Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic sub-score and mucosal TNF expression in ulcerative colitis.

BACKGROUND: Fecal calprotectin is widely used to monitor disease activity in patients with inflammatory bowel disease. Multiple commercial kits exist, however, since the analyses are not standardized, these kits cannot be used interchangeably. We aimed to perform a technical evaluation of two kits (Calpro from Calprolab, Norway and Calprest from Eurospital, Italy) and perform a tuning for detection of clinically relevant disease states in ulcerative colitis.
MATERIALS AND METHODS: For tuning against different clinical states a total of 116 patients with ulcerative colitis were recruited (67 of which were part of an earlier publication). For the technical evaluation an additional series of 80 random samples from the hospital lab were included. Technical evaluation was done by correlation and limits of agreement analysis; cut-off levels were explored by ROC analysis against clinically relevant actual states.
RESULTS: The technical evaluation revealed good correlation between assays, however a non-linear difference was found: At values below 200 mg/kg, no significant bias was found; in the interval 200-1000 mg/kg the Calprest assay measured on average 30% lower than Calpro; and at higher values Calprest measured 60% higher values than Calpro. Both assays predicted Mayo endoscopic score (MES) 0 (cutoff 28: sensitivity 0.38; specificity 0.82 for Calprest; cutoff 28: sensitivity 0.50; specificity 0.77 for Calpro), and MES 2-3 (cutoff 148: sensitivity 0.72; specificity 0.80 for Calprest; cutoff 208: sensitivity 0.64; specificity 0.80 for Calpro), but did not predict normalization of mucosal TNF transcript per se. A combination of calprotectin and MES predicted mucosal TNF transcript values reasonably well (Calpro: sensitivity 0.85, specificity 0.58; Calprest: sensitivity 0.85, specificity 0.61).
CONCLUSION: The Calpro and Calprest assays correlated well, but subtle differences were found, underlining the need for kit-specific cut-off values. Both kits were most precise in predicting active inflammation (MES 2-3), but less so for prediction of mucosal healing (MES 0) and normalization of mucosal TNF gene expression.

Introduction

Assessment of disease activity in ulcerative colitis (UC) pose several problems as there is often a discordance between patient reported symptom burden and actual state of the colon mucosa [1]. Fecal calprotectin has become an important non-invasive biomarker for surveillance of inflammatory activity in inflammatory bowel disease. To some extent, the daily clinical decisions can be made based on this analysis [2]. The overall target for the treatment of UC should be resolution of inflammation as mounting evidence indicate improved prognosis for patients who achieve deep remission (i.e. healing of mucosa) [3,4]. The state of deep remission decreases risk of long term complications and necessity for surgery [5]. In clinical practice the finding of Mayo endoscopic subscore (MES) 0–1 is considered “endoscopic remission” [3]. Recent data, however, indicates that MES 1 is not completely normal with regards to inflammatory histological state [6]. This increases the risk of relapse [7] and later complications. To achieve complete inflammatory remission, the aim should be MES 0. Another method to evaluate the activation state of the mucosal immune system is to quantify the Tumor Necrosis Factor (TNF) gene activity by tissue qPCR [8] from colon biopsies.

Several publications have aimed to assess fecal calprotectin measurements towards prediction of endoscopic findings [9-11]. There are however discrepancies between recommended cut-off values, which probably depend on technical differences between kits, as the method is not standardized. The availability of different commercial kits raises concern that cut-off values are not universal, but kit-specific. The focus of the present paper is to compare the performance of two commercial calprotektin ELISA kits: Calpro (Calprolab, Norway) and Calprest (Eurospital, Italy), and perform a tuning for detection of clinically relevant states of UC.

Materials and methods

Approval by the North Norwegian Regional Committee of Medical Ethics was granted (2012/1349). All study participants received information of the study and signed a consent form. Sixty-six participants with UC were recruited for an earlier publication [12] as part of a prospective project at the Department of Gastroenterology, University hospital of North Norway; additionally, 50 subjects with UC and 16 normal controls were recruited for the present study (please see Fig 1 and Table 1 for details). The participants were asked to deliver two separate fecal samples taken on different days during the week before endoscopy. At endoscopy participants were scored according to MES and samples for tissue qPCR were collected.

