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Normal values and reference intervals: reference systems in clinical

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4 TIAFT Bulletin 48(2)

NORMAL VALUES AND REFERENCE

INTERVALS:
Reference Systems in Clinical Chemistry and Toxicology
Torsten Arndt
Bioscientia Institut für Medizinische Diagnostik GmbH,
Konrad-Adenauer-Straße 17, D-55218 Ingelheim, Germany
torsten.arndt@bioscientia.de

1. Introduction for a long time; however, these concepts should be replaced by

Clinical chemistry and toxicology contribute to the verification
or falsification of suspected diagnoses, differential diagnosis,
prognosis, and to longitudinal control of a therapy.
Valid (accurate and precise) analytical results are required to
achieve these goals, but are rarely sufficient. In fact, it requires
a cognitive process with transformation of the results from
the technical to the nosological level with diagnosis and/ or
prognosis (Figure 1). At the biological level (which is central for
this review), the patient’s (observed) data are compared with
those from an appropriate control individual or control group.
Normal values or ranges from 'normal' groups have been used
Differential Diagnosis
Pathobiochemistry Diagnosis / Prognosis
Diagnosis
Nosological Level
reference intervals (syn. reference ranges) [1-3].
Basic knowledge about reference systems in clinical chemistry
is important, even for toxicologists, since toxicological data are
frequently embedded in a wide range of laboratory results.
The latter can indicate a malfunction or disease with impact
on the toxicological case. For example, the concentration of
glucose in NaF-plasma and/or of glycated hemoglobin A1
(HbA1C) in EDTA-blood can provide information about an
abnormal glucose metabolism which can impair car driving
abilities, e. g. of a diabetic [4]. Abnormal coagulation tests like
the thromboplastin time (Quick test) and the activated partial
thromboplastin time (aPTT) may indicate liver malfunction
and thus a delayed hepatic metabolism (detoxification) of
pharmaceuticals and other xenobiotics. Finally, an increased
serum creatinine concentration may indicate kidney injury with
a minimum (!) 50% reduction in glomerular filtration and thus
delayed urinary excretion of toxic endogenous (e. g. ammonia
from amino acid metabolism) or exogenous substances [4].

The present review aims to provide an introduction to the

Plausibility Check
Reference System Laboratory Report concept of Normal Values and the theory of Reference Values
In Vivo Effects and to inform toxicologists about these fundamental reference
Biological Level systems in clinical chemistry.
Quality Control
In Vitro Effects Analysis Result 2. Normal Values and Normal Ranges
Analysis
Technical Level The definition of normal values by Gräsbeck [5] demonstrates
worldly wisdom. In fact, it summarizes the main problem of the
Sampling
concept of normal values: "Normal values come from young,
Sample (Specimen)
Patient Pretreatment mostly female technicians, a few medical students and one
Patient old professor, all walking about. These values are then used to
evaluate data from old, bedridden patients, examined at odd
hours of the day“.
Figure. 1. From patient and sample to diagnosis and/ or
prognosis. Transformation of an analytical result into a Normal values show many shortcomings: most normal
diagnosis. Figure from [3] modified.
values studies have been insufficiently documented, e.g.

