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Analytical and Clinical Performance of Kroma iT, A Compact Fully-automated

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ANTICANCER RESEARCH
International Journal of Cancer Research and Treatment
ISSN: 0250-7005

Analytical and Clinical Performance of Kroma iT,

A Compact Fully-automated Immunochemistry
Analyzer for Fecal Occult Hemoglobin
JOSEP M. AUGE1, CRISTINA RODRIGUEZ2, MARIA PELLISE2, ANA BERNAL1, JAUME GRAU3,
ANTONI CASTELLS2, XAVIER FILELLA1 and RAFAEL MOLINA1

Departments of 1Biochemistry and Molecular Genetics, 2Gastroenterology, and 3Unit of Evaluation,

Support and Prevention, Hospital Clinic, IDIBAPS, Barcelona, Spain

Reprinted from
ANTICANCER RESEARCH 33: 5633-5638 (2013)

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ANTICANCER RESEARCH 33: 5633-5638 (2013)

Analytical and Clinical Performance of Kroma iT,

A Compact Fully-automated Immunochemistry
Analyzer for Fecal Occult Hemoglobin
JOSEP M. AUGE1, CRISTINA RODRIGUEZ2, MARIA PELLISE2, ANA BERNAL1, JAUME GRAU3,
ANTONI CASTELLS2, XAVIER FILELLA1 and RAFAEL MOLINA1

Departments of 1Biochemistry and Molecular Genetics, 2Gastroenterology, and 3Unit of Evaluation,

Support and Prevention, Hospital Clinic, IDIBAPS, Barcelona, Spain

Abstract. Background: We performed a laboratory and Worldwide, one million people each year will develop colorectal
cancer (CRC) and the incidence of this tumor is increasing. In
industrialized countries, CRC is the third most common
clinical evaluation of Kroma iT (Linear Chemicals S.L), an

malignancy in men and the second in women (1). Screening for

immunoturbidimetric analyzer for the detection of fecal

CRC and its pre-malignant lesions can identify the disease at

occult blood. Materials and Methods: After a familiarization

an earlier stage. Fecal occult blood tests (FOBT) have been

period, the imprecision, linearity of dilution and carry-over

developed to detect otherwise undetectable bleeding from

were determined and a clinical evaluation was performed

colorectal neoplasms before there are any clinical signs or

on 210 patients. Results: Within-run imprecision ranged

symptoms. Different trials have proved the effectiveness of

between 1.06% and 8.04%. Between-run imprecision ranged

FOBT screening, demonstrating a reduction in mortality of 15-

between 3.11% and 13.09%. Linearity of dilution revealed a

33% (2-4) and several screening trials have confirmed the

mean recovery of all dilutions of 95.24%, with a standard

superiority of fecal immunochemical test (FIT) screening over

deviation of 7.47%. No carry-over was detected. The

the more traditionally used guaiac-based FOBT, both with

clinical evaluation demonstrated that the mean hemoglobin

respect to attendance, as well as the detection rate of advanced

levels of the fecal immunochemical test values from patients

neoplasms (ACRN) (5-10). The latest generation FIT provides a

with advanced neoplasms (colorectal cancer plus advanced

quantitative measurement of microscopic blood loss in stool that

adenoma) were significantly higher than those of cases with

allows for selection of an optimal cut-off (6, 11). In the present

a normal colonoscopy examination. Sensitivity for advanced

study, a new FIT assay for the detection of fecal hemoglobin

neoplasms at cut-off values between 80 and 300 ng/ml (6.4-

was evaluated for its analytical and clinical performance.

