US 20140236491A1

(19) United States

(12) Patent Application Publication (10) Pub. No.: US 2014/0236491 A1
Katayev et al.
(54)

(43) Pub. Date:

METHODS FOR INDIRECT

Aug. 21, 2014

Publication Classi?cation

DETERMINATION OF REFERENCE

INTERVALS

(51)

(71) Applicant: Laboratory Corporation ofAmerica

Int- Cl
G06F 19/00

(2006.01)

(52) US, Cl,

Holdings, Burlington, NC (Us)

CPC .................................... .. G06F 19/34 (2013.01)

USPC

.......................................................... ..

702/19

(72) Inventors: Alex Katayev, Graham, NC (US); Arren

Fisher, Durham, NC (US); Dajie Luo,

(57)

Cary, NC (US)

The invention relates to methods for indirectly determining

ABSTRACT

clinical laboratory reference intervals. In one aspect, a refer

(73)

Assignee: Laboratory Corporation of America

Holdings, Burlington, NC (US)

ence interval is determined using all measurements for a

given analyte stored in a large existing database. In other

aspects, a characteristic of a subject is used to select a refer

(21) APP1- NO? 14/184,461

(22)

Filed:

ence population for inclusion in reference interval calcula

tions. In other aspects, the invention provides methods for
changing treatment plan, diagnosis, or prognosis for an indi

Feb- 19, 2014

Related U-s- Application Data

(60)

Provisional application NO' 61/766,534, ?led on Feb'

19, 2013.

vidual subject based on differences between the neW refer

ence interval and a previously utilized reference interval. In

other aspects, the invention provides systems and computer

readable media for indirectly determining reference intervals.

Pool analyte data from selected reference

_4

population
Plot data frequency against range of values

-6

Transform data if significantly skewed

-8

Calculate linear regression

10

Determine reference interval from linear portion

_ 12

of curve

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METHODS FOR INDIRECT

DETERMINATION OF REFERENCE
INTERVALS

graph for reference interval estimation. It was limited by

subjectivity of visualization and manual data manipulations.

Manual and semi-manual data manipulations using Hoff
mann s method were also used in later publications (Soldin et

RELATED APPLICATIONS

al. Pediatric Reference Intervals, AACC Press, 6th edition).

[0001] This application claims priority to US. Provisional

Application No. 61/766,534, ?led Feb. 19, 2013. The entire
disclosure of the application is incorporated herein by refer

[0008] To better serve the healthcare industry, the clinical

laboratory industry is in need of robust and reliable method
ology for determination and veri?cation of reference intervals
for clinical laboratory test results.

ence.

SUMMARY

FIELD OF THE INVENTION

[0002] The present invention relates to methods for indirect

determination of reference intervals for clinical laboratory

testing using data from existing laboratory databases.

BACKGROUND

[0003] A reference interval provides information about a

range of measurements observed in the reference population
to assist health care providers in interpretation of individual
clinical laboratory test results.

[0009]

Certain aspects of the present invention provide a

method for indirectly determining a reference interval for an

analyte, comprising: (a) pooling data from an existing data

base of measurements of the analyte from a selected reference

population; (b) plotting cumulative frequencies of data

against a range of analyte measurements from the data of the
selected reference population to determine a distribution of
the data; (c) applying a transformation to normalize data if the

[0004] Existing regulations require laboratories to provide

distribution is signi?cantly skewed; (d) calculating a linear

regression of the plotted data; and (e) determining a reference
interval for the analyte in the reference population by select

reference intervals on test result reports and review/revise

ing a range that corresponds to the linear portion of the curve.

