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© 2012 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
nature publishing group ORIGINAL CONTRIBUTIONS 99
COLON/SMALL BOWEL
INTRODUCTION
Colorectal cancer (CRC) is a major healthcare problem. World-
wide, CRC is the fourth most occurring malignancy in men and
ranks third in women ( 1 ). Furthermore, CRC is the second-most
frequent cause of cancer-related death in the Western world ( 2 ).
For an average-risk individual the lifetime risk of developing CRC
is around 5 % . For these reasons it can be concluded that CRC is
a major health problem.
Four randomized controlled trials showed that screening by
means of fecal occult blood tests (FOBT) reduces CRC-related
mortality by 15 – 33 % ( 3 – 7 ). Currently, population-based CRC
screening programs using FOBT have been implemented or are
studied in feasibility trials in many western countries. FOBTs fall
into two categories based on the detected component of blood:
the guaiac-based FOBT (gFOBT) and the more recently deve-
loped fecal immunochemical test (FIT). e rst type of FOBT
Are Fecal Immunochemical Test Characteristics
Infl uenced by Sample Return Time ? A Population-Based
Colorectal Cancer Screening Trial
Aafke H.C. van Roon , MD
1 , Lieke Hol , MD, PhD
1 , Anneke J. van Vuuren , PhD
1 , Jan Francke
1 , Martine Ouwendijk
1 , Angela Heijens
1 ,
Nicole Nagtzaam
1 , Jacqueline C.I.Y. Reijerink , MSc
2 , Alexandra C.M. van der Togt
3 , Marjolein van Ballegooijen , MD, PhD
4 ,
Ernst J. Kuipers , MD, PhD
1
,
5 and Monique E. van Leerdam , MD, PhD
1
OBJECTIVES: Fecal immunochemical tests (FIT) are preferred over guaiac-based fecal occult blood testing as
colorectal cancer (CRC) screening tool. However, hemoglobin (Hb) degradation over time may
infl uence FIT outcome. We therefore evaluated the effect of sample return time on FIT performance
characteristics in a population-based CRC screening trial.
METHODS: A representative random sample of the Dutch population ( n = 17,677), aged 50 – 74 years, was
invited for FIT screening (OC-Sensor Micro; cutoff ≥ 50 ng Hb / ml). Sample return time was defi ned
as the interval in days between fecal sampling and FIT laboratory delivery. Moreover, a random sam-
ple of positive FITs were selected to be stored at room temperature and re-tested every 3 – 4 days.
RESULTS: In total, 8,958 screenees fulfi lled our inclusion criteria. The mean sample return time was 3 days
( ± 3). Overall, 792 screenees (8.8 % ) had a positive test. Between the sample return time groups, the
positivity rate (PR) varied between 7.7 and 9.0 % . No statistically signifi cant associations were found
between PR or detection rate (DR) and the different sample return time groups ( P value = 0.84 and
0.76, respectively). For the laboratory experiment, 71 positive FITs were stored at room temperature
and re-tested with standard intervals. The mean daily fecal Hb decrease was 5.88 % per day (95 %
confi dence interval 4.78 – 6.96 % ). None of the positive FITs became negative before 10 days after
fecal sampling.
CONCLUSIONS: This population-based CRC screening trial demonstrates that both the PR and DR of FITs do not
decrease with prolonged sample return times up to 10 days. This means that a delay in sending the
FIT back to the laboratory, of up to at least 1 week, does not necessitate repeat sampling in case
of a negative test result. These data support the use of FIT-based screening as a reliable tool for
nationwide CRC screening programs.
Am J Gastroenterol 2012; 107:99–107; doi: 10.1038/ajg.2011.396; published online 22 November 2011
1 Department of Gastroenterology and Hepatology, Erasmus University Medical Centre , Rotterdam , The Netherlands ;
2 Association of Nationwide Screening
South-western Netherlands , Vlaardingen , The Netherlands ;
3 Comprehensive Cancer Centre , Rotterdam , The Netherlands ;
4 Department of Public Health, Erasmus
University Medical Centre , Rotterdam , The Netherlands ;
5 Department of Internal Medicine, Erasmus University Medical Centre , Rotterdam , The Netherlands .
Correspondence: Aafke H.C. van Roon, MD , Department of Gastroenterology and Hepatology, Erasmus University Medical Centre , ‘ s Gravendijkwal 230,
3015 CE Rotterdam , The Netherlands . E-mail: a.vanroon@erasmusmc.nl
Received 11 November 2010; accepted 28 May 2011
see related editorial on page 108
The American Journal of GASTROENTEROLOGY VOLUME 107 | JANUARY 2012 www.amjgastro.com
100
COLON/SMALL BOWEL
van Roon et al.
detects heme, which is incorporated in hemoglobin (Hb) mole-
cules, using its inherent peroxidase activity. e gFOBT reacts to
any peroxidase in feces (e.g., plant peroxidases or heme in red
meat) and is a ected by certain chemicals (e.g., high-dose vita-
min C supplements), resulting in false-positive and false-nega-
tive tests. FIT on the other hand, measures the presence of intact
globin chains in Hb molecules by means of speci c antihuman
anti bodies. erefore, FITs are in contrast with gFOBTs spe-
ci c for human blood. Furthermore, FITs are more speci c for
lower gastrointestinal bleedings, as protease enzymes gradually
digest blood from the proximal gastrointestinal tract during its
passage through the intestine, rendering it less likely that globin
chains will be recognised by the antibodies used in a FIT ( 8,9 ).