24 mg Hb/Kg feces) ranged from 45.5% to 36.4% and the
specificity ranged from 86.8% to 92.3%. The positive

Materials and Methods

predictive values for detecting colorectal cancer and
advanced adenomas were 5.4-6.4% and 27-34% respectively

Patients. A total of 210 consecutive patients at Hospital Clinic

and the negative predictive value ranged from 92.5% to

(Barcelona) who required colonoscopy for the investigation of

91.7%. Using two samples per patient, a substantial

gastrointestinal symptoms or colonic polyp surveillance were recruited

increase of sensitivity was observed, with only a slight

for this study. Patients with a previously positive FOBT, history of

decrease in specificity. Conclusion: Kroma iT analyzer is
known gastrointestinal bleeding, active rectal bleeding, menstruation,
easy to handle and safe for personnel to use. Its analytical
hematuria, and known ulcerative colitis were excluded. Patients were
asked to begin fecal sampling for the FIT five days before colonoscopy
and clinical performance makes it suitable for use in a

to ensure that two samples were collected before bowel preparation

clinical chemistry laboratory for the early detection of

commenced. No dietary restriction was required. Medications such as

advanced neoplasms.

aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) were

Correspondence to: Josep M. Auge, Biochemistry and Molecular
Genetics Department, Hospital Clinic, C/Villarroel 170, 08036
Barcelona, Spain. e-mail: jmauge@clinic.cat
Key Words: Colorectal cancer, fecal occult hemoglobin, analyzer
evaluation, Kroma iT.
withdrawn one week before preparation for colonoscopy. The study
was approved by the Hospital Clinic Ethics Committee (2013/8431),
and all examinees provided written informed consent. All participants
received an oral or telephone explanation of the tests and written
instructions on preparing the FIT. Patients were asked to prepare fecal
samples from two consecutive stool specimens using the collection kit
provided by the manufacturer (Linear Chemicals S.L. Spain).

