The fCAL turbo is an automated in vitro diagnostic test for the quantitative determination of calprotectin in human stool specimens intended as an aid in the assessment of intestinal mucosal inflammation
The fCAL turbo test is a particle enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the cobas c 503 analyzers is 30‑2000 µg/g. Samples > 2000 µg/g will be diluted automatically (1:10) by the analyzer, further extending the range to 30‑10000 µg/g.
Distinguishing organic disease from functional gastrointestinal disease
Determination of fecal calprotectin levels can be used as a reliable and simple aid in distinguishing organic from functional gastrointestinal diseases
Clinical thresholds
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges.
The result categories are based on data from clinical studies performed by BÜHLMANN and are BÜHLMANN’s recommendations. All test results should be interpreted in conjunction with information available from the patient’s clinical symptoms, medical history, and other clinical and laboratory findings:
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract. Patients with low calprotectin levels are not likely to be in need of invasive procedures to determine the inflammation cause
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status.
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease. Appropriate further investigative procedures by specialists are suggested to achieve an overall clinical diagnosis.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
• For IBD disease monitoring, multiple fecal calprotectin measurements performed at up to 4 weeks intervals have been suggested to have best diagnostic accuracy in predicting clinical relapse in patients
Special wash programming required by the instrument manufacturer:
The manufacturer of the cobas c systems considers the use of special wash steps as mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet provided by the instrument manufacturer. For further instructions, refer to the operator’s manual.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08910367190 | fCAL turbo (200 tests) | System‑ID 2149 001 | cobas c 503 |
Materials required (but not provided):
08910499190 | fCAL Calibrator Set | Code 20719 | |
08910502190 | fCAL Control Set |
| |
08910987001 | CALEX® Cap (500 pcs) |
FCAL: ACN 21490
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
Test definition | |||
Reporting time | 10 min | ||
Wavelength (sub/main) | 800/546 nm | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 100 µL | – | |
R3 | 16 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 8 µL | – | – |
Decreased | 8 µL | 8 µL | 72 µL |
Increased | 8 µL | – | – |
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to 12 weeks at 5‑15 °C |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Apply the lot-specific fCAL Calibrator values for the 6-point calibration curve: | |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve, equilibrate reagents R1 and R3 to the storage temperature of the analyzer by loading the cobas c pack at least 1 hour before calibration curve establishment. Keep calibrators at room temperature for at least 30 min before starting analysis. The calibration on cobas c 503 analyzers is performed in duplicate.
The clinical evaluation of fCAL turbo was assessed on the Master System cobas c 501 analyzer. The results are also considered applicable to cobas c 503 analyzer.
The ability of the fCAL turbo to discriminate between patients with IBD and other non-inflammatory GI disorders, including IBS, was evaluated using clinical samples collected from 295 patients and extracted using the CALEX® Cap device. One hundred and twenty seven (127) patients had a final diagnosis of IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis), 103 patients suffered from IBS and 65 patients presented with abdominal pain and/or diarrhea, or other GI-related non-inflammatory conditions. Final diagnosis was supported by endoscopic as well as other clinical findings.
The optimal cut‑off combination for these patient pools could be defined by ROC analysis at 80 µg/g and 160 µg/g calprotectin (table 3 and 5), which is slightly more stringent than a combination of a more sensitive lower cut off of 50 µg/g with lower performance in specificity, and an upper cut off 200 µg/g with slightly lower sensitivity (table 4 and 6).
Final | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 11 (8.7 %) | 8 (6.3 %) | 108 (85 %) | 127 (100 %) |
IBS | 75 (72.8 %) | 11 (10.7 %) | 17 (16.5 %) | 103 (100 %) |
Other GI | 42 (64.6 %) | 8 (12.3 %) | 15 (23.1 %) | 65 (100 %) |
Table 2: Distribution of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.3 % (85.0 %, 95.6 %) | 85.0 % (77.6 %, 90.7 %) |
Specificity (95 % CI) | 69.6 % (62.1 %, 76.5 %) | 81.0 % (74.2 %, 86.6 %) |
PPV (95 % CI) | 69.5 % (61.9 %, 76.3 %) | 77.1 % (69.3 %, 83.8 %) |
NPV (95 % CI) | 91.4 % (85.1 %, 95.6 %) | 87.7 % (81.5 %, 92.5 %) |
ROC AUC (95 % CI) | 0.912 (0.878, 0.946) |
Table 3: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD − IBS and other GI‑related disorders, at 80 μg/g and 160 μg/g clinical decision points
IBD vs. non‑IBD | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 62.5 % (54.7 %, 69.8 %) | 85.7 % (79.5 %, 90.6 %) |
PPV (95 % CI) | 65.6 % (58.2 %, 72.4 %) | 81.0 % (73.0 %, 87.4 %) |
NPV (95 % CI) | 93.8 % (87.5 %, 97.5 %) | 85.2 % (78.9 %, 90.2 %) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD – IBS and other GI‑related disorders, at 50 µg/g and 200 µg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.3 % (85.0 %, 95.6 %) | 85.0 % (77.6 %, 90.7 %) |
Specificity (95 % CI) | 72.8 % (63.2 %, 81.1 %) | 83.5 % (74.9 %, 90.1 %) |
PPV (95 % CI) | 80.6 % (73.1 %, 86.7 %) | 86.4 % (79.1 %, 91.9 %) |
NPV (95 % CI) | 87.2 % (78.3 %, 93.4 %) | 81.9 % (73.2 %, 88.7 %) |
ROC AUC (95 % CI) | 0.925 (0.892, 0.958) |
Table 5: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 67.0 % (57.0 %, 75.9 %) | 88.3 % (80.5 %, 93.8 %) |
PPV (95 % CI) | 77.9 % (70.5 %, 84.2 %) | 89.5 % (82.3 %, 94.4 %) |
NPV (95 % CI) | 90.8 % (81.9 %, 96.2 %) | 78.4 % (69.9 %, 85.5 %) |
Table 6: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 50 µg/g and 200 µg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
The presented performance characteristics have been established on the cobas c 503 analyzer, unless otherwise indicated, and should be considered as representative data. Results obtained in individual laboratories may differ.
For regulatory purposes validation was performed using extracts obtained by manual weighing and extraction. The CALEX® Cap was validated for comparability with the manual extraction method on a cobas c 501 analyzer. Please refer to the CALEX® Cap method sheet for results. Any changes to the directions for use, including special wash programming, may alter the stated performance characteristics of the assay and must be validated prior to implementation by the laboratory.
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3. Forty‑five (45) clinical samples were measured on a cobas c 503 analyzer using one lot of fCAL turbo over 1 day in one calibration cycle. Reference values, with a final calprotectin concentration interval of 39.1‑9075.4 μg/g, were established with the fCAL turbo on a cobas c 501 analyzer. Bias was determined using Passing‑Bablok linear regression and Bland‑Altman analysis.
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) |
(95 % CI) | Intercept (95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) |
|
-6.97 % (9.11 %, -4.83 %) | -20.93 % (-24.62 %, -17.25 %) | 6.99 % (3.31 %, 10.68 %) | 0.9866 | -7.276 μg/g (-12.18 μg/g, -5.32 μg/g) | -10.4 % | -5.9 % |
|
Table 9: Method comparison study results, (LoA= Limits of agreement)
Repeatability (within‑run precision): 0.2‑3.6 % CV
Total precision: 1.6‑7.6 % CV
Repeatability and total precision were established according to the CLSI guideline EP05‑A3 using a 5 days x 2 runs x 4 replicates study design using an acceptance criterion of 15 % CV. Six (6) pooled stool specimen extracts with calprotectin concentrations ranging from 49.0‑6657.9 µg/g were tested.
Sample carry-over
The sample carry‑over was established according to the CLSI guideline EP10‑A2. No statistically significant carry‑over with the fCAL turbo test on cobas c 503 instrument was detected.
Limit of Detection (LoD): 6.3 μg/g
The LoD was established according to the CLSI guideline EP17‑A2 and with proportions of false positives (α) less than 5 % and false negatives (β) less than 5 % based on 120 determinations, with 60 blank and 60 low level replicates; and a LoB of 2.6 µg/g.
Limit of Quantitation (LoQ): 13.3 μg/g
The LoQ was established according to the CLSI guideline EP17‑A2, based on 60 determinations and a precision goal of 20 % CV using an acceptance criterion of LoQ ≤ 30 µg/g.
Linearity range: 5.5‑12959.7 μg/g
The linear range of the fCAL turbo was determined according to the CLSI guideline EP06‑A. Samples with a concentration over 2000 μg/g were diluted automatically 1:10 by the analyzer. A maximum deviation from linearity of 10 % was allowed. For values below 75 µg/g an absolute difference of less than 7.5 µg/g was allowed.
The following performance characteristics have been established on the Master System cobas c 501 analyzer. The results are also considered applicable to the cobas c 503 analyzer.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | 25.4 mL | Ready to use |
Immunoparticles (R3) | 6.5 mL | Ready to use |
R1 is in position B and R3 is in position C. R3 refers to the reagent, which is pipetted at time point R3.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
Product safety labeling follows EU GHS guidance.
Contact phone: all countries: +49-621-7590
This kit contains components classified in accordance with the Regulation (EC) No. 1272/2008: 2‑methyl‑4‑isothiazolin‑3‑one hydrochloride (conc. ≥ 0.0015 %), thus the reagents may cause allergic skin reactions (H317).
The immunoparticles contain potentially infectious substances of animal origin and should be handled in accordance with Good Laboratory Practice (GLP) using appropriate precautions.
• Avoid contact of reagents with the skin, eyes or mucous membranes. If contact does occur, immediately wash with generous amounts of water; otherwise, irritation / burns can occur.
• Immunoparticles R3 contains polystyrene nanoparticles.
• Reagent Immunoparticles R3, once frozen, cannot be used anymore.
• The assay is designed for fecal extract samples prepared using the specific CALEX® Cap devices.
• Please equilibrate reagents, controls, calibrators and samples as described in this method sheet.
• Ensure that samples have no bubbles prior to running the test.
• Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Run the fCAL Control Set, Level I and Level II, in duplicate, after each new calibration, before running patient fecal sample extracts. Keep controls at room temperature for at least 30 min before starting analysis.
Validating daily runs
Run the fCAL Control Set, Level I and Level II, each day, before running patient fecal sample extracts to validate calibration curve stability. Single measurements of controls are sufficient.
The controls have assigned target values indicated on the control value sheet supplied with each lot of the fCAL Control Set. All control measurements must be within target values indicated on the control value sheet to obtain valid results for patient fecal sample extracts. If the control values are not valid, repeat quality control measurement with fresh controls. If control values remain invalid, recalibrate the instrument. If valid controls values cannot be reproduced after performing the steps described above, contact your local customer support.