More over, FITs — at least some, including the one addressed to in
this paper — provide a quantitative measurement of microscopic
blood loss in stool. is allows selection of an optimal cuto
value for a speci c target population and standardization of test
outcomes ( 10,11 ). Finally, several trials have demonstrated that
fecal immuno chemical testing is superior to the traditionally used
gFOBT (i.e., the non-rehydrated Hemoccult II) in terms of higher
attendance and diagnostic yield of advanced neoplasia at the same
or even higher speci city ( 12 – 20 ).
However, in contrast with gFOBT screening, there are con-
cerns that FITs are sensitive to delayed sample return. First, the
globin chains in Hb molecules degrade more rapidly than heme
( 21 – 23 ). Second, the degradation of Hb may occur quite fast
in moist samples as used by most FITs, in contrast to the rela-
tively dry smears used on gFOBT sample cards ( 21 ). It has been
reported that a delay between fecal sampling and arrival at the
laboratory impairs the e cacy of FITs ( 24 ). is e ect would be
a major problem for the yield of FIT-based screening programs
and could therefore create a potential obstacle for the implemen-
tation and replacement of gFOBT by FIT. However, exact data
are lacking and thus recommendations with respect to handling
of negative tests with a prolonged sample return time remain to
be determined. We therefore evaluated the e ect of FIT sample
return time on test performance characteristics in a population-
based CRC screening trial.
METHODS
Part I : study population
Between November 2006 and June 2009, a population-based
CRC screening trial was conducted in the southwest of the
Netherlands with a target population of ~ 350,000 inhabitants.
Details of the subsequent trial protocols for one- and two-sam-
ple FIT screening have been described elsewhere ( 12,25 ). Brie y,
a total of 17,677 individuals between the ages of 50 – 74 years
were randomly obtained from municipal population registers
by a computer-generated algorithm (Tenalea, Amsterdam,
the Netherlands). Selection was performed per household and
occurred before invitation. As there is no nationwide CRC
screening program in the Netherlands, the population used
for this trial was screening-na ï ve. Eventually, 14,480 indivi-
duals were invited for one-sample FIT testing, whereas 3,197
individuals were invited to undergo screening with two FITs to
be sampled on consecutive days. Exclusion criteria were asked
for on the informed consent form, which had to be lled in by
the screenee itself. Exclusion criteria were a history of CRC;
in ammatory bowel disease; a life expectancy of under 5 years;
a colonoscopy, sigmoidoscopy, or double-contrast barium
enema within the previous 3 years; and inability to give informed
consent.
Interventions
All potential participants were sent an advance noti cation
letter, which contained background information on CRC and
CRC screening. Two weeks later, this letter was followed by
a standard invitation, which included an invitation letter, one
or two FITs, an instruction lea et on how to perform fecal
sampling, an information brochure, an informed consent form,
and a reply-paid envelope. All non-respondents were sent a
reminder 6 weeks a er the standard invitation.
Fecal immunochemical test
Each invitee was sent either one or two FITs (OC-Sensor Micro,
Eiken Chemical Co., Tokyo, Japan). is quantitative FIT con-
sists of a small test tube with a fecal probe inserted into it. e
probe has a serrated tip, which is pushed several times in di er-
ent parts of the stool and then pushed back into the tube, along
a scraper, through a membrane, and thereby closing and sealing
the test tube. is action removes most of the excess feces and
leaves a semi-standard amount of stool in the probe tip serra-
tions. e tip is then located in the bottom compartment of the
tube, which contains a 2-ml Hb-stabilizing bu er. Tests do not
require dietary restrictions or medication limitations. In case of
two-sample FIT screening, two test tubes were included in the
mailing and explicit instructions were given to use them on two
bowel movements on consecutive days. All individuals were
asked to report the date of fecal sampling on the test tube(s)
and instructions were given to return the test(s) as soon as pos-
sible. If the test(s) could not be returned immediately a er fecal
sampling, e.g., in case of two-sample FIT screening, storage in a
domestic refrigerator was recommended. Participants returned
the FIT sample(s) and a signed informed consent form to the
Gastroenterology and Hepatology Laboratory at the Erasmus
University Medical Centre for analysis using the automatic
OC-Sensor Micro instrument (OC-Sensor Micro, Eiken
Chemical Co.). Samples were collected a er arrival at the labo-
ratory and immediately stored at − 20 ° C until test development,
which occurred within 1 week. e manufacturer recommends
using a positivity threshold of 100 ng Hb / ml. However, litera-
ture as well as data provided by the manufacturer itself shows
that the test results of the OC-Sensor Micro are also reliable at
a lower cuto to the level of 50 ng Hb / ml ( 26 ). We have pre-
viously shown that this low cuto value remains associated
with a substantial positive predictive value ( 10 ). For this trial,
FITs were therefore considered positive when the Hb concen-
tration in the sample was ≥ 50 ng / ml (corresponding to 10 μ g
Hb per g of feces).