0250-7005/2013 $2.00+.40 5633

 ANTICANCER RESEARCH 33: 5633-5638 (2013)
Samples. Three sample sources were used: Samples collected by the
patients, stabilizing buffer spike with human capillary blood, and
control material provided by the manufacturer. To collect the sample,
the patient inserts a probe into several different areas of the stool and
then re-inserts it firmly into the test tube container to seal it. The
probe tip with the fecal sample (approximately 20 mg) is suspended
in a standard volume of hemoglobin-stabilizing buffer (approximately
1.6 ml). Samples were stored in double ziplock bags at 4˚C until
analysis within a maximum of five days.
Analyzer. The instrument used for quantification of the FIT is Kroma
iT (Linear Chemicals S.L. Spain, distributed in Spain by Laboratorios
LETI, S.L. Unipersonal, Spain) a desktop instrument based on
immunoturbidimetry, performing up to 150 tests/h. It is self-contained
with reagent, buffer, washing and fluid-disposal bottles, and requires
access to a standard power supply. Twenty-seven of the patient-
prepared fecal sample tubes are loaded into the sample tray. The
instrument automatically mixes the fecal buffer solution with the
latex–anti-human hemoglobin antibody reagent. The latex particles
coated with anti-human hemoglobin are agglutinated when they react
with feces samples containing human hemoglobin. Following the
development cycle, there is automatic flushing of the system.
Agglutination of the latex particles is proportional to the concentration
of the hemoglobin in the sample and can be measured by turbidimetry
and compared to that of a standard calibration curve. By applying a
conversion factor of 0.08, the concentration of hemoglobin in buffer
(ng/ml) is transformed to the concentration of hemoglobin in feces
(mg/kg). The range of measurements is 50-1000 ng Hb/ml,
approximately equivalent to 4-80 mg Hb/kg feces. All events were
performed following the maintenance tasks, calibration and quality
controls as recommended by the manufacturer.
Protocol design. An evaluation protocol was designed, including a
familiarization phase, and a test phase in which imprecision, linearity
of dilution and carry-over were determined. Furthermore, a clinical
evaluation was performed to compare the hemoglobin levels for the
different colonoscopy findings, including receiver operating
characteristic (ROC) curves. The sensitivity, the specificity for
advanced neoplasm at different cut-offs, as well as the diagnostic
yield using one or two samples were assessed.
Test reproducibility. Six samples (two controls, two spiked buffers,
two patient samples) were quantified and repeatedly examined nine
more times in one day, and six samples (two controls, two spiked
buffers, two patient samples) every day for at least 10 days.
Linearity of dilution. Two samples, and one above the dynamic range
of the assay (1,000 ng/ml), were diluted with the appropriate buffer
diluent to obtain a minimum of four dilutions within the dynamic
range of the assay. Dilutions were prepared separately in one to two
steps using calibrated pipettes. Recoveries were calculated with
respect to the highest measured levels.
Carry-over. Intra-assay carry-over was determined using two
human blood-spiked buffer samples: a sample with a low test result
and a sample with a high test result (27528 ng/ml). The samples
were divided in 10 (low) aliquots (L) and 5 (high) aliquots (H). The
aliquots were loaded into the analyzer in the following order: L, L,
L, L, L, H, L, H, L, H, L, H, L, H, L. The difference between the
mean of the low measurements after a high measurement, and the
5634
mean of the low measurements after a low measurement is a
measure of the carry-over into subsequent samples. The error limit
is defined as three times the standard deviation (SD) of the low
after low mean.
Endoscopy. Colonoscopy was carried out to the cecum or up to an
obstructing carcinoma if present and without knowledge of FIT
results. All lesions were categorized, and if colorectal polyps were
detected, the polyp site was recorded and polypectomy performed.
Polyps were examined histologically, and the size and histological
type of each polyp were recorded. The locations and histologies of
carcinomas were also recorded. Polyps were categorized as advanced
adenoma (AA) or non-advanced adenoma (NAA). Adenomas
measuring 10 mm or more in diameter, with villous architecture,
high-grade dysplasia, or intramucosal carcinoma were classified as
AA. Invasive cancer was considered to be present when malignant
cells were observed beyond the muscularis mucosae. Advanced
neoplasm (ACRN) was defined as AA or invasive cancer. Tumor
staging, performed according to the TNM classification system of
the Union for International Cancer Control (UICC) (12) was based
on the most advanced lesion present.
Statistical analysis. A logarithmic transformation for graphic
representation of fecal hemoglobin values was performed. The Mann-
Whitney U-test was used to analyze differences between group
hemoglobin levels. ROC curves for the FIT were drawn as aids to
determine immunochemical fecal hemoglobin cut-off values. The
sensitivity=true positive/(true positive + false negative) and the
specificity=true negatives/(true negatives + false positives), and the
predictive values for ACRN, CRC and AA at different hemoglobin
levels were calculated. ROC curves for one test and two tests were
compared using the Delong method (13) and differences in sensitivity
and specificity using one or two samples at the same cut-off point
were calculated. We calculated Pearson correlation coefficients
between each pair of measures and Cohen’s kappa coefficient was
calculated to measure the agreement between the first and the second
FIT. Statistical analysis was performed using PASW Statistics 18,
Release Version 18.0.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad
Prism version 4.00 (GraphPad Software, San Diego, CA, USA). A
significance level of p<0.05 was regarded as a statistically significant
difference between two results.
Results
Test reproducibility. Within-run imprecision of the low control
Analytical Performance.
(111.82±1.19 ng/ml), high control (417.00±8.89 ng/ml), spiked
buffers (low 69.06±3.68 ng/ml, high 684.64±55.05 ng/ml) and
patient samples (263.64±15.77 ng/ml, 718.13±8.67 ng/mL) ranged
between 1.06% and 8.04%. Between-run imprecision of the low
control (113.92±4.31 ng/ml), high control (412.63±25.30 ng/mL),
spiked buffers (low 273.14±8.49 ng/ml, high 798.96±31.07 ng/ml)
and patient samples (255.85±15.92 ng/mL, 578.57±75.72 ng/mL)
ranged between 3.11% and 13.09%.
Linearity of dilution. Linearity of dilution revealed a mean
recovery of all dilutions of 95.24%, with a standard deviation
of 7.47% (minimum 85%, maximum 111.4%) (Figure 1).
 Auge et al: Performance of Kroma iT for Fecal Occult Hemoglobin

Figure 1. Linear regression of expected and observed dilution steps. Figure 2. Box plot of fecal hemoglobin levels according to colonoscopy
and pathology diagnosis. CRC: Colorectal cancer; AA: Advanced
adenoma; NAA: Non advanced adenoma.