inclusion and exclusion criteria for the control group are missing
or are inconsistently applied. Pre-analytical requirements have
been ignored or only partially considered. Analytical methods
have been insufficiently validated, are not comparable
between different laboratories, and are not commutable
to an international standard. Finally, the number of normal
individuals has been most often too low for valid statistical
evaluation [3]. Pediatric normal values are typical for such a
scenario. These are commonly based on an unknown number
of controls or on only a few individuals (see e.g. [6]).
Following on from this, different normal values and ranges
for the same analyte in the same sample matrix reported by
different laboratories hamper the interpretation of the results,
e.g. when moving a patient from one clinic to another or from a
clinic to a general practitioner. To overcome these shortcomings,
the Theory of Reference Values has been developed.
3. Reference Values and Reference Intervals
The Theory of Reference Values (also known as Concept
of Reference Values or Concept of Reference Intervals) was
developed and published between 1987 and 1991 by an “Expert
Panel on Theory of Reference Values” under the auspices of the
(at that time) International Federation of Clinical Chemistry
- now International Federation of Clinical Chemistry and
Laboratory Medicine (IFCC). The Approved Recommendations
are documented in a total of 6 publications of approximately 40
DIN A4 pages (see [7-12]). It is clear that the following can give
only a short introduction to this landmark work. Details can be
retrieved from references [7-12], being a truly good read.
3.1. Reference Individuals – Reference Population –
Reference Sample Group
Part 1 of these Recommendations [7] gives a brief outline
about the historical evolution of the Theory of Reference
Values. It collects also “concise, and well defined terms”
[7] which can be “adopted universally” [7] and which avoid
ambiguous descriptions of the relation between observed
Reference individuals
constitute a
Reference population
from which is selected a
Reference sample group
on which are determined
Reference values
on which is observed a
which may provide a
Reference distribution
reference system for
on which are calculated the comparison of
observed values with
Reference limits reference data.
that may define
Reference intervals
Figure. 2. From reference individuals to reference intervals.
Fundamental terms from the Theory of Reference Values and
their relation to each other. Figure from [7] modified.
TIAFT Bulletin 48(2) 5
values and reference values, such as “a reference individual is
an individual selected for comparison using defined criteria“ [7]
or “a reference population consists of reference individuals” [7].
A reference population comprises all suitable individuals with
regard to exclusion, inclusion and partition criteria.
Usually, the number of individuals of a reference population
and the individuals as such are unknown or cannot be identified
with acceptable effort. Thus, a reference sample group with “an
adequate number of reference individuals taken to represent
the reference population” [7] is collected. This reference sample
group serves for the establishment of reference values and
intervals as shown in Figure 2.
The smallest reference population or reference sample
group is a single individual, e.g. when a patient serves as a
reference for himself in follow-up examinations [7].
A central element of the concept refers to exclusion,
inclusion and partition criteria, which are discussed in Part 2
[8] of the Approved Recommendations. Many factors such as
physiological (e. g. pregnancy, physical training, food intake
prior to blood collection, cytochrome P450 (CYP) enzyme
profile etc.) and pathophysiological (e.g. chronic or acute, local
or systematic diseases and syndromes, alcohol and drug abuse)
contribute to biological variability and have to be considered
if directly or indirectly related to the target analyte and thus
to the reference values. Pathophysiological states should be
excluded by clinical examination, laboratory investigation, and
questionnaires and should be considered as exclusion criteria.
An a priori (prospective) selection of individuals should be
preferred to a posteriori selection from “big data” from large
patient populations [8].
Exclusion, inclusion, and partition (e.g. age, gender or
race) criteria should be defined with regard to the aim of the
reference value study and should also consider published data
if applicable. This is discussed in part 2 of the IFCC Approved
Recommendations [8].
Reference individuals are not compellingly healthy. This
depends on the aim of the reference study and also on the
definition of health. The World Health Organization, for
example, defines health as “a state of complete physical, mental,
and social well-being and not merely the absence of disease
or infirmity” (cited from page 340 of reference [7]). Reference
individuals may differ from this definition. For example,
alcohol-abstaining pregnant women may certainly be included
in a reference sample group for a study of alcohol abstinence
markers, but most probably not in a group for a study of
markers of impaired glucose intolerance (due to a relatively
high prevalence of gestational diabetes). Also a medication can
be acceptable if there is no direct or indirect effect on the target
analyte. Thus, it is of utmost importance to clearly define and
document the exclusion, inclusion and partition criteria when
collecting a reference sample group.
The usefulness of a given reference system is strongly
affected by the quality of the pre-analytical process. Pre-
analysis usually refers to the patient pre-treatment, sampling,
sample pre-treatment, and sample transport. Forensic
toxicologists are highly familiar with these items. For example,
blood from a decayed corpse is completely different from
that of a (healthy) driver under the influence of ethanol.
Clinical chemists also know a lot about pre-analytical culprits,
e.g. inappropriate food restriction prior to blood collection
causing lipemia and interferences (not only) in nephelo- and
turbidimetric immunoassays. Transport and agitation of
uncentrifuged blood may cause severe hemolysis and thus
6 TIAFT Bulletin 48(2)