© 2012 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
101
COLON/SMALL BOWEL
Effect of Sample Return Time on FIT Characteristics
Test result
In case of a positive test result, the general practitioner was
informed both by telephone and mail within 2 weeks. e gen-
eral practitioner informed the participant about the test result and
referred the screenee for colonoscopy. Participants with a negative
test result were informed by mail within 2 weeks.
Colonoscopy
All colonoscopies were performed in the regional hospitals by
experienced endoscopists. e maximum reach of the endoscope
with identi cation of landmarks, as well as the adequacy of bowel
preparation, was recorded. During colonoscopy, characteristics
including size, morphology, and location of any polyps, were doc-
umented. Location was de ned as rectum, sigmoid, descending,
transverse, or ascending colon or cecum, and was measured in
centimeter from the anal verge with the endoscope in straight-
ened position. Size of each polyp was estimated using an open
biopsy forceps with a span of 7 mm. All removed polyps were
evaluated by experienced gastrointestinal pathologists. In accord-
ance with the international classi cation, CRC was de ned as the
invasion of malignant cells beyond the lamina muscularis mucosa
( 27 ). Lesions with intramucosal carcinoma or carcinoma in situ
were classi ed as high-grade dysplasia.
Ethical approval
e study was approved by the Dutch National Health Council
(PG / ZP 2.727.071 and PG / ZP 2.823.158). e approval included
the random selection before invitation design. e study letters
and information brochures were approved by the Institutional
Review Board at Erasmus University Medical Centre (MEC-2005-
264 and MEC-2008-029).
Part II : laboratory experiment
For this experiment, a total of 71 positive FITs were randomly
selected a er each series of testing, re-sealed, and stored at room
temperature (20 ° C) without actively keeping laboratory condi-
tions, such as humidity and temperature, at constant levels. With
standard intervals of 3 – 4 days, all selected FIT samples were
re-tested under standard laboratory conditions. In the same
way, 31 positive FIT samples were selected and stored in a stove
at a constant temperature of 30 ° C. Because it was hypothesized
that the Hb degradation would be faster at higher temperatures,
a shorter interval of 2 – 3 days was chosen to re-test all selected
FIT samples.
Statistical analyses
Part I . e sample return time was de ned as the interval in days
between fecal sampling at home, as reported by the screenee
itself, and FIT laboratory delivery. We classi ed all positive scree-
nees based on their sample return time into three subgroups:
short ( ≤ 3 days), average (4 – 6 days), and prolonged sample return
time ( ≥ 7 days). Uni- and multivariate ordinal logistic regression
analyses were used to determine the in uence of sex, age, and
socio-economic status (SES) on sample return time. In case of
two-sample FIT screening, the positivity rates (PRs) of both
samples were compared by using the McNemar test, knowing
that the rst FIT always had been performed at least 1 day earli-
er than the second performed test. To compare the positive pre-
dictive value (PPV) and detection rate (DR), one of both tests
was randomly selected for the nal analyses. e PR was de ned
as the proportion of participants having a positive test result.
e PPV was de ned as the proportion of participants with a
positive test result having an advanced neoplasia. is was calcu-
lated as the number of screenees with an advanced neoplasia
divided by all screenees with a positive test result who under went
a successful colonoscopy. Advanced neoplasia included CRC
and advanced adenomas. An advanced adenoma was de ned
as an adenoma ≥ 10 mm, or with a histology showing either
≥ 25 % villous component, or high-grade dysplasia. e DR was
de ned as the proportion of participants in whom an advanced
neoplasia was found ( 10 – 12 ). is was calculated as the number
of screenees with an advanced neoplasia divided by all screenees
with an analyzable screening test. When more than one lesion
was present, the screenee was classi ed according to the most
advanced lesion found during the follow-up colonoscopy. e
PR, PPV, and DR were calculated and described as proportions
with 95 % con dence intervals (CIs). Di erences in proportions
between the sample return time subgroups were calculated
using the Pearson χ 2 -test. Multivariate logistic regression analy-
ses were used to determine the in uence of sample return time,
sex, age, and SES on the PR, PPV, and DR. Because a recent
Italian report demonstrated a 17 % lower probability of FITs
being positive in summer than in winter, we also included
season in the regression analysis ( 23 ). Furthermore, the out-
side temperatures were based on data of the Royal Netherlands
Meteorological Institute ( www.knmi.nl ), providing aver-
age outside temperatures per month. Association between PR
and mean outside temperature was determined. All P -values
were two-sided and considered signi cant if < 0.05. Statisti-
cal ana lysis was performed with SPSS 15.0 for Windows (SPSS,
Chicago, IL).
