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Instructions for Use
380316-C

EnglishOC-FCa Reagent

for OC-SENSOR PLEDIA
for OC-SENSOR Ceres

7. On-board stability can vary depending on the conditions of measurement, such as
tests/day and laboratory environment. It is advised to close the bottle of Latex
Reagent with a cap when the measurement is not in process.
8. Do not use reagents that have passed their expiration date.
9. The test sample may contain pathogens. Handle with care. After use, all samples
and other materials must be considered as medical waste and properly disposedof.
Example of treatment: Soak for 1 hour or longer in a sodium hypochlorite solution(available chlorine concentration 1000 ppm or greater). (Neutralize any
substances that contain acids before soaking.) Alternatively, treat in an autoclave
at 121 °C for 20 minutes. (Do not treat any items which sodium hypochlorite haveadhered to in this way.)
10. Dispose of used reagents and containers as medical waste in accordance withlocal regulations.
11. If the product is used in any way other than that specified here, the reliability ofmeasurement results cannot be guaranteed. Be sure to follow the procedure.
12. A clinical diagnosis based on the measurement results must be a comprehensive
judgment made by the attending physician, including factors such as clinicalsymptoms and other test results.

REF V-PH11
REF V-PH09

INTENDED USE

OC-FCa Reagent is in vitro diagnostic assay reagent intended for the quantitative
measurement of calprotectin, inflammatory protein, in faeces. The OC-FCa Reagent
aids in diagnostic and monitoring of inflammatory bowel diseases (IBD; e.g. ulcerative
colitis, and Crohn’s disease) in conjunction with other clinical findings. The test canbe utilized in diagnostic aid and in monitoring for symptomatic patients. The test isnoninvasive, using stool/faeces as test sample. The reagent is used on the dedicatedautomated analysers by qualified personnel in clinical laboratories and hospitals.

SUMMARY

Calprotectin is an inflammatory protein abundantly present in neutrophils.1 The
amount of calprotectin in faeces increases accompanied with intestinal mucosal
inflammation.2 Therefore measuring the amount of calprotectin in faeces can be used
for disease monitoring of IBD patients and distinguishing inflammatory bowel disease(IBD; e.g. ulcerative colitis, Crohn’s disease) from functional intestinal disorders (e.g.
Irritable bowel syndrome).3-5
Currently, the methods used for detection of calprotectin include immunoassay latex
agglutination,
ELISA,
FEIA,
chemiluminescentimmunoassay
andimmunochromatography.6
OC-FCa Reagent is an immunoassay test reagent used together with an automatic
analyser for measurement of calprotectin in faeces. OC-FCa Reagent contains the latex
particles coated with anti-human calprotectin antibodies for optical measurement of
the latex agglutination reaction.

SAMPLE COLLECTION• Use the following designated sampling device for collecting specimens.
OC-Auto Sampling Bottle 3 (REF V-PZ25, V-PZ26) (Sold separately)• Collect faecal samples by scraping the surface of the stools in different areas.
Collect the amount necessary to cover the groove of the probe.
• Check that the test sample has become fully suspended in the buffer solution inside
OC-Auto Sampling Bottle 3.
• Use the sample for measurement at least 1 hour after collection in OC-AutoSampling Bottle 3.
• Calprotectin in the OC-Auto Sampling Bottle 3 is stable at room temperature for 3days, at 2-10 °C for 14 days.7• If a specimen is received in a specimen container, it should be stored at 2-8 °C andsampled with OC-Auto Sampling Bottle 3 within 3 days.8

PRINCIPLE OF THE METHOD

The test method is based on a latex agglutination reaction. A latex reagent isprepared by coating polystyrene latex particles with anti-human calprotectin
antibodies. When this reagent is mixed with the sample, the anti-human calprotectin
antibodies on the latex particles react with the calprotectin in the sample, and the latex
aggregate is formed in the latex agglutination reaction. The change in absorbance perunit time resulting from the latex agglutination reaction is proportional to theconcentration of calprotectin in the sample. A dose response curve of theabsorbance unit (OD) vs. concentration is generated using the results obtained fromthe calibrators. The concentration of calprotectin in the patient sample is determinedfrom this curve.

PREPARATION OF REAGENTS

OC-FCa Latex Reagent for OC-SENSOR PLEDIA
Ready to use.
Before use, bring reagent to room temperature (20-25 °C) and invert the bottle
gently several times to assure uniform suspension.
Do not mix reagent with another bottle.

