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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