Part II . A linear mixed effects model was used to estimate
the mean percentage Hb decrease per day ( 28 ). We used the
log-transformed Hb values as outcome and included time
after fecal sampling, which was expressed in days, as the only
predictor. The fecal sample was included as a random inter-
cept in the model to account for the correlations between the
repeated measurements of each individual FIT sample. Hence,
the intercept was allowed to vary from sample to sample but
the slope parameter of time was assumed to be equal for
all included samples. We used the lmer package in R for the
calculations ( 29,30 ).
RESULTS
Part I : proportion of positive tests
Of the 17,677 subjects who were randomly invited for CRC
screening, 8,958 screenees (51 % ) ful lled our inclusion criteria as
they returned the FIT and wrote down the sampling date on the
The American Journal of GASTROENTEROLOGY VOLUME 107 | JANUARY 2012 www.amjgastro.com
102
COLON/SMALL BOWEL
van Roon et al.
test tube. Table 1 shows the baseline characteristics of all included
screenees in the various sample return time subgroups. e mean
sample return time was 3 ± 3 days (mean ± s.d.) and the prolonged
sample return time group had a delay, which varied between
7 and 34 days. Screenees who returned their FIT samples within
3 days were signi cantly older and more o en female (both
P -values < 0.05).
Overall, 792 screenees (8.8 % ) had a positive test result at a cut-
o value ≥ 50 ng Hb / ml and were therefore referred for colono-
scopy. Between the di erent sample return time groups, the PR
varied between 7.7 and 9.0 % ( Tab l e 2 ). e results showed a uc-
tuation of both the PR and mean Hb concentration in relation
to the sample return time. ere was no statistically signi cant
di erence between the mean Hb level and sample return time
( P -value = 0.13), although a downward trend was seen from
a sample return time of 6 days onward ( Figure 1 ). When only
the PR was taken into account, again no statistically signi -
cant di erence was observed between the PR and sample return
time ( P -value = 0.96). Other factors that were associated with PR
were in line with previous results ( 10 ). is included higher PRs
among men compared with women (odds ratio (OR) 1.71; 95 % CI
1.47 – 1.99), individuals between the ages of 60 – 64 years (OR 1.27;
95 % CI 1.04 – 1.55) and 65 – 74 years (OR 1.99; 95 % CI 1.68 – 2.36)
compared with screenees aged 50 – 60 years, and screenees from
a middle (OR 1.29; 95 % CI 1.05 – 1.60) and low SES (OR 1.32;
95 % CI 1.12 – 1.55) compared with those from a high SES. Finally,
the PR was signi cantly higher during winter season compared
with the summer (9.7 % vs. 8.0 % respectively; P -value = 0.006).
Furthermore, an OR of 0.974 (95 % CI 0.960 – 0.990) was found for
FITs being positive with each degree Celsius increase in average
outside temperature ( Figure 2 ).
As mentioned, a separate analysis was carried out for the
two-sample FIT screening group, in which di erences in PR
between the rst and second test were compared. A total of
1,874 individuals participated with two-sample FIT screening.
e rst test was positive in 169 screenees (9.0 % ; cuto level
≥ 50 ng Hb / ml), compared with a PR of 8.8 % with the second
test ( P -value = 0.74).
In a multivariate ordinal logistic regression analysis, factors that
were associated with a longer sample return time were male gender
(OR 1.25; 95 % CI 1.15 – 1.34), and age < 60 years (OR 1.31; 95 % CI
1.20 – 1.43). No correlations were seen between sample return time
and SES ( P -value = 0.072).
Table 1 . Baseline characteristics of all included screenees
Sample return time (days) Overall P value
a
≤ 3 4 – 6 ≥ 7
Number of included screenees 5,959 2,723 276 8,958
Mean age (s.d.) 61.0 (6.6) 60.5 (6.6) 60.1 (6.5) 60.8 (6.6) 0.001
Sex (male; n , % ) 2,750 (46.1) 1,349 (49.5) 136 (49.3) 4,235 (47.3) 0.011
SES ( n , % ) 0.001
Very high 1,291 (21.7) 563 (20.7) 66 (23.9) 1,920 (21.4)
High 1,233 (20.7) 637 (23.4) 83 (30.1) 1,953 (21.8)
Intermediate 1,095 (18.4) 490 (18.0) 48 (17.4) 1,633 (18.2)
Low 1,151 (19.3) 507 (18.6) 31 (11.2) 1,689 (18.9)
Very low 1,189 (20.0) 526 (19.3) 48 (17.4) 1,763
(19.7)
SES, socio-economic status.
Sample return time = the interval in days between fecal sampling at home and fecal immunochemical test (FIT) laboratory delivery.
a Pearson χ
2 -test .
Table 2 . Number of included screenees and positive tests in
relation to sample return time
Sample return
time (days)
Number of
screenees
Number of positive
FITs (PR: 95% CI)
a
Mean hemoglobin
concentration
(ng / ml) ( ± s.d.)
b
≤ 2 3,951 352 (8.9:8.1 – 9.8) 43.6 (241.9)
3 2,008 180 (9.0:7.8 – 10.3) 45.7 (247.1)
4 1,561 141 (9.0:7.7 – 10.5) 42.5 (224.2)
5 836 72 (8.6:6.9 – 10.7) 47.8 (279.2)
6 326 25 (7.7:5.3 – 11.1) 20.5 (98.8)
≥ 7 276 22 (8.0:5.3 – 11.8) 23.1 (123.9)
Total 8,958 792 (8.8:8.2 – 9.4) 42.8 (237.4)
CI, confi dence interval; FIT, fecal immunochemical test; PR, positivity rate.
