ESTABLISHMENT OF

REFERENCE INTERVALS
Reference interval:
 A pair of medical decision points that span the limits of
results expected for a given condition.
 The upper and lower reference 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
condition or disease.
 Reference intervals are sometimes referred to as
reference ranges.
 The preferred term is reference interval
because range implies the absolute maximum and
minimum values.
 Reference interval are required by professional
accreditation and regulatory standards.
 In most cases, a normal range is used as the reference
interval.
 The term ‘normal’ has been replaced by the term
‘reference’.
Who will establish reference interval?
 Reference intervals are usually established by the
scientific community or the manufacturers of reagents and
new methodologies.
REFERENCE INTERVAL STUDIES
1. 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 may require from 120 to as many as 700
study individuals.
 REFERENCE INTERVAL STUDIES

2. Verifying a reference interval (transference):

 This is done to confirm the validity of an existing reference interval
for an analyte using the same (identical) type of analytic system
(method or instrument).
 These are the most common reference interval studies performed
in the clinical laboratory and can require as few as 20 study individuals.
 TERMINOLOGY RECOMMENDED BY IFCC

Reference individual
Reference population
Reference sample group
Reference value
Reference distribution
Reference limit
Reference interval
Observed value
 Reference individual:
 An individual selected for comparison using defined criteria.
eg: age,sex,health status.
 Reference population:
 consists of all possible reference individuals. It usually has
an unknown number and is therefore a hypothetical entity.
 presence of health is not implied, allowing the construction of
reference intervals for both the healthy and the sick.
 Defining the reference population is fundamental for the
preparation of effective reference intervals.
 Reference sample group:
 an adequate number of reference individuals taken to
represent the reference population.
 Ideally they should be randomly drawn from the reference
population.
 Reference value:
 value (test result) obtained by observation or
measurement of a particular quantity on an individual
belonging to a reference sample group.
 Not to be confused with reference limit
 Reference distribution
 The statistical distribution of reference values.
 Hypotheses regarding reference distribution obtained from a reference
population can be tested using the reference distribution of the
sample group and adequate statistical methods.
 The parameters of the hypothetical distribution of the reference
population may be estimated using the reference distribution of the
reference sample group and adequate statistical methods.
 Reference limit:
 derived from the reference distribution and is used for descriptive
purposes.
 It is common practice to define a reference limit so that
a stated fraction of the reference values is less than or equal to, or
more than or equal to the respective upper or lower limit.
 A reference limit is descriptive only of reference values and should
not be confused with the term “decision limit.
 Observed value
 (patient test result) is the value of a particular type of
quantity obtained by either observation or measurement
and produced to make a medical decision.
 It can be compared with reference values, reference
distributions, reference limits or reference intervals.
Types of reference interval

1.Medical decision level:

Value for an analyte that represents the boundary between
different therapeutic approaches.
2.Normal range:
Range of results between two medical decision points that
correspond to the central 95% of results from a healthy patient
population.Of the results, 2.5% will be above the upper limit and 2.5%
will be below the lower limit of the normal range.
3. Therapeutic Range:
 Reference interval applied to a therapeutic drug.
 Reference intervals are needed for all tests in the clinical
laboratory.
 The provision of reliable reference intervals is an important task
for clinical laboratories and test manufacturers.
 The dynamic review of existing reference intervals by the
health care team (scientific community, manufacturers, and
clinical laboratory) is crucial to meeting the challenges of
providing optimal laboratory data for patient care.
Reference interval use can be grouped into 3 main
categories:
 Diagnosis of a disease or condition
 Monitoring of a physiologic condition
 Therapeutic management
 PICTURE
QUESTIONS REGARDING REFERENCE INTERVALS FOR
LABORATORY
Has the laboratory established or verified its reference intervals ?

