analytical comparison of three quantitative immunochemical fecal occult blood tests for colorectal...

Research Article

Analytical Comparison of Three Quantitative ImmunochemicalFecal Occult Blood Tests for Colorectal Cancer Screening

Lydia Guittet1, Elodie Guillaume1, Romuald Levillain2, Philippe Beley2, Jean Tichet2,Olivier Lantieri2, and Guy Launoy1

AbstractBackground: The superiority of several immunochemical fecal occult blood tests (I-FOBT) over guaiac-

based tests in colorectal cancer screening is now established. The aim of this study was to compare the

analytical performance of 3 quantitative I-FOBTs.

Methods: Stool samples from 10 healthy volunteers, initially I-FOBT negative, supplemented with human

blood, were used to compare reproducibility and stability of measurement at varying storage temperatures

(4�C, 10�C, 20�C, and 30�C) and durations before test analysis (1 to 10 days) for 3 I-FOBTs (New Hemtube/

Magstream HT, OC-Auto sampling bottle3/OC-Sensor DIANA, and FOB Gold/SENTiFOB). Concentrations

ranging from 0 to 350 mg Hb/g of feces were evaluated.

Results: The measurement reproducibility of OC-Sensor was superior to Magstream and far superior to

FOB Gold. For all tests, variability was essentially related to sampling. Detected hemoglobin (Hb) levels were

substantially lower for all tests at temperatures above 20�C. At 20�C, this loss in concentration was less

important withOC-Sensor (significant 1.7% daily decrease vs. 7.4% forMagstream and 7.8% for FOBGold). At

30�C, daily loss was 8.6% with OC-Sensor, whereas after 24 hours, only 30% of the original Hb was detected

with FOB Gold, compared to 70% with Magstream. No Hb was detected on day 5 for the latter 2 tests.

Conclusions: About reproducibility and temperature stability, OC-Sensor performed better than Mag-

stream and far better that FOB Gold.

Impact: Independently of the chosen test, the delay between sampling and test processing should be

reduced, the maximal admissible delay depending on ambient temperature. Cancer Epidemiol Biomarkers Prev;

20(7); 1492–501. �2011 AACR.

Introduction

Colorectal cancer is a major public health issue in allindustrialized countries. Screening using guaiac fecaloccult blood tests (G-FOBT) reduces specific mortalityrelated to colorectal cancer (1). Several studies haveconcluded that both the Magstream (Fujirebio) andOC-Sensor (Eiken Chemical Co.) automated immuno-chemical FOBTs (I-FOBT) offer a gain in sensitivity inthe detection of advanced neoplasias, compared with G-FOBT, at a cost of lower specificity (2–4). For both tests,ideal balance between sensitivity and specificity can bereached by variation in hemoglobin (Hb) concentration

cutoff and number of samples (5–8). For both tests, a gainin both sensitivity and specificity for the detection ofadvanced neoplasias was possible (4, 7, 8).

Since it has been established that I-FOBTs do better thanG-FOBTs, these tests are expected to beused in all nationalscreeningprogramsusingFOBT.Accordingly, the use of afecal immunochemical test has been included in U.S.guidelines for colorectal cancer screening (9). However,several I-FOBTs are available and their performance isdifficult to compare because the cutoff provided in studiesis expressed in concentration of Hb in the collecting tubedependingon the concentration ofHb in the feces, but alsoon the volume of buffer in the tube, and on the amount offeces introduced in the tube. Therefore, optimal test,optimal number of samples, or optimal Hb concentrationcutoff are for the moment indeterminate (10). Moreover,seasonal variations in positivity rates of screening pro-gramsusingOC-Sensor orMagstream I-FOBThave raisedthe question of the sensibility to temperature (11–13), andlaboratory analyses have established a decrease in Hbconcentration in OC-Sensor I-FOBTwith increasing delayin the sample (11, 14).

As a summary, the performance of I-FOBT dependsmainly on the test’s sensitivity to Hb, its reproducibility

Authors' Affiliations: 1INSERM ERI3 "Cancers & Populations," Universityof Caen Basse-Normandie (UCBN), University Hospital (CHU de Caen),Caen, France; 2IRSA (Institut inter-R�egional pour la Sant�e), La Riche,France

Corresponding Author: Lydia Guittet, INSERM ERI3 "Cancers & Popu-lations," UFR M�edecine, CHU de Caen, Avenue de la Côte de Nacre,F-14000 Caen, France. Phone: 33-231-06-51-21; Fax: 33-231-53-08-52;E-mail: [email protected]

doi: 10.1158/1055-9965.EPI-10-0594

�2011 American Association for Cancer Research.

