Establishing reference intervals for routine use

Laboratory results are often interpreted based on reference intervals from a group of healthy individuals. When determining reference intervals, many factors need to be considered and the reference population must be well defined. Individuals to be included in the study should reflect the overall healthy population, and demographics of the reference population should reflect the population for which the reference intervals are intended to be used (1, 2).

Selected inclusion criteria, including biological, clinical, and geographical factors, shall describe the individuals of the reference population and the methods used to verify their health. Tables 1 and 2 provides suggestions for inclusion criteria for humans and animals, respectively (2).

It is recommended to include a minimum of 120 reference individuals and to determine reference intervals using a nonparametric method, with an appropriate confidence interval to define upper and lower limits (2, 3). Statistical analyses are preferably carried out using a statistical analysis software, and outliers can be determined and excluded using an outlier removal method (2, 3).

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommends that laboratories develop their own reference Intervals (4), and according to ISO 15189:2012, reference intervals should be reviewed periodically (5). Clinical and Laboratory Standards Institute (CLSI) provides guidance on how to define, establish, and verify reference intervals (3). Reference interval guidelines for animal studies are given by the American Society for Veterinary Clinical Pathology (ASVCP) (2).

Ganie and colleagues at the Sher-I-Kashmir Institute of Medical Sciences, Srinagar, India studied reference data for reproductive-age Indian women using the Swelab Alfa Plus hematology system (6). Healthy individuals aged 18–40 years were requited from six regions. Statistical analyses of data from 472 included individuals were conducted using SPSS software (IBM), normality was assessed with the Kolmogorov–Smirnov test method, and outliers were identified and removed by the Dixon-Reed method. Notable variations were observed when comparing obtained data with data from Western populations, emphasizing the need for establishing normative ranges specific to Indian women.

Sherzay and coworkers at the Kabul University, Karte Sakhi, Kabul, Afghanistan studied reference data for a healthy group of men and women in Afghanistan using a Medonic hematology analyzer (7). Statistical analyses were conducted using the MedCalc software (MedCalc software), normality was assessed by the Shapiro–Wilk test, and outliers identified and removed using the method of Reed. Based on obtained results, they conclude that commonly used reference intervals are not valid for the study group for all parameters and should be revised for the Afghan population.

Lauderdale and fellow researchers at the Chicago Zoological Society–Brookfield Zoo, Brookfield, IL, USA compiled data for reference intervals for healthy common bottlenose dolphins and reference values for Indo-Pacific bottlenose dolphins, beluga whales, and Pacific white-sided dolphins (8). Hematology testing of samples originating from Mexico was conducted at the Universidad Nacional Autónoma de México (UNAM) laboratory using an Exigo veterinary hematology system, whereas samples originating from other regions were analyzed at the Cornell University Animal Health Diagnostic Center (AHDC) laboratory using an Advia 2120 hematology analyzer (Siemens). Statistical analyses of samples from AHDC and UNAM were conducted separately using SPSS 21 software (IBM). The generated reference data for hematology parameters are available alongside a range of biochemical parameters to animal care professionals through the ZooPhysioTrak iOS mobile software application. The user enters demographic data for an individual, including species, diagnostic laboratory (AHDC or UNAM for common bottlenose dolphins only), age, sex, and month. The ZooPhysioTrak application returns the appropriate reference intervals and values accounting for significant factors affecting each variable.

Due to, for example, variability in sample collection method and analyzer type or a limited number of individuals included in the reference group, values previously reported from different laboratories might not be directly comparable. To overcome this limitation, samples in the study by Lauderdale and colleagues were processed at service laboratories that are available for future analyses and protocols for sample collection and shipping are provided in the ZooPhysioTrak application.

The results from these studies indicate the importance of validating common reference intervals before taken into routine use, and if not valid, establish new reference intervals that reflect the population, for which the reference intervals will be used.

The reference intervals in the default profile configurations of Boule hematology analyzers shall be considered as indicative, and it is recommended for laboratories to establish their own reference intervals. For this, the default analysis profile (or reference group) can be edited, and additional profiles can be added from the Setup menu.

To change

1. In Profile (or Range depending on analyzer), select analysis profile (or reference group) to be changed.
2. Click Edit.
3. Edit reference intervals and click Save.

To add:

1. Select analysis profile (or reference group) and click New (or Copy).
2. Name the new analysis profile and click Save.
3. Edit reference intervals and click Save.

For more detailed description, please refer to the User manual for each analyzer.

Learn more

Contact us or visit the Boule web site to learn more about Boule hematology analyzers.

References

1. Boyd, J.C. Defining laboratory reference values and decision limits: populations, intervals, and interpretations. AJA 12, 83–90 (2010).
2. Friedrichs et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol 41, 441–453 (2012).
3. CLSI EP28 A3C: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory, 3rd Edition. Standard by Clinical and Laboratory Standards Institute (2010).
4. Solberg H.E. Approved recommendation (1987) on the theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. Clinica Chimica Acta 170, S13-S32 (1987).
5. Medical laboratories-Requirements for quality and competence. International Organization for Standardization G, Switzerland, international Standard ISO 15189:2022.
6. Ganie et al. Common Hematological Reference Indices Among Healthy Reproductive Age Indian Women‑Data Subset from Nationwide Study. Indian J Hematol Blood Transfus 40, 479–486 (2024).
7. Sherzay et al. Establishment of Reference Intervals of Blood Parameters Among the Healthy Afghan Population. J Blood Med 15, 69–75 (2024).
8. Lauderdale et al. Health reference intervals and values for common bottlenose dolphins (Tursiops truncatus), Indo-Pacific bottlenose dolphins (Tursiops aduncus), Pacific white-sided dolphins (Lagenorhynchus obliquidens), and beluga whales (Delphinapterus leucas). PLoS ONE 16(8): e0250332 (2021).