those intervals on regular basis. Many laboratories adopt

[0010]

reference intervals from other sources, such as other labora

method for providing a reference interval for an analyte to aid

in evaluation of an individual subjects test result for the

tories, manufacturers of testing reagents, or previously pub

Other aspects of the present invention provide a

lished studies. In 2008, the Clinical and Laboratory Standards

analyte, comprising: (a) selecting a reference population

Institute-approved guideline recognized the reality that, in

from an existing database based on at least one characteristic

practice, very few laboratories perform their own reference

interval studies, instead referring to studies done many

of the subject; (b) pooling data from the database for mea
surements of the analyte from the reference population; (c)
plotting cumulative frequencies of data against a range of
analyte measurements from the reference population; (d)

decades ago, when both the methods and the population were

very different. (De?ning, Establishing, and Verifying Refer

ence Intervals in the Clinical Laboratory; Approved Guide
line. Third Edition. CLSI document C28-A3. Wayne, Pa.:

Clinical and Laboratory Standards Institute; 2008). Thus, it is

applying a transformation to normalize distribution if the

initial distribution is signi?cantly skewed; (e) calculating a

linear regression of the plotted data; and (f) selecting the

apparent that many reference intervals which have been

reported for decades may not currently be accurate for a given

linear portion of the curve to determine a reference interval

laboratory due to differences in modern testing methodology

ments, such a reference interval may be used in a method

and/ or the population serviced.

[0005] There are a number of additional problems contrib
uting to resistance or reluctance to change current practice.
Conducting independent de novo studies for reference inter
val determinations using the conventional direct donor sam

further comprising: providing a biological sample from a

subject having the characteristic(s) used to select the refer

pling method is expensive and has limitations and complica

tions. The studies typically recruit healthy subjects, whereby
criteria must be de?ned for determining which subjects are

healthy.
[0006] Recruiting and obtaining informed consent from
candidate subjects and excluding subjects with subclinical
diseases can be dif?cult and expensive. Moreover, the healthy

for the analyte in the reference population. In some embodi

ence population; determining a measurement of the analyte in

the biological sample; and comparing the measurement of the

analyte in the biological sample to the reference interval.
[0011] In other aspects, the invention provides computer
readable media for determining a reference interval accord

ing to the method described, the computer readable media

comprising: (a) program code for selecting analyte data for a

speci?c reference population from an existing database; (b)

program code for plotting cumulative frequencies of the data
against the measurement of analyte; (c) program code for

reference populations likely include subjects with subclinical

calculating a linear regression equation of the plotted data; (d)

disease. Even successful studies of this type have relatively

low sample sizes (e.g. about 100-150 individuals), such that
statistical power is lacking. It is statistically more robust to

program code for applying a transformation to normalize

distribution if the initial distribution is signi?cantly skewed;

and (e) program code for selecting the linear portion of the

analyze thousands of measurements that include a number of

curve to determine a reference interval for the analyte in the

unhealthy subjects than 120 subjects assumed to be healthy.

Large sample size is essential for accuracy in determination

reference population.

of reference intervals.
[0007] An indirect method of reference interval estimation
that used test results already stored in the laboratory database
was described by Hoffmann in 1963 (Hoffmann, R G. Statis

tics in the Practice of Medicine. JAMA, 185: 864-873, Sep.

14, 1963). Hoffmann described a method using manual plot
ting of test data on graph paper and visual assessment of the

[0012] In other aspects, the invention provides a system for

determining a reference interval, comprising: (a) a compo
nent for pooling data from an existing database of measure

ments of the analyte from a selected reference population; (b)

a component for plotting cumulative frequencies of data
against a range of analyte measurements from the data of the
selected reference population to determine a distribution of
the data; (c) a component for applying a transformation to

Aug. 21,2014

US 2014/0236491Al

normalize data if the distribution is signi?cantly skewed; (d)

a component for calculating a linear regression of the plotted
data; and (e) a component for determining a reference interval
for the analyte in the reference population by selecting a range
that corresponds to the linear portion of the curve.
[0013] Other aspects of the invention are provided below.

[0027] FIG. 14 is graphical illustration of data for thyroid

stimulating hormone (TSH) in all subjects in Example 5
according to an embodiment of the invention. Panel A shows

all data and panel B with outliers removed.

[0028]

FIG. 15 is graphical illustration of linear regression

for determination of a reference interval for thyroid stimulat

ing hormone (TSH) in all subjects in Example 5 according to

BRIEF DESCRIPTION OF THE FIGURES
[0014]

an embodiment of the invention.