CancerEpidemiology,

Biomarkers& Prevention

Cancer Epidemiol Biomarkers Prev; 20(7) July 20111492

American Association for Cancer Research Copyright © 2011 on August 10, 2011cebp.aacrjournals.orgDownloaded from

Published OnlineFirst May 16, 2011; DOI:10.1158/1055-9965.EPI-10-0594

http://cebp.aacrjournals.org/

http://www.aacr.org/

of sampling and measurement, and the stability of Hb inthe collecting tube, in particular with regard to tempera-ture variations and delay from fecal sampling to testanalysis.Our study aimed to compare measurement precision

and reproducibility, together with Hb measurement sta-bility at varying storage temperatures and varying delaysbetween sampling and analysis of 3 I-FOBTs previouslyused in colorectal cancer screening programs: Magstream(New Hemtube) analyzed using a Magstream HT auto-mated instrument (Fujirebio), OC-Sensor (OC-Auto sam-pling bottle3) analyzed using an OC-Sensor DIANAinstrument (Eiken Chemical Co.; distributed by MastDiagnosis), and FOB Gold (distributed by SKD, France)analyzed using a SENTiFOB instrument (Sentinel diag-nostics).

Materials and Methods

I-FOBTsAll 3 tests use polyclonal rabbit antibodies directed

against human HbA. All tests are fully automated(Table 1). Analysis of OC-Sensor and FOB Gold is basedon immunoturbidimetry, which involves a measurementof the absorbance of light through the tube, whichincreases with the importance of Hb antibodies com-plexes. Analysis of Magstream involves the use of anautomated visual measurement of migration of aggluti-nated magnetic particles. In routine use, the crude pixelvalue generated by Magstream is converted into MSRunits, an arbitrary unit proposed by the manufacturer.For the present study, we asked Fujirebio to providesoftware (not routinely integrated in the machine) toallow for the collection of the measurements (pixelvalues). AlthoughMagstream is commercialized by Fujir-ebio as a qualitative test, a quantitative measure is pro-vided by the instrument, so we considered the test asquantitative in the analysis.

Fecal sampling methodFirst, freshly collected stools, obtained from 10

healthy subjects aged less than 50 years, were testedusing all 3 tests to confirm the initial absence of Hb.These stools were mixed and homogenized, thendivided into containers. In each of these containers, avolume of human whole blood lysate, the Hb content ofwhich had previously been measured (Advia 2120, Sie-mens), was added to obtain all prespecified Hb con-centrations in the stool. Each container was vigorouslyshaken after adjunction of blood. For each of the con-centrations, sampling of all 3 tests was done using thesame containers, ensuring that concentration was iden-tical for all tests. Finally, collecting tubes were shakenafter sampling and before analysis. This procedure wasrepeated 2 times leading to 2 distinct stool mixtures, onebeing analyzed to evaluate reproducibility and the otherto evaluate stability to storage.

Experimental planTwo distinct experiments were conducted: the first

one to evaluate and compare the reproducibility of thetests (experiment 1) and the other one to evaluate andcompare their sensitivity to temperature and delay ofstorage (experiment 2). For each of these 2 experiments,a distinct stool mixture was performed, as described asfollows.

Experiment 1(stool mixture n�1). To compare testsfor a given Hb concentration, we initially explored, foreach test, the relationship between concentration in thefeces and the value provided by the instrument for atotal of 10 values of Hb concentration in feces, varyingfrom 0 to 350 mg Hb/g of feces. This range of concen-trations was selected to: (i) cover the range of usual orproposed Hb positivity cutoffs of all tests and (ii) coverthe range of physiological bleeding of colorectal lesions(15). To assess measurement reproducibility, 10 tubeswere collected for each instrument and each concentra-tion, and all tubes were repeatedly analyzed 5 timesleading to a total of 50 readings per concentration andtest. In this experiment, all prepared I-FOBTs werestored for 3 days at 10�C before being analyzed, approx-imating to the conditions of a screening program invol-ving mailed samples.

Experiment 2 (stool mixture n�2). A second experi-mental plan was developed to assess the influence ofstorage temperature and delay between sampling andanalysis for different Hb concentrations in feces. For eachI-FOBT, 4 storage temperatures: 4�C, 10�C, 20�C, and30�C, and 5 delays between sampling and analysis: 1,3, 5, 7, and 10 days were tested for 6 Hb concentrations infeces (0, 20, 75, 100, 150, and 250) mgHb/g of feces. Due tothe large number of combinations of the 3 aforemen-tioned parameters in an exhaustive plan (360 combina-tions) and to the relatively slow operating rate of 1 of themachines (SENTiFOB, Sentinel Diagnostics), we used anoptimal experience plan, determined using the ADX(Analysis and Design of eXperiments) tool developedby SAS software. Four factors were introduced in thisexperimental plan (I-FOBT, storage temperature, delay,and concentration), as well as their 2-level interactions.Optimization of the design was achieved by D-optimaloptimization (maximization of the determinant of theinformation matrix), to do a linear regression evaluatingthe mean daily decrease in standardized concentrations(to avoid the impact of the slope of the relationshipbetween fecal concentration and buffer concentrationof blood) in the collecting tubes. However, due to thenonlinear relationship between concentration of blood inthe feces and the collecting tube (roughly logarithmic)and to the semiquantitative nature of the Magstream test(see Results), each test was assessed individually and nooverall analysis was conducted (see Statistical Methodsnext).

