1.5 upper RL); intermediate (AFP ments may assist in diagnosis and may be used to rule
1000–10000 ng/mL, hCG 5000–50000 IU/L, LDH 1.5–10 out non-ST elevation myocardial infarction [68]. The
upper RL) and poor (AFP >1000 ng/mL, hCG >50000 IU/ prerequisites to calculate delta changes from serial
L, LDH >10 upper RL) [62]. This help in the selection measurements are a well-accepted clinical algorithm
of the appropriate therapy [63]. Interestingly, this classi- with defined time points (e.g. baseline, 3 h, 6 h for cTn)
fication combines two strategies of defining decision
and the knowledge of the intra-individual biological
limits: for AFP and hCG, absolute cutoffs are used,
variation of the measurand (within-individual CV [CVi]).
whereas for LDH, multiples of the upper reference
Although the source of biological variation data is typic-
limit(s) are used.
ally from a healthy reference population, its application
to disease assumes that biological variation is the same
Critical values in chronic disease as in health [69], and this has been
The term “critical value,” also known as critical result, adopted as a surrogate for clinically significant changes.
panic value, or alert value, represents a pathophysio- The choice of statistical model used for clinical monitor-
logical state different from normal that poses a risk to a ing varies according to clinical circumstance [70].
patient’s life unless immediate action is taken [64].
Currently, the use of the term, panic value, is discour-
What is an acceptable risk?
aged, because it suggests emotional stress, and because
it is contrary to the process of communicating informa- The arbitrary setting of the risk level to be flagged, pri-
tion clearly [55]. marily a clinical decision, is based on the balance of
There is an overall risk of mortality in any tertiary set- false positive alarms that are acceptable compared to
ting compared to the general community, but the the clinical outcome benefit gained from true positive
increase in that risk that is associated with laboratory alarms. It is important that the rationale behind any
results can be further used as a flag for clinical action. choice of risk level be explained. Whilst they are primar-
As results deviate from normality, they represent a loss ily clinician-defined, it is essential that laboratory profes-
of homeostasis and the risk of decompensation and, sionals are involved in their definition for several
ultimately, of death. Any result that is outside the RI is reasons. Firstly, the laboratory will need to manage the
associated with a relative increase in the risk of mortal- reporting mechanisms. Secondly, some assays are not
ity [65], but the odds ratio for death varies from analyte standardized, and the risk levels could be method-
to analyte [66]. The perceived risk of death should be
dependent. Thirdly, it is important that results outside
understood, for example, a 25 versus 50% mortality risk
CDLs are not due to preanalytical or analytical artifacts,
in patients who have been admitted in a hospital, and it
and that this potential is managed and communicated
becomes a specifically stated arbitrary setting that can
when necessary.
be agreed upon by clinicians and laboratorians. It is
There are many other points to mention, including
important to note that in some circumstances (e.g.
that the laboratory may report to several clinical set-
hyponatremia), the abnormality may directly reflect the
tings that may have different clinical questions. Gamma
cause of death and the need to focus treatment on cor-
recting the abnormality is implied, while in others (e.g. glutamyl-transferase thresholds can be set to determine
hypernatremia), the abnormality may be an indirect alcohol abstinence or alcohol abuse [71]. Similarly, an
reflection of the cause of death (e.g. dehydration and alanine aminotransferase cutoff could be set to flag
hypovolemia) and correction of the abnormality is sec- everyone in the emergency department with possible
ondary to addressing the underlying mechanisms [67]. paracetamol overdose (e.g. >1000 U/L) [72], everyone in
the blood bank with possible subclinical viral hepatitis
(e.g. >20 U/L) [73], everyone in the lipid clinic with pos-
Reference change values sible fatty liver (e.g. >26 U/L) [74], or everyone in a
The progress of a disease or recovery from it is health screen who may be possibly obese (e.g. >40 U/L)
often reflected by the dynamics of test results (delta [75] (although there are easier ways to define obesity).
6 Y. OZARDA ET AL.

Harmonization of CDLs aspects are accurately defined and described, because

the preanalytical phase is known to have the highest
The implementation of shared electronic health records
error rate in the total testing process [76].
has highlighted the need for standardized or harmon-
As RI studies are generally well-designed and con-
ized reporting of laboratory test results [47]. Most of the
trolled, and efforts have been made to standardize
criteria checked during the harmonization of RIs, such
them according to guidelines, it is accepted that prea-
as method specificity, partitioning based on age or gen-
nalytical problems occur less often. However, it should
der, and preanalytical differences, apply to the harmon-
be noted that this extra attention and effort may result
ization of CDLs [47]. In addition, the clinical settings
in narrower RIs than if the usual preanalytical processes
could form an additional obstacle for the harmonization
were to be used.
of CDLs. The measurand could have a central and well-
Age and gender are acknowledged as major factors
defined role in the decision-making for a specific dis-
affecting RIs. The main factors that cause the difference
ease or clinical situation, but there could be several
in RIs between the genders are muscle mass, body
CDLs, such as for diagnosis of anemia (e.g. hemoglobin
mass index (BMI), different organs (uterus, ovaries,
130 g/L in men, 120 g/L in nonpregnant women, 110 g/L
breast, testis, and prostate), related hormonal control
in children) or for performing a blood transfusion (e.g.
systems, and higher iron demands in females related to
hemoglobin 70–80 g/L or according to the clinical situ-
blood loss with menstruation, pregnancy and breast-
ation; if the patient is stable, transfusion might not be
feeding [19].
needed even at these concentration levels) [1].