MATERIALS PROVIDED
Productcode

Product name

V-PH11 OC-FCa Reagent
V-PH09 OC-FCa Reagent

Contents

2 x 8 mL2 x 15 mL
2 x 8 mL2 x 15 mL

Storage

C om p a ti b l e
a n a l y se r

2-10 °C

PLEDIA

2-10 °C

Ceres

OC-FCa Buffer for OC-SENSOR PLEDIA
Ready to use.
Before use, bring the buffer to room temperature (20-25 °C).
Do not mix buffer from another bottle.
Do not use precipitated buffer, as precipitate may cause problems on analysers.
OC-FCa Latex Reagent for OC-SENSOR Ceres
Ready to use.
Before use, invert the bottle gently several times to assure uniform suspension.
Do not mix reagent with another bottle.

MATERIALS REQUIRED BUT NOT PROVIDED
Productcode

Product name

V-PH12 OC-FCa Calibrator
V-PH13 OC-FCa Control LV1
V-PH14 OC-FCa Control LV2
V-PH15 OC-FCa Control LV3
V-PH19
OC-SENSOR Sample Diluent
V-PH08V-PZ25 OC-Auto Sampling Bottle 3
OC-Auto Sampling Bottle 3 without
V-PZ26barcode6 x 1 mL
2 x 5 mL
2 x 5 mL
2 x 5 mL3 x 45 mL
2 x 20 mL
100 bottles

C om p a ti b l e
a n a l y se r
2-8 °C PLEDIA, Ceres
2-8 °C PLEDIA, Ceres
2-8 °C PLEDIA, Ceres
2-8 °C PLEDIA, Ceres
2-8 °CPLEDIA
2-8 °C
Ceres
1-30 °C PLEDIA, Ceres

100 bottles

1-30 °C PLEDIA, Ceres

Contents

Storage

OC-FCa Buffer for OC-SENSOR Ceres
Ready to use.
Do not mix buffer from another bottle.
Do not use precipitated buffer, as precipitate may cause problems on analysers.
Note: OC-SENSOR Ceres is equipped with refrigerated reagent slots so that thereagents do not need to be brought to room temperature.

TEST PROCEDURE

Perform measurements according to the instruction manual of the analyser.
1) Set the OC-FCa R-2 Latex Reagent and OC-FCa R-1 Buffer on the designatedlocations in the analyser.
2) Input the parameters into the analyser.
3) Check water, wash solution, and drain tank volumes.
4) Create calibration curve.
5) Set control materials, OC-FCa Controls as internal control and start measurement.
Make sure the control values are within the acceptable range.
6) Set samples sampled with OC-Auto Sampling Bottle 3 and start measurement.

REQUIRED MATERIALS NOT PROVIDED BY THE MANUFACTURER
Prepare these materials before measurement••••

Wash solution: Sodium hypochlorite 0.15% (0.10%–0.30% is acceptable)
Purified water for wash: Distilled or de-ionized water (1.0-10.0 MΩcm is acceptable)
Sample cups
Printer paper: Thermal printer paper which fits the analyser

RESULTSThe reaction of each sample is compared to the calibration curve that has been createdpreviously. The concentration of calprotectin (μg/g faeces) is reported as a result.

REAGENTS

Reagents are stable until the date printed on the label assuming the container
remains unopened at a storage temperature of 2-10 °C.

LIMITATIONS• Patients taking NSAIDs (non-steroidal anti-inﬂammatory drug) regularly may raise
faecal calprotectin levels.9• Samples exceeding 50,000 μg/g of calprotectin concentration may result in
decreased measurement value due to Hook effect.
• Patient with IBD fluctuate between active (inflammatory) and inactive (remission)
stages of the disease. These stages should be considered when the result of the
faecal calprotectin is interpreted.10• Other intestinal diseases such as colorectal cancer and gastrointestinal infections
can result in increase of calprotectin level. A clinical diagnosis based on themeasurement results must be a comprehensive judgment made by the attending
physician, including factors such as clinical symptoms and other test results.10-12

R-2 Latex Reagent (Suspension of latex particles coated with anti-human calprotectinmouse antibodies)for OC-SENSOR PLEDIA ･････8 mL x 2 bottles; approx. 50 tests/bottle
for OC-SENSOR Ceres ･････8 mL x 2 bottles; approx. 115 tests/bottle
Stable for 4 weeks if stored back in a refrigerator after use with cap closed.
Stable for 4 weeks on-board with cap closed after use on PLEDIA and Ceres.
R-1 Buffer (50 mM N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES))
for OC-SENSOR PLEDIA ･････15 mL x 2 bottles; approx. 50 tests/bottle
for OC-SENSOR Ceres ･････15 mL x 2 bottles; approx. 115 tests/bottle
Stable for 4 weeks if stored back in a refrigerator after use with cap closed.
Stable for 4 weeks on-board with cap closed after use on PLEDIA and Ceres.