In our experimental plan, 32 combinations of storagetemperature, delay between sampling and analysis, andfecal Hb concentration were used for each test. The

Analytical Comparison of Immunochemical FOBTs

www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 20(7) July 2011 1493





Tab

le1.

Tech

nica

lcom

parison

oftheI-FO

BTs

OC-S

enso

rFO

BGold

Mag

stream

Buffervo

lumea

2mL

1.7mL

1mL

Feca

lsam

plingvo

lumea

10mg

10mg

0.3mg

Antibod

iesus

edRab

bitan

ti-hu

man

HbA

(polyc

lona

l)Rab

bitan

ti-hu

man

HbA

(polyc

lona

l)Rab

bitan

ti-hu

man

HbA(polyc

lona

l)

Prove

nan

alytical

rang

e50

to1,00

0ng

/mL(equiva

lent

to10

to20

0mg

/g)

50to

1,00

0ng

/mL

from

20ng

/mL(M

SR

¼1;

commercialized

asaqua

litativetest)

Rea

dingtech

nique

Anti-hu

man

Hban

tibod

iesaread

sorbed

onlatexpartic

les.

Inthepres

ence

ofblood

instoo

ls,they

provo

kethe

antig

en–an

tibod

yreac

tionan

dag

glutination,

cons

eque

ntly

referred

toas

thelatexag

glutinationtest.Cha

nges

insa

mpleturbidity

bylatexag

glutinationare

mea

suredop

tically.

Mag

netic

gelatin

partic

lesareattach

edto

anti-hu

man

Hb

antib

odies.

Collectingtubes

aretilted60

�from

theho

rizon

tal

pos

ition

,en

ablingfree

mag

netic

partic

lesto

slidedow

nthe

slop

eof

thewell,thus

form

ingamea

surable

line.

Thehigh

ertheprese

nceof

human

Hb,thesh

orterthelineis.Adigita

lpicture

ofthelineis

take

nbythemac

hine

.

Pixels:

328

Pixels:

197

Pixels:

132

Neg

ative

Pos

itive

Stron

gpos

itive

Abso

rban

ce57

0nm

Abso

rban

ce66

0nm

Collectingtube

FOB

Gold

OC-A

utosa

mplingbottle

3New

Hem

Tube

Autom

ated

analyzer

SENTiFO

BDiana

Mag

Strea

mHT

Rates

ofread

ing

75tubes

/hou

r28

0tubes

/hou

r90

0tubes

/hou

rUsu

althresh

old

175ng

Hb/m

linthebuffer

100ng

Hb/m

linthebuffer

211pixe

ls(M

SR

¼1.0)

Adjustab

lethresh

olddue

toqu

antitativena

ture

ofthetest.

Theo

retic

ally

nona

djustab

lethresh

oldsinc

ethetest

isco

mmercialized

asaqua

litativetest

bytheman

ufac

turers.

aTa

kenfrom

doc

umen

tsprovided

bytheman

ufac

turer.

Guittet et al.

Cancer Epidemiol Biomarkers Prev; 20(7) July 2011 Cancer Epidemiology, Biomarkers & Prevention1494





experimental plan can be deducted from Table 2 whichpresents mean measurement obtained for each test,according to temperature, concentration, or delay, andtherefore in which nonempty cells correspond to evalu-ated combinations of temperature*concentration*time*t-est. Ten tubes were collected for each of the includedsituations, each tube being analyzed 5 times.Temperature of storage was monitored using a system

of electronic measurement of temperature every 5 min-utes, and radio frequency recording (AOIP instrument).All tubes, whatever test or concentration, were stored inthe same conditions for each temperature (dedicatedfridge for 4�C and 10�C temperatures with a range ofvariations of temperature of �2�C, air-conditioned roomfor 20�C temperaturewith a range of variations from 20�Cto 22�C, and a dedicated incubator for 30�C temperaturewith a range of variations of �1�C).