Sample return time = the interval in days between fecal sampling at home and
FIT laboratory delivery.
FIT (OC-Sensor Micro; cutoff value ≥ 50 ng Hb / ml).
PR (i.e., the proportion of participants having a positive test result).
No statistically signifi cant difference was found between either the PR or mean
hemoglobin concentration and sample return time, in which the sample return
time group ≤ 2 days was taken as reference.
a Univariate logistic regression analysis: P value = 0.96.
b ANOVA on the log-transformed data: P value = 0.13.
© 2012 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
103
COLON/SMALL BOWEL
Effect of Sample Return Time on FIT Characteristics
not signi cantly decrease the PR, PPV, or DR ( P -values 0.33, 0.54,
and 0.36, respectively).
Part II : laboratory experiment
In total, 71 positive FIT samples were randomly selected, stored
at room temperature, and re-tested with standard intervals of 3 – 4
days over a period of 3 weeks. In total, 69 (97 % ) of the screenees
from whom these positive FITs had been obtained, underwent
a successful colonoscopy. e samples included for this part of
the trial had a sample return time of 2 – 7 days. e initial Hb
concentration of the selected tests varied between 53 and 1,894
ng / ml. Figure 3a shows the Hb concentrations of the repeated
measurements on a logarithmic scale vs. the time in days a er
fecal sampling at home. Furthermore, Figure 3b demonstrates
in more detail all fecal samples with initial Hb concentrations
between 50 and 500 ng / ml on a normal scale. During storage at
room temperature, the mean Hb concentration in the fecal sam-
ples decreased by 5.88 % per day (95 % CI 4.78 – 6.96 % ). A er cor-
rection for sample return time, it was only a er 10 days that the
rst Hb concentrations dropped below the 50 ng / ml cuto level,
which resulted in a conversion from a positive test outcome into
a negative test result. e corresponding three samples had initial
Hb values between 53 and 58 ng / ml. ese three screenees had
a negative colonoscopy (i.e., two screenees with no lesions and
one screenee with a hyperplastic polyp). e remaining FIT sam-
ples became negative by a further lengthening of the interval. Two
weeks a er fecal sampling, 21 / 71 samples (30 % ) became negative.
By extending the sample return time towards 14 days, in total six
non-advanced adenomas, ve advanced adenomas and one CRC
would have been missed.
Moreover, another 68 positive FIT samples were stored in a stove
at a constant temperature of 30 ° C and re-tested every 2 – 3 days
over a period of 3 weeks. e collected positive tests had a sam-
ple return time of 2 – 6 days. e initial Hb concentration of the
selected fecal samples varied between 52 and 3,196 ng / ml. When
stored in a stove at 30 ° C, the mean Hb level decreased by 18.07 %
per day (95 % CI 16.88 – 19.24 % ). One week a er fecal sampling,
22 / 68 samples (32 % ) became negative. Moreover, this percentage
increased toward 84 % (57 / 68 samples) when the samples were
stored for a period of 2 weeks.
Follow-up and test performance characteristics
In total, 92 % (732 / 792) of all positive FIT screenees underwent
a successful colonoscopy, 294 (40 % ) of them were diagnosed
with an advanced neoplasia (252 advanced adenomas and 42
CRCs). No statistically signi cant correlation was found between
the PPV and sample return time: the PPV was 41 % in the
sample return time group ≤ 2 days vs. 33 % in the group with a
sample return time ≥ 7 days ( P -value = 0.66). Table 3 shows the
number of advanced neoplasia, as well as the PPV and DR for the
di erent sample return time groups.
Furthermore, the DR of advanced neoplasia per 100 screenees
was calculated. Between the di erent sample return time groups,
the DR varied between 2.5 and 3.7 % with an overall DR of 3.3 %
(294 / 8,958). e DR did not signi cantly decrease when the
sample return time was increased ( P -value = 0.85). Factors that
were associated with higher DRs were in line with previous results
( 10 ). In a multivariate logistic regression analysis, this included
in particular higher DRs among men compared with women
(OR 1.93; 95 % CI 1.52 – 2.46), individuals between the ages of
60 – 64 years (OR 1.40; 95 % CI 1.01 – 1.94) and 65 – 74 years
(OR 2.31; 95 % CI 1.76 – 3.03) compared with screenees aged
50 – 60 years, and screenees from a middle (OR 1.53; 95 % CI
1.10 – 2.13) and low SES (OR 1.40; 95 % CI 1.08 – 1.83) compared
with those from a high SES. e DR of advanced neoplasia
was signi cantly higher during winter season (OR 1.30; 95 % CI
1.03 – 1.65) compared with the summer.
