THE REFERENCE VALUE CONCEPT

The Reference Value Concept:

Its Definition, Function, Formulation and Misconceptions
Joemar P. Pelegrino
University of San Agustin

THE REFERENCE VALUE CONCEPT

The Reference Value Concept:

Its Definition, Function, Formulation and Misconceptions
A reference is the basis in determining the condition of the patient through laboratory
analysis, diagnosis and medical or clinical decision making. This reference is often to be used in
most clinical laboratory as a guide, based on the results from the test conducted to the patient, in
making their diagnosis this reference includes specific values, observations and interpretations
related to what test is performed to the patient whether it falls in the typical range or not,
A reference is important in every medical decision making. When a person is taken to a
hospital for diagnosis, he/she wont get the results of the test as immediately as possible. Doctors
will provide the person first the required test before making the actual diagnosis. These test will
provide results and analytical information regarding the condition of the patient which will then
be interpreted by the doctor who will give the actual state of the patient this is where reference
will take its role. The reference will serve as a guide to interpret the result that will effectively
guide the doctors a correct, accurate and precise diagnosis.
This reference does not come first-handedly. In fact, they came or derive from the series
of test, studies, and trials conducted by several or even numerous institution and/or groups before
it will be used in medical and laboratory purposes. This test has different scope and factors which
primarily depend to the institution, group or individual who conducted the test this is the
reason why some reference values is different from one sources and to the other sources.
Even though, a reference is somewhat a common knowledge when it comes to medical or
laboratory field, there are still some misconceptions surrounding it that mistakenly define what is
a reference actually is. Notice how I minimize the use of the word normal or any related words

THE REFERENCE VALUE CONCEPT

to define a reference? This because they are never been the same: in terms of values,
interpretation or on other means other than they are somewhat closely related. Also, according to
Ralph Grasbeck on his paper The Evolution of the Reference Values Concept (2004), he
pointed out that the concept of a reference, from the time it was made, is essentially
philosophical.
During the course of this research paper will primarily devoted to explain the
mathematical and statistical definition of a reference; its pivotal and integral importance of the
reference in the event of decision making; how the reference is derived or formulated from a
series of test and studies and finally to clear out any misconception regarding the concept of the
reference.
Definition
According to Merriam-Webster dictionary, literally a reference defines as the act of
mentioning something in speech or in writing : the act of referring to something or someone; the
act of looking at or in something for information. Reference being referred to in the dictionary
can be easily describe to the one found on, for example, a research paper or any academicals and
curricular writings on a footnote of a page or on a separate part of the paper. This reference is
credited to the validation and credibility of the paper and most importantly how reliable the piece
of information the author has written on that paper. In medical terminology, a reference serve as
a guide in medical diagnosis.
IFCC Recommendation
The International Federation of Clinical Chemistry (IFCC) and Laboratory Medicine is
established to set the standard procedures set in the field of Clinical Laboratory around the globe.

THE REFERENCE VALUE CONCEPT

In 1987, the Committee of Standards of the IFCC had published a paper entitled Approved
recommendation on the theory of reference value. There, is recommended to use the term
reference values in applying or relating to the concept of a reference. Additionally, there are also
several terms considered such as reference individual, reference limit, reference interval, and
observed values. This term becomes thus becomes the formal terminology in respect to the
recommendation made by the IFCC.
The following terms are defined from the Stage 2, Draft 14 of the Provisional
Recommendation on the Theory of Reference Values by the Committee of Standards of the
IFCC (1978).
Reference Value. Reference value is the value obtained by observation or measurement
of a particular type of quantity on a reference individual. (R. Grsbeck, Siest, Wilding,
Williams, & Whitehead, 1979). The reference value is derived from the series of test conducted
from a specific study with respect to the data obtained from an individual or a population.
Reference Individual. A reference individual is an individual selected using defined
criteria (R. Grsbeck et al., 1979). This individual is picked according to what study is being
conducted or what reference value is to be gathered. According to Grsbeck (2004), the subject
that was opt to become the reference individual may came from a wide selection of individuals
depending on the selection criteria, specimen collection, analytical procedures and mas noted
on the IFCC recommendation, the individuals state of health.
Reference Population. For even accurate and precise determination of the reference
value, most studies uses a reference population instead of a reference individual. A reference
population consists of all possible reference individuals (R. Grsbeck et al., 1979). In addition