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecal extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX® Cap device.
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or refrigerated. Stool specimens should be refrigerated at 2‑8 °C and extracted within 3 days of receipt at the laboratory. Do not store samples at elevated temperatures.
Stability in CALEX
For CALEX® Cap extract stability refer to the instruction for use provided with the CALEX® Cap (REF: 08910987 001).
Laboratories are responsible for validating use of alternate storage temperatures/timeframes.
Follow the instruction for use provided with the CALEX® Cap kit. Fecal sample extracts prepared using the CALEX® Cap will have a final dilution of 1:500 and are ready to use.
Important: Centrifuge the CALEX® Cap device for 10 minutes at 1000‑3000 g prior to running the fCAL turbo procedure.
The fCAL turbo is an automated in vitro diagnostic test for the quantitative determination of calprotectin in human stool specimens intended as an aid in the assessment of intestinal mucosal inflammation
The fCAL turbo test is a particle enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the
Distinguishing organic disease from functional gastrointestinal disease
Determination of fecal calprotectin levels can be used as a reliable and simple aid in distinguishing organic from functional gastrointestinal diseases
Clinical thresholds
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges.
The result categories are based on data from clinical studies performed by BÜHLMANN and are BÜHLMANN’s recommendations. All test results should be interpreted in conjunction with information available from the patient’s clinical symptoms, medical history, and other clinical and laboratory findings:
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract. Patients with low calprotectin levels are not likely to be in need of invasive procedures to determine the inflammation cause
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status.
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease. Appropriate further investigative procedures by specialists are suggested to achieve an overall clinical diagnosis.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
• For IBD disease monitoring, multiple fecal calprotectin measurements performed at up to 4 weeks intervals have been suggested to have best diagnostic accuracy in predicting clinical relapse in patients
Special wash programming required by the instrument manufacturer:
The manufacturer of the cobas c systems considers the use of special wash steps as mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet provided by the instrument manufacturer. For further instructions, refer to the operator’s manual.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08910367190 | fCAL turbo (200 tests) | System‑ID 2149 001 |
Materials required (but not provided):
08910499190 | fCAL Calibrator Set | Code 20719 | |
08910502190 | fCAL Control Set |
| |
08910987001 | CALEX® Cap (500 pcs) |
FCAL: ACN 21490
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
Test definition | |||
Reporting time | 10 min | ||
Wavelength (sub/main) | 800/546 nm | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 100 µL | – | |
R3 | 16 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 8 µL | – | – |
Decreased | 8 µL | 8 µL | 72 µL |
Increased | 8 µL | – | – |
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to 12 weeks at 5‑15 °C |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Apply the lot-specific fCAL Calibrator values for the 6-point calibration curve: | |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve, equilibrate reagents R1 and R3 to the storage temperature of the analyzer by loading the cobas c pack at least 1 hour before calibration curve establishment. Keep calibrators at room temperature for at least 30 min before starting analysis. The calibration on
The clinical evaluation of fCAL turbo was assessed on the Master System cobas c 501 analyzer. The results are also considered applicable to
The ability of the fCAL turbo to discriminate between patients with IBD and other non-inflammatory GI disorders, including IBS, was evaluated using clinical samples collected from 295 patients and extracted using the CALEX®
The optimal cut‑off combination for these patient pools could be defined by ROC analysis at 80 µg/g and 160 µg/g calprotectin (table 3 and 5), which is slightly more stringent than a combination of a more sensitive lower cut off of 50 µg/g with lower performance in specificity, and an upper cut off 200 µg/g with slightly lower sensitivity (table 4 and 6).
Final | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 11 (8.7 %) | 8 (6.3 %) | 108 (85 %) | 127 (100 %) |
IBS | 75 (72.8 %) | 11 (10.7 %) | 17 (16.5 %) | 103 (100 %) |
Other GI | 42 (64.6 %) | 8 (12.3 %) | 15 (23.1 %) | 65 (100 %) |
Table 2: Distribution of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.3 % (85.0 %, 95.6 %) | 85.0 % (77.6 %, 90.7 %) |
Specificity (95 % CI) | 69.6 % (62.1 %, 76.5 %) | 81.0 % (74.2 %, 86.6 %) |
PPV (95 % CI) | 69.5 % (61.9 %, 76.3 %) | 77.1 % (69.3 %, 83.8 %) |
NPV (95 % CI) | 91.4 % (85.1 %, 95.6 %) | 87.7 % (81.5 %, 92.5 %) |
ROC AUC (95 % CI) | 0.912 (0.878, 0.946) |
Table 3: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD − IBS and other GI‑related disorders, at 80 μg/g and 160 μg/g clinical decision points
IBD vs. non‑IBD | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 62.5 % (54.7 %, 69.8 %) | 85.7 % (79.5 %, 90.6 %) |
PPV (95 % CI) | 65.6 % (58.2 %, 72.4 %) | 81.0 % (73.0 %, 87.4 %) |
NPV (95 % CI) | 93.8 % (87.5 %, 97.5 %) | 85.2 % (78.9 %, 90.2 %) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD – IBS and other GI‑related disorders, at 50 µg/g and 200 µg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.3 % (85.0 %, 95.6 %) | 85.0 % (77.6 %, 90.7 %) |
Specificity (95 % CI) | 72.8 % (63.2 %, 81.1 %) | 83.5 % (74.9 %, 90.1 %) |
PPV (95 % CI) | 80.6 % (73.1 %, 86.7 %) | 86.4 % (79.1 %, 91.9 %) |
NPV (95 % CI) | 87.2 % (78.3 %, 93.4 %) | 81.9 % (73.2 %, 88.7 %) |
ROC AUC (95 % CI) | 0.925 (0.892, 0.958) |
Table 5: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 67.0 % (57.0 %, 75.9 %) | 88.3 % (80.5 %, 93.8 %) |
PPV (95 % CI) | 77.9 % (70.5 %, 84.2 %) | 89.5 % (82.3 %, 94.4 %) |
NPV (95 % CI) | 90.8 % (81.9 %, 96.2 %) | 78.4 % (69.9 %, 85.5 %) |
Table 6: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 50 µg/g and 200 µg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
The presented performance characteristics have been established on the
For regulatory purposes validation was performed using extracts obtained by manual weighing and extraction. The CALEX® Cap was validated for comparability with the manual extraction method on a cobas c 501 analyzer.
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3. Forty‑five (45) clinical samples were measured on
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) | Slope (95 % CI) | Intercept (95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) | r |
-20.9 % (-24.6 %, -17.3 %) | 7.0 % (3.3 %, 10.7 %) | 0.9866 | -7.28 μg/g (-12.18 μg/g, -5.32 μg/g) | -10.4 % | -5.9 % |
|
Table 9: Summary of Bland‑Altman and Passing‑Bablok regression analyses obtained on cobas c 503 analyzer vs. established reference values on cobas c 501 analyzer (LoA= Limits of agreement)
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) | Slope (95 % CI) | Intercept (95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) | r |
6.4 % (4.8 %, 7.9 %) | -3.7 % (-6.3 %, -1.0 %) | 16.4 % (13.7 %, 19.0 %) | 1.075 | -1.122 μg/g (-5.722 μg/g, 4.137 μg/g) | 6.1 % | 6.8 % |
|
Table 10: Summary of Bland‑Altman and Passing‑Bablok regression analyses obtained on cobas c 303 analyzer vs. established reference values on cobas c 501 analyzer (LoA= Limits of agreement)
Repeatability (within‑run precision):
cobas c 503 analyzer: 0.2‑3.6 % CV
Total precision:
cobas c 503 analyzer: 1.6‑7.6 % CV
Repeatability and total precision were established according to the CLSI guideline EP05‑A3 using a 5 days x 2 runs x 4 replicates study
Sample carry-over
The sample carry‑over was established according to the CLSI guideline EP10‑A2. No statistically significant carry‑over with the fCAL turbo test on
Limit of Detection (LoD):
cobas c 503 analyzer: 6.3 μg/g
The LoD was established according to the CLSI guideline EP17‑A2 and with proportions of false positives (α) less than 5 % and false negatives (β) less than 5 % based on 120 determinations, with 60 blank and 60 low level replicates; and
Limit of Quantitation (LoQ):
cobas c 503 analyzer: 13.3 μg/g
The LoQ was established according to the CLSI guideline EP17‑A2, based on 60 determinations and a precision goal of 20 % CV using an acceptance criterion of LoQ ≤ 30 µg/g.
Linearity range:
cobas c 503 analyzer: 18.7‑12988 μg/g
The linear range of the fCAL turbo was determined according to the CLSI guideline EP06‑A. Samples with a concentration over 2000 μg/g were diluted automatically 1:10 by the analyzer. A maximum deviation from linearity of 10 % was allowed. For values below 75 µg/g an absolute difference of less than 7.5 µg/g was allowed.
The following performance characteristics have been established on the Master System cobas c 501 analyzer. The results are also considered applicable to the
The extraction reproducibility was established according to the CLSI guideline EP05‑A3 using a 2 days x 2 operators x 3 CALEX® Cap lots x 2 extractions x 3 replicates study design. Twelve clinical stool specimens, including specimens with solid, semi-solid and liquid consistency, with calprotectin concentrations in the range of 42.7‑3440.0 μg/g, were tested.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | 25.4 mL | Ready to use |
Immunoparticles (R3) | 6.5 mL | Ready to use |
R1 is in position B and R3 is in position C. R3 refers to the reagent, which is pipetted at time point R3.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
Product safety labeling follows EU GHS guidance.
Contact phone: all countries: +49-621-7590
This kit contains components classified in accordance with the Regulation (EC) No. 1272/2008: 2‑methyl‑4‑isothiazolin‑3‑one hydrochloride (conc. ≥ 0.0015 %), thus the reagents may cause allergic skin reactions (H317).
The immunoparticles contain potentially infectious substances of animal origin and should be handled in accordance with Good Laboratory Practice (GLP) using appropriate precautions.
Avoid contact of reagents with the skin, eyes or mucous membranes. If contact does occur, immediately wash with generous amounts of water; otherwise, irritation
Immunoparticles R3 contains polystyrene nanoparticles.
Reagent Immunoparticles R3, once frozen, cannot be used anymore.
The assay is designed for fecal extract samples prepared using the specific
Please equilibrate reagents, controls, calibrators and samples as described in this method sheet.
Ensure that samples have no bubbles prior to running the test.
Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Run the fCAL Control Set, Level I and Level II, in duplicate, after each new calibration, before running patient fecal sample extracts. Keep controls at room temperature for at least 30 min before starting analysis.
Validating daily runs
Run the fCAL Control Set, Level I and Level II, each day, before running patient fecal sample extracts to validate calibration curve stability. Single measurements of controls are sufficient.
The controls have assigned target values indicated on the control value sheet supplied with each lot of the fCAL Control Set. All control measurements must be within target values indicated on the control value sheet to obtain valid results for patient fecal sample extracts. If the control values are not valid, repeat quality control measurement with fresh controls. If control values remain invalid, recalibrate the instrument. If valid controls values cannot be reproduced after performing the steps described above, contact your local customer support.
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecal extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX®
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or refrigerated. Stool specimens should be refrigerated at 2‑8 °C and extracted within 3 days of receipt at the laboratory. Do not store samples at elevated temperatures.
Follow the instruction for use provided with the CALEX® Cap. Fecal sample extracts prepared using the CALEX® Cap will have a final dilution of 1:500 and are ready to use.
Liquid stool samples can be pipetted directly into the CALEX® Cap. Unscrew the blue cap and pipet 10 μL of stool sample into the device. Recap the CALEX® Cap and proceed with vortexing step according to the extraction procedure described and illustrated in the instruction for use delivered with the CALEX® Cap.
Important: Centrifuge the CALEX® Cap for 10 minutes at 1000‑3000 g prior to running the fCAL turbo procedure.
Fecal calprotectin in extracts obtained by the CALEX® Cap is stable at room temperature (23 °C) for 7 days, at 2‑8 °C for 15 days and at -20 °C for at least 23 months.
CALEX® Cap extracts can be frozen directly and stored within the CALEX® Cap. Extracts can be subject to four freeze‑thaw cycles. Prior to measurement, allow frozen extracts to equilibrate to room temperature, vortex thoroughly for 10 seconds and centrifuge according to the instruction for use of the assay.
The fCAL turbo is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. fCAL turbo aids in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC) and aids in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other laboratory and clinical findings.
The fCAL turbo test is a particle‑enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract. Low fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for additional diagnostic work‑up. Specifically, for patients with a clinical and laboratory presentation suggesting a non‑inflammatory disorder such as IBS, fecal calprotectin values of < 80 µg/g can be used to support a decision to defer invasive testing.
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status. This decision should be made by the clinician in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings.
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease. Elevated fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for further investigative procedures, including invasive procedures performed by specialists, to achieve an overall clinical diagnosis, in particular of IBD.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
• False negative results could occur in patients who have granulocytopenia due to bone marrow depression.
• Some patients taking non‑steroidal anti‑inflammatory drugs (NSAID) will have elevations in their fecal calprotectin levels.
• Results may not be clinically applicable to children less than 4 years of age who have mildly increased fecal calprotectin levels.
• Patients with IBD fluctuate between active (inflammatory) and inactive stages of the disease. These stages must be considered when interpreting results of the fecal calprotectin assay.
Special wash programming required by the instrument manufacturer:
The manufacturer of the cobas c systems considers the use of special wash steps as mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry-over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet provided by the instrument manufacturer. For further instructions, refer to the operator’s manual.
Analyzer(s) on which cobas c pack(s) can be used | |||
08910367190 | fCAL turbo (200 tests) | System‑ID 2149 001 |
Materials required (but not provided):
08910499190 | fCAL Calibrator Set |
| |
08910502190 | fCAL Control Set |
| |
08910987001 | CALEX® Cap (500 pcs) |
FCAL: ACN 21490
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
Test definition | |||
Reaction time | 10 min | ||
Wavelength (sub/main) | 800/546 nm | ||
Reagent pipetting | Diluent | ||
R1 | 100 µL | – | |
R3 | 16 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 8 µL | - | - |
Decreased | 8 µL | 8 µL | 72 µL |
Increased | 8 µL | - | - |
NOTE:
For technical reasons, it is necessary to specify a dummy unit (µg/mL) on cobas c analyzers. Values can be converted to the correct unit (µg/g) via the host computer.
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to 12 weeks at 5‑15 °C |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Apply the lot-specific fCAL Calibrator values for the 6-point calibration curve: | |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve, equilibrate reagents R1 and R3 to the storage temperature of the analyzer by loading the cobas c pack at least 1 hour before calibration curve establishment. Keep calibrators as well as controls and patient samples at room temperature for at least 30 min before starting analysis. The calibration on
Traceability: This method has been standardized against internal reference material.
The clinical evaluation of fCAL turbo was assessed on the master system cobas c 501 analyzer. The results are also considered applicable to cobas c
The ability of the fCAL turbo to discriminate between patients with IBD and other non‑inflammatory gastrointestinal (GI) disorders, including IBS, was evaluated using clinical samples collected from 337 adult and pediatric patients. One hundred and thirty five patients had a final diagnosis of IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis), 130 patients suffered from IBS and 72 patients presented with abdominal pain and/or diarrhea, or other GI‑related non‑inflammatory conditions. Final diagnosis was supported by endoscopic as well as other clinical findings.
Final | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 12 (8.9 %) | 15 (11.1 %) | 108 (80 %) | 135 (100 %) |
IBS | 99 (76.2 %) | 15 (11.5 %) | 16 (12.3 %) | 130 (100 %) |
Other GI | 51 (70.8 %) | 7 (9.7 %) | 14 (19.4 %) | 72 (100 %) |
Table 2: Distributions of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 74.3 % (67.7 %, 80.1 %) | 85.1 % (79.5 %, 89.8 %) |
PPV (95 % CI) | 70.3 % (62.9 %, 76.9 %) | 78.3 % (70.4 %, 84.8 %) |
NPV (95 % CI) | 92.6 % (87.4 %, 96.1 %) | 86.4 % (80.9 %, 90.9 %) |
ROC AUC (95 % CI) | 0.916 (0.884, 0.947) |
Table 3: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD − IBS and other GI‑related disorders, at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 76.2 % (67.9 %, 83.2 %) | 87.7 % (80.8 %, 92.8 %) |
PPV (95 % CI) | 79.9 % (72.7 %, 85.9 %) | 87.1 % (79.9 %, 92.4 %) |
NPV (95 % CI) | 89.2 % (81.9 %, 94.3 %) | 80.9 % (73.4 %, 87.0 %) |
ROC AUC (95 % CI) | 0.929 (0.898, 0.960) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
Reference range
Stool samples were obtained from 141 apparently healthy normal adults (> 21 years of age) with no symptoms or signs of gastrointestinal disease. The test results were categorized by the assay cut‑offs.
Distribution of results within fCAL turbo diagnostic ranges | ||||
< 80 μg/g | 80-160 μg/g | > 160 μg/g | Total | |
Number of subjects (%) | 106 (75.2 %) | 18 (12.8 %) | 17 (12.1 %) | 141 (100 %) |
Table 5: Distribution of healthy subjects results within fCAL turbo diagnostic ranges
The presented performance characteristics have been established on a cobas c
For regulatory purposes, validation was performed using extracts obtained by manual weighing and extraction. The CALEX® Cap was validated for comparability with the manual extraction method on a cobas c 501 analyzer. Any changes to the directions for use, including special wash programming, may alter the stated performance characteristics of the assay and must be validated prior to implementation by the laboratory.
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3. Forty‑five (45) clinical samples were measured on a
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) |
(95 % CI) |
(95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) |
|
6.4 % (4.8 %, 7.9 %) | -3.7 % (-6.3 %, -1.0 %) | 16.4 % (13.7 %, 19.0 %) | 1.075 (1.049, 1.105) | -1.122 μg/g (-5.722 μg/g, 4.137 μg/g) | 6.1 % | 6.8 % | 1.000 |
Table 6: Summary of Bland-Altman and Passing-Bablok regression analyses obtained on cobas c 303 analyzer vs. established reference values on cobas c 501 analyzer. (LoA = Limits of agreement)
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) |
(95 % CI) |
(95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) |
|
-20.9 % (-24.6 %, -17.3 %) | 7.0 % (3.3 %, 10.7 %) | 0.9866 (0.9657, 1.005) | -7.28 μg/g (-12.18 μg/g, -5.32 μg/g) | -10.4 % | -5.9 % | 0.998 |
Within-laboratory precision
Repeatability and within‑laboratory precision were established according to the CLSI guideline EP05-A3. Six pooled stool specimen extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 5 days, in 2 runs per day, with 4 results generated per run. One reagent lot was used in the study.
ID | Mean [μg/g] | Within-run | Between-run | Between-day | Total | ||||
SD | CV | SD | CV | SD | CV | SD | CV | ||
P1 | 59.3 | 1.8 | 3.0 | 1.8 | 0.9 | 0.6 | 1.0 | 1.92 | 3.2 |
P2 | 85.4 | 1.9 | 2.2 | 2.7 | 3.1 | 0.0 | 0.0 | 3.25 | 3.8 |
P3 | 184.1 | 1.8 | 1.0 | 0.9 | 0.5 | 2.8 | 1.5 | 3.44 | 1.9 |
P4 | 733.9 | 1.9 | 0.3 | 20.9 | 2.8 | 39.0 | 5.3 | 44.27 | 6.0 |
P5 | 1738.4 | 5.7 | 0.3 | 37.1 | 2.1 | 36.0 | 2.1 | 52.00 | 3.0 |
P6 | 6758.5 | 20.2 | 0.3 | 86.0 | 1.3 | 242.8 | 3.6 | 258.31 | 3.8 |
Table 8: Within-laboratory precision study results on the cobas c 303 analyzer
ID | Mean [μg/g] | Within-run | Between-run | Between-day | Total | ||||
SD | CV | SD | CV | SD | CV | SD | CV | ||
P1 | 49.0 | 1.8 | 3.6 | 1.5 | 3.2 | 1.6 | 3.2 | 2.8 | 5.7 |
P2 | 64.5 | 1.8 | 2.7 | 1.6 | 2.4 | 4.3 | 6.7 | 4.9 | 7.6 |
P3 | 163.4 | 1.3 | 0.8 | 2.6 | 1.6 | 4.3 | 2.6 | 5.2 | 3.2 |
P4 | 735.2 | 1.8 | 0.2 | 10.6 | 1.4 | 27.4 | 3.7 | 29.4 | 4.0 |
P5 | 1743.0 | 6.6 | 0.4 | 29.2 | 1.7 | 75.1 | 4.3 | 80.8 | 4.6 |
P6 | 6657.9 | 27.2 | 0.4 | 105.3 | 1.6 | 0.0 | 0.0 | 108.7 | 1.6 |
Table 9: Within-laboratory precision study results
Extraction reproducibility‑CALEX®Cap: 8.1‑19.7 % CV
The extraction reproducibility was established according to the CLSI guideline EP05‑A3 using a 2 days x 2 operators x 3 CALEX® Cap lots x 2 extractions x 3 replicates study design on the cobas c 501 analyzer. Twelve clinical stool specimens, including specimens with solid, semi‑solid and liquid consistency, with calprotectin concentrations in the range of 42.7‑3440.0 μg/g, were tested.