Fig 1

Overview of available samples in the study.

UC: patients with ulcerative colitis; NC: normal controls. Abbreviations: UC: Ulcerative Colitis; NC: Normal Controls; ROC: Receiver operating characteristic; SJG; Scandinavian Journal of Gastroenterology.

Table 1

Available samples and key metadata in the different analyses.

Series		Ulcerative colitis				Normal control	TotalN
Day to day variation*	N	8	18	10	4	11	51*
	MES	0	1	2	3	0
Assay comparison	N	43	42	43	42	16	186**
		Quartile 1	Quartile 2	Quartile 3	Quartile 4	Mean (Min–max)
	Calpro	0–25	26–137	138–595	596–1485	64 (24–195)
	Calprest	0–35	36–120	121–386	387–3000	52 (24–145)
ROC	n	104**	104**	96	96		104
	Analysis	MES = 0 by Calprotectin	MES ≥2 by Calprotectin	TNF by Calprotectin	TNF by Calprotectin+MES
	Pos/neg	42/62	25/79	27/69	27/69

*43 and

**66 of these samples were used in an earlier publication [12]. Calprotectin values are mg/kg. MES: Mayo endoscopic sub-score. ROC: receiver operating characteristics. TNF: Tumor necrosis factor gene expression.

Due to missing values the actual number of observations in each analysis vary (Table 1). For day-to-day variation, dual samples were available from 51 individuals, obtained within 1 week (43 of these were part of the previous publication [12]). These dual samples were measured on both kits, and the variation between results analysed.

For head to head comparison of the two ELISA assays, the UC and normal control samples mentioned above were used. In addition, 80 samples from the routine calprotectin workflow at the hospital lab were picked at random, extracted, and analysed on both assays. These samples were only used for technical evaluation; thus no clinical information was obtained. For the most part those samples would be from IBD patients (both UC and Crohn’s disease), or samples used for diagnostic work-up in cases of chronic diarrhoea.

For 104 of the UC samples, MES were also available, which were used for ROC curves (67 of these observations were part of a previously published paper [12]). For 96 patients, a combination of TNF and calprotectin and MES values were available.

Both kits contain extraction devices for accurate sampling of the desired amount of fecal matter. The samples were mixed and extracted according to the manufacturer's instructions for Calpro (Calprolab, Norway) and Calprest (Eurospital, Italy). The extracts were analysed on an automated system (Dynex DS2 ELISA processor, Dynex, Germany).

The method for TNF measurements have been published earlier [13]–in brief: biopsies were immediately immersed in RNA-later (Qiagen, Hilden, Germany) for storage. The biopsies were then disintegrated using a Magnalyzer (Roche, Germany) ceramic bead shaker system and RNA was isolated using the Allprep DNA/RNA Mini Kit (Qiagen) on a Qiacube instrument (Qiagen). Quantity and purity of the extracted RNA were determined using the Qubit 3 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was then produced with QuantiTect Reverse Transcription Kit (Qiagen), and qPCR was run using TaqMan assays for TNF with ACTB as housekeeping gene (previously published [13]; primers/probes ordered from Eurogentec, Liège, Belgium; Mastermix QuantiNova Probe RT-PCR Kit from Qiagen). Absolute quantification was done by a standard curve, based on a diluted PCR product. The cut-off value for normalisation is defined by the 95% upper CI limit for healthy controls.

Statistics

Samples with readings above the standard curves were set at limit (2000 mg/kg for Calpro and 3000 mg/kg for Calprest). Day-to-day variation and head-to-head comparison was performed by Pearson correlation and Bland-Altman limits of agreement analysis on raw data and ln-transformed values. For single predictor, Receiver operating characteristic analysis (ROC), the raw data was used. A combined predictor of [MES + calprotectin] was constructed. To give the two base variables equal weight, both were standardized: [MES+calprotectin] = [MES/max MES] + [ln(calprotectin)/ln(max calprotectin*)]; for each assay (*defined by the standard curve for each assay). These constructed variables span values from 0 to 2. Optimal cut-off was defined by maximal Youden J. Statistical analysis was performed in IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY, USA. Plots were produced in GraphPad Prism version 7.05 for Windows (GraphPad Software, La Jolla, CA, USA).

