**American Society for Veterinary Clinical Pathology**

2424 American Lane

Madison, WI 53704

**Telephone**: +1-608-443-2479

**Fax:** +1-608-443-2474

**email**: info@asvcp.org

**Decision limits** are required for interpretation of laboratory
test values. Decision limits may be **cut-points** or **reference
intervals**. At least one type of decision limit is required to
answer relevant clinical problems or questions.

Cut-points
are used to differentiate health from disease or, of greater clinical
relevance, to differentiate diseases with similar presenting problems.
Diagnostic sensitivity and diagnostic specificity are determined at different
cut-points. **Receiver-operating-characteristic (ROC) curves** can be used to help identify optimal cut-points for different clinical
needs. If disease prevalence is known, the predictive values of a positive
and negative test can be calculated.

Alternatively, clinicians may rely upon reference intervals for interpreting patient test values. Due to the many combinations of factors affecting test results, including instrumentation, reagents, technologists, collection and processing of samples, animal related factors, etc, reference intervals should be confirmed in each laboratory. The reference intervals reported by the laboratory typically are the lowest and highest value expected for the central 95% of similar healthy individuals.

International Federation of Clinical Chemists (IFCC) guidelines are recommended for use when developing reference intervals in veterinary diagnostic laboratories. The IFCC discourages use of the term reference "range" due to the statistical definition of range as a single number, ie, the difference between the upper and lower limit. It is almost impossible for each veterinary diagnostic laboratory to develop and maintain reliable, current, method-related reference intervals for each test and for each subset of animals of potential clinical interest. Frequent changes in technology and the variables of species, breed, age, sex, animal husbandry, diet, and geographic location may create significant differences among laboratories. In spite of this challenge, we recommend, at minimum, that a veterinary laboratory provide the following information to clinicians for every test:

**Reference intervals**for at least one well-defined subset of each major species. It should be stated clearly whether reference intervals were determined de novo using current methodology or whether reference intervals from a previous method were modified following comparison of old and new methods. It also should be noted if the reference intervals were historic or extrapolated from the literature.**Reference interval calculations.**The number of reference observations used for calculating reference intervals, whether data did, or did not, have normal distribution, and the parametric or non-parametric method used to calculate the 2.5 and 97.5 percentiles should be indicated. If observations were excluded, these should be listed with explanations.**Confidence intervals (tolerance limits)**for the lower and upper reference limit. This calculation requires a minimum number of observations.-
**Additional information upon request.**The laboratory should be able to provide users with additional information about the relevance and reliability of each test method. This information should include the signalment and state of health of the reference individuals, how this was determined, fasting state, sampling time, collection, storage, and processing of samples, etc. In addition, information regarding handling of outliers and observations excluded for other reasons should be described. This information should allow clients to determine whether the reference observations represent the patient(s) in question, eg, regarding breed, age, sex, and management.

The IFCC recommends a minimum of 120 observations for reference intervals. With this sample size, confidence intervals can be calculated for the lower and upper reference limits independent of whether observations have a normal (Gaussian) or non-normal distribution. The confidence interval around the lower and upper limits for 60 observations with normal distribution is similar to that for 100-120 observations with non-normal distribution. Confidence intervals indicate the reliability of the reference limits and whether a test is able to meet clinical expectations.

Although
reference intervals often are developed using fewer observations, until
>40 observations are available the best estimates for the central 95%
reference intervals are the lowest and highest values observed.

References

- Expert Panel on Theory of Reference Values (EPTRV), International Federation of Clinical Chemistry, Scientific Committee, Clinical Section. The theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. Clin Chem Acta 1984;137:97F-114F.
- Richard Jones and Brian Payne. Data for diagnosis and monitoring. In: Clinical Investigation and Statistics in Laboratory Medicine, Jones R and Payne B (eds), Association of Clinical Biochemists, ACB Venture Publications, London, 1997.