REFERENCE INTERVALS AND ITS SIGNIFICANCE IN

CLINICAL CHEMISTRY

BY
MLS. JOHN FAVOUR MANA
(INTERN)

DEPARTMENT OF CHEMICAL PATHOLOGY

FEDERAL MEDICAL CENTRE BIDA, NIGER STATE

SUPERVISED BY:
MLS ABDULAZEEZ ANIBASA
11TH OCTOBER, 2023
1
 OUTLINE
• TERMINOLOGIES
• INTRODUCTION
• HISTROCIAL BACKGROUD
• TYPES/ FEATURES REFERENCES INTERVALS
• PROCESS/ METHOD FOR VERIFYING REFERENCES INTERVALS
• DATA ANALYSIS FOR TRANSFERENCE
• FACTORS AFFECTING REFERENCES INTERVALS
• SIGNIFICANCE OF REFERENCE INTERVALS IN CLINICAL
CHEMISTRY
• CONCLUSION
• RECOMMENDATION
• TAKE HOME MESSAGE
• REFERENCES

2
 LIST OF ABBREVIATION
• CLSI: Clinical and Laboratory Standards Institute
• IFCC: International Federation of Clinical Chemistry
• ISO: International Organization for Standardization
• NCCLS: National Committee for Clinical Laboratoryoratory
Standards
• QMS: Quality Management System

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 TERMINOLOGIES 1/2
• REFERENCE INDIVIDUAL is an individual selected for comparison
using defined criteria.
• REFERENCE POPULATION consists of all possible reference
individuals.
• REFERENCE SAMPLE GROUP is an adequate number of reference
individuals taken to represent the reference population.
• REFERENCE VALUE is the value (test result) obtained by observation or
measurement of a particular quantity on an individual belonging to a
reference sample group. (Higgins, 2009)
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 TERMINOLOGIES 2/2

• REFERENCE DISTRIBUTION is the statistical distribution of reference

values.
• REFERENCE LIMIT is derived from the reference distribution and is
used for descriptive purposes.
• REFERENCE INTERVAL is the range between and including two
reference limits.
• REFERENCE RANGE the difference between the highest and lowest
value in a number set. (Higgins, 2009)
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Fig 1:The concept of reference interval. Adapted from Higgins, 2009 6
 INTRODUCTION 1/4

• A reference typically refers to a source of information or a point

of comparison that is used to provide context, support, or
validation.
• Therefore a reference interval is the value between and
including two the range of values that is deemed normal for a
physiological measurement in healthy persons.

(Horowitz, 2008)

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 INTRODUCTION 2/4

• Reference intervals are important tools for accurate interpretation of

results for Laboratory testing. It is a basis for comparison for a
physician or Medical Laboratory Scientists to interpret a set of test
results for a particular patient clinical care.
• They can be used in clinical trials for appropriate assessment of health
status, monitoring disease progression, and reporting of possible
toxicity and adverse events.
(Ozarda, 2016)

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 INTRODUCTION 3/4

• These intervals are established by testing a large population of healthy

individuals and determining the intervals within the majority the results
fall.
• Reference intervals can vary from Laboratory to another and maybe
influenced by factors such as age, sex, population demographics.

(Horowitz, 2008)

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 INTRODUCTION 4/4

Hence, the standard definition of a reference intervals for a particular

measurement is defined as the interval between which 95% of values
of a reference population fall into, in such a way that 2.5% of the time
a value will be less than the lower limit of this interval, and 2.5% of
the time it will be larger than the upper limit of this interval, whatever
the distribution of these values.
(Bangert et al., 2009)

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 HISTORICAL BACKGROUND 1/2

• The concept of ‘reference intervals’ as known today was developed by

Gräsbeck and Saris in the late sixties and presented at a congress of the
Scandinavian Society in 1969.
• Previously, reference intervals were usually named ‘normal values’ or
‘normal ranges’ without a clear definition of the term.
• Twenty years later, the International Federation of Clinical Chemistry
(IFCC) expert panel published the first official IFCC document on the
theory of reference values. (Ceriotti, et al., 2009)
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 HISTORICAL BACKGROUND 2/2

• Today, Laboratory Quality Management System (QMS) provides for all

processes that allow proper control of Medical Laboratory procedures,
which is a key element in ISO quality system standard (ISO 9001, 15189),
and is a requirement of most accreditation bodies/agencies. Hence,
verifiable Reference intervals is part of this requirement.