Statistical analysisReproducibility of I-FOBTs (experiment 1). For each

test and each of the experimental concentrations, theintertube and intratube variances were determined byusing a random effect model (SAS PROC MIXED). Thisprocedure enabled us to compute the variation coeffi-cients due to sampling (intertube) and reading (intra-tube).Stability to temperature and duration of storage

(experiment 2). In an exploratory analysis, we com-puted the mean measurement obtained for each test,according to temperature, concentration, or delay(Table 2). Then, for each temperature and processingdelay, the mean measurement was standardized on theinitial fecal concentration to allow comparison of var-iations in concentration due to storage duration andtemperature between tests. For FOB Gold and OC-Sensor, the buffer concentration was directly propor-tional to fecal concentration (Fig. 1). The theoreticalrelationship between fecal Hb concentration (Cf),expressed in mg/g, and concentration of Hb in thebuffer (Cb), expressed in ng/mL, is given by the follow-ing formula: Cb ¼ ðCf * qsÞ=vb, where qs is the quantityof stools introduced in the tube (in mg) and vb is thevolume of buffer (in mL). However, as we did notcheck independently the values of qs and vb providedby the manufacturers, the buffer concentration pro-vided by the automated analyzer was simply dividedby the initial theoretical fecal concentration, to stan-dardize data. For Magstream, the pixel value providedby the automated analyzer had an inverse logarithmrelationship to fecal Hb concentration, and the meanpixel value was 330 in the absence of Hb in the sample(Fig. 1). The following transformation was thereforeapplied to data: (330 pixel)/log(initial fecal concentra-tion). However, this transformation was less adapted tosmaller concentrations. For each test, we evaluated themean daily decrease in standardized Hb concentrationaccording to temperature using mixed effect linearmodels.

Statistical analysis was done using SAS software, ver-sion 9.1 (SAS Institute).

Results

Experiment 1Relationship between measurements and fecal concen-

tration of Hb. First, the relationship of the values pro-vided by each automated analyzer (3 days’ storage at10�C) was assessed according to the initial fecal Hbconcentration. A linear relationship between measure-ment and fecal Hb content was observed for OC-Sensorand FOB Gold, but not for Magstream (Fig. 1).

For Magstream, the pixel values were bounded inthe range 130 to 330 pixels. Above a concentration inthe tested stools of 250 mg Hb/g, the pixel measure-ments in the tube did not change with concentration(the mean pixel measurement was 130 pixels). There-fore, the test could only be considered as quantitativewithin a fecal Hb concentration range of 20 to 200 mgHb/g of feces.

Instrument measurements have been plotted in Figure1 and the expected value is represented as a dotted lineaccording to linear modeling for FOB Gold and OC-Sensor, and loess (local polynomial fitting) modelingfor Magstream. We defined overlap between concentra-tions whether no cutoff could perfectly separate instru-ment measurements between 2 successive fecalconcentrations. For Hb concentrations up to 150 mgHb/g of feces, there was no overlap in the range ofmeasurements obtained with the different Hb concentra-tions in feces selected in our protocol for OC-Sensor. Forboth Magstream and FOB Gold, overlap was observedbetween concentrations in the entire range of these testedconcentrations.

ReproducibilityReproducibility could be explored in the entire concen-

tration range for OC-Sensor and FOB Gold (Fig. 2). How-ever, it was only explored for concentrations below 250 mgHb/g of feces for Magtream, because for higher concen-trations, the absence of measurement variation betweenconcentrations using this test would lead to artificiallygood reproducibility.

The best reproducibility (smaller total variation coeffi-cient) was observed with OC-Sensor, with the exceptionof concentrations below 75 mg Hb/g of feces, for whichMagstream offered better reproducibility. However,variability for small concentrations using Magstreamwas reduced due to lack of quantitative measurementsabove 330 pixels. The worst reproducibility was observedwith the FOB Gold test. Indeed, the mean total variationcoefficient observed between 75 and 250 mg Hb/g of feceswas 0.07 for OC-Sensor, 0.10 for Magstream, and 0.18 forFOB Gold.

For all tests, measurement variability involved inter-tube variability rather than intratube variability (thevariation coefficients associated with sampling were far







Tab

le2.

Mea

nmea

suremen

tac

cordingto

test*con

centratio

n*temperature*delay

Tes

tOC-S

enso

rFO

BGold

Mag

stream

Con

centratio

nof

hemog

lobin

inthefece

s,mg

/g0

2075

100

150

250

020

7510

015

025

00

2075

100

150

250

Exp

erim

ent

Temperature

Day

s1

10� C

30

8533

449

867

01,08

66

124

552

664

877

1,86

332

129

921

018

614

713

22

4�C

110

84

151

147

30

789

6328

113

213

35

100

464

641

2,22

132

47

1207

1255

259

148

134

1058

762

532

010

� C1

01,28

767

61,09

924

03

579

163

2,12

131

514

55

1,21

94

129

70

101

796

130

1041

970

54

205

152

130

20� C

154

765

716

462

031

513

33

9539

360

092

012

45

984

8868

819

918

67

436

697

331

310

032

659

531

422

330

� C1

340

565

420

23

733

531

228

35

220

438

213

317

311

796

289

612

317

315

108

317

810

315

Guittet et al.






greater than those associated with reading). Intratubevariability was low for all tests (the mean coefficient ofvariation due to reading between 75 and 250 mg Hb /g offeces was 0.025, 0.016, and 0.060 for FOB Gold, OC-Sensor, and Magstream, respectively).The difference between these sources of variation was

the highest for FOB Gold, for which the intertube varia-bility was particularly important compared with theother tests. The mean coefficient of variation due tosampling between 75 and 250 mg Hb/g of feces was0.15 for FOB Gold, whereas it was only 0.10 for OC-Sensor and 0.07 for Magstream.Intertube variability associated with FOB gold tended

to decrease as the Hb concentration in feces increased.