Sample carry-over
The sample carry-over was established according to the CLSI guideline EP10‑A2. No statistically significant sample carry-over with the fCAL turbo test on cobas c
Limit of Blank (LoB)
cobas c 503 analyzer: 2.6 μg/g
The LoB was established according to the CLSI guideline EP17-A2 with 4 negative (extraction buffer) samples. The samples were measured over 3 days in 5 replicates each day to produce 60 blank values. The LoB was calculated using non-parametric analysis. The study was performed with 1 reagent lot.
Limit of Detection (LoD)
cobas c 503 analyzer: 6.3 μg/g
The LoD was established according to the CLSI guideline EP17-A2 with 4 stool specimen extracts with concentrations corresponding to 1‑5 times the LoB value. The samples were measured over 3 days in 5 replicates each day to produce 60 values. The LoD was calculated using parametric analysis. The study was performed with 1 reagent lot.
Limit of Quantitation (LoQ)
cobas c 503 analyzer: 30 μg/g
The LoQ was established according to the CLSI guideline EP17-A2 with 4 low level stool specimen extracts. The samples were measured over 3 days in 5 replicates each day to produce a total of 15 replicates per sample. The study was performed with 1 reagent lot. The LoQ was defined as the lowest calprotectin concentration, which can be determined with a precision of < 20% CV using an acceptance criterion of LoQ ≤ 30 µg/g.
Gastroenterologists are often faced with the diagnostic difficulty of differentiating individuals with functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), from those with inflammatory bowel disease (IBD). Many symptoms are common to both conditions, whereas other clinical features such as a predominance of diarrhea and rectal bleeding will increase the likelihood of inflammatory disease. The clinical differentiation between these conditions remains problematic and may result in delayed diagnosis. Furthermore, many individuals with IBS must undergo invasive procedures (endoscopy) to rule out an organic disorder. This has significant implications for health care costs as well as exposing individuals to the inherent risks associated with invasive procedures.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | 25.4 mL | Ready to use |
Immunoparticles (R3) | 6.5 mL | Ready to use |
R1 is in position B and R3 is in position C. R3 refers to the reagent, which is pipetted at time point R3.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
Product safety labeling follows GHS guidance.
Contact phone: 1-800-428-2336
• The immunoparticles contain potentially infectious substances of animal origin and should be handled in accordance with good laboratory practice (GLP) using appropriate precautions.
• R1 contains MOPS (3-(N-morpholino)propanesulfonic acid) (< 1 %), that can be irritating to eyes and skin. Handle with due caution.
• Avoid contact of reagents with the skin, eyes or mucous membranes. If contact does occur, immediately wash with generous amounts of water; otherwise, irritation / burns can occur.
• Immunoparticles R3 contains polystyrene nanoparticles.
• Reagent immunoparticles R3, once frozen, cannot be used anymore. Freezing R3 will lead to reduced sensitivity and precision in low‑level samples and in the worst case to decreased measurement levels.
• The assay is designed for fecal extract samples prepared using the specific CALEX® Cap. Application of other extraction buffers could lead to incorrect results.
• Please equilibrate reagents, controls, calibrators and samples as described in this method sheet.
• Ensure that samples have no bubbles prior to running the test.
• Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Run the fCAL Control Set, Level I and Level II, in duplicate, after each new calibration, before running patient fecal sample extracts. Keep controls at room temperature for at least 30 min before starting analysis.
Validating daily runs
Run the fCAL Control Set, Level I and Level II, each day, before running patient fecal sample extracts to validate calibration curve stability. Single measurements of controls are sufficient.
The controls have assigned target values indicated on the control value sheet supplied with each lot of the fCAL Control Set. All control measurements must be within target values indicated on the control value sheet to obtain valid results for patient fecal sample extracts. If the control values are not valid, repeat quality control measurement with fresh controls. If control values remain invalid, recalibrate the instrument. If valid control values cannot be reproduced after performing the steps described above, contact Roche US Technical Support 1-800-428-2336.
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecal extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX® Cap.
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or refrigerated. Stool specimens should be refrigerated at 2-8 °C and extracted within 3 days of receipt at the laboratory. Do not store samples at elevated temperatures.
Stool sample extraction and extract stability
Follow the instruction for use provided with the CALEX® Cap. Fecal sample extracts prepared using the CALEX® Cap will have a final dilution of 1:500 and are ready to use. Liquid stool samples can be pipetted directly into the CALEX® Cap. Unscrew the blue cap and pipet 10 μL of stool sample into the
Important: Centrifuge the CALEX® Cap for 10 minutes at 1000‑3000 g prior to running the fCAL turbo procedure.
CALEX® Cap extracts can be kept at room temperature for up to 2 hours, and after centrifugation, at 2-8 °C for up to 3.5 days (84 hours). For longer storage, freeze CALEX® Cap extracts at -20 °C. CALEX® Cap extracts can be subjected to 4 freeze-thaw cycles. Allow frozen extracts to equilibrate to room temperature for up to 2 hours before measurement. Prior to measurement, CALEX® Cap extract should be vortexed thoroughly for 10 seconds and centrifuged for 10 minutes at 1000‑3000 g.
Please note that extracts can be stored and frozen directly within the CALEX® Cap.
The fCAL turbo is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. fCAL turbo aids in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC) and aids in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other laboratory and clinical findings. The fCAL turbo is intended to be run on cobas c 501/502 systems.
The fCAL turbo test is a particle‑enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the cobas c 501/502 analyzers is 30‑2000 µg/g. Samples > 2000 µg/g will be diluted automatically (1:10) by the analyzer, further extending the range to 30‑10000 µg/g.
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract. Low fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for additional diagnostic work‑up. Specifically, for patients with a clinical and laboratory presentation suggesting a non‑inflammatory disorder such as IBS, fecal calprotectin values of < 80 µg/g can be used to support a decision to defer invasive testing.
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status. This decision should be made by the clinician in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings.
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease. Elevated fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for further investigative procedures, including invasive procedures performed by specialists, to achieve an overall clinical diagnosis, in particular of IBD.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
• False negative results could occur in patients who have granulocytopenia due to bone marrow depression.
• Some patients taking non-steroidal anti-inflammatory drugs (NSAID) will have elevations in their fecal calprotectin levels.
• Results may not be clinically applicable to children less than 4 years of age who have mildly increased fecal calprotectin levels.
• Patients with IBD fluctuate between active (inflammatory) and inactive stages of the disease. These stages must be considered when interpreting results of the fecal calprotectin assay.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on Roche/Hitachi cobas c systems. The latest version of the carry‑over evasion list can be found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets. For further instructions refer to the operator’s manual. cobas c 502 analyzer: All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08910332 190 | fCAL turbo (200 tests) | System‑ID 07 7626 2 | cobas c 501/502 |
08910499 190 | fCAL Calibrator Set | Code 719 | |
08910502 190 | fCAL Control Set |
| |
08910987 001 | CALEX® Cap (500 pcs) |
For cobas c 501 analyzer:
FCAL: ACN 462
For cobas c 502 analyzer:
FCAL: ACN 8462
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
cobas c 501/502 test definition | |||
Assay type | 2 Point End | ||
Reaction time / Assay points | 10/36‑55 | ||
Wavelength (sub/main) | 800/546 nm | ||
Reaction direction | Increase | ||
Units | µg/g | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 100 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 10 µL | – | – |
Decreased | 10 µL | 10 µL | 90 µL |
Increased | 10 µL | – | – |
Note
For technical reasons it is necessary to specify a dummy unit (µg/mL) on cobas c analyzers. Values can be converted to the correct unit (µg/g) via the host computer.
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to 3 months at 5‑12 °C |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Apply the lot-specific fCAL Calibrator values for the 6-point calibration curve: | |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve equilibrate reagents R1 and R2 to the storage temperature of the analyzer by loading the cobas c pack at least 1 hour before calibration curve establishment. Keep calibrators as well as controls and patient samples at room temperature for at least 30 min before starting analysis. The calibration on cobas c 501/502 is performed in duplicates.
A calibration curve stability of 2 months, established for reagents stored on board, is defined in the instrument application.
Traceability: This method has been standardized against internal reference material.
The ability of the fCAL turbo to discriminate between patients with IBD and other non‑inflammatory gastrointestinal (GI) disorders, including IBS, was evaluated using clinical samples collected from 337 adult and pediatric patients. One hundred and thirty five patients had a final diagnosis of IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis), 130 patients suffered from IBS and 72 patients presented with abdominal pain and/or diarrhea, or other GI‑related non‑inflammatory conditions. Final diagnosis was supported by endoscopic as well as other clinical findings.
Final diagnosis | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 12 (8.9 %) | 15 (11.1 %) | 108 (80 %) | 135 (100 %) |
IBS | 99 (76.2 %) | 15 (11.5 %) | 16 (12.3 %) | 130 (100 %) |
Other GI | 51 (70.8 %) | 7 (9.7 %) | 14 (19.4 %) | 72 (100 %) |
Table 2: Distributions of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 74.3 % (67.7 %, 80.1 %) | 85.1 % (79.5 %, 89.8 %) |
PPV (95 % CI) | 70.3 % (62.9 %, 76.9 %) | 78.3 % (70.4 %, 84.8 %) |
NPV (95 % CI) | 92.6 % (87.4 %, 96.1 %) | 86.4 % (80.9 %, 90.9 %) |
ROC AUC (95 % CI) | 0.916 (0.884, 0.947) |
Table 3: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD − IBS and other GI‑related disorders, at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 76.2 % (67.9 %, 83.2 %) | 87.7 % (80.8 %, 92.8 %) |
PPV (95 % CI) | 79.9 % (72.7 %, 85.9 %) | 87.1 % (79.9 %, 92.4 %) |
NPV (95 % CI) | 89.2 % (81.9 %, 94.3 %) | 80.9 % (73.4 %, 87.0 %) |
ROC AUC (95 % CI) | 0.929 (0.898, 0.960) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
Reference range
Stool samples were obtained from 141 apparently healthy normal adults (> 21 years of age) with no symptoms or signs of gastrointestinal disease. The test results were categorized by the assay cut‑offs.