(Ceriotti, et al., 2009)

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 TYPES OF REFERENCE INTERVALS

1. Population- based reference intervals: Derived from healthy population

2. Patient-specific reference intervals: Based on an individual patient’s
previous test results. Reflects the patient’s own physiological baseline
3. Therapeutic-based reference intervals: Use to adjust the dosage of
medication or treatment for individual patients.

(Fiseha et al., 2022)

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 FEATURES OF REFERENCE VALUES 1/3

All reference intervals should have a lower and upper limit, values above
upper or below lower limit may be abnormal.
X Y

LOWER UPPER
LIMITS LIMITS
NORMAL
VALUES

(Fiseha et al., 2022)

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 FEATURES OF REFERENCE VALUES 2/3
• The upper & lower limits are set to define a specified percentage
(usually 95%) of the values for a population: this means that a
percentage (usually 5%) of patients will fall outside of the reference
interval in the absence of any disease condition.
• Normal value is the range of results between 2 medical decision point
that correspond to the central 95% of results from healthy population.
• 2.5% of the results will be above the upper limit and 2.5% will be below
the lower limit of the normal values.
(NCCLS, 2000)
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 FEATURES OF REFERENCE VALUES 3/3
• Developing reference intervals often has a financial impact on vendors
and marketers of Laboratory products.
• The main 2 types of reference intervals studies are:
1. Establishing a reference interval, &
2. Verifying a reference intervals.

(NCCLS, 2000)
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 ESTABLISHING A REFERENCE INTERVAL

A new reference interval is established when there is no existing analyte or

methodology in the clinical or reference Laboratory with which to conduct
comparative studies. It is a costly and Labor-intensive study that will
involve Laboratory resources at all levels and many require from 120 to as
many as over 700 study individuals.

(NCCLS, 1999)

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 LIMITATIONS OF ESTABLISHING REFERENCE
INTERVALS

• Population variability • False positive and negative results

• Small sample size • Non-standardized method
• Temporal variability • Ethical concerns
• Disease spectrum • Limited scope.
• Individual variation
• Co-morbidity

(Miller et al., 2016)

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 VERIFYING A REFERENCE VALUES
(TRANSFERENCE)
• This is done to confirm the validity of an existing reference interval for
an analyte using the same type of analytical system and population.
• These are the common reference interval studies performed in the
clinical Laboratory and can require as few as 20 study individuals.

(NCCLS, 1999)

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 PROCESS FOR VERIFYING A REFERENCE
INTERVAL 1/3
• Define the analyte (measured) for which the reference interval is
being verified.
• Define the method used, and analytical specificity.
• Define important pre-analytical considerations together with any
actions in response to the interference.

(Jones and Barker., 2008)

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 PROCESS FOR VERIFYING A REFERENCE
INTERVAL 2/3
• Define the principle behind the reference interval (i.e. central 95%
etc.)
• Describe the data source(s), including: number of subjects, nature of
subjects.
• Define considerations of partitioning based on age, sex etc. depending
on the analyte of interest.

(Jones and Barker., 2008)

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 PROCESS FOR VERIFYING A REFERENCE
INTERVAL 3/3
• Define the number of significant figures, i.e. the degree of
rounding (3-4 decimal places).
• Define the clinical relevance of the reference limits.
• Consider the use of common reference intervals (i.e. without
reference to age, sex, etc. as seen in plasma Glucose levels).
• Quality control.