Finally, the same conclusions could be drawn for a higher cut-
o value of 100 ng Hb / ml: increasing the sample return time did
234567
0
1
5
50
500
5000
Sample return time (days)
Hemoglobin concentration (ng/ml)
Figure 1 . Hemoglobin (Hb) concentration of all included fecal immuno-
chemical tests (FITs) for the different sample return time groups.
FIT (OC-Sensor Micro; cutoff value ≥ 50 ng Hb / ml). Sample return
time = the interval in days between fecal sampling at home and FIT
laboratory delivery. 䊊 = Hb concentration of one analyzed FIT sample.
• = Arithmetic mean Hb concentration per sample return time group.
No statistically signifi cant difference was found between the mean
Hb concentration and sample return time, in which the sample return
time group of 2 days or less was taken as reference. ANOVA was used on
the log-transformed data: P value = 0.13.
0
4
8
12
16
20
0
January
February
March
April
May
June
July
August
September
October
November
December
2
4
6
8
10
12
Temperature (°C)
Positivity rate (%)
Positivity rate (%) Average outside temperature (°C)
Figure 2 . Positivity rate vs. average outside temperature.
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104
COLON/SMALL BOWEL
van Roon et al.
DISCUSSION
Screening for CRC by means of a FIT forms an attractive
alternative to the most common and traditionally used gFOBT
(i.e., the non-rehydrated Hemoccult II) because of higher
attendance and diagnostic yield of advanced neoplasia ( 12 –
20 ). On the basis of modeling of data from various screening
trials, annual FIT screening has recently been reported to have
an impact on CRC-related mortality, which may amount to
a similar level as colonoscopy screening ( 31 ). Worldwide,
these ndings have raised strong interest in FIT testing as a
primary screening tool for CRC. In Europe, several countries
are considering to switch from gFOBT to FIT screening,
whereas others are preparing to newly introduce CRC screening
with FITs. e same applies for certain regions in Canada,
whereas in the United States a comparative trial is being
prepared between FIT and colonoscopy screening. However,
one important obstacle for the implementation of FIT screen-
ing is the possible limited stability of the test: due to globin
degra dation test sensitivity might drop with prolonged intervals
bet ween fecal sampling and arrival at the laboratory. How-
ever, our results demonstrate that with almost 10,000 FITs
analyzed, both the PR as well as the DR of advanced neopla-
sia does not signi cantly decrease with sample return times of
up to 7 days. Moreover, our trial results were con rmed by
Table 3 . Follow-up results of positive FIT screenees
Sample return
time (days)
Number of
positive tests
Number of
successful
colonoscopies ( % )
Number of patients with
advanced neoplasia
(PPV % ) PPV OR (95 % CI)
DR of advanced
neoplasia per 100
screenees ( % ) DR OR (95 % CI)
≤ 2 352 325 (92) 134 (41) 1 3.4 1
3 180 170 (94) 61 (36) 0.80 (0.54 – 1.17) 3.0 0.89 (0.66 – 1.21)
4 141 126 (89) 57 (45) 1.18 (0.78 – 1.78) 3.7 1.08 (0.79 – 1.48)
5 72 67 (93) 26 (39) 0.90 (0.53 – 1.55) 3.1 0.91 (0.60 – 1.40)
6 25 23 (92) 9 (39) 0.92 (0.39 – 2.18) 2.8 0.81 (0.41 – 1.60)
≥ 7 22 21 (95) 7 (33) 0.71 (0.28 –
1.81) 2.5 0.74 (0.34 – 1.60)
Total 792 732 (92) 294 (40) 3.3
CI, confi dence interval; DR, detection rate; FIT, fecal immunochemical test; OR, odds ratio; PPV, positive predictive value.
FIT (OC-Sensor Micro; cutoff value ≥ 50 ng Hb / ml).
Sample return time = the interval in days between fecal sampling at home and FIT laboratory delivery.
Advanced neoplasia = all colorectal cancers and advanced adenomas.
Advanced adenoma = adenoma ≥ 10 mm, adenoma ≥ 25 % villous component, or high-grade dysplasia.
5 7 10 15 20 25 30
0.5
5
50
500
ab
Time after fecal sampling (days)
Hemoglobin concentration (ng/ml)
5
500
400
300
200
100
50
0
71015202530
Time after fecal sampling (days)
Hemoglobin concentration (ng/ml)
Figure 3 . Laboratory experiment — hemoglobin (Hb) concentration of repeated fecal immunochemical test (FIT) measurements on a ( a ) logarithmic scale
and ( b ) normal scale. FIT (OC-Sensor Micro; cutoff value ≥ 50 ng Hb / ml). 䊊 = Hb concentration < 50 ng / ml (i.e., negative test result).
© 2012 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGY
105
COLON/SMALL BOWEL
Effect of Sample Return Time on FIT Characteristics
trial individuals from the same age were recruited from an
asymptomatic average-risk population and identical FITs
were used (OC-Sensor Micro; cuto value ≥ 50 ng Hb / ml).
