Scandinavian Journal of Clinical & Laboratory Investigation, 2015; Early Online: 1–7

ORIGINAL ARTICLE

Validation of a new extraction device for measuring faecal calprotectin in inflammatory bowel disease, and comparison to established extraction methods

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VENDEL KRISTENSEN1,2,*, TRINE LAURITZEN3,*, LARS-PETTER JELSNESSJØRGENSEN4,5 & BJØRN MOUM2,6 1Unger-Vetlesen’s

Institute, Department of Medicine, Lovisenberg Diakonale Hospital, Oslo, Norway, 2Institute of Clinical Medicine, University of Oslo, Norway, 3Department of Medical Biochemistry, Vestre Viken Hospital Trust, Drammen, Norway, 4Department of Gastroenterology, Østfold Hospital Trust, Fredrikstad, Norway, 5Department of Health and Social Sciences, Østfold University College, Fredrikstad, Norway, and 6Department of Gastroenterology, Oslo University Hospital, Oslo, Norway

Abstract Objective: Faecal (f-) calprotectin is a widely used surrogate marker of intestinal inflammation. F-calprotectin analysis is labour demanding partly due to a time consuming extraction step. The aim of this project was to validate a new extraction method for f-calprotectin. Material and methods: A prospective multicentre study included 135 patients with an established diagnosis of inflammatory bowel disease. The patients submitted a faeces sample, which was extracted with both the conventional method at the participating laboratory and the new extraction device. The extracts were analyzed by an automated ELISA instrument. Results: Method comparison of the traditional method and the new device showed a slope of 1.01 (0.93–1.07) with intercept of ⫺ 2.2 (⫺ 4.9–0.6). The Spearman rank correlation coefficient was 0.96. Conclusions: The new extraction device is a reliable and time saving alternative to the conventional extraction method. Keywords: Biological markers, colitis, ulcerative, Crohn disease, enzyme-linked immunosorbent assay, faeces, inflammatory bowel disease, leukocyte L1 antigen complex

Introduction Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn’s disease (CD), which are chronic diseases characterized by flares of intestinal inflammation [1]. The current gold standard for evaluating intestinal inflammation is colonoscopy with biopsies [2–4]. Frequently performing this invasive procedure is however costly, time-consuming, invasive, inconvenient and unpleasant for the patients [2,5]. Calprotectin is a calcium-binding protein found in cytosol in neutrophils, accounting for approximately 60% of the protein in the cytosol fraction of these cells [6]. In the presence of calcium the protein is stable, and it remains stable in faeces samples for a week at room temperature [7]. Previous studies have demonstrated that faecal (f-) calprotectin is a reliable biomarker of intestinal inflammation [3,8–11]. Moreover,

elevated f-calprotectin has been shown to predict future relapse in IBD [12], although this prediction seems more reliable in patients suffering from UC rather than CD [13]. The latter may be due to poor correlation between f-calprotectin and mucosal inflammation in CD patients with strictly ileal disease [14]. On the other hand, CD is a less uniform disease, sometimes with scattered inflammation and isolated ulcerations, which also might contribute to more variation in f-calprotectin and potentially uneven distribution of f-calprotectin in the faeces. It has also been demonstrated that routine measurements of f-calprotectin in the management of UC can predict clinical relapse [15]. The level of f-calprotectin can also be valuable in predicting nonresponders to medical treatment and need of future colectomy in acute severe UC [16].

*These two authors contributed equally to this study. Correspondence: Vendel Kristensen, Unger-Vetlesen’s Institute, Department of Medicine, Lovisenberg Diakonale Hospital, 0440 Oslo, Norway. E-mail: [email protected] (Received 15 September 2014 ; accepted 15 March 2015) ISSN 0036-5513 print/ISSN 1502-7686 online © 2015 Informa Healthcare DOI: 10.3109/00365513.2015.1030765

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V. Kristensen et al.

