InterandIntraAssayCoefficientsofVariability

Inordertoexpresstheprecision,orrepeatability,ofimmunoassaytestresults,researchersinthesocialand
behavioralsciencestypicallyreporttwomeasuresoftheCoefficientofVariability(CV)intheirpublications:
theInterAssayCVandtheIntraAssayCV.TheCVisadimensionlessnumberdefinedasthestandard
deviationofasetofmeasurementsdividedbythemeanoftheset.Sincetheusageofthetermintraassay
CVmayvarysomewhatbetweenfieldsofstudy,someclarificationofterminologyandmethodsisinorder.
Inlargerstudieswithmanysamplestobetested,itisnecessarythatsamplesberunonmultipleassay
plates.Eachplateisrunwithitsowncalibratorsforthestandardcurve,andsalivacontrolswithknown
concentrationsoftheanalyteusuallyahighandalowvaluearealsoincludedoneachplate.Theinter
assayCVisanexpressionofplatetoplateconsistencythatiscalculatedfromthemeanvaluesforthehigh
andlowcontrolsoneachplate,asillustratedinExample1below(takenfrominternalSalimetricsdata).
Moststudiesmeasureeachsampleinduplicateforeachanalyte.Thedegreetowhichtheduplicateresults
differcanbeexpressedbycalculatingthestandarddeviationofthetworesultsandconvertingittotheCV.
Testingeachsamplewithgreaternumbersofreplicateswouldproducestatisticallybetterresultsforthe
standarddeviationandcalculationoftheCV,butthispracticewouldbeprohibitivelyexpensiveforlarge
studies.TheintraassayCVreportedinthesestudiesisanaveragevaluecalculatedfromtheindividualCVs
foralloftheduplicates,evenifthetotalnumberofsamplesrequirestheuseofmultipleassayplates.(1)An
illustrationofthecalculationoftheintraassayCVfrom40samples(usinginternalSalimetricsdata)is
providedinExample2.
SalimetricsoffersthefollowingcommentsoncalculatingandinterpretingCVs:

CVsshouldbecalculatedfromthecalculatedconcentrationsratherthantherawopticaldensities.
Interassay%CVsoflessthan15aregenerallyacceptable.
Intraassay%CVsshouldbelessthan10.
Thesescoresreflecttheperformanceoftheassayinthehandsoftheuser;theyarenotthesameas
themeasuresusedbytheassaymanufacturertoinsureconsistencyinthemanufacturingprocess.
ItisimportantthattheCVsarereportedforconcentrationsthatreflecttherangeofresultsfoundin
thespecimens.

ExperimentalresultswithpoorintraassayCVs(>10%)frequentlyreflectpoorpipettingtechniqueonthe
partoflaboratorytechnicians.(1)Inaddition,mishandlingduetothehighviscosityofsalivacanmake
pipettingespeciallydifficult.Salimetricsrecommendsfreezingeachsampleonce,followedbyvortexingand
centrifugation,whichwillhelpprecipitateandremovemucins.Prewettingeachpipettetipinthesolution
tobepipettedwillalsohelpimproveCVs.(Remembertochangetipsbetweeneachsample,standard,or
control.)Pipettesmustalsobeproperlycalibratedandmaintainedforbestresults.Foradiscussionofthe
importanceofproperpipetting,seethearticleAssayVarianceandControlfoundinthisnewsletter,and
followtheselinkstorelatedarticlesfrompipettemanufacturers.(26)

Example1.CalculatingInterAssayCV:TheAverageCoefficientofVariationfromPlateControl
Means.
Inthisexamplethesamehighandlowcortisolcontrolsareruninquadruplicateontendifferentplatesto
monitorplatetoplatevariation.Theplatemeansforhighandlowarecalculatedandthenusedto
calculatetheoverallmean,standarddeviation,and%CV.Overall%CV=SDofplatemeansmeanof
platemeansx100.Theaverageofthehighandlow%CVisreportedastheinterassayCV.
Control

Result1

Result2

high

1.199

1.132

high

1.071

0.957

high

1.101

0.973

high

1.007

0.911

high

1.093

0.942

high

0.913

0.957

high

1.077

0.986

high

0.978

0.900

high

1.106

0.993

high

0.993

0.986

high

1.045

1.068

high

1.037

0.936

high

1.075

1.022

high

1.164

0.958

high

1.032

1.054

high

1.046

1.017

high

0.918

0.905

high

0.973

0.892

high

0.986

0.923

high

0.950

0.904

Plate

PlateMean
(g/dL)