However, the number of included subjects in that study was
considerably smaller (3,767 vs. 8,958 screenees in our trial),
only allowing for calculations with rather wide CIs. Second,
the PRs were remarkably di erent for the average sample return
time group (6.0 vs. 8.3 % in our study, respectively), and pro-
longed sample return time group (4.1 vs. 8.0 % ). e only
likely explanation for these di erences was the storage con-
ditions used at the laboratory. In the previous Dutch trial, all
included samples were stored in a laboratory refrigerator at
4 ° C, compared with storage at − 20 ° C in our trial. e pre-
viously mentioned Israelian trial also reported a drop in FIT
results below the 100 ng Hb / ml threshold a er prolonged
storage at 4 ° C ( 26,33 ).
e present study has some limitations. Although the
number of participants was high, the number of screened
individuals with a sample return time of 6 days or more was
relatively small which limited power of the study. ese rela-
tively low number of screenees with a strongly delayed sample
return time, in turn resulted in relatively even lower numbers
of screened individuals with an advanced neoplasm. erefore,
a type II error, i.e., ruling out an actual di erence between the
di erent sample return time groups, could not be excluded and
larger series are necessary to con rm our observations. Second,
advice was given to store the FITs in a domestic refrigerator if
the test(s) could not be returned instantly a er fecal sampling.
However, we were not able to verify if screenees obeyed these
instructions. erefore, the home conditions could have been a
potential bias in our results, because keeping the FIT samples
refrigerated would have postponed the Hb degradation proc-
ess. On the other hand, the organization of this trial mimics
the reality and we therefore believe that our results are still
applicable for a nationwide FIT-based CRC screening program.
ird, the FIT performance characteristics for di erent sample
return times only pertain to screenees who had a positive test
result (fecal Hb concentrations ≥ 50 ng / ml) and subsequently
underwent a follow-up colonoscopy. ese results can there-
fore not be used to evaluate the FIT sensitivity for advanced
neoplasia subdivided per sample return time. Fourth, only a
limited number of FIT samples were used for the laboratory
experiment. However, we used the repeated measurements only
to create more insight in the Hb degradation process and we
did not use these results to compare the mean Hb decrease per-
centage for di erent subgroups (i.e., CRC, advanced adenomas,
and non-advanced adenomas). Fi h, the Hb-stabilizing bu er
only consists of 2 ml, which is su cient for a maximum of 10
repeated measurements. On the basis of the promising labora-
tory results by Vilkin et al. ( 26 ), we wanted to spread all re-tests
over a period of at least 3 weeks and we were therefore not able
to perform the re-tests every day.
Despite these limitations, we are able to conclude that this
population-based CRC screening trial demonstrates that with
almost 10,000 FITs analyzed, both the PR and DR do not
a laboratory experiment in which 71 positive FIT samples
were randomly selected, stored at room temperature, and
re-tested with standard intervals of 3 – 4 days. Our data show
that no clinical signi cant lesions would have been missed
within the rst 10 days a er fecal sampling. It has been shown
that non-advanced adenomas have a lower baseline Hb level
than advanced adenomas and CRCs ( 10,24,32 ). As such, FIT
samples from screenees with non-advanced adenomas may
sooner convert to negative than samples from patients with
advanced neoplasia. Furthermore, our data do show the impor-
tance of not further lengthening the sample return time, for
instance towards 14 days. By adapting this strategy, 14 scree-
nees would have tested false-negative, including 6 screenees
with advanced neoplasia.
Our main results con rm the laboratory data reported by
Israelian investigators who observed no signi cant Hb degra-
dation over a period of 21 days when FIT samples were stored
at 20 ° C ( 26,33 ). However, a fall in the Hb concentration of
3.7 % ( ± 1.8 % ) per day was observed when tests were kept at
ambient summer room temperature (on average 28 ° C). A rst
explanation for the discrepancy in main outcome between the
Israelian vs. the current study (i.e., an interval of 21 vs. 10 days,
respectively, for the rst tests become negative), is the extreme
high initial Hb concentrations found in the Israelian trial,
787 – 1,032 ng Hb / ml compared with 53 – 1,894 ng Hb / ml in the
present study. ese di erences can be explained by the fact
that the Israelian study was performed among high-risk and
symptomatic individuals, whereas our trial only included scree-
nees in an asymptomatic average-risk population and is thus
more applicable to general population-based CRC screening.
Although di erent cuto values were used (100 vs. 50 ng Hb / ml,
respectively), it is not surprising that our samples — with initial
Hb concentrations close to the cuto threshold — became nega-
tive within a shorter time interval. A second explanation for
the somewhat di erent outcomes with respect to the daily Hb
decrease at higher temperatures (i.e., 3.7 % in the Israelian study
vs. 18.1 % in the present study), might be the actual temperature
at which the positive FIT samples were stored. In contrast with
our trial, room temperature was not kept at a constant level
in the Israelian study but uctuated over the day and was, on
average, somewhat lower than the constant 30 ° C in the present
study. Nevertheless, the same conclusion can still be drawn
from both trials, i.e., the Hb degradation process increases at
higher outside temperatures.