Distribution of results within fCAL turbo diagnostic ranges | ||||
< 80 μg/g | 80-160 μg/g | > 160 μg/g | Total | |
Number of subjects (%) | 106 (75.2 %) | 18 (12.8 %) | 17 (12.1 %) | 141 (100 %) |
Table 5: Distribution of healthy subjects results within fCAL turbo diagnostic ranges
The presented performance characteristics have been established on a cobas c 501 analyzer. For regulatory purposes validation was performed using extracts obtained by manual weighing and extraction. The CALEX® Cap was validated for comparability with the manual extraction method. Please refer to the CALEX® Cap IFU for results. Any changes to the directions for use, including special wash programming, may alter the stated performance characteristics of the assay and must be validated prior to implementation by the laboratory. Refer to clinical chemistry analyzer specific application notes for the performance characteristics on other clinical chemistry analyzers.
Please refer to the CALEX® Cap IFU for fCAL turbo performance with the CALEX®Cap device.
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3. Two hundred and forty eight clinical samples were measured according to the instructions for use with the fCAL turbo and with BÜHLMANN fCAL® ELISA assay, with 220 samples yielding results within the measuring range for both tests. Measurements were performed over 18 days using 3 reagent lots.
Bland‑Altman analysis | Passing-Bablok regression analysis | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) |
(95 % CI) |
(95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) |
|
-0.5 % (-3.6 %, 2.7 %) | -46.5 % (-51.8 %, -41.1 %) | 45.5 % (40.2 %, 50.9 %) | 1.025 (0.990, 1.058) | -4.5 μg/g (-8.7, 0.3) | -3.1 % | -0.3 % | 0.972 |
Table 6: Method comparison study results, (LoA= Limits of agreement)
Reproducibility (multisite precision evaluation study)
Reproducibility was established according to the CLSI guideline EP05-A3 by performing measurements at 3 laboratory sites. Eight pooled stool specimen extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 5 days, in 1 run per day, with 5 results generated per run. One reagent lot was used in the study.
ID | Mean [μg/g] | n | Within-run (repeatability) | Between-day | Between-site | Total precision | ||||
SD | %CV | SD | %CV | SD | %CV | SD | %CV | |||
S01 | 47.2 | 75 | 3.6 | 7.6 % | 2.4 | 5.0 % | 0.0 | 0.0 % | 4.3 | 9.1 % |
S02 | 91.1 | 75 | 3.5 | 3.8 % | 3.5 | 3.8 % | 2.8 | 3.1 % | 5.7 | 6.2 % |
S03 | 185.4 | 75 | 5.1 | 2.7 % | 2.7 | 1.4 % | 5.5 | 3.0 % | 7.9 | 4.3 % |
S04 | 276.9 | 75 | 6.4 | 2.3 % | 4.5 | 1.6 % | 9.7 | 3.5 % | 12.5 | 4.5 % |
S05 | 674.5 | 75 | 12.9 | 1.9 % | 1.2 | 0.2 % | 22.8 | 3.4 % | 26.3 | 3.9 % |
S06 | 1519.6 | 75 | 25.3 | 1.7 % | 17.8 | 1.2 % | 62.3 | 4.1 % | 69.6 | 4.6 % |
S07 | 3343.8 | 75 | 54.6 | 1.6 % | 35.6 | 1.1 % | 100.0 | 3.0 % | 119.4 | 3.6 % |
S08 | 5475.6 | 75 | 72.1 | 1.3 % | 35.8 | 0.7 % | 154.2 | 2.8 % | 173.9 | 3.2 % |
Table 7: Reproducibility study results – within‑run, between‑day, between‑site and total precision estimates
Between‑lot precision
Between‑lot precision was established according to the CLSI guideline EP05-A3. Eight pooled stool extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 5 days, in 1 run per day, with 5 results generated per run. Three reagent lots were used in the study.
ID | Mean [μg/g] | n | Within-run (repeatability) | Between-day | Between-lot | Total precision | ||||
SD | %CV | SD | %CV | SD | %CV | SD | %CV | |||
S01 | 45.2 | 75 | 3.22 | 7.1 % | 1.36 | 3.0 % | 3.70 | 8.2 % | 5.09 | 11.3 % |
S02 | 86.4 | 75 | 3.69 | 4.3 % | 1.19 | 1.4 % | 5.66 | 6.6 % | 6.86 | 7.9 % |
S03 | 175.8 | 75 | 5.04 | 2.9 % | 0.29 | 0.2 % | 9.90 | 5.6 % | 11.11 | 6.3 % |
S04 | 263.9 | 75 | 7.55 | 2.9 % | 0.00 | 0.0 % | 9.98 | 3.8 % | 12.52 | 4.7 % |
S05 | 647.4 | 75 | 15.47 | 2.4 % | 0.00 | 0.0 % | 15.28 | 2.4 % | 21.74 | 3.4 % |
S06 | 1460.7 | 75 | 33.66 | 2.3 % | 11.64 | 0.8 % | 41.01 | 2.8 % | 54.32 | 3.7 % |
S07 | 3234.5 | 75 | 71.23 | 2.2 % | 8.90 | 0.3 % | 130.29 | 4.0 % | 148.76 | 4.6 % |
S08 | 5303.1 | 75 | 97.42 | 1.8 % | 11.18 | 0.2 % | 163.87 | 3.1 % | 190.97 | 3.6 % |
Table 8: Between‑lot precision study results
Within-laboratory precision
Repeatability and within‑laboratory precision were established according to the CLSI guideline EP05-A3. Eight pooled stool specimen extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 20 days, in 2 runs per day, with 2 results generated per run. One reagent lot was used in the study.
ID | Mean [μg/g] | n | Repeatability | Between-run | Between-day | Within-laboratory | ||||
SD | %CV | SD | %CV | SD | %CV | SD | %CV | |||
S01 | 42.9 | 80 | 3.6 | 8.3 % | 1.2 | 2.7 % | 1.1 | 2.5 % | 3.9 | 9.1 % |
S02 | 98.4 | 80 | 2.5 | 2.6 % | 1.8 | 1.8 % | 2.2 | 2.2 % | 3.7 | 3.8 % |
S03 | 166.5 | 80 | 4.3 | 2.6 % | 0.8 | 0.5 % | 1.9 | 1.2 % | 4.8 | 2.9 % |
S04 | 267.6 | 80 | 3.9 | 1.4 % | 2.5 | 0.9 % | 1.8 | 0.7 % | 5.0 | 1.9 % |
S05 | 642.0 | 80 | 20.1 | 3.1 % | 14.9 | 2.3 % | 0.0 | 0.0 % | 25.1 | 3.9 % |
S06 | 1414.2 | 80 | 19.6 | 1.4 % | 11.1 | 0.8 % | 3.5 | 0.2 % | 22.8 | 1.6 % |
S07 | 3251.4 | 80 | 40.8 | 1.3 % | 21.4 | 0.7 % | 19.7 | 0.6 % | 50.1 | 1.5 % |
S08 | 5405.6 | 80 | 40.2 | 0.7 % | 56.6 | 1.0 % | 34.5 | 0.6 % | 77.5 | 1.4 % |
Table 9: Within‑laboratory precision study results
Gastroenterologists are often faced with the diagnostic difficulty of differentiating individuals with functional gastrointestinal disorders, such as irritable bowel syndrome, from those with inflammatory bowel disease. Many symptoms are common to both conditions, whereas other clinical features such as a predominance of diarrhea and rectal bleeding will increase the likelihood of inflammatory disease. The clinical differentiation between these conditions remains problematic and may result in delayed diagnosis. Furthermore, many individuals with IBS must undergo invasive procedures (endoscopy) to rule out an organic disorder. This has significant implications for health care costs as well as exposing individuals to the inherent risks associated with invasive procedures.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | 1 x | Ready to use |
Immunoparticles (R2) | 1 x | Ready to use |
R1 is in position B and R2 is in position C.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
The immunoparticles contain potentially infectious substances of animal origin and should be handled in accordance with good laboratory practice (GLP) using appropriate precautions.
• R1 contains MOPS (3-(N-morpholino)propanesulfonic acid) (< 1 %), that can be irritating to eyes and skin. Handle with due caution.
• R2 contains polystyrene nanoparticles.
• R1 and R2 contain 2-Methyl-2H-isothiazol-3-one, a preservative agent, which is below the allowable limits (0.01 %).
• Do not mix reagents R1 and R2 of different reagent lots.
• Reagent R2, once frozen, cannot be used anymore. Freezing R2 will lead to reduced sensitivity and precision in low‑level samples and in the worst case to decreased measurement levels.
• The assay is designed for fecal extract samples prepared using the specific CALEX® Cap devices. Application of other extraction buffers could lead to incorrect results.
• Please equilibrate reagents, controls, calibrators and samples as described.
• Ensure that samples have no bubbles prior to running the test.
• Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Assay the fCAL Control Set, Level I and Level II, in duplicate, after each new calibration, before running patient fecal sample extracts. Keep controls at room temperature for at least 30 min before starting analysis.
Validating daily runs
Assay the fCAL Control Set, Level I and Level II, each day, before running patient fecal sample extracts to validate calibration curve stability. Single measurements of controls are sufficient.
The controls have assigned value ranges indicated on the QC datasheet supplied with each lot of the fCAL Control Set. All control measurements must be within the indicated value ranges to obtain valid results for patient fecal sample extracts. If the control values are not valid, repeat quality control measurement with fresh controls. If control values remain invalid, recalibrate the instrument. For U.S., contact BUHLMANN Diagnostics Corp, BDC at (844) 300-9799 (Mon-Fri 8:00AM - 5:00PM EST) if valid control values cannot be reproduced, after performing the steps described above. For rest of the world, contact BÜHLMANN Laboratories AG, at +41 61 487 12 12 (Mon-Fri 8:00AM - 5.00PM CET).
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecals extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX® Cap device.
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or on cold packs. Stool specimens should be refrigerated at 2‑8 °C and extracted within 3 days of receipt at the laboratory.
Stability in CALEX® Cap extracts
For CALEX® Cap extract stability refer to the instruction for use provided with the CALEX® Cap (REF: 08910987 001).
Laboratories are responsible for validating use of alternate storage temperatures/timeframes.
Follow the instruction for use provided with the the CALEX® Cap kit. Fecal sample extracts prepared using the CALEX® Cap will have a final dilution of 1:500 and are ready to use.
Important: Centrifuge the CALEX® Cap device for 10 minutes at 1000‑3000 g prior to running the fCAL turbo procedure.