odd or even “lethal” potassium concentrations and pathological
lactate dehydrogenase (LDH) activities in serum or plasma from
healthy controls, not to speak of interferences of the atypical
red-colored samples in optical tests according to Warburg (see
Textbooks on Clinical Chemistry).
This shows that inappropriate pre-analytical conditions
may highly affect the analysis and thus the reference intervals
drawn from an invalid reference range study. Part 3 of the IFCC
Approved Recommendations [9] presents a multitude of factors
which may have to be considered and which, if applicable, be
standardized in a reference value study:
• Reference individual treatment before (blood) sampling:
food intake (amount, day-time, time gap between last meal
and sampling), medication and use of pharmacologically
active substances including alcohol, caffeine and nicotine
(amount, dosing regime, time between last dose and
specimen collection), exclusion of illegal drug use,
synchronisation of activity and sleep, physical activity
(short-term, long-term, mild, work-related, lifestyle etc.)
and mental stress.
• Specimen collection (sampling): environmental conditions
(temperature, humidity, geographic altitude), day-time
(absolute and relative to meals and sleeping periods),
season, body posture (upright, sitting, supine).
• Specimen type (sample type): arterial or venous blood,
spot urine or 24h-urine, collection site, site preparation
(disinfection), blood flow promotion (warming, pressure,
muscle work), collection equipment, collection type (free-
flow, suction).
• Specimen (sample) handling: transport conditions,
clotting time, centrifugation time and conditions, storage
conditions, preparation for analysis.
This (incomplete) selection shows that collecting a reference
sample group can be demanding.
3.2. Reference distribution – Reference interval –
Reference interval limits
Once a representative reference sample group has been
examined by a valid and well-documented analysis method
(IFCC recommendation part 4 [10]), the data have to be checked
statistically for normal distribution, reference interval limits
have to be calculated and reference intervals defined (IFCC
recommendation part 5 [11]). Different statistical models have
been described. For details see reference [11].
In short: reference intervals usually comprise the central
segment with 95% of the reference values from the reference
sample group. The limits of the reference interval are defined
according to the use of the laboratory marker. If increased
and decreased values are diagnostically relevant, the 2.5% and
97.5% percentiles are applied. This is the most common case.

0.0% Percentile Reference Values Sorted by Increasing Value 100% Percentile

0.0% Percentile 100% Percentile

B
Reference Interval
2.5% Percentile 97.5% Percentile

0.0% Percentile 100% Percentile

C
Reference Interval
True- or False- 2.5% Percentile 97.5% Percentile True- or False-
Positives Positives

0.0% Percentile Borderline Values 100% Percentile

D
Reference Interval
True- or False- 2.5% Percentile 97.5% Percentile True- or False-
Positives Positives

0.0% Percentile Borderline Values 100% Percentile

E Reference Interval
0.0% Percentile 95.0% Percentile True- or False-
Positives

Figure. 3. From reference values to reference intervals. Sorting the reference values (Grey) from the reference sample group
according to increasing values (A). Defining the reference interval limits, usually the 2.5% and 97.5% percentile of the reference
values (B), in special cases the 0.0% and 95.0% percentiles (E). The reference interval (Green) comprises in each case 95% of the
reference values and is defined by the reference interval limits which belong to the reference interval (B-D). Values outside the
reference interval (Red) may be false-positives or may indicate malfunction or disease (C-E). The ratio between true-positives and
false-positives strongly depends on the diagnostic specificity of the laboratory marker (see Textbooks of Clinical Chemistry).
Introducing a borderline range (Yellow) can reduce the number of false-positives (D,E) or of false negatives (not illustrated).