INTERNAL QUALITY CONTROLWARNINGS AND PRECAUTIONSEach laboratory should establish a quality control program to monitor the performanceof R-2 Latex Reagent. It is recommended to use the following materials for the quality
control in your laboratory.
OC-FCa Controls: Liquid, ready-to use controls sold separatory
LV1 (REF V-PH13)
LV2 (REF V-PH14)
LV3 (REF V-PH15)

1. For in vitro diagnostic use only.
2. Before performing measurement, be sure to read the instruction manual of theanalyser.
3. Fully prepare and adjust the analyser before measurement.
4. Use OC-FCa Calibrator (REF V-PH12) to create calibration curve. Follow its
instruction for use when to create a new calibration curve.
5. If the measurement results exceed the measurement range, use OC-SENSOR
Sample Diluent (REF V-PH19, V-PH08) to dilute the sample and performmeasurement again.
6. Store all reagents at 2-10 °C and avoid freezing.

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Instructions for Use
380316-C
Studies demonstrated that the reference intervals to be 10-33 μg/g (N=128),13 and 3-35μg/g (N=82)14 in the normal population, 59–276 μg/g (N = 82) in patients with ulcerative
colitis, and 110–353 (N=49) in patients with Crohn's disease,14 respectively as 95%
confidence intervals.

PERFORMANCE CHARACTERISTICS

1. Accuracy
When samples of known concentration (170, 250, 500, 1360 µg/g) were measured, thevalue obtained was within ±15% of the indicated value. Example of measurement
resulted in 95-103%, 98-100%, 97-100%, and 96-100% for the samples 170, 250, 500,
and 1360 µg/g, respectively.

REFERENCE

2. Repeatability and Precision
When the same samples were measured in 2 replicates, twice a day, for 20 days
(2x2x20), the coefficient of variation (CV) for the values obtained was 10% or less,using ANOVA analysis. Examples of measurement of controls and faecal samples(N=80 each) using OC-SENSOR PLEDIA and Ceres are shown below.
OC-SENSOR PLEDIA
Samples
LV1
LV2
S1
S2

Mean
(μg/g)
252.7
496.0
53.0
284.3

OC-SENSOR Ceres
Samples
LV1
LV2
S1
S2

Mean
(μg/g)
252.7
508.5
49.7
307.3

Repeatability
SD
CV
2.8
1.1%
4.1
0.8%
3.0
5.6%
2.6
0.9%

Between-Run
SD
CV
2.2
0.9%
2.3
0.5%
1.5
2.8%
1.3
0.4%

Between-Day
SD
CV
2.9
1.2%
4.4
0.9%
0.7
1.4%
2.7
0.9%

Precision
SD
CV
4.6
1.8%
6.5
1.3%
3.4
6.4%
3.9
1.4%

Repeatability
SD
CV
2.4
1.0%
4.9
1.0%
1.7
3.4%
3.3
1.1%

Between-Run
SD
CV
1.1
0.4%
0.3
0.0%
0.8
1.5%
0.9
0.3%

Between-Day
SD
CV
3.1
1.2%
6.1
1.2%
1.2
2.5%
3.6
1.2%

Precision
SD
CV
4.1
1.6%
7.8
1.5%
2.2
4.5%
4.8
1.6%

1.
2.
3.
4.
5.
6.
7.
8.

9.
3. Measurement range
Up to 2720 µg/g (faeces)
Examples of measurement using OC-SENSOR PLEDIA result showed LOD of 9 µg/g,
LOQ of 23 µg/g; OC-SENSOR Ceres showed LOD of 5 µg/g, and LOQ of 22 µg/g. The
values can vary due to faecal samples and whether/how calprotectin is spiked.

10.
11.

4. Cut-off value
Results for the OC-FCa Reagent greater than 50 μg/g (faeces) should be considered
positive results. It is recommended for each laboratory to establish the cut-off value
suitable for purposes in screening programs or diagnostic use.6

12.
13.

5. Acceptable range between-reagent batches
R-2 Latex Reagent
When control samples with known concentration (250 and 500 μg/g) were measured,
the value obtained was within 100±15% of the indicated value.

14.

