ESTABLISHMENT OF REFERENCE

INTERVAL

Dr. S. Rathore

MD (Sco)Dept. Of Biochemistry

Pt. J. N. M. Medical College,Raipur

INTRODUCTION Reference interval:

A pair of medical decision points that span the limits of results expected for a given condition

Reference interval are required by professional accreditation and regulatory standards

In most cases, a “normal range” is used as the test’s “reference Interval”

For some analytes, the reference interval is defined as “less than” or “greater than” a certain value Example: total cholesterol: <200mg is desirable

WHY IT IS NEEDED….. To make medical diagnoses Assess physiologic function Manage therapy When interpreting laboratory data,

clinicians compare the measured test result from a patient with a reference interval

Reference intervals are sometimes referred to as reference ranges; the preferred term is reference interval because range implies the absolute maximum and minimum values

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

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 and/or instrument).

These are the most common reference interval studies performed in the clinical laboratory and can require as few as 20 study individuals.

TYPES OF REFERENCE INTERVAL Medical decision level:

Value for an analyte that represents the boundary between different therapeutic approaches

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

Therapeutic range: Reference interval applied to a therapeutic drugReference intervals are needed for all tests in

the clinical laboratoryThe 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 three main categories: Diagnosis of a disease or condition Monitoring of a physiologic condition And therapeutic management Examples:

QUESTIONS REGARDING REFERENCE INTERVALS FOR LABORATORY Has the laboratory established or verified its

reference intervals (normal values)?

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, cerebrospinal fluid [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

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 RANGE

1 •Define an appropriate list of biological variations and analytic interferences from medical literature

2 •Choose selection and partition (e.g., age or gender) criteria

3 •Complete a written consent form and questionnaire to capture selection criteria

4 • Categorize the potential reference individuals based on the questionnaire findings

5 •Exclude individuals from the reference sample group based on exclusion criteria

6 • Define the number of reference individuals in consideration of desired confidence limits and statistical accuracy

7 • Standardize collection and analysis of reference specimens for the measurement of a given analyte consistent with the routine practice of patients

8 • Inspect the reference value data and prepare a histogram to evaluate the distribution of data

9 •Identify possible data errors and/or outliers and then analyze the reference values

SELECTION OF REFERENCE INDIVIDUALS A set of selection criteria determines which

individuals should be included in the group of reference individuals

Such selection criteria include Statement describing the source population Specifications of criteria for health The disease of interest

Often, separate reference values for each sex and for different age groups, as well as other criteria, are necessary

Our group of reference individuals therefore may have to be divided into more homogeneous subgroups. For this purpose, specific rules for the division, called stratification of partitioning criteria, are needed

It is important to distinguish between selection and partitioning criteria. First, selection criteria are applied to obtain a group of reference individuals Thereafter,this group is divided into subgroups usingpartitioning criteria. Whether a specific criterion (e.g., gender) is a selection or a partitioning criterion depends on the purpose of the actual project.

PREANALYTIC AND ANALYTIC CONSIDERATIONS

ANALYSIS OF REFERENCE VALUES Nonparametric method:

Statistical test that makes no specific assumption about the distribution of data

Nonparametric methods rank the reference data in order of increasing size

Because the majority of analytes are not normally (gaussian) distributed , nonparametric tests are the recommended analysis for most reference range intervals

Parametric method: Statistical test that assumes the observed

values, or some mathematical transformation of those values, follows a (normal) gaussian distribution

Confidence interval: Range of values that include a specified

probability, usually 90% or 95%

For example, consider a 95% confidence interval for slope = 0.972–0.988 from a method comparison experiment. If this same experiment were conducted 100 times, then slope would fall between 0.972 and 0.988 in 95 of the 100 times

Confidence intervals serve to convey the variability of estimates and quantify the variability.

Bias: Difference between the observed mean and

the reference mean

Negative bias indicates that the test values tend to be lower than the reference value

Whereas positive bias indicates test values are generally higher. Bias is a type of constant systematic error

With the development of statistical software packages such as EP Evaluator MedCalc, GraphPad Prism Minitab JMP SAS/STAT Now reference intervals are rarely determined manually

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 Parameters are broadly defined as diagnostic

efficiency, which can be broken down into sensitivity, specificity, and predictive values

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 an individual does not have a given disease or

condition if the test is within the reference interval

ReferencesTietz Textbook of Clinical Chemistry and Molecular Diagnostics

Clinical Chemistry: Principles, Techniques, and Correlations - Bishop, Michael L, MS.