FIG. 1 is a schematic illustration of a method for

DETAILED DESCRIPTION

determining a reference interval for an analyte according to

an embodiment of the invention.
[0015] FIG. 2 is a schematic illustration of a method for
determining a reference interval based on at least one char
acteristic of an individual subject, according to an embodi
ment of the invention.
[0016] FIG. 3 is a schematic illustration of a system for
determining a reference interval according to an embodiment

of the invention.
[0017] FIG. 4 is a graphical illustration of the data for

eosinophils in subjects 13-18 years old in Example 1 accord

ing to an embodiment of the invention, where panel A shows
all data and panel B shows data with outliers removed.
[0018] FIG. 5 is a graphical illustration of determination of
a reference interval for eosinophils in subjects 13-18 years old
in Example 1 according to embodiments of the invention,

showing linear regression by Cooks Distance in panel A and

by exhaustive search method in panel B.
[0019]

FIG. 6 is a graphical illustration of data for hemo

globin at high altitudes (subjects 8-30 days old) in Example 2

according to an embodiment of the invention, where panel A
shows all data and panel B shows data with outliers removed.
[0020] FIG. 7 is a graphical illustration of data for hemo

globin at low altitudes (subjects 8-30 days old) in Example 2

according to an embodiment of the invention, where panel A
shows all data and panel B shows data with outliers removed.
[0021] FIG. 8 is a graphical illustration of determination of

reference intervals for hemoglobin at high (panel A) and low

(panel B) altitudes in Example 2 according to embodiments
of the invention.
[0022] FIG. 9 is a graphical illustration of data for platelets
in subjects age 18-100 years old in Example 3 according to an
embodiment of the invention, where the data is selected from
the database at an early time point. Panel A shows all data and

panel B with outliers removed.

[0023] FIG. 10 is a graphical illustration of data for plate
lets in subjects age 18-100 years old in Example 3 according
to an embodiment of the invention, where the data is selected
from the database at a later time point. Panel A shows all data

and panel B with outliers removed.

[0024] FIG. 11 is a graphical illustration of determination
of reference intervals for platelets in subjects 18-100 years
old in Example 3 according to embodiments of the invention.
Panel A shows linear regression for the early time point and
panel B for the later time point.
[0025] FIG. 12 is graphical illustration of data for neutro
phils in subjects 13-18 years old in Example 4 according to an
embodiment of the invention. Panel A shows data before
transformation and panel B after BoxCox transformation.
[0026] FIG. 13 is graphical illustration of determination of
reference intervals for neutrophils in subjects 13-18 years old
in Example 4 according to embodiments of the invention.
Panel A shows linear regression of data without transforma
tion, and panel B shows linear regression of data after Box
Cox transformation.

[0029] The following description recites various aspects

and embodiments of the present invention. No particular
embodiment is intended to de?ne the scope of the invention.

Rather, the embodiments merely provide non-limiting

examples of various methods and systems that are at least
included within the scope of the invention. The description is
to be read from the perspective of one of ordinary skill in the
art; therefore, information well known to the skilled artisan is
not necessarily included.
DEFINITION AND ABBREVIATIONS

[0030]

The following terms, unless otherwise indicated,

shall be understood to have the following meanings:

[0031] As used herein, the terms a, an, and the can
refer to one or more unless speci?cally noted otherwise.

[0032]

The term or is not to be construed as identifying

mutually exclusive options. For example, the phrase X con

tains A or B means that X contains A and not B, X contains

B and notA, orX contains bothA and B. That is, the term or
is used to mean and/ or unless explicitly indicated to refer to

alternatives only or the alternatives are mutually exclusive,

although the disclosure may support a de?nition that refers to
only alternatives and and/or. As used herein another can
mean at least a second or more.

[0033]

As used herein, the terms subject, individual,

and patient are used interchangeably. The use of these

terms does not imply any kind of relationship to a medical
professional, such as a physician.