Results

Inter-assay variation

51 dual samples were extracted and analysed on both assays. Samples were collected within 1 week, but not on the same day. Both assays showed a fair correlation between the two samples with Pearson r = 0.744; P < 0.0001 for Calprest, and Pearson r = 0.729 P < 0.0001 for Calpro (logarithmic transformation applied, please see Fig 2A and 2B). Bland-Altman plots showed that the variance of both assays were evenly distributed with no clinically relevant bias and a fairly equal limit of agreement (Fig 2C and 2D). These variations cover the total variance from sampling on different days, sample handling, extraction, and the ELISA method itself.

Fig 2

Day-to-day variation.

Calprotectin was measured in 51 sets of 2 fecal samples collected within 1 week from each participant. Samples were extracted and run on both calprotectin assays. Values were then ln transformed. Panels A and B show scatterplots of the two samples for both assays. Dashed line is line of identity. Panels C and D show Bland-Altman plots of the same samples.

Method comparison

For direct comparison of the two assays, 186 samples from different individuals were extracted and the same extract was run using both ELISA assays. A direct correlation plot shows a systematic skewing towards higher measurements in the high end using the Calprest kit. This reflects that the standard curve for Calprest extends to 3000, while Calpro stops at 2000. Thus a higher dynamic for the most elevated calprotectin values is expected using the Calprest assay (Fig 3A). Using log-transformed values, a good correlation can be seen (Fig 3B).

Fig 3

Method comparison.

One-hundred-eightysix samples measured with both assays. Panel A shows the raw data. Below 1000 there is a fairly linear correlation, but in the higher range the Calprest assay measures higher values than Calpro. After ln transformation of the raw data, a significant linear trend can be seen in panel B. Dashed line is line of identity.

A Bland-Altman plot shows a tendency of lower mid-range values for the Calprest compared to the Calpro values (Fig 4A and 4B). This plot also shows that the range can be divided into sections: values of 0–200; 201–1000; and above 1000 (Fig 4C). Mean bias between the methods can be calculated as follows: segment 0–200: Calprest measures 3.9% lower than Calpro (transformed mean bias -0.04); 201–1000: Calprest measures 30% lower than Calpro (transformed mean bias -0.36); above 1000: too few datapoints, and the difference in maximal scale value makes Bland Altman analysis difficult to interpret in this segment. Calprest measures values 60% higher than Calpro (transformed mean bias of 0.47).

Fig 4

Bland-Altman.

Analysis of 186 samples measured with both assays. The raw data in panel A shows shifting bias along the x-axis. This tendency is confirmed in the Bland-Altman plot of ln transformed raw data in panel B. Three sections with distinct bias pattern can be defined: calprotectin values <200; values in the interval 200–1000; and values >1000. Bland-Altman plots with mean bias and limits of agreement of each of these segments are presented in panel C.

ROC analysis

For ROC analysis 104 observations were available for the MES, and 96 for the TNF gene expression measurement. A ROC curve for prediction of deep remission defined by MES of 0 is shown in Fig 5A; Both assays had a significant AUC: 0.73 (0.63–0.82; P<0.0005) for Calpro, and 0.73 (0.64–0.83; P<0.0005) for Calprest, respectively. The Calprest kit may be marginally better at predicting MES 0 (Youden J 0.45; cut-off 100; sensitivity 0.86; specificity 0.60) vs Calpro (Youden J 0.41; cut-off 98; sensitivity 0.83; specificity 0.58). For both methods, however, a relatively low specificity is noted, and using these cut-off values may yield a considerable part of endoscopies with MES 1. However, for higher specificity, a cut-off of 28 is recommended for both kits (Calprest: specificity 0.82, sensitivity 0.38; Calpro: specificity 0.77, sensitivity 0.50).

Fig 5

ROC curves.