(Jones and Barker., 2008)

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 STATISTICAL METHODS FOR REFERENCE
INTERVAL
• Non-Gaussian distribution
• Percentile method
• Gaussian curve
• Normal distribution
• Log-normal distribution
• Logarithmized sample values
• Bimodal distribution
(Jones and Barker., 2008)

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 NON-GAUSSIAN DISTRIBUTION 1/3
• Non-Gaussian refers to data or distributions that do not follow a
Gaussian distribution (i.e. normal distribution).
• For data that is non-Gaussian (e.g. skewed or curved), the data can be
mathematically transformed, e.g. to logarithms. If it yields a Gaussian
distribution, the geometric mean ± 2 SD can be used for reference
interval determination.

(Walton, 2012)
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 NON-GAUSSIAN DISTRIBUTION 2/3

However, in most instances, non-parametric statistical methods,

specifically percentiles, are used to create reference intervals from non-
Gaussian data. The top 97.5 and bottom 2.5 percentiles are used as the
upper and lower limits of the reference interval (includes only the middle
95% of data, just like Gaussian data).

(Walton, 2012)

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NON-GAUSSIAN DISTRIBUTION 3/3

Figure 2: A Non-Gaussian distribution. Adapted from Walton, 2012)

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 PERCENTILE METHOD

Figure 3: Percentile method. Adapted from Martins, 2017.

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 BIMODAL DISTRIBUTION 1/2

• A bimodal distribution has two modes. In other words, the outcome of

two processes with different distributions are shown together in one set of
data. It is also known as double-peaked distribution.
• Two reference ranges can be established for the two different groups of
people, making it possible to assume a normal distribution for each
group. This bimodal pattern is commonly seen in tests that differ between
men and women
(Hessing , 2022)

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BIMODAL DISTRIBUTION 2/2

Figure 4: Bimodal distribution. Adapted from Hessing, 2022.

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 DATA ANALYSIS TO VERIFY TRANSFERENCE
The CLSC allows less vigorous studies to verify a reference interval with
as few as 20 subject specimen.
The manufacturer’s reported 95% reference limits may be considered
valid if no more than 10% of the test subjects fall outside the original
reported limits. If more than 10% of the values fall outside of the
proposed interval, an additional 20 or more specimens should be
analyzed.
(CLSI, 2008)

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 DATA ANALYSIS TO VERIFY TRANSFERENCE
Most patient data are not normally distributed because, these data may be
skewed or exhibit multiple centiles (Bimodal, trimodal etc.). Therefore,
the analysis of patient data require “non-parametric” approach, and this is
encountered in Reference Interval Studies.
When possible, clinical Laboratory rely on manufacturers to determine
reference intervals. This avoids the expensive and lengthy process of
establishing a reference range interval on a minimum of 120 healthy
people. (CLSI, 2008)

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 DATA ANALYSIS TO VERIFY TRANSFERENCE

If the second attempts at verification fails, the Laboratory should re-

examine the analytic procedure and identify and difference between the
Laboratory population & the population used by the manufacturer for their
analysis. If no differences are identified, the Laboratory. May need to
establish the reference interval using at least 120 individuals and at most
700 individuals.
(CLSI, 2008)

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DATA ANALYSIS TO VERIFY TRANSFERENCE

Fig 5: Reference interval Histogram adapted from CLSI, 2008

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 FACTORS AFFECTING REFERENCES VALUES

• Age • Exercise
• Gender • Time of sampling
• Diet • Type of specimen
• Ethnicity • Geographic locations
• Physical size • Analytical methods
• Pregnancy

(Fiseha et al., 2022)

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 SIGNIFICANCE OF REFERENCE INTERVALS IN
CLINICAL CHEMISTRY 1/3
• Diagnostic Tool: Reference intervals provide a basis for determining
whether a patient's test results are within a normal, healthy range or
indicate a potential medical issue.
• Treatment Monitoring: Doctors use reference intervals to monitor
patients' progress during treatment.
• Research and Clinical Trials: Reference intervals serve as a standard in
clinical research and drug trials, helping researchers assess the impact of
new treatments on patients' health. (Koerbin et al., 2016)
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 SIGNIFICANCE OF REFERENCE INTERVALS IN
CLINICAL CHEMISTRY 2/3
• Quality Control: Laboratories use reference intervals to validate the
accuracy and precision of their testing methods. Consistently producing
results within established reference intervals is a sign of quality in
Laboratory testing.
• Population Health: Reference intervals are useful for studying population
health trends and variations. They can help identify regional or
demographic differences in health.
(Koerbin et al., 2016)
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 SIGNIFICANCE OF REFERENCE INTERVALS IN
CLINICAL CHEMISTRY 3/3
• Risk Assessment: Reference intervals help assess an individual's risk for
certain diseases. For example, cholesterol levels outside the reference
interval may indicate a higher risk of heart disease.
• Patient Education: Doctors can use reference intervals to educate patients
about their health and the significance of their test results.