Many studies of specific RIs for geriatric age groups
have had methodological issues, which means that
Significant figures used in CDLs careful consideration is required before applying these
to clinical care [77].
The issues regarding the number of significant figures
The preanalytical effect of age, gender, race, ethni-
in an analytical result are the same as discussed above
city, time of day and collection processes that have
for RIs. The number of significant figures used in CDLs
been acknowledged as factors with a modifying effect
should ideally be the same as that used in the clinical
on RIs may be equally relevant to CDLs. Gender can
study that determined that limit. Problems can arise
affect the decision limits for HDL-C (<1.0 mmol/L in
when reporting unit changes occur. For example, the
men and <1.3 mmol/L in women) in the definition of
CDL for the diagnosis of diabetes is 6.5% using National
metabolic syndrome [78]. Race may also change clinical
Glycohemoglobin Standardization Program (NGSP) units
decision thresholds, and the typical decision thresholds
and 48 mmol/mol using IFCC units, while an IFCC result
for overweight (BMI >25 and <30 kg/m2) and obesity
of 47 mmol/mol converts to an NGSP result of 6.4505%
(> ¼30 kg/m2) may vary with race; for example, there is
(both nondiabetic), but the rounded NGSP value
evidence that Indian adults should have much lower
becomes 6.5% (diabetic).
thresholds [79]. Diurnal effects should be considered in
setting clinical decision thresholds, and there has been
Preanalytical, analytical, and a recent shift from fasting to nonfasting lipid studies
postanalytical factors because they have been generally found to be equiva-
lent in their ability to act as clinical decision points [80].
Preanalytical factors
Collection conditions during trials that establish clinical
General awareness of preanalytical problems that may decision thresholds should continue to be applied in
cause false positive results is needed. These include clinical practice, although this is not always the case; for
sample contamination, inadequate transport conditions, example, in the global trial that established internation-
incorrect timing of sample collection (e.g. for toxico- ally agreed-upon criteria for gestational diabetes, glu-
logical testing), and delay in sample processing. cose samples were stated to be collected on ice, a
Generally, the preanalytical considerations for RIs practice not often observed in routine phlebotomy [81].
involve biological (i.e. sampling time in relation to bio- While RIs for pediatric bilirubin can be defined, for
logical rhythms, fasting or non-fasting and physical early identification of biliary atresia the focus changes
activity) and methodological factors (i.e. sample collec- from the RI to the CDL that is applied to the direct bili-
tion techniques, type of additives, collection with or rubin measurement to distinguish biliary obstruction
without tourniquet and sampling equipment, specimen from conjugation defects [82].
handling, transportation, time and speed of centrifuga- Creatinine RIs have long been defined by gender [83]
tion, and storage conditions). For reproducibility and and are now increasingly defined by age [84]. Without
standardization, it is essential that the preanalytical considering the expected differences in muscle mass
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 7

between individuals, it is difficult to achieve the primary important for both RIs and CDLs. Neither universal CDLs
purpose of serum creatinine measurement, which is to (e.g. for lipids and HbA1c) nor common CDLs (e.g. for
gauge renal function. Serum creatinine should now be routine analytes) can be clinically reliable without trace-
routinely translated to an estimation of eGFR using ability and analytical quality standards [94]. When
acknowledged formulae that account for the impact of adopting RIs derived externally, a laboratory can per-
age and gender on muscle mass. While appropriate form a local validation (e.g. 20 healthy people) [95],
serum creatinine RIs could also be translated to eGFR although these procedures are not available for adjust-
RIs [85], the primary purpose for eGFR is to allow appli- ing CDLs and we are reliant on other techniques to con-
cation of the profession-defined CDLs for eGFR that firm method suitability (e.g. external quality assurance).