A typical example may be the glucose concentration in serum,
which can indicate hypo-, normo- or hyperglucosemia. Other
parameters may indicate disease or malfunction only if they
are increased, e. g. tumor markers or carbohydrate-deficient
transferrin as a marker of chronic alcohol abuse (see Textbooks
of Clinical Chemistry). In these cases, the 0.0% and 95.0%
percentiles may be more adequate as reference interval limits.
In any case, the reference interval limits always belong to the
reference interval [11].
3.3. Presentation of Analysis Results and Reference
Intervals
A patient’s (observed) result provides useful diagnostic
information only after comparison with an adequate reference
system, e.g. by data from the same patient in a follow-up study,
by a reference interval or by a warning or decision limit.
The reference system must be adequate. Thus, the
laboratory report should contain enough information, e.g.
about the available sets of reference values, the definition of
the reference individuals (and subclassification/ partitioning),
the methods of selection and preparation of the reference
individuals, sample pre-treatment, analysis method, and the
number of reference individuals in each subclass (part 6 of the
Approved Recommendations [12]).
In common practice, this information is usually restricted to
the name of the analyte, the sample matrix, the analysis method,
the analysis result, the reference interval limits, and the unit
of measurement for observed results and reference interval.
Adequate age- and gender-dependent reference intervals are
selected computer-assisted from a set of reference intervals in
the laboratory information management system (LIMS).
This minimum information is considered absolutely essential.
Less information, e.g. analysis results without unit and/ or
reference systems are not acceptable and should be rejected.
4. Alternative Concepts
The difficulties in collecting a sufficient number of reference
individuals ahead (a priori) of a reference study, led to the
promotion of so-called posteriori concepts which means
deducing reference intervals from analysis data of the routine
laboratory [13,14]. However, most of these models are based
on large analysis series, nowadays frequently referred to as "big
data“, which are not available in small laboratories and which
are also frequently not available for esoteric tests or diseases.
The protocol C28-A3 [15,16] from the Clinical and Laboratory
Standards Institute (CLSI) could be a practical alternative at least
for the verification of given reference intervals by data from
one's own laboratory as frequently requested in accreditation
protocols. Thus, it would be sufficient to analyse samples from
only 20 reference individuals and to compare these data with
the reference interval given by the supplier of an analytical test
kit or with appropriate data from the literature. The reference
interval can be considered adequate if at maximum 2 out of
the 20 analysis results are outside. If 3 or 4 results are outside,
another 20 reference individuals have to be examined. If no
more than 4 results out of the 40 are outside the proposed
reference interval, this can be considered adequate. If there
are more than 4 results outside, the reference interval has to
be rechecked or an alternative reference interval has to be
searched for.
Finally, intra-individual reference values should be
mentioned [17]. In theory, these can solve many of the
TIAFT Bulletin 48(2) 7
problems of a priori and posteriori concepts since a given
individual serves as his/her own control (reference individual).
In doing so, many variables, e.g. those belonging to genetics like
the CYP enzyme system being important for the metabolism
of xenobiotics, remain life-long constant. However, this concept
poses many questions. To mention just a few: Which analytes
should be monitored? What about new analytes which have not
been known during e.g. childhood of an individual? What about
significance of an increase or decrease in the concentration of
an analyte? Considering a mean life expectancy of 80 years,
who can perform and guarantee analyses over 8 decades with
an inter-assay and inter-laboratory imprecision less than the
biological variability? Last but not least, who will carry the costs?
5. Conclusions
The Theory of Reference Values has strongly contributed
to a better understanding and use of reference systems
in clinical chemistry. Still, there is much to be done, e.g. in
implementing information about the reference values or about
the measurement uncertainty into the laboratory reports.
I would like to end this short review with citing a statement
from H. E. Solberg (Oslo, Norway) one of the pioneers of
the Theory of Reference Values in part 1 of the Approved
Recommendations on the Theory of Reference Values [7]:
“Any useful concept of reference values must have application
beyond the scope of clinical chemistry and haematology. The
concept proposed in this document provides a basis to pro-
duce, present and apply reference values and related terms to
measurements which concern both static and dynamic states.
Moreover, it is assumed that the production of meaningful
laboratory data usually requires collaborative effort of
laboratory workers, statisticians, physiologists, physicians and
patients. Universal adoption of the terminology recommended
in this document will enhance the understanding of reference
values and the interpretation of observed values.”
6. Acknowledgments
This review is based on an earlier article by the same author
[18] (in German). Most parts of the original article have not
only been translated into English but extensively revised. The
copyright for the German article is with the author. The author
is grateful to Dr. Adrian Sewell, Ingelheim for stylistic comments.
7. References
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for laboratory medicine [editorial]. Clin Chem Lab Med
2004;42:686-691.
[2] Gräsbeck R. The evolution of the reference value concept.
Clin Chem Lab Med 2004;42:692-697.
[3] Stamm D. Normalwerte und Referenzwerte. In: Greiling
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71-111.
[4] Textbooks of Clinical Chemistry.
[5] Gräsbeck R, Saris NE. Establishment and use of normal
values. Scand J Clin Lab Invest 1969;26(Suppl 110):62-63.
[6] Soldin SJ, Wong EC, Brugnara C, Soldin OP (eds.). Pediatric
reference intervals. 7th edition, AACC Press, Washington DC,
U.S.A., 2011.
[7] International Federation of Clinical Chemistry (IFCC) and
 8 TIAFT Bulletin 48(2)
International Committee for Standardization in Haematology
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[10] International Federation of Clinical Chemistry (IFCC).
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View publication stats
of Reference Values (EPTRV) and International Committee
for Standardization in Haematology (ICSH). Approved
recommendation (1987) on the theory of reference values. Part
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[13] Haeckel R, Wosniok W, Arzideh F. A plea for intra-laboratory
reference limits. Part 1. General consideration and concepts of
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[14] Arzideh F, Wosniok W, Gurr E, Hinsch W, Schumann G et al.
A plea for intra-laboratory reference limits. Part 2. A bimodal
retrospective concept for determining reference limits from
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[15] Appold K. Determining laboratory reference intervals: CLSI
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[18] Arndt T. Normalwerte und Referenzintervalle – Zur
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8. Further Reading
Clin Chem Lab Med 2004;42(7):685-873 – A special issue
of Clinical Chemistry Laboratory Medicine on the Theory of
Reference Values and other reference systems in Laboratory
Medicine/ Clinical Chemistry.