[0034]

As used herein, the term reference population is

used to refer to all the subjects having measurements for an

analyte of interest within a database whose data are selected
for inclusion in the calculation of a reference interval. The

entire population represented in the database may be

included, or speci?c characteristics of the subjects may be
selected for inclusion, to ?lter the data for determination of a

speci?c reference interval.

[0035]

As used herein, the term reference interval refers

to a central range of measurements for an analyte that is

observed in a reference population and reported by a labora

tory along with an individual test result to aid a health care

provider in interpretation of that individual result. Typically

(but not necessarily), a reference interval has referred to the
central 95% of values obtained from the reference population

of subjects.
[0036]

As used herein, the term biological sample is used

to refer to any ?uid or tissue that can be isolated from an

individual. For example, a biological sample may be whole

blood, plasma, serum, other blood fraction, urine, cerebrospi

nal ?uid, tissue homogenate, saliva, amniotic ?uid, bile,

mucus, peritoneal ?uid, lymphatic ?uid, perspiration, tissues,

tissue homogenate, buccal swabs, chorionic villus samples,
and the like.

Aug. 21,2014

US 2014/0236491A1

[0037]

As used herein, the term like biological sample is

used to refer to comparisons between the same types of bio

logical samples described above. For example, a measure
ment of analyte in a blood sample is compared to a reference
interval determined from measurements of the analyte in

other blood samples.

[0038] As used herein, the term analyte is used to refer to
a substance of interest in an analytical procedure. It is the

substance being analyzed in the biological sample.

[0039] As used herein, the terms normal distribution or
Gaussian distribution refers to a continuous probability
distribution, also known as the bell-shaped curve. Skewed
distribution, by contrast, as used herein refers to a probabil
ity distribution in which an unequal number of observations
lie below or above the mean and the curve is not bell-shaped

(see e. g., FIGS. 12 and 13). The terms skewed distribution

and signi?cantly skewed distribution will be understood to
those skilled in the art. In some embodiments, signi?cantly
skewed refers to a dataset where the mean is located in the ?rst

or ?fth quintile of the distribution.

[0040] As used herein, the term characteristic refers to
any feature or trait that can distinguish sub-groups of subjects

within the entire starting reference population for inclusion in

the speci?c reference population. For example, age, gender,

race, and geographic location are characteristics that may be
designated in a reference population. A more speci?c refer
ence population corresponds to a more individualized refer
ence interval.

Methods for Determining Reference Intervals

[0041]

Certain aspects of the present invention provide a

method for indirectly determining a reference interval for an

analyte, comprising: (a) pooling data from an existing data

base of measurements of the analyte from a selected reference

population; (b) plotting cumulative frequencies of data

against a range of analyte measurements from the data of the
selected reference population to determine a distribution of
the data; (c) applying a transformation to normalize data if the

distribution is signi?cantly skewed; (d) calculating a linear

regression of the plotted data; and (e) determining a reference
interval for the analyte in the reference population by select
ing a range that corresponds to the linear portion of the curve.
[0042] In some embodiments, maximum allowable error is
restricted to account for a known individual biological varia

tion for the analyte in selecting the range that corresponds to

the linear portion of the curve. In some embodiments, the

for the analyte in the reference population. In some embodi

ments, such a reference interval may be used in a method

comprising: providing a biological sample from a subject

having the characteristic(s) used to select the reference popu
lation; determining a measurement of the analyte in the bio
logical sample; and comparing the measurement of the ana
lyte in the biological sample to the reference interval.
[0044] In some embodiments, the selected reference popu

lation includes at least 100, 120, 140, 160, 180, 200, 220, 240,

260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800,
900, 1,000, 1,500, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000,
20,000, 40,000, 60,000, 80,000, or 100,000 different indi
viduals. In some embodiments, a different course of treat

ment, diagnosis, or prognosis is determined or selected for the

subject based on the reference interval as compared to the
course of treatment, diagnosis, or prognosis using a different
reference interval previously utilized for the same analyte. In
some embodiments, the reference population is selected
according to at least two characteristics of the individual

subject.
[0045] FIG. 1 illustrates a method for determining a refer
ence interval for an analyte according to an embodiment of

the invention. In this method (2), analyte data is pooled from

a selected reference population (4). The data is plotted against
the range of measurement values represented (6), and at least
one transformation is applied if the data is initially signi?
cantly skewed (8).A linear regression of the data is calculated
(10), and the reference interval is determined from the linear
portion of the resulting curve (12).
[0046]

FIG. 2 illustrates a method for determining a refer

ence interval based on at least one characteristic of an indi

vidual subject, according to an embodiment of the invention.