THE REFERENCE VALUE CONCEPT

to the factors in selecting a reference individual, selecting individuals to be included in the

population as reference ranged from every possible circumstances to which the reference value is
consistently obtainable from that population and this includes different ages, gender, height,
weight etc.
It is noted by the IFCC (R. Grsbeck et al., 1979) that the number of individuals included
in the reference population is not fixed and it depends to the criteria set by the study as it is
suggested that the reference population is a hypothetical entity and it may or may not affect the
reference value that is being gathered in that specific study.
Reference Sample Group. When the reference value had already been gathered to an
individual, a group or a population, it is now being sorted out. A reference sample group is an
adequate number of reference individuals selected to represent the reference population (R.
Grsbeck et al., 1979). This groups value is considered as the best value than the other data
gathered with less dispersion and it is more precise and consistent when compared. This group is
vital in obtaining the reference value
Reference Limit. Reference limit is derived from the reference distribution and is used
for descriptive purposes (R. Grsbeck et al., 1979). It is noted by the IFCC that defining a
reference limit is a common practice in order to compare the reference value obtained in the
study between the limit made from the probability or the hypothesis.
Reference Distribution. Reference distribution is the distribution of reference value
(R. Grsbeck et al., 1979). IFCC recommends that this distribution of the value can be tested by
different statistical tools and methods in order to test the hypotheses relating to the reference
distribution of an individual or population.

THE REFERENCE VALUE CONCEPT

Reference Interval. Derive from the value gathered in the reference limits, a reference
interval is the interval between, and including two reference limits (R. Grsbeck et al., 1979).
Mathematical Definition
Additionally side from what is stated from the IFCC recommendation, reference limit
accoding to the book entitled Chemical Laboratory Chemistry (2011) is considered to be a
numerical value and is formulated from the data gathered from the reference distribution. In
other words, reference limit is expected to be a numerical in nature (Sunheimer & Graves, 2011).
Furthermore as suggested by the similar book, reference expresses a well-defined selection of
subjects used to mathematically determine the numerical values equivalent to reference limits
and thus reference interval (Sunheimer & Graves, 2011). Boyd (2010) also denote the definition
of reference interval as it is traced by threshold values between which the test results of a
specified percentage of apparently healthy individuals would fall (Boyd, 2010). The specified
percentage being referred by Boyd is usually 95%.
Statistical Definition
The statistical definition has a similar tone as the previous definition. According to
Dijkman, Jaap H M, et. al. (2009), the reference intervals are gathered from the data and results
generated from the reference sample (an individual or a population). Finally, in the book Tietzs
Fundamentals of Clinical Chemistry and Molecular diagnosis, it defined Reference values as
results of a certain type of quantity obtained from a single individual or group of individuals
corresponding to a stated description, which must be spelled out and made available for use by
others.
The Role and Importance of a Reference

THE REFERENCE VALUE CONCEPT

In Describing the Depression of Variables

The major role of the reference value is to serve as a guide in medical diagnosis.
According to Geffr et al. (2009), the reference values are used to describe the depression of
variable in healthy individuals. This values purpose is to determine the observed value gathered
to a specific individual (eg. patient) if it belong to the healthy population as indicated to the
reference. This kind of reference value used in that determination according to Geffr are usually
reported as a population-based intervals which includes 95% of the healthy population where the
specific individual belong this can be correlated to a local based reference value.
Boyd, J. C. (2010) state that the role of the reference value as it is established for each
laboratory test in order to clearly show or describe or to delineate the range of values that
would usually be encountered in a healthy population. What this means that not only that the
reference value has a numerical variables but also it shows how to interpret the data as precisely
as possible the diagnosis can be made to determine the condition of a patient by only using the
reference value as a guide. However, it doesnt rule out that a further diagnosis that to be made
by the doctor that is not needed it only shows that an initial diagnosis can be formulated
through the use of the reference value. Thus, the final diagnosis will be interpreted only by the
doctor assigned to the patient.
In Medical Diagnosis
According to the second edition of Tietzs Applied Laboratory Medicine (Scott,
Gronowski, Eby, & Tietz, 2007), the books preface state that 80% of medical decisions
encompass or incorporate laboratory data in the decision process. This laboratory data which
composed of the patients own laboratory test results and reference values are considered to be