Prediction of endoscopic inflammatory activity. Panel A: Mucosal healing defined by Mayo endoscopic subscore (MES) = 0 by calprotectin (actual state: positives 42; negatives 62). Panel B: Significant inflammatory activity defined by MES 2–3 by calprotectin (actual state: positives 25; negatives 79). Panel C: Normalized mucosal TNF gene expression (<7500 copies/ug total RNA) by calprotectin (actual state: positives 27; negatives 69). Panel D: Normalized TNF gene expression (<7500 copies/ug total RNA) by the sum of calprotectin and MES (both standardized by ln transformation and division with the maximal scale value). For all plots: blue curve for Calprest; red curve for Calpro.

A ROC curve for prediction of significant inflammation, defined by MES of 2–3, is shown in Fig 5B. Both assays again have significant AUC’s: 0.79 (0.70–0.89; P<0.0005) for Calpro, and 0.80 (0.70–0.90; P<0.0005) for Calprest. Optimal cut-off by maximal Youden J: 138 (J = 0.48; sensitivity = 0.72; specificity = 0.76) for Calpro, and 73 (J = 0.53; sensitivity = 0.88; specificity = 0.65) for Calprest. Cut-off for specificity 0.80: 208 (J = 0.44; sensitivity = 0.64; specificity = 0.80) for Calpro and 148 (J = 0.52; sensitivity = 0.72; specificity = 0.80) for Calprest.

We also tested if mucosal TNF gene expression could be predicted by fecal calprotectin. Using earlier defined cut-off for normal TNF gene expression of <7500 copies/ug total RNA, the samples were categorized as normal or high. We then entered this as actual state variable in a ROC curve for both assays (Fig 5C). The curves did not yield significant AUC’s: 0.58 (0.46–0.69; P = 0.24) for Calpro, and 0.57 (0.46–0.68; P = 0.29) for Calprest.

Finally, we tested a combination of MES and calprotectin (Fig 5D) For Calpro combined with MES, AUC was 0.72 (0.62–0.82; P = 0.001); for Calprest combined with MES the AUC was 0.71 (0.61–0.81; P = 0.001). Optimal cut-off by max Youden J was 0.81 for [MES+Calpro] (J = 0.43; sensitivity = 0.85; specificity = 0.58); and 0.79 for [MES+Calprest] (J = 0.46; sensitivity = 0.85; specificity = 0.61). For specificity of 0.80, a cut-off of 0.51 for [MES+Calpro] (J = 0.13; sensitivity 0.33; specificity = 0.80); and a cut-off of 0.51 for [MES+Calprest] (J = 0.24; sensitivity 0.44; specificity 0.80) were found.

Discussion

We have performed an inter-assay variation analysis and head-to-head comparison of two commercial calprotectin kits, and explored cut-off values for MES 0, MES 2–3, and mucosal TNF transcript–features that are important to assess in follow-up of UC patients. The kits both performed well but the optimal cut-off values varied somewhat, and must be determined for each manufacturer. Additionally, subtle differences were detected both in linearity and level, which probably rely on differences in standards and/or antibodies provided in the kits. It is possible to predict MES 0 to a certain extent, but calprotectin cannot yield both high sensitivity and specificity with the same cut-off. Eventually, calprotectin can be used to select patients for endoscopic evaluation to confirm MES 0. The calprotectin tests alone could not predict normalization of mucosal TNF expression, but by adding MES, the combined variables performed better in predicting mucosal TNF normalization, however still at quite low performances when using cut-off for high specificity (Youden 0.13 and 0.24 for Calpro and Calprest, respectively). Direct measurement of TNF expression still remains necessary for evaluation of immunological remission.

Day to day variation in calprotectin measurements is illustrated by the spread in Fig 2A and 2B. This is in agreement with earlier reports investigating repeated sampling and other factors, like time of day and different extraction devices [14,15]. The much lower error reported in similar studies is not comparable since the same samples were measured twice, and thus only reflect technical intra-assay error [16,17]. Since fecal samples mostly are collected by the patient, factors regarding collection are difficult to standardize. Serial samples (at least 2) will increase reliability. In our practice, two consecutive normal calprotectin measurements prompt endoscopy to ensure full remission. This approach saves endoscopy resources while still enabling close follow-up.