The fCAL turbo is an automated in vitro diagnostic test for the quantitative determination of calprotectin in human stool specimens intended as an aid in the assessment of intestinal mucosal inflammation.
The fCAL turbo test is a particle‑enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the cobas c 501/502 analyzers is 30‑2000 µg/g. Samples > 2000 µg/g will be diluted automatically (1:10) by the analyzer, further extending the range to 30‑10000 µg/g.
Distinguishing organic disease from functional gastrointestinal disease
Determination of fecal calprotectin levels can be used as a reliable and simple aid in distinguishing organic from functional gastrointestinal diseases.
Clinical thresholds
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges.
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract.
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
Special wash programming required by the instrument manufacturer:
The manufacturer of the cobas c systems considers the use of special wash steps as mandatory
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08910332190 | fCAL turbo (200 tests) | System‑ID 07 7626 2 | cobas c 501/502 |
Materials required (but not provided):
08910499190 | fCAL Calibrator Set | Code 719 | |
08910502190 | fCAL Control Set | ||
08910987001 | CALEX® Cap (500 pcs) |
For cobas c 501 analyzer:
FCAL: ACN 462
For cobas c 502 analyzer:
FCAL: ACN 8462
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
cobas c 501/502 test definition | |||
Assay type | 2 Point End | ||
Reaction time / Assay points | 10/36‑55 | ||
Wavelength (sub/main) | 800/546 nm | ||
Reaction direction | Increase | ||
Units | µg/g | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 100 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 10 µL | – | – |
Decreased | 10 µL | 10 µL | 90 µL |
Increased | 10 µL | – | – |
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Apply the lot-specific fCAL Calibrator values for the 6-point calibration curve: | |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve, equilibrate reagents R1 and R3 to the storage temperature of the analyzer by loading the cobas c pack at least 1 hour before calibration curve establishment. Keep calibrators
The ability of the fCAL turbo to discriminate between patients with IBD and other non-inflammatory GI disorders, including IBS, was evaluated using clinical samples collected from 295 patients and extracted using the CALEX® Cap. One hundred and twenty seven (127) patients had a final diagnosis of IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis), 103 patients suffered from IBS and 65 patients presented with abdominal pain and/or diarrhea, or other GI-related non-inflammatory conditions. Final diagnosis was supported by endoscopic as well as other clinical findings.
The optimal cut‑off combination for these patient pools could be defined by ROC analysis at 80 µg/g and 160 µg/g calprotectin (table 3 and 5), which is slightly more stringent than a combination of a more sensitive lower cut‑off of 50 µg/g with lower performance in specificity, and an upper cut‑off 200 µg/g with slightly lower sensitivity (table 4 and 6).
Final | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 8 (6.3 %) | 108 (85 %) | 127 (100 %) | |
IBS | 75 (72.8 %) | 11 (10.7 %) | 17 (16.5 %) | 103 (100 %) |
Other GI | 42 (64.6 %) | 8 (12.3 %) | 15 (23.1 %) | 65 (100 %) |
Table 2: Distribution of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 85.0 % (77.6 %, 90.7 %) | |
Specificity (95 % CI) | 69.6 % (62.1 %, 76.5 %) | 81.0 % (74.2 %, 86.6 %) |
PPV (95 % CI) | 69.5 % (61.9 %, 76.3 %) | 77.1 % (69.3 %, 83.8 %) |
NPV (95 % CI) | 91.4 % (85.1 %, 95.6 %) | 87.7 % (81.5 %, 92.5 %) |
ROC AUC (95 % CI) | 0.912 (0.878, 0.946) |
Table 3: Clinical performance characteristics of the
IBD vs. non‑IBD | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 62.5 % (54.7 %, 69.8 %) | 85.7 % (79.5 %, 90.6 %) |
PPV (95 % CI) | 65.6 % (58.2 %, 72.4 %) | 81.0 % (73.0 %, 87.4 %) |
NPV (95 % CI) | 93.8 % (87.5 %, 97.5 %) | 85.2 % (78.9 %, 90.2 %) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD – IBS and other GI‑related disorders, at 50 µg/g and 200 µg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.3 % (85.0 %, 95.6 %) | 85.0 % (77.6 %, 90.7 %) |
Specificity (95 % CI) | 72.8 % (63.2 %, 81.1 %) | 83.5 % (74.9 %, 90.1 %) |
PPV (95 % CI) | 80.6 % (73.1 %, 86.7 %) | 86.4 % (79.1 %, 91.9 %) |
NPV (95 % CI) | 87.2 % (78.3 %, 93.4 %) | 81.9 % (73.2 %, 88.7 %) |
ROC AUC (95 % CI) | 0.925 (0.892, 0.958) |
Table 5: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
50 μg/g | 200 μg/g | |
Sensitivity (95 % CI) | 94.5 % (89.0 %, 97.8 %) | 80.3 % (72.3 %, 86.8 %) |
Specificity (95 % CI) | 67.0 % (57.0 %, 75.9 %) | 88.3 % (80.5 %, 93.8 %) |
PPV (95 % CI) | 77.9 % (70.5 %, 84.2 %) | 89.5 % (82.3 %, 94.4 %) |
NPV (95 % CI) | 90.8 % (81.9 %, 96.2 %) | 78.4 % (69.9 %, 85.5 %) |
Table 6: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 50 µg/g and 200 µg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3.
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) | Slope (95 % CI) | (95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) | r |
-46.0 % (-51.6 %, -40.3 %) | 47.3 % (41.6 %, 53.0 %) | 1.139 (1.104, 1.172) | -18.3 μg/g (-24.4 μg/g, -13.2 μg/g) | -9.0 % | 2.4 % |
|
Table 9: Method comparison study results, (LoA= Limits of agreement)
The method comparison study was performed according to the CLSI guideline EP09‑A3. One hundred and sixty eight (168) clinical samples were extracted using three lots of the CALEX® Cap and measured using one lot of fCAL turbo over 18 days in one calibration cycle. Reference values, with a final calprotectin concentration interval of 30.5‑1573.8 µg/g, were established using the manual extraction method and extract measurement with the BÜHLMANN fCAL® ELISA. Extracts were measured in single determinations in both methods. Bias was determined using Passing‑Bablok linear regression and Bland‑Altman analysis.
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean (95 % CI) | Lower (95 % CI) | Upper (95 % CI) | Slope (95 % CI) | Intercept (95 % CI) | Bias at 80 μg/g (95 % CI) | Bias at 160 μg/g (95 % CI) | r |
11.1 % (5.5 %, 16.6 %) | -60.7 % (-70.3 %, -51.2 %) | 82.8 % (73.3 %, 92.4 %) | 1.336 (1.265, 1.429) | -31.7 μg/g (-44.1 μg/g, -19.4 μg/g) | -6.0 % | 13.8 % |
|
Table 10: Method comparison study result, (LoA= Limits of agreement)
Reproducibility (multisite precision evaluation study): 3.2‑9.1 % CV
Reproducibility was established according to the CLSI guideline EP05-A3 using a 3 laboratory sites x 5 days x 5 replicates study design. Eight pooled stool specimen extracts with calprotectin concentrations ranging from 47.2‑5475.6 µg/g were tested.
Between‑lot precision: 2.4‑8.2 % CV
Between‑lot precision was established according to the CLSI guideline EP05‑A3 using a 3 lots x 5 days x 5 replicates study design. Eight pooled stool specimen extracts with calprotectin concentrations ranging from 45.2‑5303.1 µg/g were tested.
Repeatability: 0.7‑8.3 % CV
Within-laboratory precision: 1.4‑9.1 % CV
Repeatability and within‑laboratory precision were established according to the CLSI guideline EP05‑A3 using the standardized 20 days x 2 runs x 2 replicates study design. Eight pooled stool specimen extracts with calprotectin concentrations ranging from 42.9‑5405.6 µg/g were tested.
Extraction reproducibility‑CALEX®Cap: 8.1‑19.7 % CV
The extraction reproducibility was established according to the CLSI guideline EP05‑A3 using a 2 days x 2 operators x 3 CALEX® Cap lots x 2 extractions x 3 replicates study design. Twelve clinical stool specimens, including specimens with solid, semi‑solid and liquid consistency, with calprotectin concentrations in the range of 42.7‑3440.0 μg/g, were tested.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | Ready to use | |
Immunoparticles | Ready to use |
R1 is in position B and
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
Product safety labeling follows EU GHS guidance.
Contact phone: all countries: +49-621-7590
Reagent
The assay is designed for fecal extract samples prepared using the specific CALEX® Cap
Please equilibrate reagents, controls, calibrators and samples as described
Ensure that samples have no bubbles prior to running the test.
Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Validating daily runs
The controls have assigned
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecal extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX®
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or
Follow the instruction for use provided with the CALEX® Cap. Fecal sample extracts prepared using the CALEX® Cap will have a final dilution of 1:500 and are ready to use.
Liquid stool samples can be pipetted directly into the CALEX® Cap. Unscrew the blue cap and pipet 10 μL of stool sample into the device. Recap the CALEX® Cap and proceed with vortexing step according to the extraction procedure described and illustrated in the instruction for use delivered with the CALEX® Cap.
Important: Centrifuge the CALEX® Cap for 10 minutes at 1000‑3000 g prior to running the fCAL turbo procedure.
Fecal calprotectin in extracts obtained by the CALEX® Cap is stable at room temperature (23 °C) for 7 days, at 2‑8 °C for 15 days and at -20 °C for at least 23 months. CALEX® Cap extracts can be frozen directly and stored within the CALEX® Cap. Extracts can be subject to four freeze‑thaw cycles. Prior to measurement, allow frozen extracts to equilibrate to room temperature, vortex thoroughly for 10 seconds and centrifuge according to the instruction for use of the assay.
The fCAL turbo is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. fCAL turbo aids in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC) and aids in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other laboratory and clinical findings.
The fCAL turbo test is a particle‑enhanced turbidimetric immunoassay (PETIA), which allows for automated quantification of calprotectin in fecal extracts on cobas c systems. Fecal samples are extracted with extraction buffer using the CALEX® Cap extraction device and applied at a final dilution of 1:500. The extracts are incubated with reaction buffer and mixed with polystyrene nanoparticles coated with calprotectin-specific antibodies (immunoparticles). Calprotectin available in the sample mediates immunoparticle agglutination. Sample turbidity, measured by light absorbance, increases with calprotectin-immunoparticle complex formation and is proportional to calprotectin concentration. The detected light absorbance allows quantification of calprotectin concentration via interpolation on an established calibration curve.