describe the risk associated with the various stages of
chronic kidney disease. Creatinine reporting is therefore
Postanalytical factors: reporting CDLs and RIs
associated with gender and age-related RIs, while eGFR
reporting is associated with CDLs that take gender and The CLSI guideline EP28-A3c states that “To avoid con-
age into account. fusion, the working group encourages laboratories to
In the case of nutritional factors such as vitamin D, report either decision limits or RIs, but not both, with a
Vitamin B12, and ferritin, where the reference popula- clear indication of which has been used” [2]. The stand-
tion has a high prevalence of deficiency, the use of cor- ard also states that “When decision limits determined
rect RIs is generally problematic because of widespread by national or worldwide consensus exist, these limits,
subclinical deficiency, and these are typically handled as rather than RIs, should be reported,” thus prioritizing
CDLs [86–88]. the preference for CDLs over RIs. Current practice is to
report CDLs for the following measurands: total choles-
terol, LDL-C, HDL-C, non-HDL-cholesterol (non-HDL-C),
Analytical factors
triglycerides, apolipoprotein A (Apo A), apolipoprotein B
In 1999, the IFCC participated in a consensus meeting (Apo B) and HbA1c. However, in the section of the med-
in Stockholm as part of a global effort to define analyt- ical decision limits of the CLSI guideline EP28-A3c, an
ical quality specifications [89]. The hierarchy of example of a report shows that the CDLs for total chol-
approaches to determine analytical quality require- esterol and HDL-C are listed in the column entitled “RI”
ments was headed by clinical outcome studies, but [2]. With many routine laboratory reports, RIs and CDLs
because these were seldom available, the second level are also listed in the “RI” column. This practice disre-
of the hierarchy was strategies supported by clinical gards the basis of the terminology and confuses the dis-
opinion and biological variability. These principles sug- tinction between RIs and CDLs. If this column of the
gested that CDLs based on clinical outcome were super- CLSI guideline EP28-A3c were to be updated and enti-
ior to RIs that were based on biological variation [90]. tled “Reference”, not “RI”, the reference comparator
Fifteen years later in Milan, at a European Federation of could then be either a RI or a CDL. As C-RIDL, we would
Laboratory Medicine (EFLM) meeting, approaches based like to emphasize that the distinction between RIs and
on clinical outcome and biological variation were CDLs should be clear in the report; such a distinction
endorsed [91] and specifically applied to the postanalyt- will have valuable effects on postanalytical quality. A
ical phase, including CDLs and RIs [92]. clearer distinction could also be facilitated with new
Analytical quality affects the reliability of both RIs presentation formats including graphical ele-
and CDLs. The biological variability theory suggests ments [96–98].
that the desirable bias for RI classification takes into The aim of report interpretation is to lead to clinical
account intraindividual and interindividual variability decisions that benefit clinical outcomes, and the basis
[<0.25  (CVi2 þ CVg2)1/2] and that it will prevent an for those decisions usually starts with the rationale for
unacceptable increase in the proportion of healthy flagging an important result.
individuals flagged as outside RIs. Analytical quality will The rationale for flagging elevated cholesterol
similarly affect the application of CDLs, although the related results is based on the two main purposes for
impact is defined not by the statistics of the reference this testing; primary and secondary prevention. In pri-
population distribution but by the clinical risk defini- mary prevention, risk thresholds and lower treatment
tions as well as the prevalence of disease [93]. goals for preventative statin therapy are usually defined
Increasing measurement uncertainty generally causes through epidemiological studies; while in secondary
greater clinical uncertainty; similarly, the impact of prevention, clinical studies have defined even stricter
uncorrected measurement bias will lead to clinical bias. treatment goals. It is also not possible to tailor the
The traceability of method calibration is vitally report to a patient’s individual clinical need if the
8 Y. OZARDA ET AL.

clinical background of the patient has not been shared decision to exempt these men from further screening
with the laboratory. Nevertheless, the issue of variable could be made to allow greater focus on men with
interpretation could be covered with an interpretative higher concentrations.
comment (including a table or link) that details the clin-
ical situation and the agreed-upon corresponding deci-
Concluding remarks
sion thresholds.
Desirable HDL-C values, according to the European The principles for describing RIs and CDLs should be
Guidelines, are 1.03 mmol/L (40 mg/dL) for males kept separate, and this distinction should be clear in
and 1.20 mmol/L (46 mg/dL) for females [99]. HDL-C the laboratory report. We believe that this would greatly
is an example of how physiology, which is mainly a RI improve the postanalytical quality of interpretation and
concern, also impacts the risk point. In the case of HDL- facilitate the national and international adoption of
C, decision limits can be used to categorize people common RIs (including harmonized RIs) and CDLs.
at increased risk (<1.04 mmol/L) or decreased risk The IFCC C-RIDL workplan includes participation in
(>1.55 mmol/L) of coronary artery disease based on the creation of guidelines for the definition of CDLs.
data from large population studies [99]. For example, it The requirements for the creation of CDLs could include
is well-established that the Turkish population has a aspects relating to:
high prevalence of coronary heart disease associated
with some known risk factors [100]. Turks have distinct- 1. the process; that is, the involvement of both clini-
ively low concentrations of HDL-C that are associated cians and laboratory professionals,
with elevated hepatic lipase activity and fasting trigly- 2. the definition; that is, the precise definition of the
ceridemia [101]. Therefore, it would be better to state clinical question (and clinical decision) to
only the population-based RIs in the RI column of the be addressed,
laboratory report, and to state the CDL clearly as a com- 3. the clinical confidence with which the question
ment, for example at the bottom of the report, when a needs to be answered, and finally,
parameter has a well-defined CDL [102]. 4. the mechanisms for the broader implementation
PSA is a good example for contrasting the applica- of CDLs at the national and international level.
tion of RIs and CDLs. The first RI for PSA, defined as
<4.0 lg/L, was based on studies of apparently healthy
men [103]. It was soon found that this upper reference Disclosure statement
limit was not specific in older men, and possibly too No potential conflict of interest was reported by the authors.
specific (and insensitive) in younger men, so age-related
PSA RIs were developed and adopted [104]. The consid-
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