Validate, Transfer or Establish: What Are You Doing with Your Reference Intervals?

Reference intervals are absolutely necessary for proper interpretation of laboratory tests, and yet obtaining appropriate reference intervals can be the bane of the laboratory. I mentioned establishing, validating or transferring reference intervals in an earlier blog post, but didn’t talk about exactly what these are and when to use which one.

Establishing a reference interval is exactly what it says. A reference interval must be established if a new assay has never been performed in the lab and there is no current reference interval to start with. Most often, laboratory developed tests (LDTs) that are developed from scratch will require the establishment of a reference interval. To do this, ideally 120 samples from healthy individuals for each sub-population (gender or age sub-group) is used, although there are methods available using smaller sample sizes. Samples used to establish reference intervals may be collected a priori, meaning they are collected from individuals for the express purpose of establishing a reference interval, with well-defined inclusion or exclusion criteria used, or a posteriori, meaning they are samples collected and analyzed first, with exclusion criteria applied after statistical analysis.

Validating a reference interval is the easiest way to obtain one, and is what is hoped for when a new method is introduced. Validation is usually used when a new instrument or method replaces an old one, and reference intervals are currently in place. A patient correlation study is done using at least 20 patients. The data is analyzed with regression, bias and correlation statistics. If the bias and regression are acceptable, the reference interval that is currently in place will also work with the new assay. The interval has been validated and can be used with the new method.

When a validation study is done for a new method and the results of the data analysis are NOT acceptable to validate the assay, then a transference study is necessary. A transference study is simply an extended correlation. More than 20 patients are used, enough to determine the amount of bias between the two methods. Then the old reference interval is adapted to fit the new method, using the amount of bias determined. For example if the new method measures 15 percent higher than the old method, the reference intervals will be increased 15 percent across the board. Transference is recommended to be performed once. If another new method is brought in for that analyte, rather than transfer the reference interval again, a new interval should be established.

All three of these methods for obtaining reference intervals are useful in the right situations. It is important to know when to use which method.

 

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-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

Reference Ranges

According to Wikipedia, reference ranges in health-related fields are generally defined as “the prediction interval between which 95% of values of a reference group fall into, in such a way that 2.5% of the time a sample value will be less than the lower limits of this interval, and 2.5% of the time it will be larger than the upper limit of this interval, whatever the distribution of these values.”

In other words, reference ranges are important! They provide the necessary context for medical analysis and diagnosis. Without a reference range (also sometimes referred to as reference value or reference interval) medical professionals have no comparison group for which to make diagnosis and advise treatment.

In all instances where reference ranges are used, context is key. In sub-Saharan Africa many labs use European established reference ranges which represent a primarily Caucasian population. This is because reference ranges specific to populations in sub-Saharan Africa do not universally exist. This presents a problem as many factors can contribute to what is considered “normal” in different populations. Genetics, dietary patterns, pregnancy, gender, age, ethnic origin, and prior exposure to pathogens all can influence reference range values.

Establishing accurate reference ranges for a given population takes time and an enormous amount of resources. It is often recommended that laboratories establish their own reference ranges based upon the population that they serve. This is cost and resource prohibitive for many laboratories in the developing world. In absence of region specific reference ranges, it is recommended that each lab validate existing ranges using their own population. However, even this can be prohibitive in resource (both physical and human) limited settings.

This can lead to egregious errors in disease diagnosis and treatment. Clement Zeh, Collins Odihiambo and Lisa Mills write that reference range research thus far reveals that African populations differ from their European/Caucasian counterparts with lower hemoglobin, red blood cell counts, hematocrit, mean corpuscular volume, platelet counts, and neutrophil counts  and higher monocyte and eosinophil counts (see http://www.intechopen.com/books/blood-cell-an-overview-of-studies-in-hematology/laboratory-reference-intervals-in-africa for their chapter on Laboratory Reference Intervals in Africa).

In addition to diagnosis and treatment of individuals, reference ranges are crucial components in drug and vaccine studies. Historically, clinical trials of drugs and vaccines have relied upon ranges developed in the Western world. This can have significant impact upon the research data resulting in health risks to study participants, poor data, and huge amounts of resources wasted.

Thus, while it is costly and time consuming, reference ranges specific to populations in countries in the developing world need to be established. This would help both the treatment of individuals, and the testing, study and development of important vaccines and drugs.

-Marie Levy