THE REFERENCE VALUE CONCEPT

the vital tool; an integrated part in clinical judgment (Dijkman, Fraser, Treasure, & Kapke,
2009). On the study conducted by the Indian J. Med Reasearch in 2012 on the reference values in
making medical decision, it conclude that the success of all medical decision making depends
as much on selecting and properly using the reference values and their limits. This affirms the
vital role of not only the reference value but all laboratory data in the foundation of clinical and
medical decision making only if the right values is used on interpreting the data.
Formulation of the Reference Value
Depending how the study is being conducted, there are different ways to obtain the
reference value. As explain earlier in the definition of reference population given by the IFCC,
the number of people comprising the reference is undetermined and thus depends on the
hypothesis on the study. Most studies stick with the constant percentage of 95% which
corresponds to the overall number of apparently healthy individuals (Boyd, 2010). However
due to a large number of individual that will comprise the 95% of the 100%, the central
laboratory reference which holds the standard reference values and intervals of a specific country
has a fixed number of approximately 2,000 generally healthy clinical trial subjects as stated by
Dijkman (2009).
Factors that influenced the Reference Value
Recommendation provided by CLIA 88. The Clinical Laboratory Improvement
Amendments of 1988 regulations have set the standard of all United States-based clinical
laboratory that test human specimens for health assessment or to diagnose, prevent, or treat
disease (CLIA Home, n.d.). Whats included also in the CLIA 88 is the recommended factor
to be made prior to the identification/gathering of the reference value and/or interval. There are

THE REFERENCE VALUE CONCEPT

four factors stated on the CLIA 88 and this include the selection of appropriate reference
individuals, pre-analytical variables, analytical methods to be used in obtaining the data to be
gathered and the statistical methods (Sunheimer & Graves, 2011).
Boyd, J. C. (2010) state a different factor in the determination of reference intervals. The
first factor is Choice of population to study and he stresses out that this factor is one
extremely important factor than the rest. Choosing the population to study is where a researcher
able to choose to which the reference is originated as most of the time as it is believed that the
environment/location of the population affect the observed value and sometimes this reference
value is not applicable to be used from the distant population with a different climate or
environment. The second up to the fourth factor is the same as what the CLIA 88 recommends
the pre-analytical variables. This variable include age, diet, gender, race, posture, medications,
physical activity, fasting status and variables of the study to name a few. The third factor speaks
about the analytical variation which refers to how the test procedure is performed. Boyd, J.C.
(2010) also state that the third factor can have major impact on the test results of reference
intervals. Finally, the fourth factor is the calculation of the reference limit or the statistical
method. There are two kinds of statistical method: the parametric approach and the nonparametric approach.
Geffr factors affirms both of CLIAs and Boyds by stating that the most critical steps
in the determination of reference values are the selection of reference individuals. Additionally,
he also spoken out that inclusion and exclusion criteria and the use of quality-controlled
analytical procedures must be extensively documented (Geffr et al., 2009).
Similarly but in contrast, Druyan et al (1998) stated that the validity of a reference ranges
or limits mostly depends on the reliability and limitations of the analytical methods. And