Intertube variability associated with OC-Sensor wasstable between 75 and 250 mg Hb/g of feces, but washigh for concentrations below 75, or above 250 mg Hb/gof feces. The latter concentrations were higher than theupper limits of good performance recommended by themanufacturer. Intertube variability using Magstreamincreased as did fecal Hb concentration up to 150 mgHb/g of feces, then decreased due to a nonlinear relation-ship between pixels and concentration.

Experiment 2Stability. Mean measurements according to test,

temperature, concentration, and time are provided inTable 2. For all 3 tests, measurement was stable over

OC-Sensor

Hb

conc

entr

atio

n in

the

buffe

r, ng

/mL

0

0 50 100 150 200 250 300 350

500

1,00

01,

500

FOB Gold

Hb

conc

entr

atio

n in

the

buffe

r, ng

/mL

0 50 100 150

Initial fecal Hb concentration, µg/g

200 250 300 3500

500

1,00

01,

500

2,00

02,

500

3,00

0 Magstream

0 50 100 150 200 250 300 350

150

200

Pix

el v

alue 25

030

0

Figure 1. Value returned by the automated analyzers according to test and fecal Hb concentration. Manufacturer's thresholds are presented as a continuousline (175 ng/mL for FOB Gold; 100 ng/mL for OC-Sensor; and 211 pixels for Magstream). Data modeling is presented as a dotted line (linear modeling:FOB Gold, OC-Sensor; loess modeling: Magstream).

OC-Sensor

0.2

0.15

0.1

Var

iatio

n co

effic

ient

0.05

0

50 100 150 200 250* 300* 350*

0.2

0.15

0.1

Var

iatio

n co

effic

ient

0.05

0

50 100 150* 200*

Initial fecal Hb concentration, µg/g

250* 300* 350*

FOB Gold

0.2

0.15

0.1

Var

iatio

n co

effic

ient

0.05

0

50 100 150 200 250** 300** 350**

Magstream

Figure 2. Variation coefficients due to sampling or reading according to fecal Hb concentration. X, variation coefficients due to sampling. ^, variationcoefficients due to reading. *, variation coefficients total. *, quantification of Hb implied dilution during reading. **, not calculable.







time, independently of storage temperature when no Hbwas added to feces. Relationship between instrumentmeasurements (pixel or buffer concentration) and fecalHb concentration was verified (data not shown). Figures3 and 4 plot meanmeasurement permachine according tostorage duration or temperature. A decrease in measure-ment was observed for all tests at increased storagedurations, and at temperatures of 20�C and 30�C, butnot 4�C or 10�C.

Figure 5 provides the mean of ratio of measurementsstandardized on the experimental fecal concentration foreach combination of storage duration and temperatureand for each concentration (see Materials and Methods).The stability of the Hb measurement provided by all 3tests was good at 4�C and 10�C for OC-Sensor and Mag-stream. However, an increase in concentration providedby FOB Gold was observed within the first days, parti-cularly at a storage temperature of 4�C. A similar (yet

smaller) increase in concentration detected by OC-SensorandMagstream over the first days cannot be excluded onthe basis of our data.

At a storage temperature of 20�C, a substantialdecrease in Hb concentration was observed over timeusing all 3 tests. The best stability was observed usingOC-Sensor with a significant daily decrease in measure-ment of 1.7% (P < 0.01) at 20�C, compared with a dailydecrease of 7.4% and 7.8% with Magstream and FOBGold, respectively (P < 10�3). However, the decreaseobserved with Magstream depended on the initial Hbconcentration. It was greater with the smaller concentra-tion of 20 mg Hb/g of feces.

At 30�C, the performance of OC-Sensor was far betterthan that of FOB Gold and Magstream. Indeed, the meandaily decrease in measurement observed with OC-Sensorwas 8.6% (P < 10�3), whereas the mean decrease inmeasurement observed on the first day was 30% for

OC-Sensor

0 5 10 15 20 25 30

Mea

n of

buf

fer

conc

entr

atio

, ng/

mL

020

040

060

080

01,

000

0 5 10 15 20 25 30

Temperature of storage, °C

Mea

n of

buf

fer

conc

entr

atio

n, n

g/m

L

020

040

060

080

01,

000

1,20

0

FOB Gold

0 5 10 15 20 25 30

Mea

n of

pix

el v

alue

150

200

250

300

350

Magstream

Figure 4. Comparison of mean measurements and SD according to test: temperature of storage.