Comparison between values obtained from same samples on the two kits revealed a non-linear relationship. At values below 200 mg/kg, no significant systematic bias was detected, but mid-range values 200–1000 mg/kg are on average 30% lower in the Calprest assay compared to the Calpro assay. The lack of standardization prevent evaluation of which is more “right” [2]. The higher values measured by Calprest in samples above 1000 mg/kg is probably a consequence of a higher dynamic range for Caprest because of assay and standard curve differences. Compared to similar studies, the differences between Calpro and Calprest are relatively minute [17-19]. In comparison the Calpro assay performed quite similar but slightly superior to the HK325 (Hycultbiotech, Netherlands), and the EK-CAL ELISA (Bühlmann Laboratories AG, Switzerland) [20]

Our suggested cut-off for MES 0 is comparable to the findings of Kristensen et al [21] but considerably lower than that suggested for other assays [10] which underlines the necessity of kit-specific cut-off level. Interestingly, our earlier publication using mean of two samples yielded a better AUC for MES 0, and slightly different cut-off values for the Calpro kit [12]; this likely reflect that dual sampling yield better precision in a system with high day to day variation.

Detection of significant inflammation (MES 2–3), on the other hand, shows different cut-off levels reflecting the difference in detected values by the assays. Moreover, the difference in standard curves especially affect the mid-high range samples which show higher dynamic in the Calprest assay. This underlines the necessity of kit-specific cut-off values. A similar conclusion must be drawn from similar studies demonstrating highly different cut-off values [17,18,22]

The level of mucosal TNF transcript was poorly predicted by calprotectin values. It has been shown earlier that even in endoscopically normalised mucosa, not all patients have normalised TNF transcript level [8]. Combined with MES, however, the ROC analysis showed improvement, though not at a level that can stand alone as evaluation of immunological remission. Most likely, this represents a deeper level of remission, and, so far, the only way to assess this feature is by measuring TNF directly in biopsies.

Weaknesses of the study are primarily that the endoscopic evaluation was not done by central reader, but graded by different clinicians. Moreover, a relatively low number of samples were used in the inter-assay evaluation (n = 51); however, the plots illustrate the high day to day variation, and the importance of repeated samples still stands. Regarding the ROC curves, the number of observations are reasonable, however not high enough to validate the proposed cut-off values by e.g. split-half analysis. The main point being that cut-off values are kit-specific still stands, though.

Conclusions

In spite of a high day-today variation, calprotectin remains one of the best non-invasive measures of the inflammatory state in ulcerative colitis. However, even between these two highly correlated ELISA kits for calprotectin there are important differences, also in the interval containing cut-off values for clinical decisions. Consequently, due to the lack of standardisation for calprotectin measurement, cut-off values should be regarded as kit-specific. Both kits were most precise in predicting active inflammation (MES 2–3), but less so for prediction of mucosal healing (MES 0) and normalization of mucosal TNF gene expression. The Calprest assay has a higher dynamic range, the use of which is still to be determined. Calprotectin alone could not predict mucosal TNF transcript normalization.

Raw data and metadata is available in SPSS format.

(SAV)

Click here for additional data file.

24 Jul 2019

PONE-D-19-17736

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic subscore and mucosal TNF expression in ulcerative colitis

PLOS ONE

Dear Dr Goll,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Standardization is a frequent problem with assays such as fecal calprotectin. Diagnostic cut-off determined with one assay do not necessary apply for another assay.

It is useful to compare different producer’s tests and I like the attempt to use an independent parameter as TNF transcripts. Unfortunate the authors only compare two of the many available assays for fecal calprotectin, extending to more would have increased the usefulness of the paper.

I wonder if the authors have thought about directly comparing the assays with spiked samples? Using a given amount of WBC and stool from non-human species could have provided exact limit of detection/quantification and could have normalized the assays.

It seem rather unclear how many samples/patients that have been used in the present paper, I would suggest providing a flowchart.

Have the authors got permission to use data from the authors and journal of the previous paper (Scand J Gastroenterol. 260 2018 Jul 3;53(7):825–30).

