Biological Variation

Dr WA Bartlett
Biochemical Medicine
Ninewells Hospital & Medical School
Dundee
Scotland

Objectives
Identification the nature of biological

variation.
Appreciation of the significance of
biological variation in clinical
measurements.
Attain insight into the determination and
application of indices of biological
variation.

Identification the nature of

biological variation.
What is meant by the term
biological variation in the context
of clinical biochemistry?
A component of the variance in
biochemical measurements
determined by the physiology of
the subjects observed.

Components of Variance in
Clinical Chemistry
Measurements
Analytical variance.
Within Subject biological variance.
Between Subject biological variance.

Biological Variation
All clinical chemistry measurements

change with time.

Knowledge of temporal changes useful in
diagnosis and interpretation.
Rate of change may be useful in prognosis.
Understanding of the sources of biological
variation in non-diseased subjects is
fundamental to the development of
reference data.

Sources of Biological Variation

Biological Rhythms (time)
Homeostasis
Age
Sex
Ethnicity
Pathology
Stimuli

Practical significance of
biological variation.
What is the significance of this result?
Is the performance of the analytical

method appropriate (imprecision,

accuracy)?
When should I measure it again?
Has this result changed significantly over
time?
Changes in variability be used as a tool?

Models of Biological Variation

Assume values represent random

fluctuation around a homeostatic setting

point.
More general model allows correlation
between successive results. (Time series
and non-decayed biological variation)

Quantifying Biological
Variation
How are you going to quantify biological
variation?
You have to dissect out the components
of variance: total = Analytical + Individual + Group

Quantifying Biological
Variation

Analytical =

Individual =
Group =

Average variance of replicate assays

within run analytical variance
Average biological within subject
variance.
Average Variance around the
homeostatic setting point
Variance of true means among subjects.
Variance in homeostatic setting points

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Analytical
Variance

Subject 1

Within Subject
Variance

Subject 2

Between Subject
Variance

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Subject 3

Quantifying Biological
Variation
How do you do the experiment?
Subjects
How many?
Collect specimens Number? Frequency?
Analyse specimens
Analyse data

Minimise Analytical ?

Outliers? Statistics?
Apply results of analysis.

Quantifying Biological
Variation
Estimates of biological variation are
similar regardless of: Number of subjects
Time scale of study (Short v Long?)
Geography
A lot of information can be obtained
from small studies.

Within Subject Variation (CVI,%) for Serum Sodium and Urea

No. of Time
Sexb status
Na+ Urea
subjects

11
11
62
11
10
14
111
37
274
15
9
15
16

0.5 h
8h
1d
2 weeks
4 weeks
8 weeks
15 weeks
22 weeks
6 months
40 weeks
2d 6 weeks
8 weeks

m
m
m
m
F
m
m
F
m

H
H
H
H
H
H
H
H
H
H
RF
HP
DM

0.6
0.5
0.6
0.7
0.9
0.5
0.6
0.5
0.5
0.7
0.8
0.8
0.8

2.2
6.0
4.8
12.3
14.3
11.3
15.7
11.1
11.2
13.9
6.5
14.5
13.0

Collection of Specimens.
Conditions should minimise pre-analytical

variables.
Healthy subjects.
Usual life styles.
No drugs (alcohol, smoking?).
Phlebotomy by same person.
Same time of day at regular intervals.
Set protocol for sample transport, processing &
storage.

Analysis of Specimens
Need to minimise analytical imprecision.
Ideal : -

Single lots of reagents and calibrants.

Single analyst and analytical system.
Single or very small number of
batches.

Preferred Protocol: Cotlove et al

Healthy subjects.
Specimens taken at set time intervals.
Specimens processed & stored frozen.
When ALL specimens are available: -

Analysis of all samples in a single run.

Simultaneous replicate analysis.
Quality control to monitor drift

Preferred Protocol: Cotlove et al

Advantage: Minimisation of Analytical

Disadvantages: Limits the number of specimens and subjects

that can be studied.
Analyte must be stable on storage.

Other Protocols: Costongs et al

Collection and storage as before.
Singleton assay of all samples in a single

run.
Duplicate assay of QC or

estimate Analytical

patient pool to

Other Protocols: Costongs et al

Disadvantages: True estimate of Analytical ?
Integrity of QC materials
Viral infections of pools
Vial to vial variability in QC

Other Protocols: Costongs/Moses et al

Samples assayed once or in duplicate on

the day of collection

Disadvantage: individual confounded by between batch
variance.

Advantage: Useful if analyte is unstable.

Analysis of Data
2 Stages
Identification of outliers
Nested analysis of variance

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Analytical
Variance

Subject 1

Within Subject
Variance

Subject 2

Between Subject
Variance

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Subject 3

Applications of BV Data
Setting of analytical goals.
Evaluating the significance of change in

serial results.
Assessing the utility of reference
intervals.
Assessing number of specimens required
to estimate homeostatic set points.