OC-Sensor

0 2 4 6 8 10

020

0

Mea

n of

buf

fer

conc

entr

atio

n, n

g/m

L

400

600

800

1,00

0

0

Duration of storage, d

2 4 6 8 10M

ean

of b

uffe

r co

ncen

trat

ion,

ng/

mL

020

040

060

080

01,

000 FOB Gold

0 2 4 6 8 10

Mea

n of

pix

el v

alue

150

200

250

300

350

Magstream

Figure 3. Comparison of mean measurements and SD according to test: duration of storage.

Guittet et al.






Magstream and 70% for FOB Gold. The decreaseobserved with OC-Sensor at 30�C depended on the initialconcentration, being more important for small concentra-tions. On day 7, 38% of an initial concentration of 150 mgHb/g of stool was still detected with OC-Sensor. On day10, 25% of an initial concentration of 250 mg Hb/g of stoolwas still detected by the same test. On the contrary, fromday 5, no Hb content was detected by FOB Gold orMagstream in the samples.

Discussion

Our results show that the precision and the reprodu-cibility of Hb measurements in feces are better with OC-Sensor than with Magstream, and better with Magstream(in the range of concentrations where the test can beconsidered as quantitative) than with FOB Gold. Aboutstability at varying temperatures, our results show thatindependently of the test used, there is a substantial lossin Hb measurement as from 20�C. This loss is moreimportant for FOB Gold than for the other tests. At20�C and 30�C, denaturation of Hb is less importantand occurs less quickly in the buffers used with OC-Sensor than with Magstream devices.Independently of the test used, intratube variability

was lower than intertube variability. Similar findingswere found in an experimental study conducted in U.K., although intratube variabilitywas assessed in solutionof Hb, and intertube in artificially positive stools (16). Theintertube variability estimated in our experiment was aconsequence of both the tube characteristics and thebiologist’s reproducibility in using the test (samplingof feces using the immunochemical tests probes). In realscreening settings, such a tube effect would probably begreater because a patient is certainly less reproducible in

his sampling technique than a biologist, and patients donot mix their stool to homogenize their blood contentbefore performing the test. Moreover, the tube effectmeasured in screening programs including several tubesper patient also includes the effect of the intermittency ofthe bleeding associated with colonic lesions. To the con-trary, the intratube variability measured in our experi-ment should be similar to that which occurs in realscreening settings, as it is solely an effect of the automatedanalyzer itself.

The lower intertube variability when using OC-Sensorcould be explained by a more accurately calibrated quan-tity of stool incorporated in the sample (notmeasurable inour study). In addition, more overlap occurred betweenthe evaluated concentrations with FOB Gold and Mag-stream than with OC-Sensor. Accordingly, the loss inprecision of these tests (including intertube and intratubevariability) could have consequences on positivity ratesin real screening settings.

Temperature-related Hb degradation is expected;however, it can be delayed by the use of a suitablestabilizing agent in the buffer. Such stabilizing agentsare included in all 3 tests. Nevertheless, the stability ofHbmeasurement at varying temperatures and over timewas better with OC-Sensor than with Magstream andfar better than FOB Gold. The superiority of OC-Sensorwas also observed in the NHS evaluation report (16). Ata temperature of 20�C, the decrease observed with FOBGold and Magstream was at least twice that observedwith OC-Sensor. At 30�C, the performance of Mag-stream and FOB Gold was very poor, whereas theOC-Sensor was much better and remained reliable, evenat this high temperature. I-FOBT sensitivity variationsrelated to storage duration have previously beendescribed in both laboratory experiments and genuine

Magstream

Mea

n of

rat

io (

(330

-Pix

el)/

log(

feca

l con

cent

ratio

n))

2 4 6 8 10

510

15

FOB Gold

2 4

Duration of storage, d

6 8 100

24

68

10

Mea

n of

rat

io (

buffe

r co

ncen

trat

ion/

feca

l con

cent

ratio

n)

1

2 4

OC-Sensor

6 8 10

Mea

n of

rat

io (

buffe

r co

ncen

trat

ion/

feca

l con

cent

ratio

n)

23

45

6

Figure 5. Comparison of the evolution of the measured concentration in the buffer according to storage temperature and duration. *, mean ratiotemperature ¼ 4�C; modelization (—) linear (OC-Sensor); polynomial (FOB Gold, Magstream). D, mean ratio temperature ¼ 10�C; modelization (- - -)linear (OC-Sensor); polynomial (FOB Gold, Magstream). þ, mean ratio temperature ¼ 20�C; modelization (. . ..) linear (OC-Sensor); polynomial (FOB Gold,Magstream). �, mean ratio temperature ¼ 30�C; modelization (_ . _) linear (OC-Sensor); polynomial (FOB Gold, Magstream).