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Reviewer #1: Regarding the manuscript by Goll et al.

Today F-calprotectin assays are widely used and is an important part of the workup of patients with suspected IBD. Evaluation assays and the correlation with TNF expression are thus important.

I think the data is clearly presented and I have only minor comments.

Abstract line 25: I am not completely enthusiastic about the use of calibration in this context. Calibration is more often used for assay calibrations and here the authors are relating the values to clinical states which is much more complex and also more valuable. I believe that the word calibration here may be misinterpreted by some readers. I thus suggest that the authors try and change calibration to another word.

Page 3, line 45: To some extent, part of… Please remove either as this is a duplication

Page 3 line 56. Add a space before (9-11)

Page 4: The extraction process: How similar were the sample extractions. Same sample? Were the samples mixed before sampling?

Page 5 and 6 method comparison: Most likely the authors had results that were above and below the measuring ranges of the assays. How were such samples handled? Values set at the limits? Analyzed rediluted? Omitted?

Page 7 line 164: Add a space between andhead-to

Table 1: Please add a table legend that also should include the units for the F-calprotectin assays.

Figure 3: please add the units

Please a sentence about availability of data. The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository

Reviewer #2: Material and methods

When the authors refer to the two Calprptectin kits, they have to specify which methodology they use, and they put it in results, at the end of Inter-assay variation.

Nor do they indicate the cut-off from which they consider the TNF activity of the biopsies to be positive.

The total number of samples used for the comparative study is not understood, as they indicate that data from another study have been used, but everything is very unclear. In addition, the controls for which they were used are not well known, as no results are given either in Table 1.

Results

The first fragment of results should actually be included in Material and methods (lines 102 to 112), because there are not results on it.

I do not understand how lowering the cut-off increases specificity and lowers sensitivity (lines 144 and 145).

Discussion

The results have been compared with few previous results, hence the scarce bibliography.

Table 1

The legends of the abbreviations are missing.

Errata

Some errors should be corrected, as in Line 60: Calpro (Calpro Norway), and some words together, without spaces.

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28 Aug 2019

PONE-D-19-17736

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic subscore and mucosal TNF expression in ulcerative colitis

PLOS ONE

Dear Dr Goll,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Standardization is a frequent problem with assays such as fecal calprotectin. Diagnostic cut-off determined with one assay do not necessary apply for another assay.

It is useful to compare different producer’s tests and I like the attempt to use an independent parameter as TNF transcripts. Unfortunate the authors only compare two of the many available assays for fecal calprotectin, extending to more would have increased the usefulness of the paper.

Response: Although we agree on this point, only a limited number of the samples are available as of today, and rerunning on other assays would reduce the number of samples in the analysis substantially.

I wonder if the authors have thought about directly comparing the assays with spiked samples? Using a given amount of WBC and stool from non-human species could have provided exact limit of detection/quantification and could have normalized the assays.

Response: Being of technical interest, such experiments would have limited use for the clinical interpretation of measured values. Besides, this would be the type of validation already performed by the company’s marketing the tests.

It seem rather unclear how many samples/patients that have been used in the present paper, I would suggest providing a flowchart.

Response: Thanks for the suggestion. We have provided a flowchart figure and tried to clean up the description of patients and samples.

Have the authors got permission to use data from the authors and journal of the previous paper (Scand J Gastroenterol. 260 2018 Jul 3;53(7):825–30).

Response: As can be seen in the flow chart figure, all samples in the present study were collected in Tromsø by the group of authors for the present paper.

We would appreciate receiving your revised manuscript by Sep 07 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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Kind regards,

Pal Bela Szecsi, M.D. D.M.Sci.

Academic Editor

PLOS ONE

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Reviewer #2: Partly

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Reviewer #2: Yes

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Reviewer #2: Yes

Response: The raw data was submited in a supplementary file

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Regarding the manuscript by Goll et al.

Today F-calprotectin assays are widely used and is an important part of the workup of patients with suspected IBD. Evaluation assays and the correlation with TNF expression are thus important.

I think the data is clearly presented and I have only minor comments.