Applications of BV Data
Assessment of reporting strategies.
Selecting the best specimen.
Comparing utility of available tests.

Setting of analytical goals.

Accepted analytical goal for imprecision: CVGoal = CVI

therefore: CVAnalytical = CVGoal

= of the Individual if achieved.

(Harris. Am J Clin Pathol 1979:72;274)

Utility of Analytical Goals

Assessment of methods and equipment.
Should be addressed in early stages of

method development.
Index of Fiduciality: CVAnalytical /CVGoal

If <1 analytical goal met

(Fraser Clin Chem 1988:34;995)

Evaluating the significance

of change in serial results.
Critical Difference or Reference Change value

indicates the value by which 2 serial results

must differ to be considered statistically
significant: CD = 2 * Z * (CVA2 + CVI2)
Probabilty = 95% Z = 1.96
Probability = 99% Z = 2.58

Only valid if the variance of Individual is

homogenous.

(Costongs J Clin Chem Clin Biochem 1985;23:7-16)

Multipliers for (CVA2 + CVI2) to Obtain Critical

Difference at Different Levels of Probability
Multiplier
(2 * Z)
Probability of
false alarm

3.64 2.77 2.33 1.81 1.47 1.19 0.95

Probability

99%

0.01 0.05 0.10 0.20 0.30 0.40

95%

90%

80%

70%

60%

50%

Significance of Change?
63 year old patient: Cholesterol 1 = 6.60 mmol/L
Cholesterol 2 = 5.82 mmol/L
Significant change ?

Cva = 1.6%

CVI = 6.0%

RCV = 2 * Z * (CVA2 + CVI2)

95%RCV = 1.414 * 1.96 * (1.6 + 6.60 ) = 17.2%
99%RCV = 1.414 * 2.58 * (1.6 + 6.60 ) = 22.6%
Actual Change = ((6.60 5.82)/6.60)*100= 11.8%

Dispersion =Z* (SD2A + SD2I)

Dispersion of first result = result 1.96 SD : 95% level 6.60 = 5.80 7.40
99% level 6.60 = 5.54 7.66
Dispersion of 2 result
95% level = 5.82 = 5.11 6.53
99% level = 5.82 = 4.89 6.75
Overlap: therefore neither significantly or highly
significantly different
Can use the formula to ascertain the probability that
change is significant. Calculate Z using the (((6.65.82)/6.6)*100%) as RCV and look up in tables. 82% in
this case.

USE of RCV
Handbooks

reports, 95% and 99%

probabilities that change is significant.
(> or >> * or **)
Delta checking, exemption reporting.
95% auto validate, 99% refer for clinical

validation or renanalysis.

Index of Heterogeneity
Measure of the heterogeneity of

variance within

the study population: ratio of the observed CV of the set of subjects

variances (SDA+I2) to the theoretical CV ( / 2/n-1)
for the set.
The ratio should =1

(1SD = 1/ /2n )
Large ratio = more heterogeneity.
(Costongs J Clin Chem Clin Biochem 1985;23:7-16)

Assessing the utility of

reference intervals.
Utility of population based reference data?
Ratio of Within to Between subject variances.

Index of Individuality = CVI / CVG

Population Ref Intervals: Index <0.6 = Limited in Value
Index >1.4 = Applicable

Biological Variation &Utility of Reference

Intervals

Number of specimens
required to estimate
homeostatic set points.
n = ( Z. CVA+ I/D)
where: Z = number of Standard deviates for a
stated probablity (e.g. 1.96 for 95%).
D = desired % closeness homeostatic set
point.

Number of specimens required to

estimate homeostatic set points: Cholesterol testing
How many samples (n) required to
estimate set point within 5% given: CVI = 4.9% CVA = 3% (Recommended)
Substitute equation: n = ( Z. CVA+ I/D)

n = [1.96(32 + 4.92)/5]2 = 5.07

RCV at 95% and Number. of Specimens Required

to Assess the Homeostatic Set Point at Different
Levels of Imprecision
CVA
CVI
RCVa
Number of
(%)
(%)
(%) specimensb
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
15.0
20.0
a

4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
4.7
2

14.1
15.4
17.1
19.0
21.1
23.4
25.7
28.1
30.6
43.5
56.9
2

4
5
6
7
9
11
13
16
19
38
65

Assessment of reporting
strategies
Results may be reported in different

formats
e.g. 24h Urinary creatinine output: CVI for concentration = 23.8%
CVI for output per collection = 13.0%
CD for concentration = 66.0%
CD for output = 36.2%

Selecting best Specimen.

e.g early morning urines for albumin

versus 24h collections.

Random hormone measurements versus

timed measurements.

Comparing Available Tests

Creatinine v Creatinine Clearance
FT4 v TSH in replacement situations
FT4 v Total T4

Reference Intervals
Dr WA Bartlett
Birmingham Heartlands & Solihull
NHS Trust (Teaching)