Measuring calprotectin in faeces is a non-invasive, but time consuming procedure. Patients collect a stool sample in a closed container and mail or deliver it to the laboratory. Following a process of faeces sample extraction, f-calprotectin is quantified using enzyme linked immunosorbent assays (ELISA). The extraction procedure, originally including weighing and diluting, is both cumbersome and time consuming for the laboratory personnel. Bypassing the step of weighing every single faeces sample, volume-based extraction devices such as the ‘Faeces sample preparation kit’ (Roche device) (Roche Diagnostics GmbH®) are widely used. But still, the extraction procedure is labour intensive and accounts for most of the manual work load necessary for analysis of f-calprotectin. For rationalization purposes alternative extraction devices have been developed and are now under consideration. Calpro EasyExtract© is a new device merging extraction and dilution of the faeces sample into one process. Bypassing multiple steps in the preparation process, this may be a labour saving procedure for the laboratory. But before this can be recommended as a routine preparation method, this extraction device must be proven reliable and ELISA results comparable with established extraction procedures. The aim of this project was to validate the Calpro EasyExtract© extraction device compared to the routinely used Roche device, in a situation where both extraction methods were performed by experienced laboratory personnel. As a secondary aim, we investigated if the correlation between the two extraction methods was identical for UC and CD.

Material and methods Patients and design The project is part of a prospective multicentre observational study at eight different centres in the South-Eastern and Western parts of Norway. Patients aged 18 years or older, with the ability to read and write Norwegian and having a previously verified diagnosis of IBD, (clinically, endoscopically, radiologically and histologically), were included during routine follow-up. Exclusion criteria were cognitive impairment, dementia, or kidney failure (defined as an estimated glomerular filtration rate below 60 mL/min). Both clinical and socio-demographic data were registered, including the Harvey-Bradshaw Index for clinical activity in CD [17] and Simple Clinical Colitis Activity Index (SCCAI) for clinical activity in UC [18]. The patients submitted a stool sample for analysis of f-calprotectin. The personnel responsible for registering the clinical data were blinded to the results of the f-calprotectin analyses.

Extraction and f-calprotectin analysis Stool samples were delivered in person or sent by mail to Gas-Cal-Lab©, Oslo University Hospital, where they were stored at ⫺ 20°C until extraction. Extraction was performed by experienced laboratory personnel with both the Roche device and EasyExtract simultaneously (Calpro AS, Oslo, Norway). ELISA analysis of the extracts was performed at the Department of Medical Biochemistry, Vestre Viken Hospital Trust. The Roche device is a volume-based extraction device where a faeces sample is placed in a sample chamber carrying approximately 100 mg of faeces, and an extraction tube containing a spiral coil is thereafter attached. According to the manufacturer’s instructions, 4.9 mL of extraction buffer was added and the sample vortexed for approximately 3 minutes. The sample was then left to settle for a few minutes, and the supernatant transferred to another tube for analysis. The EasyExtract is also volume based. A rod with small grooves carrying approximately 30 mg of faeces is inserted into a faeces sample. The grooves are filled with faeces, and the rod is inserted through a narrow chamber back into a tube prefilled with 1.5 mL extraction buffer. Excessive faeces is brushed off in this process.These samples were also vortexed 3 minutes before analysis. The EasyExtract tubes were designed to fit into the automated ELISA instrument. For a subgroup of samples we also applied the manual weighing method described by Tøn et al. [19]. The extracts were then refrigerated for a maximum of 2 days until analysis. All samples were analyzed with a commercially available ELISA assay (Calpro AS, Oslo, Norway) in duplicates, by an automated ELISA instrument following the manufacturer’s instructions. The personnel responsible for extraction and analysis were blinded to the patients’ diagnosis and additional clinical information. The short-term imprecision was determined by repeated extractions (10–20) of samples with known high and low calprotectin values and the coefficient of variation (CV) was calculated after analysis. The samples were analyzed on the same day, under similar conditions. For evaluating if the consistency of the faeces would affect the imprecision, a modified version of the Bristol stool scale (BSS) [20] was applied; classifying the faeces as (a) hard/lumpy (BSS type 1 and 2), (b) normal (BSS type 3 and 4), (c) soft/mushy (BSS type 5 and 6), and (d) watery (BSS type 7). Within each category, one sample was chosen, five parallel extractions were made, and the CV was calculated after analysis. The stability of the ELISA assay was assessed by long-term imprecision. Stable control material provided by the manufacturer was analyzed daily during the last year prior to project start and the CV was calculated.