1.090

0.998

0.976

0.985

1.019

1.021

1.055

1.037

0.922

10

0.941

High

Control

Result1

Result2

low

0.113

0.115

low

0.108

0.083

low

0.112

0.089

low

0.101

0.085

low

0.129

0.106

low

0.099

0.108

low

0.100

0.097

low

0.099

0.083

low

0.093

0.093

low

0.099

0.094

low

0.102

0.100

low

0.108

0.108

low

0.112

0.106

low

0.132

0.105

low

0.114

0.102

low

0.105

0.109

low

0.108

0.104

low

0.110

0.104

low

0.113

0.107

low

0.099

0.095

Plate

PlateMean
(g/dL)

0.105

0.097

0.111

0.095

0.095

0.104

0.114

0.108

0.106

10

0.103

Low

MeanofMeans

1.005

MeanofMeans

0.104

StdDevofMeans

0.051

StdDevofMeans

0.006

%CVofMeans

5.1

%CVofMeans

6.3

InterassayCV(n=10)=averageofhighandlowcontrolCV=5.1+6.3/2=5.7%

Example2.CalculatingIntraAssayCV:TheAverageCoefficientofVariationbetweenDuplicates.
Inthisexamplecortisolconcentrationsaremeasuredinduplicatefor40samples.The%CVfor
eachsampleiscalculatedbyfindingthestandarddeviationofresults1and2,dividingthatbythe
duplicatemean,andmultiplyingby100.TheaverageoftheindividualCVsisreportedasthe
intraassayCV.
Sample

Result1

Result2

DuplicateMean
(g/dL)

StandardDev

%CV

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

0.132
0.351
0.282
0.237
0.178
0.119
0.136
0.155
0.145
0.135
0.212
0.214
0.144
0.066
0.117
0.116
0.174
0.119
1.372
1.052
1.357
0.957
0.485
0.186
0.136
0.181
0.253
0.225
0.094
0.094
0.119
0.314
0.134
0.628
0.071
0.073
0.059
0.061
0.201
0.149

0.128
0.361
0.306
0.218
0.215
0.128
0.125
0.150
0.152
0.148
0.210
0.210
0.125
0.065
0.102
0.110
0.169
0.114
1.287
1.024
1.248
0.904
0.425
0.162
0.134
0.181
0.258
0.199
0.098
0.113
0.112
0.294
0.131
0.614
0.076
0.076
0.040
0.059
0.175
0.155

0.130
0.356
0.294
0.227
0.197
0.123
0.131
0.152
0.149
0.142
0.211
0.212
0.135
0.066
0.109
0.113
0.172
0.117
1.329
1.038
1.303
0.930
0.455
0.174
0.135
0.181
0.256
0.212
0.096
0.103
0.116
0.304
0.133
0.621
0.074
0.074
0.050
0.060
0.188
0.152

0.002
0.007
0.017
0.013
0.026
0.006
0.007
0.004
0.005
0.009
0.001
0.003
0.013
0.001
0.011
0.004
0.004
0.004
0.060
0.019
0.077
0.038
0.042
0.017
0.002
0.001
0.003
0.018
0.003
0.013
0.005
0.014
0.002
0.010
0.004
0.002
0.013
0.001
0.018
0.004

1.7
2.0
5.6
5.9
13.5
5.2
5.7
2.6
3.4
6.2
0.6
1.5
9.7
1.0
9.9
3.8
2.3
3.2
4.5
1.9
5.9
4.0
9.3
9.7
1.4
0.3
1.2
8.7
2.7
13.0
4.1
4.7
1.7
1.6
4.9
3.2
27.1
2.4
9.8
2.8

IntraassayCV(n=40)=average%CV=

5.2

References
1. Schultheiss,O.C.,Stanton,S.J.(2009).Assessmentofsalivaryhormones.In:HarmonJones,E.,Beer,J.S.,
eds.MethodsinSocialNeuroscience.NewYork:GuilfordPress.

2. Mannonen,S.,TiusanenT.,Suovaniemi,O.(2000).Majorsourcesoferrorofairdisplacementpipettors.
Biohit.PDF
3. Yltupa,S.(1996).Optimizingyourpipettingperformance.Biohit.PDF
4. Mannonen,S.,Nieminen,P.Kaasinen,J.,Andersin,K.(2006).Raisingthestandardofmechanical
pipetting.Biohit.PDF
5. Hemmings,I.(2009).Theimportanceofregularinlabcheckingofpipettes.Biohit.PDF
6. http://www.fisher.co.uk/techzone/pdfs/pipetting_techniques.pdf