THE REFERENCE VALUE CONCEPT

10

additionally, factors including sampling, analytical sensitivity, accuracy, and precision are stated
as the one who has a high effect in the determination of data being collected and the value of the
reference ranges.
Clarification on the Concept
One of the most common misconception on the concept of a reference value is that it is
similarly compared to a normal value. Grsbeck, R. (2004) explains that the term reference
value only requires that the criteria for the selection of the individual are given. The selection of
the individual as stated by Grsbeck also include the individuals state of health and this also
including disease. Technically this means that the reason behind the reference value differ from
the normal value is it goes beyond further beyond the normal by giving out also values when the
person is experiencing a specific kind of disease.
The Clinical Laboratory Chemistry book also states that using the word normal was
often imprecise and confusing as the normal values does not always indicate a lack of disease
since the limits and intervals that was derived from apparently normal healthy subject may not
have represented a certain segments of the population (Sunheimer & Graves, 2011).
Normality is approximate according to Boyd, J.C. (2010) and also emphasizes that the
normal sperm concentration is influenced by many non-disease-related factors. Thus, even
with the shift of the sperm concentration (whether decreasing or increasing) due to age,
endocrine status, physical activity, duration of abstinence, volume of ejaculate, and history of
fertility it is still considered within the normal range even if the difference between normal value
is significantly high.

THE REFERENCE VALUE CONCEPT

11

Thus, Grsbeck who is one of the author of the IFCC recommendation and part of the
first to create the concept of reference value had resolve that the reference value can be derived
from all kinds of individuals depending to the criteria being given and this includes the state of
health of the individual regardless if he/she is in good or poor health (Ralph Grsbeck, 2004).
The reference value of a specific test is not fixed. As stated by Grsbeck, the concept of
the reference value is essentially philosophical. This means that the conception or the creation
of the value is came from the establishment and use of relevant data for interpreting medical
observations. Furthermore, the value may change with the advancement of analytical, clinical
and medical methods.
Conclusion
Summary
Reference is vital in identifying the condition of a person through laboratory diagnosis.
This references, in literal form, are the source of information. Likewise, these references are
product of series of test, studies and trials. Statistically and mathematically, a reference is derived
from the results and observations conducted and generated from the samples collected from a
sample individual or population composing of specific criteria based on the factors as
recommended by the one who conducted the survey. Furthermore, a reference is made with a
specific function like in this case, medical judgment and diagnosis in which the reference value
and all laboratory data is considered as the core in that area.
The role of the reference is inevitable in medical diagnosis or decision making. It is used
to describe or interpret the irregularities of the sample that is tested in the laboratory that is

THE REFERENCE VALUE CONCEPT

12

considered abnormal to the common regular value. Most medical decision process is
incomplete without the basis of a laboratory results and its success depends primarily on it.
The misconception of the concept includes the reference value must is compared normal
value as they are different in concept and in meaning and also the concept is considered to be
philosophical as the values are created from the use of various available methods to come up the
theoretical value and this value might change over time.
Thesis Revisited
Therefore, a reference is the integral groundwork in determining the condition of the
patient through laboratory diagnosis and medical decision making. It serves as a guide in medical
diagnosis. It is important in every medical diagnosis. It is derived from a series of test and
studies and finally it is essentially philosophical and is entirely different from normal