screening settings (11–14). The interaction between sto-rage duration and temperature has been quantified forthe OC-Sensor test in a laboratory experiment: The dailydecrease in fecal Hbmeasurement was 0.3% at 4�C, 2.2%at 20�C, and 3.7% at 28�C (14). Our findings (2% dailydecrease at 20�C and 9% daily decrease at 30�C) areconsistent with this observation, although the decreaseobserved at 30�C seemed more important. This could beexplained by a difference in initial Hb concentration infeces, the relative decrease being higher for small initialconcentrations. Such differences in sensitivity related tostorage temperature and duration, and affecting thereliability of colorectal screening test programs, couldhave an important impact on screening organizationand test choice, particularly in countries where ambienttemperatures are high. It would seem that, wheneverpossible, the delay between sampling and test proces-sing should be reduced to 3 days, or CRC screeningprograms should be stopped during the summer incountries with long period of very high temperatures(>30�C). In addition, patients should be advised to storefecal samples in the refrigerator at home before for-warding them by post. Acceptability of such a recom-mendation needs further investigations.

Our study has several limitations associated withexperimental conditions and data analysis constraints.Since it was based on laboratory preparation of positivefecal samples, it is possible that these artificial positivesamples behave differently than native positive samples,despite the fact that we used human feces. However,decrease in Hb from real positive samples has also beenshown with OC-Sensor (11). On the one hand, mixed ornonmixed stool samples could behave differently in theapplication of I-FOBT. On the other hand, the mixture ofstool samples from several subjects done in our protocolallowed us to avoid differential bias between concentra-tions and tests in case ofundetectedHbpreviouslypresentin 1 or several fecal samples. In addition, sampling errorwas probably underestimated since the tests were con-ducted by trained biologists, rather than by subjects fromthe general population. For example, no miss manipula-tion of tubes occurred, such as opening the wrong side ofthe FOB Gold test or spilling the OC-Sensor or FOB Goldbuffer. Targeted concentrations were not checked in thesamples by independent Hb measurement methods.Nevertheless, within each of the experiments (for exam-ple, storage evaluation), calibrated stool samples wereobtained from the same negative FOB-tested stools.Finally, the nonlinear relationship between pixel valueand concentration of Hb in the buffer for the Magstreamtest implied transformation of crude data. However, weselected the transformation which minimized underlyingassumptions and independently of the results.

However, the fact that this study was entirely labora-tory-performed enabled every parameter to be controlled,as from the introduction of blood into the stool. This is notthe case for studies exploring the stability of fecal samplessent to the laboratory after having been performed by the

patient at home, measurement of initial Hb concentrationin the stool, and storage duration or temperature fromhome to laboratory being unknown (11, 14). This alsoenabled the 3 tests to be compared on the same stools.Moreover, the use of blood lysate guaranteed the bestpossible conditions for the antibody–antigen reaction.Finally, our laboratory study was designed to fit as mostas possible real screening settings with the choice of arange of concentrations adapted to physiological bleedingof lesions and discussed cutoffs, temperatures close tousual weather conditions, and delays compatibles withmailing of samples bypost to a central analysis center, as itis the case in FOBT-based screening programs.

Our results show better analytical and stability perfor-mances of OC-Sensor or Magstream than FOB Gold.Comparison between OC-Sensor and Magstream wasmade more complex since: (i) the area of optimal perfor-mance was different for both tests, OC-Sensor detectingsmaller Hb concentrations, and (ii) the relationshipbetween measurement of OC-Sensor and Magstreamwas not linear. The improved stability offered by OC-Sensor compared with Magstream and the semiquanti-tative nature of the Magstream offer strong arguments infavor of OC-Sensor. Nevertheless, several studies haveshown the good performance of Magstream in popula-tion surveys, even using only 1 sample at the man-ufacturer’s threshold (17, 18). Costs of the tests shouldalso be considered, together with adaptation of screeningprograms to limit the effect of sensitivity of tests totemperature on their performances (12). Further studiesand cost-effectiveness analysis are needed to compareMagstream and OC-Sensor in real screening settings,appropriately taking into account temperature and dura-tion of storage.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Contributions

L. Guittet, E. Guillaume, R. Levillain, P. Beley, and G. Launoy elabo-rated the experimental plan. P. Beley and R. Levillain performed thelaboratory analyses. L. Guittet and E. Guillaume performed the statisticalanalysis. Results were analyzed and discussed by all authors. Finally, L.Guittet, E. Guillaume, and G. Launoy wrote the article, which was care-fully corrected by all authors.