In this method (22), analyte data is pooled from a reference
population that is based on at least one characteristic of an

individual test subject as represented by asterisk * (24). The

data is plotted against the range of measurement values rep
resented (26), and at least one transformation is applied if the

data is initially signi?cantly skewed (28). A linear regression

of the data is calculated (30), and the reference interval is

determined from the linear portion of the resulting curve (3 2).

This individualized reference interval may then be used to
evaluate the analyte in an individual subject. In this method

(34), a biological sample from the individual subject is pro

vided (36) and the analyte is measured in the sample (38).

selected reference population includes at least 100, 120, 140,

Analytes

160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380,
400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 4,000,
6,000, 8,000, 10,000, 15,000, 20,000, 40,000, 60,000,

[0047] In some embodiments, the invention provides a

method for indirectly determining a reference interval for an

80,000, or 100,000 different individuals.

[0043] Other aspects of the present invention provide a
method for providing a reference interval for an analyte to aid
in evaluation of an individual subjects test result for the

number of measurements of that analyte. In some embodi

ments, the invention provides a method for determining an

analyte, comprising: (a) selecting a reference population

from an existing database based on at least one characteristic

of the subject; (b) pooling data from the database for mea
surements of the analyte from the reference population; (c)
plotting cumulative frequencies of data against a range of

analyte measurements from the reference population; (d)

analyte using data from an existing database having a large

analyte reference interval for evaluating a laboratory test
result. In some embodiments, the invention provides a
method for determining an analyte reference interval to aid in
evaluating a laboratory test result. In some embodiments, the

invention provides a method for determining an analyte ref

erence interval to aid in making a medical decision. In some

embodiments, the invention provides a method for veri?ca

applying a transformation to normalize distribution if the

tion of existing laboratory reference intervals.

initial distribution is signi?cantly skewed; (e) calculating a

linear regression of the plotted data; and (f) selecting the

[0048] The analyte may be any substance (such as a bio

molecule or compound), parameter, ratio, or other relation

linear portion of the curve to determine a reference interval

ship that is measurable within the body or in biological

Aug. 21,2014

US 2014/0236491A1

samples removed from the body, such as those described

above. The invention is not limited to any particular analyte or

set of analytes.
[0049] In some embodiments, analytes include hormones,
lipids, proteins, nucleic acids, or combinations or fragments
thereof. In some embodiments, the analytes are small mol
ecules such as creatinine, ATP, or glucose. In some embodi
ments, the analytes are larger entities such as platelets or red

blood cells (such as hematocrit).

[0050] In some embodiments, analytes include polypep
tides or oligopeptides, including antibodies or antibody frag
ments. In some such embodiments, the peptides or oligopep
tides are indicative of a likelihood of showing responsiveness
or resistance to a course of treatment, such as a drug-based
course of treatment.

[0051]

invention provides a method for selecting a speci?c reference

population from the database. In some embodiments, the
reference population is restricted based on multiple charac
teristics; this can generate a more individualized reference

interval. In some embodiments, the data is ?ltered according

to speci?c characteristics of interest to achieve a desired

reference population. For example, only data from female

subjects are included, or only subjects in a speci?c age range,
or both. Any number of characteristics may be used to narrow

the reference population.