In a recent Italian report, it was demonstrated that the Hb con-
centrations measured during summer were signi cantly lower
than those during winter ( 23 ). An increase in temperature of
1 ° C resulted in a 0.7 % reduced probability of FITs being positive.
Our results con rmed a signi cantly reduced PR and DR during
summer time, with an odds of 0.974 (95 % CI 0.960 – 0.990), for
FITs being positive with each degree Celsius increase in average
outside temperature.
In contrast with our results, another Dutch study found
that the PR signi cantly decreased with each extra day
of delay with an OR of 0.9 (95 % CI 0.8 – 1.0) ( 24 ). In this
The American Journal of GASTROENTEROLOGY VOLUME 107 | JANUARY 2012 www.amjgastro.com
106
COLON/SMALL BOWEL
van Roon et al.
decrease with prolonged sample return times up to 10 days.
is means that a delay in sending the FITs back to the labora-
tory, of up to at least 1 week, does not necessitate repeat testing
in case of a negative test result. Our data support the use of
FIT-based screening as a reliable tool for nationwide CRC
screening programs.
ACKNOWLEDGMENTS
We thank the members of the advisory board (A Cats, JWW
Coebergh, RAM Damhuis, EH Eddes, and J van Krieken),
H ‘ t Mannetje for retrieval of demographic data of all potential
participants in the target population, E van der Donk (Tenalea)
for the random selection of invitees, CWN Looman and GJJM
Borsboom for statistical advise and the analysis concerning our
laboratory experiments, all general practitioners in the region,
gastroenterologists and surgeons of the Erasmus University
Medical Centre, IJsselland Hospital, St Franciscus Gasthuis Hospital,
Vlietland Hospital, Haven Hospital, Ikazia Hospital, Medical
Centre Rijnmond-South and Albert Schweitzer Hospital, residents,
secretaries, nurses, and all participants of the trial.
CONFLICT OF INTEREST
Guarantor of the article: Conceived the idea for the trial: E.J.
Kuipers, M. van Ballegooijen and M.E. van Leerdam; designed the
protocol: E.J. Kuipers, M. van Ballegooijen and M.E. van Leerdam;
responsible for the database design: M.E. van Leerdam, L. Hol and
A.H.C. van Roon; data entry: L. Hol and A.H.C. van Roon.
Speci c author contributions: Conceived the idea for the study: E.J.
Kuipers, M. van Ballegooijen, and M.E. van Leerdam; designed the
protocol: E.J. Kuipers, M. van Ballegooijen, and M.E. van Leerdam;
supervised the execution of the study: E.J. Kuipers; performed the
retrieval of the population sample and the randomization in collabo-
ration with the Association of Nationwide Screening South-western
Netherlands, Vlaardingen and Tenalea, Amsterdam: L. Hol and
A.H.C. van Roon; responsible for the retrieval of the target popula-
tion from the municipal registries and all mailings: J.C.I.Y. Reijerink
and H. ‘ t Mannetje; responsible for the analysis of all FIT samples:
A.J. van Vuuren, J. Francke, M. Ouwendijk, A. Heijens, and N.
Nagtzaam; responsible for the database design: M.E. van Leerdam,
L. Hol, and A.H.C. van Roon; responsible for data entry: L. Hol and
A.H.C. van Roon; coordinated the daily process: A.C.M. van der
Togt; dra ed the report: A.H.C. van Roon; performed the statistical
analyses: A.H.C. van Roon, C.W.N. Looman, and G.J.J.M Borsboom;
and all the collaborators listed above were given an opportunity
to comment on the paper and approved the nal dra of the
manuscript for submission.
Financial support: is trial was funded by the Dutch Cancer
Society (EMCR 2006-3673), the Dutch Ministry of Health, Health
Care Prevention Program — Implementation (ZonMw 63300022
and ZonMw 120720011), Olympus Medical Systems Europe GmbH,
Hamburg, Germany, the Breenco Foundation, and Eiken Chemical
Co., Tokyo, Japan. None of them was involved in the study design,
collection, analysis, and interpretation of the data and in the writing
of the report.
Potential competing interests: None.
Study Highlights
WHAT IS CURRENT KNOWLEDGE
3 Colorectal cancer (CRC) is a major healthcare problem.
3 Fecal immunochemical tests (FIT) are preferred over
guaiac-based fecal occult blood tests for CRC screening.
3 There are concerns that due to globin degradation FIT
sensitivity drops with prolonged sample return times.
3 Exact data whether FIT characteristics are infl uenced by
sample return time are lacking.
3 Recommendations with respect to handling of negative FIT
results with a prolonged sample return time remain to be
determined.
WHAT IS NEW HERE
3 The positivity rate and detection rate do not signifi cantly
decrease with FIT sample return times of up to one week.
3 In a validation experiment performed at room temperature,
conversion of test outcomes only occurred 10 days or
longer after fecal sampling.
3 Delayed sample return up to one week, does not necessitate
repeat testing in case of a negative FIT result.
3 These data support the use of FIT-based screening as
a reliable tool for nationwide CRC screening programs.
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