Grant Support

The study was funded by the French National Cancer Institute (InCA).Instruments were provided on loan by manufacturing companies. Supportingcompanies had no access to crude data. Preliminary analysis was provided tothem for the purposes of information; however, in no manner whatsoever did itinfluence our analysis or the drafting of this article.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 7, 2010; revised April 18, 2011; accepted May 3, 2011;published OnlineFirst May 16, 2011.

Guittet et al.






References1. Hewitson P, Glasziou P, Watson E, Towler B, Irwig L. Cochrane syste-

matic review of colorectal cancer screening using the fecal occult bloodtest (hemoccult): an update. Am J Gastroenterol 2008;103:1541–9.

2. Guittet L, Bouvier V, Mariotte N, Vallee JP, Ars�ene D, Boutreux S, et al.Comparison of guaiac based and an immunochemical faecal occultblood test in screening for colorectal cancer in a general average riskpopulation. Gut 2007;56:210–4.

3. Van Rossum LG, Van Rijn AF, Laheij RJ, van OijenMG, Fockens P, vanKrieken HH, et al. Random comparison of guaiac and immunochem-ical fecal occult blood tests for colorectal cancer in a screeningpopulation. Gastroenterology 2008;135:82–90.

4. Hol L, Wilschut JA, van Ballegooijen M, van Vuuren AJ, van der Valk H,Reijerink JC, et al. Screening for colorectal cancer: random compar-ison of guaiac and immunochemical faecal occult blood testing atdifferent cut-off levels. Br J Cancer 2009;100:1103–10.

5. Levi Z, Rozen P, Hazazi R, Vilkin A, Waked A, Maoz E, et al. Aquantitative immunochemical fecal occult blood test for colorectalneoplasia. Ann Intern Med 2007;146:244–55.

6. Grazzini G, Visioli CB, Zorzi M, Ciatto S, Banovich F, Bonanomi AG,et al. Immunochemical faecal occult blood test: number of samplesand positivity cutoff. What is the best strategy for colorectal cancerscreening? Br J Cancer 2009;100:259–65.

7. Guittet L, Bouvier V, Mariotte N, Vallee JP, Levillain R, Tichet J, et al.Performances of immunochemical faecal occult blood test in colorectalcancer screening in average-risk population according to positivitythreshold and number of samples. Int J Cancer 2009;125:1127–33.

8. van Rossum LG, van Rijn AF, Laheij RJ, van Oijen MG, Fockens P,Jansen JB, et al. Cutoff value determines the performance of a semi-quantitative immunochemical faecal occult blood test in a colorectalcancer screening programme. Br J Cancer 2009;101:1274–81.

9. U.S. Preventive Services Task Force. Screening for colorectal cancer:U.S. preventive services task force recommendation statement. AnnInt Med 2008;149:627–37.

10. Lieberman DA. Clinical practice. Screening for colorectal cancer. NEngl J Med 2009;361:1179–87.

11. van Rossum LG, van Rijn AF, van Oijen MG, Fockens P, Laheij RJ,Verbeek AL, et al. False negative fecal occult blood tests due todelayed sample return in colorectal cancer screening. Int J Cancer2009;125:746–50.

12. Grazzini G, Ventura L, Zappa M, Ciatto S, Confortini M, Rapi S, et al.Influence of seasonal variations in ambient temperatures on perfor-mance of immunochemical faecal occult blood test for colorectalcancer screening: observational study from Florence district. Gut2010;59:1511–5.

13. Australian Government, Department of Health and Aging. Nationalbowel cancer screening program: screening retest fact sheet.[accessed 2010 Sept 13]. Available from: www.cancerscreening.gov.au/internet/screening/publishing.nsf/content/bw-retest

14. Rozen P, Waked A, Vilkin A, Levi Z, Niv Y. Evaluation of a desk topinstrument for the automated development and immunochemicalquantification of fecal occult blood. Med Sci Monit 2006;12:MT27–32.

15. Ciatto S, Martinelli F, Castiglione G, Mantellini P, Rubeca T, GrazziniG, et al. Association of FOBT-assessed faecal Hb content with coloniclesions detected in the florence screening programme. Br J Cancer2007;96:218–21

16. NHS Evaluation report. Immunochemical faecal occult blood tests;2009 Nov. Report No.: CEP09042.

17. Morikawa T, Kato J, Yamaji Y, Wada R, Mitsushima T, Shiratori Y. Acomparison of the immunochemical fecal occult blood test and totalcolonoscopy in the asymptomatic population. Gastroenterology2005;129:422–8.

18. Guittet L, Bouvier V, Mariotte N, Vallee JP, Levillain R, Tichet J, et al.Comparison of a guaiac and an immunochemical fecal occult bloodtest for the detection of colonic lesions according to lesion type andlocation. Br J Cancer 2009;100:1230–5.







analytical comparison of three quantitative immunochemical fecal occult blood tests for colorectal...

Documents