 Reference intervals are important to allow a clinician to assess patient

results against an appropriate population.
 The reference range must be established or verified for each analyte
and specimen source (e.g., blood,urine,CSF, when appropriate.
 For many analytes (e.g., therapeutic drugs and CSF total protein )
literature references or a manufacturer’s package insert information may
be appropriate.
QUESTIONS REGARDING REFERENCE INTERVALS FOR
LABORATORY
Does the laboratory evaluate the appropriateness of its reference
intervals,and take corrective action if necessary?
Criteria for evaluation of reference intervals include
 Introduction of a new analyte to the test repertoire
 Change in analytic methodology
 Change in patient population
If it is determined that the range is no longer appropriate for the
patient population, corrective action must be taken.
Steps to establish a reference interval
 Define an appropriate list of biological variation and
analytic interferences from medical literature.

 Choose selection and partition criteria.

 Complete a written consent form and questionnaire to

capture selection criteria
Categorize the potential reference individuals based on the
questionnaire findings

•Exclude individuals from the reference sample group based

on exclusion criteria
Define the number of reference individuals in consideration
of desired confidence limits and statistical accuracy
Standardize collection and analysis of reference specimens
for the measurement of a given analyte consistent with the
routine practice of patients

Inspect the reference value data and prepare a histogram to

evaluate the distribution of data

Identify possible data errors and/or outliers and then analyze

the reference values
Selection of reference individual

 All group of reference individuals should be defined.

 Condition under which reference specimen were obtained
and processed should be known and condition should be
same for all patient specimen.
 The analyte of interest should be same for patient and
reference individuals.
 Results produced using adequate standardized methods
under sufficient analytical quality control.
 For detailed approach stages in pathogenesis or disease
should be demarcated for diagnosis.
 It is important to distinguish selection and partitioning criteria.
 Selection criteria applied to obtain a group of reference
individuals. This group is subdivided into subgroups using
partitioning criteria. Whether specific gender is a selection or
partitioning criteria depends on the purpose of the actual
project.
Pre analytic and analytic considerations for reference interval

Pre analytic factors Analytic factors

 Subject preparation  Precision
 Prescription medications  Accuracy
 Collection time  Linearity
 Sample storage  Interference
 Stress  lot to lot reagents
 Food / beverage ingestion  Recovery
 Analysis of reference values
Non parametric method Parametric method

statistical test that makes no specific  Statisticaltest that assumes
assumption about distribution of data. the observed values or some
 Non parametric methods rank the mathematical transformation
reference data in order of increasing of those values, follows a
size. (normal) gaussian
 Majority of analytes are not normally distribution
distributed, non parametric tests are
recommended analysis for most
reference range intervals
 Data analysis to verify a reference interval
( transference)
 When possible, clinical laboratories rely on assay manufacturers or on

published primary literature to determine reference intervals.

 This avoids the expensive and lengthy process of establishing a
reference range interval on a minimum of 120 healthy people.
 The CLSI allows less vigorous studies to verify a reference interval
with as few as 20 subject specimens.
 Method verification studies can be used if the test method and study
subjects are similar to the vendor’s reference data and package insert
information.
 The main assumption in using transference studies is that
the reference method is of high quality and the subject
populations are similar.
 The manufacturer’s reported 95% reference limits may be
considered valid if no more than 10% of the tested subjects
fall outside the original reported limit.
Diagnostic efficiency
 Diagnostic sensitivity

Ability of a test to detect a given disease or condition.
 Diagnostic specificity
 Ability of a test to correctly identify the absence of a given
disease or condition.
 Positive predictive value:

chance of an individual having a given disease or condition

if the test is abnormal.
 Negative predictive value:

chance of an individual does not have a given disease or

condition if the test is within the reference interval.
COMMON PROBLEMS ENCOUNTERED WHEN
MONITORING
REFERENCE INTERVALS
 Changes in reagent formulations by the vendor
(e.g., new antibody)
 Erroneous initial reference interval either by the vendor or
laboratory
 Minor changes in reagents due to lot-to-lot variations
 Differences between reference interval and test populations
THANK YOU