The measuring range for the fCAL turbo assay on the cobas c 501/502 analyzers is 30‑2000 µg/g. Samples > 2000 µg/g will be diluted automatically (1:10) by the analyzer, further extending the range to 30‑10000 µg/g.
Calprotectin concentration | Interpretation | Follow-up |
< 80 µg/g | normal | none |
80‑160 µg/g | gray-zone/borderline | follow-up within 4‑6 weeks |
> 160 µg/g | elevated | repeats as needed |
Table 1: fCAL turbo diagnostic ranges
Calprotectin values < 80 μg/g
Fecal calprotectin values < 80 µg/g are not indicative of active inflammation in the gastrointestinal tract. Low fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for additional diagnostic work‑up. Specifically, for patients with a clinical and laboratory presentation suggesting a non‑inflammatory disorder such as IBS, fecal calprotectin values of < 80 µg/g can be used to support a decision to defer invasive testing.
Calprotectin values between and equal to 80 and 160 μg/g
Mid‑fecal calprotectin levels between and equal to 80 and 160 µg/g, also called gray‑zone levels, are not directly indicative of an active inflammation requiring immediate follow‑up with invasive testing. However, the presence of inflammation cannot be excluded. Re‑evaluation of fecal calprotectin levels after 4-6 weeks is recommended to determine the inflammatory status. This decision should be made by the clinician in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings.
Calprotectin values > 160 μg/g
Fecal calprotectin values > 160 µg/g are indicative of neutrophil infiltrate in the gastrointestinal tract; therefore, this may signal the presence of active inflammatory disease. Elevated fecal calprotectin levels can be used in conjunction with the patient’s clinical symptoms, medical history and other clinical and laboratory findings to determine the need for further investigative procedures, including invasive procedures performed by specialists, to achieve an overall clinical diagnosis, in particular of IBD.
• Test results should be interpreted in conjunction with information available from clinical assessment of the patient and other diagnostic procedures.
• False negative results could occur in patients who have granulocytopenia due to bone marrow depression.
• Some patients taking non-steroidal anti-inflammatory drugs (NSAID) will have elevations in their fecal calprotectin levels.
• Results may not be clinically applicable to children less than 4 years of age who have mildly increased fecal calprotectin levels.
• Patients with IBD fluctuate between active (inflammatory) and inactive stages of the disease. These stages must be considered when interpreting results of the fecal calprotectin assay.
Special wash programming required by the instrument manufacturer:
The manufacturer of the cobas c systems considers the use of special wash steps as mandatory to avoid carry-over, when certain test combinations are run together.
The latest version of the carry‑over evasion list can be found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets provided by the instrument manufacturer. For further instructions refer to the operator’s manual. cobas c 502 analyzer: All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08910332190 | fCAL turbo (200 tests) | System‑ID 07 7626 2 | cobas c 501/502 |
Materials required (but not provided):
08910499190 | fCAL Calibrator Set | Code 719 | |
08910502190 |
| ||
08910987001 | CALEX® Cap (500 pcs) |
For cobas c 501 analyzer:
FCAL: ACN 462
For cobas c 502 analyzer:
FCAL: ACN 8462
Ready for use
Loading of reagents
The reagents supplied are ready to use. Mix gently before loading onto the instrument. Avoid bubble formation.
cobas c 501/502 test definition | |||
Assay type | 2 Point End | ||
Reaction time / Assay points | 10/36‑55 | ||
Wavelength (sub/main) | 800/546 nm | ||
Reaction direction | Increase | ||
Units | µg/g | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 100 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (H2O) | ||
Normal | 10 µL | – | – |
Decreased | 10 µL | 10 µL | 90 µL |
Increased | 10 µL | – | – |
Note
For technical reasons it is necessary to specify a dummy unit (µg/mL) on cobas c analyzers. Values can be converted to the correct unit (µg/g) via the host computer.
fCAL turbo | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | store for up to |
Do not freeze reagents!
Calibrators | S1: fCAL Calibrator 1 S2: fCAL Calibrator 2 S3: fCAL Calibrator 3 S4: fCAL Calibrator 4 S5: fCAL Calibrator 5 S6: fCAL Calibrator 6 |
Calibration mode | Spline |
Calibration frequency | Full calibration |
To generate a reproducible calibration curve, equilibrate reagents R1 and
Traceability: This method has been standardized against internal reference material.
The ability of the fCAL turbo to discriminate between patients with IBD and other non‑inflammatory gastrointestinal (GI) disorders, including IBS, was evaluated using clinical samples collected from 337 adult and pediatric patients. One hundred and thirty five patients had a final diagnosis of IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis), 130 patients suffered from IBS and 72 patients presented with abdominal pain and/or diarrhea, or other GI‑related non‑inflammatory conditions. Final diagnosis was supported by endoscopic as well as other clinical findings.
Final | Distribution of patients results in numbers (%) within fCAL turbo diagnostic ranges | |||
< 80 μg/g | 80‑160 μg/g | > 160 μg/g | Total | |
IBD | 12 (8.9 %) | 15 (11.1 %) | 108 (80 %) | 135 (100 %) |
IBS | 99 (76.2 %) | 15 (11.5 %) | 16 (12.3 %) | 130 (100 %) |
Other GI | 51 (70.8 %) | 7 (9.7 %) | 14 (19.4 %) | 72 (100 %) |
Table 2: Distributions of patients results within fCAL turbo diagnostic ranges
IBD vs. non‑IBD | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 74.3 % (67.7 %, 80.1 %) | 85.1 % (79.5 %, 89.8 %) |
PPV (95 % CI) | 70.3 % (62.9 %, 76.9 %) | 78.3 % (70.4 %, 84.8 %) |
NPV (95 % CI) | 92.6 % (87.4 %, 96.1 %) | 86.4 % (80.9 %, 90.9 %) |
ROC AUC (95 % CI) | 0.916 (0.884, 0.947) |
Table 3: Clinical performance characteristics of the fCAL turbo in discriminating IBD from non‑IBD − IBS and other GI‑related disorders, at 80 μg/g and 160 μg/g clinical decision points
IBD vs. IBS | Clinical decision point | |
80 μg/g | 160 μg/g | |
Sensitivity (95 % CI) | 91.1 % (85.0 %, 95.3 %) | 80.0 % (72.3 %, 86.4 %) |
Specificity (95 % CI) | 76.2 % (67.9 %, 83.2 %) | 87.7 % (80.8 %, 92.8 %) |
PPV (95 % CI) | 79.9 % (72.7 %, 85.9 %) | 87.1 % (79.9 %, 92.4 %) |
NPV (95 % CI) | 89.2 % (81.9 %, 94.3 %) | 80.9 % (73.4 %, 87.0 %) |
ROC AUC (95 % CI) | 0.929 (0.898, 0.960) |
Table 4: Clinical performance characteristics of the fCAL turbo in discriminating IBD from IBS at 80 μg/g and 160 μg/g clinical decision points
CI − confidence interval
PPV − positive predictive value
NPV − negative predictive value
ROC AUC − area under receiver operating characteristic curve
Reference range
Stool samples were obtained from 141 apparently healthy normal adults (> 21 years of age) with no symptoms or signs of gastrointestinal disease. The test results were categorized by the assay cut‑offs.
Distribution of results within fCAL turbo diagnostic ranges | ||||
< 80 μg/g | 80-160 μg/g | > 160 μg/g | Total | |
Number of subjects (%) | 106 (75.2 %) | 18 (12.8 %) | 17 (12.1 %) | 141 (100 %) |
Table 5: Distribution of healthy subjects results within fCAL turbo diagnostic ranges
The presented performance characteristics have been established on a cobas c 501 analyzer. For regulatory purposes validation was performed using extracts obtained by manual weighing and extraction. The CALEX® Cap was validated for comparability with the manual extraction method
Method comparison
The method comparison study was performed according to the CLSI guideline EP09-A3. Two hundred forty‑eight (248) clinical samples were measured according to the instructions for use with the fCAL turbo and with BÜHLMANN fCAL® ELISA assay, with 220 samples yielding results within the measuring range for both tests. Measurements were performed over 18 days using 3 reagent lots.
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean | Lower | Upper | Slope | y‑Intercept | Bias at 80 μg/g | Bias at 160 μg/g | r |
-0.5 % (-3.6 %, 2.7 %) | -46.5 % (-51.8 %, -41.1 %) | 45.5 % (40.2 %, 50.9 %) | 1.025 (0.990, 1.058) | -3.1 % | -0.3 % | 0.972 |
Table 6: Method comparison study results, (LoA= Limits of agreement)
The method comparison study was performed according to the CLSI guideline EP09-A3. Two hundred forty‑one (241) clinical samples, extracted using the CALEX® Cap and manual weighing and extraction method, were measured according to the instructions for use with the fCAL turbo, with 202 samples yielding results within the measuring range of the assay. Measurements were performed over 18 days using 3 CALEX® Cap lots.
Bland‑Altman analysis | Passing-Bablok regression | ||||||
Mean | Lower | Upper |
|
| Bias at 80 μg/g | Bias at 160 μg/g | r |
7.7 % (3.5 %, 12.0 %) | -51.9 % (-59.1 %, 44.6 %) | 67.4 % (60.1 %, 74.6 %) | 1.149 1.100, 1.201) | -8.3 μg/g (-17.1 μg/g, -2.0 μg/g) | 4.6 % | 9.7 % | 0.921 |
Table 7: Method comparison study results, (LoA= Limits of agreement)
Reproducibility (multisite precision evaluation study)
Reproducibility was established according to the CLSI guideline EP05-A3 by performing measurements at 3 laboratory sites. Eight pooled stool specimen extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 5 days, in 1 run per day, with 5 results generated per run. One reagent lot was used in the study.
ID | Mean [μg/g] | n | Within-run (repeatability) | Between-day | Between-site | Total | ||||
SD | % CV | SD | % CV | SD | % CV | SD | % CV | |||
S01 | 47.2 | 75 | 3.6 | 7.6 % | 2.4 | 5.0 % | 0.0 | 0.0 % | 4.3 | 9.1 % |
S02 | 91.1 | 75 | 3.5 | 3.8 % | 3.5 | 3.8 % | 2.8 | 3.1 % | 5.7 | 6.2 % |
S03 | 185.4 | 75 | 5.1 | 2.7 % | 2.7 | 1.4 % | 5.5 | 3.0 % | 7.9 | 4.3 % |
S04 | 276.9 | 75 | 6.4 | 2.3 % | 4.5 | 1.6 % | 9.7 | 3.5 % | 12.5 | 4.5 % |
S05 | 674.5 | 75 | 12.9 | 1.9 % | 1.2 | 0.2 % | 22.8 | 3.4 % | 26.3 | 3.9 % |
S06 | 1519.6 | 75 | 25.3 | 1.7 % | 17.8 | 1.2 % | 62.3 | 4.1 % | 69.6 | 4.6 % |
S07 | 3343.8 | 75 | 54.6 | 1.6 % | 35.6 | 1.1 % | 100.0 | 3.0 % | 119.4 | 3.6 % |
S08 | 5475.6 | 75 | 72.1 | 1.3 % | 35.8 | 0.7 % | 154.2 | 2.8 % | 173.9 | 3.2 % |
Table 8: Reproducibility study results – within‑run, between‑day, between‑site and total precision estimates
Between‑lot precision
Between‑lot precision was established according to the CLSI guideline EP05-A3. Eight pooled stool extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 5 days, in 1 run per day, with 5 results generated per run. Three reagent lots were used in the study.
