Establishment of Reference

Values in the Clinical

Chemistry Laboratory
Moutasem Elsheikh
Sep. 28, 2010

Background

A persons
health is determined by comparison, thus health is

relative.
Medical interpretation by comparison could be done by:
A) Intuitive assessment base on clinical experience.
B) Reasoning using advanced knowledge of normal and
pathological anatomy, physiology, biochemistry, etc.
To arrive at a diagnosis for an individual, laboratory tests are
compared to data from healthy persons, patients with relevant
diseases, and previous values from the same subject.

Definition

What is a Reference Range/value/limit/interval, observed

values, normal value, or critical value?
It is a set of values of some measurement that a
healthcare professional can use to interpret a set of
results for a particular patient (Wikipedia).

Definition

Reference Value is a value obtained by observation or

measurement of a particular type of quantity on a

reference individual.
Reference Individual is an individual selected for
comparison using a defined criteria.
Observed Value is a value of a particular type of quantity,
obtained by observation or measurement and produced
to make a medical decision by comparing them with a
reference value (observed value=lab result=test value).

Comparison Criteria

To make a valid comparison between observed and reference

values, the following should be met:

1. Reference individuals should be clearly defined.
2. Patient examined should resemble reference individuals.
3. Specimen collection/processing for analysis should be similar.
4. Quantities compared should be of the same type.
5. Standardized methods under adequate analytical QC should
be used to produce lab. results.

Selection of Reference
Individuals

A selection criteria should be applied to select a group of

reference individuals whom could be partitioned into more
homogeneous subgroups (base on gender, age, ethnic
group, etc).
Reference individuals must be healthy?
WHO definition of health: a state of complete physical,
mental, and social well-being and not merely the absence of
disease or infirmity.

Selection Criteria for Reference

Individuals

a) Direct: individuals are selected using a defined criteria.

b) Indirect: analytical values in a laboratory database are
used (simple, inexpensive).
In the Indirect method, analytical values are plotted and
mathematical methods are used to estimate the normal interval
by extracting it from total distribution.

Selection Criteria for

Reference Individuals

c) Random: each and every individual has an equal

chance of
being chosen.
d) Non-random: each and every individual has an unequal
chance of being chosen.

Exclusion Criteria for

Reference Individuals

Medical Interview
Questionnaire
Supplementary investigation

Physical Exam

Partitioning
of
the
Reference
Group

Afterselecting a group of reference individuals, they could be subgrouped or partitioned into more homogenous groups based on:
A) Age.
B) Gender.
C) Genetic Factors (e.g. Ethnic origin).
D) Physiological Factors (e.g. stage in menstrual cycle, stage
in pregnancy, exercise).
E) Other factors (e.g. environmental, socioeconomic).
Partitioning is necessary if differences between subgroups are statistically significant.

Sample Collection

Preparation of the reference individual prior to specimen

collection, procedure for specimen collection, handling
and storing of the specimen prior to analysis should be
standardized to eliminate bias or variability from these
factors.

TEITZ Text book of Clinical Chemistry and Molecular Diagnosis, page 432

Sample analysis-Analytical
Procedures and QC

The same method used to estimate observed values should

be used to establish reference values to ensure
comparability.
The following should be checked prior to sample analysis:
A) Method reliability (precision, accuracy, analytical
range, detection limit, and analytical specificity).
B) QC program (control of pre-analytical variables,
analytical variables, using stable control materials,
etc).

Pre-analytical VariablesSnapshot

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ok
Sm

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Po

ur

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Me

TEITZ Text book of Clinical Chemistry

And Molecular Diagnosis
pages 450,452,457,461

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Ex

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cis

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op

Sample Size

A sample
size of 120 has been

recommended by the International

Federation of Clinical Chemistry (IFCC) and the Clinical and
Laboratory Standards Institute [formally National Committee for
Clinical Laboratory Standards (NCCLS)].
A smaller sample size could be used for analytes that does not
vary widely with age, gender, and other partitioning factors
(Sodium is constant while creatinine is variable)

Statistical Analysis of Data

A reference range for an analyte is calculate roughly as

the values that 95% of the data fall into or the mean + 2
STDEV. This relies on the fact that almost all biological
phenomena follows a Gaussian Distribution.
Mean

Factors to consider when

establishing a Reference
Interval:

1) Makeup of the reference individuals (age, gender, etc).

2) Inclusion and Exclusion criteria for reference individuals.
3) Physiological and environmental conditions under which
reference individuals were studied and sampled.
4) Specimen collection procedure including patient prep.
5) Analytical method used including details of its precision
& accuracy.

52

Minimu
m

89

Maximu
m

96

Points

70

Mean

71

Median

9.31

StdDev

86.63

Varianc
e

Statistical Analysis of Data-Example

Chemical Pathology 2ed Year

20

Mean 70.18
StDev 9.308
N
96

10

0.95

10

20

30

40

50
Score

60

70

80

90

100

99.9
99

Skewne
ss

-0.81

StDev
9.308
Kurtosis

95
90
80
70
60
50
40
30
20
10
5
1
0.1

40

50

60

70
Score

80

Std
Error

-0.20

Probability Plot of CHEM 2EDY

Percent

Frequency

15

90

100

Mean

70.18

N
AD
P-Value

96
0.740
0.052

http://en.wikipedia.org/wiki/File:Reference_ranges_for_blood_tests_-_by_units.png

Reference Intervals

University of California San Francisco, Departments of Pathology & Laboratory Medicine

http://labmed.ucsf.edu/labmanual/mftlng-mtzn/test/index-normal.html

References

1. Establishment
and use of Reference Values. Textbook
of
Clinical Chemistry and Molecular Diagnostics.
Chapter 16:
Establishment and use of reference values, page 425.

2. Reference Intervals (normal range). Clinical

Chemistry,
Principles, Procedures and Correlation (M.L. Bishop).
Chapter 3, page 57.

Backup Slides

Percentile Rank in relation to normal

distribution