Abstract line 25: I am not completely enthusiastic about the use of calibration in this context. Calibration is more often used for assay calibrations and here the authors are relating the values to clinical states which is much more complex and also more valuable. I believe that the word calibration here may be misinterpreted by some readers. I thus suggest that the authors try and change calibration to another word.

Response: In biomarker research, calibration is more widely used as compared to the more technical usage in assay design. However, we have rephrased from “calibration” to “tuning” throughout the manuscript.

Page 3, line 45: To some extent, part of… Please remove either as this is a duplication

Response: Point taken

Page 3 line 56. Add a space before (9-11)

Response: Thank you

Page 4: The extraction process: How similar were the sample extractions. Same sample?

Response: The extraction devices provided with these two kits are very similar, while differences in extraction buffer may exist.

Were the samples mixed before sampling?

Response: Yes, the samples were mixed

We have rephrased the description on p 5 in the revised manuscript

Page 5 and 6 method comparison: Most likely the authors had results that were above and below the measuring ranges of the assays. How were such samples handled? Values set at the limits? Analyzed rediluted? Omitted?

Response: For low/high samples, values were set at the limits; we added this in the revised manuscript in line 102

Page 7 line 164: Add a space between andhead-to

Response: Thank you

Table 1: Please add a table legend that also should include the units for the F-calprotectin assays.

Response: We have added this to the legend.

Figure 3: please add the units

Response: We have added this to the figure

Please a sentence about availability of data. The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository

Response: We added a sentence in the remarks at the end of the manuscript. Line 224

Reviewer #2: Material and methods

When the authors refer to the two Calprptectin kits, they have to specify which methodology they use, and they put it in results, at the end of Inter-assay variation.

Response: Duly noted. We have added methodology to “aim” in line 63 as well as in “methods” line 78

Nor do they indicate the cut-off from which they consider the TNF activity of the biopsies to be positive.

Response: Thanks for this comment. The assay is in-house, rendering the exact cutoff value of less interest for the readers. However, it is defined as upper 95% CI of healthy controls. In our lab the cut-off is 7500 copies/ug total RNA in extracts from colon mucosa – this is already stated in the ROC section of results; we have now clarified the method section in this regard. Line 99

The total number of samples used for the comparative study is not understood, as they indicate that data from another study have been used, but everything is very unclear.

Response: Point taken, we have added a flow diagram to clarify

In addition, the controls for which they were used are not well known, as no results are given either in Table 1.

Response: The 16 normal controls were entered only in the technical evaluations (day-to-day variation and assay comparison). For a technical evaluation, the source of fecal matter is less important.

Results

The first fragment of results should actually be included in Material and methods (lines 102 to 112), because there are not results on it.

Response: This can be disputed, but we have moved the paragraph to the methods section and cleaned up a bit to clarify the sample overview.

I do not understand how lowering the cut-off increases specificity and lowers sensitivity (lines 144 and 145).

Response: This depends on the direction of the predictor variable and the target variable. In the final ROC analysis a lower MES/calpro value yields higher probability of normal TNF. Thus lowering cut-off will increase specificity and lower sensitivity.

Discussion

The results have been compared with few previous results, hence the scarce bibliography.

Response: We have expanded the discussion a bit, citing 5 more earlier works.

Table 1

The legends of the abbreviations are missing.

Response: This has been added

Errata

Some errors should be corrected, as in Line 60: Calpro (Calpro Norway), and some words together, without spaces.

Response: Thank you; these errors have been corrected

________________________________________

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: response to reviewers 280819.docx

Click here for additional data file.

17 Sep 2019

PONE-D-19-17736R1

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic subscore and mucosal TNF expression in ulcerative colitis

PLOS ONE

Dear Dr Goll,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript have greatly improved, but some minor issues still remain.

Please address the point reviewer #2 has raised.

We would appreciate receiving your revised manuscript by Nov 01 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Pal Bela Szecsi, M.D. D.M.Sci.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Abstract

- Line 35: In the sentence "and at higher values Calprest measured higher values than Calpro", the authors should indicate the average increase increase of Calprest over Calpro in % at values >1000 mg/Kg.