(Koerbin et al., 2016)

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 CONCLUSION
Reference intervals are essential tools in clinical Laboratory that aid in
accurate diagnosing, proper treatment and patients monitoring. They provide
valuable context for interpreting individual test results.

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 RECOMMENDATION
• Chemical Pathology department in FMC Bida should verify the
Reference Interval for all test analytes.
• Therefore, Medical Laboratory scientist should continue to participate
in quality improvement activities that deal with relevant areas and
outcomes of patients care.
• Because, according to ISO 1518 (4.12.4), Medical Laboratory Shall
implement quality indicator (quality control) to systematically monitor
and evaluate Laboratory’s contribution to patient care.

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 TAKE HOME MESSAGE

When we talk about “ Reference Intervals’’ we talk of processes of “Quality

Control’’; and when we talk about “Quality Control, we are talking about “
process Improvement” which is one of the 12 Quality System Essentials in
the Medical Laboratory for inputs (patient samples) that are turned into
outputs (patient test results). Therefore, patient test results SHOULD
always be accompanied with verifiable Reference Intervals for meaningful
clinical patients care.

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 REFERENCE
Bangert, S., Marshall, W. J., & Leonard, W. (2008). Clinical
biochemistry: metabolic and clinical aspects. Elsevier. 4(3): 10-11.
Ceriotti, F., Hinzmann, R., & Panteghini, M. (2009). Reference
intervals: the way forward. Sage Journal. 1(46):1.
Clinical and Laboratory Standard Institute (CLSI). (2008) Defining,
establishing, and verifying reference intervals in the clinical
Laboratoryoratory; approved guideline, 3rd ed. CLSI document. C28-
P3.
Fiseha, T., Alemayehu, E., Adem, O. M., Eshetu, B., & Gebreweld, A.
(2022). Reference intervals for common clinical chemistry
parameters in healthy adults of Northest Ethiopia. Plos ONE.
17(11):10-13.
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 REFERENCE
Hessing, T. (2022). Bimodal distribution. Six sigma. 1-3.
Higgins, C. (2009). An introduction to reference intervals (1) – some
theoretical considerations. Acutecaretesting.org.
Horowitz, G. (2008). Reference intervals: practical Aspects. The
electronic Journal of the International Federation of Clinical C
hemistry. 19(2):95-105.
Jones, G., & Barker, A. (2008). Reference intervals. Clinical
Biochemistry Review. 1(29):93-97.
Koerbin, G., Cavanaugh, J., Potter, J. M., Abhayaratna, W. P., &
West, N. P. (2016). Estimation of reference intervals for aminotransferases
and derivation of de-identified reference intervals for gamma glutamyl
transferase in a healthy Australian population. Clinical Biochemistry.
49(13-14):1037-1040.
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 REFERENCE
Martins, J. W. (2017). Sampling Distribution, confidence interval,
percentile. Oneclass.com. 2
National Committee for Clinical Laboratoryoratory Standards (NCCLS).
(2000). How to define and determine reference intervals in the clinical
Laboratoryoratory; approved guideline. Wanye publishing. C28-A2.
Özarda, Y. (2016). Reference intervals: current status, recent developments
and future considerations. Biochemical Medicine. 1(26):5–16.
Reed, A., Henry, R., Mason, W. (1971) Influence of Statistical Method
Used on Resulting Estimate of Normal Range. Clinical
Chemistry. 17:275-276.
Walton, R. M. (2012). Subject-based reference values: biological variation,
individuality, and reference change values. Veterinary Clinical
pathology. 2(41):175-181.
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