Dale I, Fagerhol MK, Naesgaard I. Purification and partial characterization of a highly
immunogenic human leukocyte protein, the L1 antigen. Eur J Biochem. 1983;134(1):16.
Røseth AG, Fagerhol MK, Aadland E, Schjønsby H. Assessment of the neutrophil
dominating protein calprotectin in feces. A methodologic study. Scand J
Gastroenterology. 1992;27(9):793-798.
Tibble J, Teahon K, Thjodleifsson B, et al. A simple method for assessing intestinal
inflammation in Crohn's disease. Gut. 2000;47(4):506-513.
Tibble JA, Sigthorsson G, Foster R, Forgacs I, Bjarnason I. Use of surrogate markers ofinflammation and Rome criteria to distinguish organic from nonorganic intestinal disease.
Gastroenterology. 2002;123(2):450-460.
Schoepfer AM, Trummler M, Seeholzer P, Seibold-Schmid B, Seibold F. Discriminating
IBD from IBS: comparison of the test performance of fecal markers, blood leukocytes,
CRP, and IBD antibodies. Inflamm Bowel Dis. 2008;14(1):32-39.
Freeman K, Willis BH, Fraser H, Taylor-Phillips S, Clarke A. Faecal calprotectin to detectinflammatory bowel disease: a systematic review and exploratory meta-analysis of test
accuracy. BMJ Open. 2019;9(3):e027428. Published 2019 Mar 8.
Hiraoka S, Takashima S, Inokuchi T, et al. The novel latex agglutination turbidimetricimmunoassay system for simultaneous measurements of calprotectin and hemoglobinin feces. Intest Res. 2019;17(2):202-209.
Tøn H, Brandsnes, Dale S, et al. Improved assay for fecal calprotectin. Clin Chim Acta.
2000;292(1-2):41-54.
Tibble JA, Sigthorsson G, Foster R, et al. High prevalence of NSAID enteropathy asshown by a simple faecal test. Gut. 1999;45(3):362-366.
Reenaers C, Bossuyt P, Hindryckx P, Vanpoucke H, Cremer A, Baert F. Expert opinion
for use of faecal calprotectin in diagnosis and monitoring of inflammatory bowel disease
in daily clinical practice. United European Gastroenterol J. 2018;6(8):1117-1125.
van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of patientswith suspected inflammatory bowel disease: diagnostic meta-analysis. BMJ.
2010;341:c3369. Published 2010 Jul 15.
Lin JF, Chen JM, Zuo JH, et al. Meta-analysis: fecal calprotectin for assessment ofinflammatory bowel disease activity. Inflamm Bowel Dis. 2014;20(8):1407-1415.
Tibble J, Sigthorsson G, Foster R, Sherwood R, Fagerhol M, Bjarnason I. Faecal
calprotectin and faecal occult blood tests in the diagnosis of colorectal carcinoma andadenoma. Gut. 2001;49(3):402-408.
Costa F, Mumolo MG, Bellini M, et al. Role of faecal calprotectin as non-invasive marker
of intestinal inflammation. Dig Liver Dis. 2003;35(9):642-647.

NOTICEIn case of occurrence of any serious incident that has occurred in relation to the device
shall be reported to the authorised representative, the manufacturer, and thecompetent authority of the Member State in which the user and/or the patient isestablished.

6. Interference
Following substances were added to sample and the test results showed nointerference or cross-reactivity.
- Toilet cleaners
0.01% Poly(oxyethylene)alkylether- Co-existing substances
20 mg/dL Bilirubin (free and conjugated)
3.5 g/dL Protein
1500 FTU Chyle (Formazin turbidity)
4.0 g/dL Glucose
125 mg/dL Haemoglobin
100 mg/dL Barium sulphate

EXPLANATION OF SYMBOLS

7. Sample Stability
Performance testing with the sample collection device demonstrated that samples
stored at 2 °C for 31 days had 88±23.4%, at 25 °C for 3 days had 80±33.4%, and at 30 °C
for 3 days had 66±42.4% of calprotectin recovery (in-house data, recovery rate shownas mean±2SD).
8. Comparison between analysers
When OC-SENSOR analysers, Ceres and PLEDIA are compared, there were no
significant differences in measurement. Example of measurement of 168 samples
resulted with the slope of 1.03 and r (correlation coefficient) of 0.9996 in regression
analysis.
9. Traceability and Standard Material
No international reference material or reference measurement procedures are available
for calprotectin. The calibrators and controls are traceable to the internal referencematerial.
10. Clinical Performance
Meta-analysis data is summarized from articles using various immunochemical faecal
calprotectin tests as general information. Data from this product is not included.
Diagnostic sensitivity and specificity for IBD with different cut-offs12
50 μg/g

100 μg/g

250 μg/g

Sensitivity

0.92

0.84

0.80

Specificity

0.60

0.66

0.82

PLR

2.33

2.95

4.17

NLR

0.13

0.23

0.22

N (patients)

693

559

763

PLR: positive likelihood ratio
NLR: negative likelihood ratio

Diagnostic sensitivity and specificity for different disorders12
IBD

UC

CD

Sensitivity

0.85

0.88

0.81

Specificity

0.81

0.82

0.81

N (patients)

1471

744

727

(at the cut-offs with the highest
diagnostic accuracy)

Differentiation of IBD and IBS at different cut-offs6
50 μg/g

100 μg/g

Sensitivity

0.96

0.92

Specificity

0.77

0.86

Date of issuance: 2022-05-26

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