Table I. Fecal hemoglobin levels by Kroma iT of all samples according Table II. Sensitivity and specificity of the results. CRC: Colorectal cancer;
to colonoscopy and pathology diagnosis. CRC: Colorectal cancer; AA: AA: advanced adenoma.

Sensitivity CRC + AA
advanced adenoma; NAA: non-advanced adenoma.

Fecal hemoglobin (ng/ml)

Hb Concentration CRC AA Sensitivity Specificity
Diagnosis No. of samples (mean±SD)
80 ng/ml 66.7% 45.5% 48.0% 86.8%
p-Value*

Normal 122 75.43±480.4 100 ng/ml 66.7% 40.9% 44.0% 88.4%

Other 170 130.4±572.4 0.91 150 ng/ml 66.7% 40.9% 44.0% 89.8%
Adenomas 200 ng/ml 66.7% 38.6% 41.0% 91.1%
NAA 78 238.0±907.4 0.95 250 ng/ml 50.0% 36.4% 38.0% 91.9%
AA 44 818.9±1943 <0.01 300 ng/ml 50.0% 36.4% 38.0% 92.3%
CRC 6 1895±2573 <0.01
CRC + AA 50 948.0±2028 <0.01

*In relation to normal group.

FOBT results. For the entire population undergoing

Carry-over. The mean and standard deviation after low
measurements and high measurements was 2.6±3.6 ng/ml and
3±4.5 ng/ml respectively, therefore the intra-assay carry-over
met the set requirements.
Patient and colonoscopy results. Out of 210 patients (103
Clinical Performance.
colonoscopy, the mean, standard deviation and median FIT
measure of all analyzed samples were 231.8±950.7 and 1.0
(interquartile range: 1.0-14.8) ng/ml respectively. The FIT
results according to colonoscopy and pathology diagnosis are
provided in Table I. The FIT values from patients with
advanced neoplasms (CRC plus AA) were significantly higher
than in cases those with a normal colonoscopy examination
(Figure 2). The positivity rate at 80, 100, 150, 200, 250 and
300 ng/ml was 17.6%, 15.5%, 15%, 12.6%, 11.6% and 11.4%,

males, 107 females) with a mean age of 59±15 years respectively.

colonoscopy detected advanced neoplams in 25 patients
(11.9%). These included three CRCs and 22 AAs. NAAs
were found in 39 (18.6%) patients and other lesions such as neoplasms. Figure 3 displays the ROC curve for advanced
Sensitivity, specificity and predictive values for advanced

hyperplastic or inflammatory polyps (n=16), diverticulosis
(n=20), haemorrhoids (n=41), angiodisplasia (n=2),
inflammatory bowel disease (n=1) and minor irrelevant
lesions (n=5) were found in 85 (40.5%) patients; in 61 (29%)
patients, no findings were detected and the colonoscopy was
reported as normal.
neoplasms obtained with the FIT measurements for each
participant. We measured the sensitivity and specificity of the
FIT results at different hemoglobin thresholds (Table II). At the
80 ng/ml fecal hemoglobin threshold, the sensitivity and
specificity for detecting all advanced neoplasms were 48.0%
(95% confidence interval CI=33.7-62.6%) and 86.8% (95%

5635
 ANTICANCER RESEARCH 33: 5633-5638 (2013)

Figure 3. Receiver operating characteristic curve for advanced

Figure 4. Receiver operating characteristic curves of first, second, the

neoplasm. AUC: Area under the curve.

highest and mean results for advanced neoplasm. AUC: Area under the

CI=82.9-90.1%). At the 300 ng/ml fecal hemoglobin threshold,

curve.

the sensitivity and specificity for detecting all advanced Table III. Sensitivity, specificity and kappa coefficient of the first and
neoplasms were 38.0% (95% CI=24.7-52.8%) and 92.5% (95%
CI=89.3-94.9%), respectively. The 80 ng/ml fecal hemoglobin
second result and combinations among them for advanced neoplasm.