THE REFERENCE VALUE CONCEPT

13

References
Boyd, J. C. (2010). Defining laboratory reference values and decision limits: populations,
intervals, and interpretations. Asian Journal of Andrology, 12(1), 8390.
https://doi.org/http://dx.doi.org/10.1038/aja.2009.9
Burtis, C. A., Bruns, D. E., Sawyer, B. G., & Tietz, N. W. (Eds.). (2015). Tietz fundamentals of
clinical chemistry and molecular diagnostics (Seventh edition). St. Louis, Missouri:
Elsevier/Saunders.
CLIA Home. (n.d.). Retrieved September 24, 2016, from http://wwwn.cdc.gov/clia/
Cox, P., Wilken, D., Palko, T., & Palko, T. (2011). Palkos medical laboratory procedures (3rd
ed). Boston: McGraw Hill Higher Education.
Crapo, R. O. (2005). Role of reference values in making medical decisions. Indian Journal of
Medical Research, 122(2), 1002.
Dijkman, J. H. M., Fraser, C. G., Treasure, F. P., & Kapke, G. F. (2009). Local Laboratory
Reference Intervals in Clinical Trials. Drug Information Journal, 43(2), 139150.
Druyan, M. E., Bass, D., Puchyr, R., Urek, K., Quig, D., Harmon, E., & Marquardt, W. (1998).
Determination of reference ranges for elements in human scalp hair. Biological Trace
Element Research, 62(3), 18397. https://doi.org/http://dx.doi.org/10.1007/BF02783970
Geffr, A., Friedrichs, K., Harr, K., Concordet, D., Trumel, C., & Braun, J.-P. (2009). Reference
values: a review. Veterinary Clinical Pathology, 38(3), 288298.
Grsbeck, R. (2004). The evolution of the reference value concept. Clinical Chemistry and
Laboratory Medicine, 42(7), 692697. https://doi.org/10.1515/CCLM.2004.118

THE REFERENCE VALUE CONCEPT

14

Grsbeck, R., Siest, G., Wilding, P., Williams, G. Z., & Whitehead, T. P. (1979). Provisional
recommendation on the theory of reference values (1978). Part 1. The concept of
reference values. Clinical Chemistry, 25(8), 15061508.
Higgins, C. (2013). Understanding laboratory investigations: a guide for nurses, midwives, and
healthcare professionals (3rd ed). Chichester, West Sussex; Hoboken, NJ: WileyBlackwell.
Kitchen, S., Olson, J. D., & Preston, F. E. (Eds.). (2013). Quality in laboratory hemostasis and
thrombosis (2nd ed). Chichester, West Sussex: Wiley-Blackwell.
Koch, B., Schper, C., Ewert, R., Vlzke, H., Obst, A., Friedrich, N., Glser, S. (2011). Lung
function reference values in different German populations. Respiratory Medicine, 105(3),
35262. https://doi.org/http://dx.doi.org/10.1016/j.rmed.2010.10.014
Marshall, W. J., & Bangert, S. K. (Eds.). (2008). Clinical biochemistry: metabolic and clinical
aspects (2. ed., reprint). Edinburgh: Churchill Livingstone.
Pagana, K. D., & Pagana, T. J. (2006). Mosbys manual of diagnostic and laboratory tests. St.
Louis, Mo.: Mosby Elsevier.
Pagana, K. D., & Pagana, T. J. (2009). Mosbys diagnostic and laboratory test reference. St.
Louis, Mo.: Mosby.
Reference | Definition of Reference by Merriam-Webster. (n.d.). Retrieved September 22, 2016,
from http://www.merriam-webster.com/dictionary/reference
Sacks, D. B., Bruns, D. E., Goldstein, D. E., Maclaren, N. K., & al, et. (2002). Guidelines and
recommendations for laboratory analysis in the diagnosis and management of diabetes
mellitus. Diabetes Care, 25(4), 75086.

THE REFERENCE VALUE CONCEPT

15

Scott, M. G., Gronowski, A. M., Eby, C. S., & Tietz, N. W. (Eds.). (2007). Tietzs applied
laboratory medicine (2nd ed., Expanded and updated). Hoboken, N.J: Wiley-Interscience.
Shirin, K., Shafiq, S., Kazmi, S. A., Imad, S., & Khan, M. A. (2015). Development of In-house
Standard Reference Material for Estimation of Metals in Whole Blood. Pakistan Journal
of Medical Research, 54(1), 2933.
Sorgon, R. J. (2016). Reference intervals of hemoglobin, hematocrit, red blood cell count, and
blood indices among reference individuals in Iloilo City. University of San Agustin, 139p.
Sunheimer, R. L., & Graves, L. (2011). Clinical laboratory chemistry. Boston: Pearson.
Toman, B. (2005). Bayesian Approach to Assessing Uncertainty and Calculating a Reference
Value in Key Comparison Experiments. Journal of Research of the National Institute of
Standards and Technology, 110(6), 605612.