ID | Mean [μg/g] | n | Within-run (repeatability) | Between-day | Between-lot | Total | ||||
SD | % CV | SD | % CV | SD | % CV | SD | % CV | |||
S01 | 45.2 | 75 | 7.1 % | 1.4 | 3.0 % | 3.7 | 8.2 % | 5.1 | 11.3 % | |
S02 | 86.4 | 75 | 3.7 | 4.3 % | 1.2 | 1.4 % | 5.7 | 6.6 % | 6.9 | 7.9 % |
S03 | 175.8 | 75 | 5.0 | 2.9 % | 0.3 | 0.2 % | 9.9 | 5.6 % | 11.1 | 6.3 % |
S04 | 263.9 | 75 | 7.6 | 2.9 % | 0.0 | 0.0 % | 10.0 | 3.8 % | 12.5 | 4.7 % |
S05 | 647.4 | 75 | 15.5 | 2.4 % | 0.0 | 0.0 % | 15.3 | 2.4 % | 21.7 | 3.4 % |
S06 | 1460.7 | 75 | 33.7 | 2.3 % | 11.6 | 0.8 % | 41.0 | 2.8 % | 54.3 | 3.7 % |
S07 | 3234.5 | 75 | 71.2 | 2.2 % | 8.9 | 0.3 % | 130.3 | 4.0 % | 148.8 | 4.6 % |
S08 | 5303.1 | 75 | 97.4 | 1.8 % | 11.2 | 0.2 % | 163.9 | 3.1 % | 3.6 % |
Table 9: Between‑lot precision study results
Within-laboratory precision
Repeatability and within‑laboratory precision were established according to the CLSI guideline EP05-A3. Eight pooled stool specimen extracts with calprotectin concentrations covering the measuring range of the test and clinical decision points were tested over 20 days, in 2 runs per day, with 2 results generated per run. One reagent lot was used in the study.
ID | Mean [μg/g] | n | Repeatability | Between-run | Between-day | Within-laboratory | ||||
SD | % CV | SD | % CV | SD | % CV | SD | % CV | |||
S01 | 42.9 | 80 | 3.6 | 8.3 % | 1.2 | 2.7 % | 1.1 | 2.5 % | 3.9 | 9.1 % |
S02 | 98.4 | 80 | 2.5 | 2.6 % | 1.8 | 1.8 % | 2.2 | 2.2 % | 3.7 | 3.8 % |
S03 | 166.5 | 80 | 4.3 | 2.6 % | 0.8 | 0.5 % | 1.9 | 1.2 % | 4.8 | 2.9 % |
S04 | 267.6 | 80 | 3.9 | 1.4 % | 2.5 | 0.9 % | 1.8 | 0.7 % | 5.0 | 1.9 % |
S05 | 642.0 | 80 | 20.1 | 3.1 % | 14.9 | 2.3 % | 0.0 | 0.0 % | 25.1 | 3.9 % |
S06 | 1414.2 | 80 | 19.6 | 1.4 % | 11.1 | 0.8 % | 3.5 | 0.2 % | 22.8 | 1.6 % |
S07 | 3251.4 | 80 | 40.8 | 1.3 % | 21.4 | 0.7 % | 19.7 | 0.6 % | 50.1 | 1.5 % |
S08 | 5405.6 | 80 | 40.2 | 0.7 % | 56.6 | 1.0 % | 34.5 | 0.6 % | 77.5 | 1.4 % |
Table 10: Within‑laboratory precision study results
The extraction reproducibility was established according to the CLSI guideline EP05‑A3. Twelve clinical stool specimens, selected to reflect different stool consistencies: solid, semi-solid and liquid, with calprotectin concentrations covering the measuring range of the test and clinical decision points, were extracted in duplicate by 2 operators on 2 days using 3 CALEX® Cap lots. Each stool extract was tested in 3 replicates using 1 reagent lot of the fCAL turbo.
ID | Mean [µg/g] | n | Within-run | Between-extraction | Between-day | |||
|---|---|---|---|---|---|---|---|---|
SD | % CV | SD | % CV | SD | % CV | |||
S1 | 42.7 | 72 | 3.2 | 7.5 | 4.6 | 10.8 | 0.0 | 0.0 |
S2 | 71.5 | 72 | 3.9 | 5.4 | 6.9 | 9.6 | 9.4 | 13.1 |
S3 | 111.3 | 72 | 3.3 | 2.9 | 14.2 | 12.7 | 0.0 | 0.0 |
S4 | 119.8 | 72 | 2.9 | 2.4 | 7.2 | 6.0 | 5.8 | 4.8 |
S5 | 213.0 | 72 | 3.2 | 1.5 | 27.9 | 13.1 | 0.0 | 0.0 |
S6 | 297.2 | 72 | 3.7 | 1.2 | 24.5 | 8.2 | 13.5 | 4.6 |
S7 | 561.2 | 72 | 5.5 | 1.0 | 18.6 | 3.3 | 66.1 | 11.8 |
S8 | 610.0 | 72 | 4.7 | 0.8 | 74.3 | 12.2 | 28.2 | 4.6 |
S9 | 940.4 | 72 | 12.2 | 1.3 | 152.7 | 16.2 | 34.8 | 3.7 |
S10 | 1558.4 | 72 | 7.8 | 0.5 | 152.0 | 9.8 | 39.9 | 2.6 |
S11 | 2041.6 | 72 | 27.2 | 1.3 | 150.3 | 7.4 | 133.8 | 6.6 |
S12 | 3440.0 | 72 | 48.7 | 1.4 | 177.7 | 5.2 | 321.5 | 9.3 |
ID | Mean [µg/g] | n | Between-lot | Between-operator | Total precision | |||
|---|---|---|---|---|---|---|---|---|
SD | % CV | SD | % CV | SD | % CV | |||
S1 | 42.7 | 72 | 2.7 | 6.3 | 0.0 | 0.0 | 6.2 | 14.5 |
S2 | 71.5 | 72 | 0.0 | 0.0 | 0.0 | 0.0 | 12.2 | 17.1 |
S3 | 111.3 | 72 | 6.8 | 6.1 | 7.8 | 7.0 | 17.9 | 16.1 |
S4 | 119.8 | 72 | 0.0 | 0.0 | 0.0 | 0.0 | 9.7 | 8.1 |
S5 | 213.0 | 72 | 0.0 | 0.0 | 9.0 | 4.2 | 29.5 | 13.8 |
S6 | 297.2 | 72 | 18.0 | 6.1 | 12.3 | 4.1 | 35.6 | 12.0 |
S7 | 561.2 | 72 | 0.0 | 0.0 | 0.0 | 0.0 | 68.9 | 12.3 |
S8 | 610.0 | 72 | 0.0 | 0.0 | 0.0 | 0.0 | 79.6 | 13.1 |
S9 | 940.4 | 72 | 0.0 | 0.0 | 97.5 | 10.4 | 184.9 | 19.7 |
S10 | 1558.4 | 72 | 98.6 | 6.3 | 146.2 | 9.4 | 236.4 | 15.2 |
S11 | 2041.6 | 72 | 88.9 | 4.4 | 10.5 | 0.5 | 221.9 | 10.9 |
S12 | 3440.0 | 72 | 0.0 | 0.0 | 0.0 | 0.0 | 370.5 | 10.8 |
Table 11: CALEX® Cap extraction reproducibility study results
Gastroenterologists are often faced with the diagnostic difficulty of differentiating individuals with functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), from those with inflammatory bowel disease (IBD). Many symptoms are common to both conditions, whereas other clinical features such as a predominance of diarrhea and rectal bleeding will increase the likelihood of inflammatory disease. The clinical differentiation between these conditions remains problematic and may result in delayed diagnosis. Furthermore, many individuals with IBS must undergo invasive procedures (endoscopy) to rule out an organic disorder. This has significant implications for health care costs as well as exposing individuals to the inherent risks associated with invasive procedures.
Reagents | Quantity | Preparation |
Reaction Buffer (R1) | Ready to use | |
Immunoparticles (R3) | Ready to use |
R1 is in position B and
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
Product safety labeling follows GHS guidance.
Contact phone: 1-800-428-2336
• The immunoparticles contain potentially infectious substances of animal origin and should be handled in accordance with good laboratory practice (GLP) using appropriate precautions.
• R1 contains MOPS (3-(N-morpholino)propanesulfonic acid) (< 1 %), that can be irritating to eyes and skin. Handle with due caution.
• Immunoparticles R3 contains polystyrene nanoparticles.
• Reagent Immunoparticles R3, once frozen, cannot be used anymore. Freezing R3
• The assay is designed for fecal extract samples prepared using the specific
• Please equilibrate reagents, controls, calibrators and samples as described
• Ensure that samples have no bubbles prior to running the test.
• Evaporation of calibrators and controls on the analyzer could lead to incorrect results. Run the assay immediately after loading the analyzer.
For quality control, use control materials as listed in the \"Order information\" section.
Validating instrument accuracy after calibration
Validating daily runs
The controls have assigned
Measuring patient samples
Once a calibration curve is established and validated with the quality controls, patient fecal extracts may be measured. Allow patient fecal sample extracts to equilibrate to room temperature for at least 30 minutes before starting analysis. Perform patient fecal extract measurement according to this instruction for use and the instrument manual.
The fCAL turbo is designed for calprotectin quantification in fecal sample extracts. Fecal samples are collected, extracted and diluted to a final concentration of 1:500 using the CALEX®
Specimen transport and storage
Stool specimens should be received for processing by the laboratory within 3 days of collection. Stool specimens may be shipped at room temperature or
Follow the instruction for use provided with the CALEX®
Important: Centrifuge the CALEX®
Please note that extracts can be stored and frozen directly within the CALEX® Cap.
fCAL turbo
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