[0056]

In some embodiments, the data is used only from the

subjects on whom the test of interest was ordered in combi

nation with another speci?c test. In certain embodiments the

data is used when results for the other test meet prede?ned

criteria (e.g. the result is within the prede?ned limits). In

In some embodiments, the analytes include viruses

some embodiments, it may be desirable for the reference

or biomarkers indicative of infection. In some embodiments,

population to be geographically restricted. For example, as

described in more detail herein, measurements of hemoglo
bin from subjects in areas with high elevation (e.g. Colorado)

the analytes include antibodies (or fragments thereof), such as

antibodies or antibody fragments that are indicative of human
allergic responses, e. g., human IgE antibodies, or are indica

tive of immuno-rejection during organ transplant, or are

indicative of the ef?cacy of a vaccination protocol, or are

antibodies related to cellular signaling.

[0052] In some embodiments, the analytes include biomar
kers, such as biomarkers indicative of a disease or condition,
e.g., an autoimmune disease. In some embodiments, the

are signi?cantly different than from subjects at sea level. In

this manner, mining the database for a more speci?c reference
population can provide a more individualized reference inter
val. In such embodiments, resources are not wasted on deter

mining who is healthy or unhealthy. Data is extracted for all

subjects who meet the designated reference population crite
na.

biomarkers may include biological measurements unrelated

to chemistry, e.g. height, weight, skull dimensions, etc. In

some embodiments, the analytes include bacteria or parasites.
In some embodiments, the analytes include polynucleotides
that are indicative of adverse drug reactions. The analytes can

also include biomarkers for various diseases, cytokines,

chemokines, and growth factors. They can also include small
molecules, such as steroid hormones and inorganic molecules
such as salts and other electrolytes.

Indirect Sampling of Speci?c Populations

[0053] In some embodiments, the invention provides a
method for indirectly determining a reference interval for an

Statistical Methods

[0057] In some embodiments, the invention provides sta

tistical methods for analysis of analyte data from a speci?c
reference population. In some embodiments, the invention

provides methods for plotting data and removing outliers

from the reference population dataset. In some embodiments,
the invention provides methods for calculating linear regres
sion of the plotted data. In other embodiments, the invention
provides a method in which a transformation is applied to
normalize distribution if the initial distribution is non-Gaus
sian.
[0058] In some embodiments, the invention allows the user

analyte from an existing database containing a large number

of measurements of that analyte. Larger sample sizes corre
spond to higher statistical power. In some embodiments, the
majority of the database comprises data from an outpatient
population rather than a population associated with a hospital
by including only the data for tests ordered from non-acute
settings. In some embodiments, the invention provides a
method for veri?cation of an existing laboratory reference
interval.
[0054] In some embodiments, the invention provides a
method for determining an individualized reference interval.

population. As described in more detail below, in some

embodiments the reference interval does not exclude greater
than 2.5% from both upper and lower limits when central 95%
is used. In some embodiments, the linear portion of the linear
regression curve is selected to derive a reference interval for

In some embodiments, the invention provides a method for

the analyte in the reference population.

determining an updated individualized reference interval. In

[0059] In some embodiments, the reference interval is pro

vided to a health care provider for assistance in evaluating the
analyte measurement for a particular subject. In some

some embodiments, the invention provides a method for

determining a sub-population reference interval. In some
embodiments, the invention provides a method for determin

ing an updated reference interval essentially concurrent with

generation of the individual lab test report. In some embodi
ments, the invention provides a method for determining an

to account for biologic variation of analytes by setting a

maximum allowable error at the linear regression step, such
that the reference interval has increased clinical relevance and
re?ects the reference population with respect to normal

physiological variation of the analyte of interest and does not

exclude a signi?cant number of subjects in the reference

embodiments, following selection of the reference popula

tion, numerical laboratory test results for the given analyte

updated global reference interval.

that are stored in the laboratory database are loaded in the

program data source. Where multiple laboratories or data
bases are networked, data may be loaded from only one

[0055]

location or from two or more locations.

In some embodiments, the reference interval is indi

vidualized based on at least one characteristic of the subject of

[0060]

interest. According to a preferred embodiment of the inven

tion, there is no need to de?ne healthy or parse healthy

?ed number of decimal places. In some embodiments, outly

subjects from unhealthy subjects. In some embodiments, the

In some embodiments, data are rounded to a speci

ing observations may be removed. In some embodiments the

outliers are removed using Chauvenet criteria. Other outlier