- Lines 37 and 38: the authors use the terms "reasonable precision" and "transcript values reasonably well" subjectively, rather than objective identifiers in quantitative terms.

- Line 58: the authors continue to use the term 'calibrating'. Instead, it would be more appropriate to use the terms "adjustment" or "setting", better than tuning.

Material and methods

- Line 66: delete "Patients with UC were recruited first" because it is repeated later; add "as part of a prospective project at the Department of Gastroenterology, University hospital of North Norway" behind (12), on line 70.

- Line 97: add manufacturer and locality of TaqMan assays for TNF.

- Line 11: GraphPad Software, La Jolla, CA, USA, best put in parentheses.

Results

- Lines 145-146: I disagree that when the cut-off goes down, the sensitivity drops and the specificity increases. When lowering the cut-off, more cases of UC are diagnosed (sensitivity increases), but also other non-inflammatory diseases, with a greater number of false positives (specificity decreases). These data should be reviewed.

I do not understand how you have obtained these results; it is not understandable or reasonable. In fact, to increase specificity, the cut-off increase above 100 later (lines 150-151).

- Line 146: correct "maksimal".

- Line 153: separate "totalRNA".

- Lines 160 and 161: it is noteworthy that the Youden indices obtained with the MES + Calprotectin in both trials are very low (0.13 and 0.24), and the authors do not refer to it in the Discussion.

Table I: The result of the 16 controls has not been included in the trial comparison.

Figure 2: The authors should add the line of the line and refer in the text that the correlation between the calprotectin results of two samples from the same patient is weak by both methods (Pearson's r of 0.53 and 0.55).

Figure 5: the authors must add in the ROC curves the kit that corresponds to each color (blue and red), and also in the legend, in brackets TNF <7500 after “Normalized mucosal TNF gene expression”.

Conclusion

Add some mention to the prediction of inflammation or its cure (MES), as you indicated in the summary.

References

- Reference 11 (line 260), change 1936.e1. for 36.e1.

- Reference 20: the year is incorrect; volume and pages are missing and the authors are incompletely cited.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Oct 2019

please see attached file "response to reviewers"

Submitted filename: Response to reviewers round 2.docx

Click here for additional data file.

15 Oct 2019

PONE-D-19-17736R2

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic sub-score and mucosal TNF expression in ulcerative colitis

PLOS ONE

Dear Dr Goll,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Some minor issues still needs your attention, please correct.

We would appreciate receiving your revised manuscript by Nov 29 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Pal Bela Szecsi, M.D. D.M.Sci.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have greatly improved the manuscript quality, there are only minor details to modify before publication. These are the following:

Authors' contributions: delete Summary (line 17).

Keywords: delete "calibration".

Lines 145-145. Now I understand the objective of the authors: different cut-off points according to the MES searched (positive/negative). That is correct, but clinically this is difficult to establish. Laboratory tests usually have a single cut-off point, and results that are above/below are considered positive/negative (or vice versa). Otherwise, a grey area of results is established, difficult to classify.

I accept your explanation.

Results

Add a full stop, new paragrah behind of"...for Calprest." (line 160).

Conclusions

Add to the end of the Conclusions the statement that the authors include in the Abstract, but that does not appear in the manuscript text (already indicated in the previous review): "Both kits were more accurate in the prediction of active inflammation (MES 2-3), but less for the prediction of mucosal healing (MES 0) and normalization of the expression of the mucosal TNF gene ".

Figure 2: I was referring to the straight line of identity, which you have already added by the authors.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Oct 2019

Please see response in the file attached "response to reviewers round 3"

Submitted filename: Response to reviewers round 3.docx

Click here for additional data file.

24 Oct 2019

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic sub-score and mucosal TNF expression in ulcerative colitis

PONE-D-19-17736R3

Dear Dr. Goll,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Pal Bela Szecsi, M.D. D.M.Sci.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

15 Nov 2019

PONE-D-19-17736R3

Head to head comparison of two commercial fecal calprotectin kits as predictor of Mayo endoscopic sub-score and mucosal TNF expression in ulcerative colitis

Dear Dr. Goll:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pal Bela Szecsi

Academic Editor

PLOS ONE