threshold increased sensitivity but reduced specificity. The Hb concentration

sensitivity for detecting cancer was considerably higher than
100 ng/ml 200 ng/ml 300 ng/ml
that for detecting all clinically- advanced neoplasms. In the
studied group, the positive predictive value for detecting CRC Sensitivity (1st) 44.0% 40.0% 36.0%
and AA was 5.4-6.4% and 27-34%, respectively, and the Specificity (1st) 89.6% 91.4% 91.9%
negative predictive value was 92.5-91.7%. Sensitivity (2nd) 44.0% 44.0% 40.0%
Diagnostic yield using one or two samples. The correlation Specificity (2nd) 87.6% 90.3% 91.9%
kappa coefficient 0.618 0.633 0.593
coefficient of the first and second FIT was 0.479. These Sensitivity (highest) 56.0% 52.0% 48.0%
moderate correlations most probably reproduce daily variations Specificity (highest) 84.3% 87.6% 88.6%
in blood loss, otherwise the kappa coefficient showed a Sensitivity (mean) 52.0% 48.0% 44.0%
substantial agreement between the first and the second FIT. We Specificity (mean) 85.4% 88.6% 91.4%
measured the hemoglobin content of each of two consecutive
fecal samples but considered them to represent one test, to
which we assigned the highest of the two FIT results. Figure 4
displays the ROC curves for advanced neoplasms obtained with hemoglobin. The test used in the current study is easy to
the first, second and the highest of both FIT measurements for perform, and the results are independent of operator
each participant. No statistical differences were observed experience. The technical evaluation included study of
among them. Table III shows the sensitivity and specificity for reproducibility, linearity of dilution and carry-over.
advanced neoplasms of first, second, the highest and the mean The clinical evaluation in a mixed group of individuals
of both FITs at different cut-off values, as well as the kappa demonstrated at a threshold of 80-300 ng/ml (6.4-24 mg
coefficient between first and second sample. Hb/kg feces) adequate sensitivity for advanced neoplasms
of 45.5%-36.4% and acceptable specificity of 86.8%-
Discussion 92.3%. The positivity rate at different cut-offs was quite
high compared to the median in risk screening (14, 15) and
This colonoscopy-controlled study allowed for a detailed would lead to an excessive colonoscopy rate. However, the
evaluation of an automated desktop instrument for studied population was not a true screening population
quantitative, immunochemical determination of fecal occult because all the patients had symptoms or were indicated

5636
 Auge et al: Performance of Kroma iT for Fecal Occult Hemoglobin
for colonoscopy. From this study, we confirmed that a FIT
threshold of 200 ng Hb/mL (16 mg Hb/kg feces) allowed
detection of the majority of the carcinomas and 38.6% of
AAs, giving together a sensitivity of 41%. It provided an
acceptable specificity of 91% and using two samples for
each patient and choosing the highest result, the sensitivity
for advanced neoplasm increased to 52%, providing a
specificity of 87.6%. These results are suitable for
screening and are consistent with results from other studies
that used immunochemical tests to screen larger
populations (16-19).
In conclusion, the current study provides useful data
regarding the potential application of automated FIT in
screening the population for CRC. The compact fully-
automated immunochemistry analyzer evaluated for fecal
hemoglobin measurement demonstrates an adequate analytical
and clinical performance. We have demonstrated that the
Kroma iT assay (Linear Chemicals S.L.) is a robust, specific,
and accurate tool for the detection of advanced neoplasm and
provides the basis for a large-scale screening program.
Conflicts of Interest
Laboratorios LETI, S.L. Unipersonal and Linear Chemicals, S.L.
provided instruments, reagents and technical support.
Potential Financial Conflicts of Interest
Grants received: JM. Auge (Laboratorios LETI, S.L. Unipersonal and
Linear Chemicals, S.L.).
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Received October 11, 2013
Revised November 7, 2013
Accepted November 11, 2013
5637
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