Validation of calprotectin extraction Table I. Characteristics of the study participants. Age mean (95% CI) Gender male, n (%) Crohn’s disease, n (%) Duration of disease years mean (95% CI) (missing) Harvey Bradshaw index mean (95% CI) (missing) (CD only) SCCAI mean (95% CI) (UC only) F-calprotectin (Roche) median (IQR) μg/g F-calprotectin (EasyExtract) median (IQR) μg/g

Table II. Imprecision data of both f-calprotectin extraction methods.

39.8 (37.8–41.8) 65 (48.1) 71 (52.6) 11.4 (9.7–13.0) (5) 5.0 (4.0–6.1) (1) 3.1 (2.5–3.8) 71 (212) 77 (225)

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Roche CV%

EasyExtract CV%

25.5 6

11 6

18 9 16 8

8 14 16 6

F-calprotectin level Low sample High sample Faeces consistency Hard/lumpy Normal Soft/mushy Watery

Abbreviations: CV, coefficient of variation

Abbreviations: CI, confidence interval; CD, Crohn’s disease; UC, ulcerative colitis; SCCAI, simple clinical colitis activity index; IQR, interquartile range

Statistical considerations The statistical analyses were performed using SPSS version 22 (SPSS Inc., Chicago, IL) and Microsoft Office Excel 2007. Method comparisons were performed using the Passing Bablok regression test and the Spearman rank correlation coefficient. A probability value of ⬍ 0.05 was considered statistically significant.

Ethical considerations The study was performed according to the declaration of Helsinki and was approved by the Regional Committee for Medical and Health Research Ethics (REC South East). All participants signed an informed consent prior to enrolment.

Results Patient characteristics A total of 135 patients with IBD were consecutively included during 2013 and 2014. Patient characteristics are presented in Table I.

Method imprecision Table II shows short-term imprecision data for the two extraction devices. The CV varied within all evaluated categories. The long-term imprecision for the low control (80 μg/g) was found to be 3.5% and for the high control (400 μg/g) 7%, both controls provided by the manufacturer. Method comparison – Roche device vs. EasyExtract Passing Bablok regression and Spearman rank correlation coefficient between values obtained by the two extraction procedures were calculated for all samples together, and for the following subgroups: a low subgroup consisting of the samples where extraction with the Roche device concluded with f-calprotectin ⬍ 200 μg/g, a high subgroup consisting of the samples where extraction with the Roche device concluded with f-calprotectin ⱖ 200 μg/g, and four subgroups depending on the consistency of the faeces samples as described earlier. We also did method comparison in the subgroups CD patients and UC patients. The results are presented in Table III, and demonstrate significant correlation in all subgroups with an overall slope close to 1 and insignificant intercept.

Table III. Passing Bablok regression analysis and Spearman rank correlation coefficient for all samples, for samples below and above 200 μg/g, for Crohn’s disease patients and ulcerative colitis patients separately, and for samples subgrouped according to faeces consistency. n All patients ⬍ 200 μg/ga ⱖ 200 μg/ga UC CD Hard/lumpy Normal Soft/mushy Watery

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135 96 39 64 71 18 45 64 8

Slope (95% CI) 1.01 0.97 0.90 1.05 0.97 0.91 1.18 0.97 0.80

(0.93–1.07) (0.87–1.07) (0.70–1.12) (0.96–1.21) (0.84–0.99) (0.77–1.27) (1.03–1.46) (0.90–1.04) (0.70–1.32)

Intercept (95% CI) ⫺ 2.2 ⫺ 1.2 72.9 ⫺ 3.4 ⫺ 1.7 ⫺ 3.7 ⫺ 6.4 ⫺ 1.6 ⫺ 3.2

(⫺ 4.9–0.6) (⫺ 4.5–1.2) (⫺ 62.8–160.0) (⫺ 8.3–(–0.2) (⫺ 5.1–4.4) (⫺ 25.8–13.6) (⫺ 19.1–0.9) (⫺ 4.6–0.8) (⫺ 32.1–1.9)

Spearman correlation

p-value

0.96 0.90 0.78 0.96 0.94 0.96 0.95 0.95 0.98

⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001

af-calprotectin values according to the Roche device Abbreviations: CI, confidence interval; UC, ulcerative colitis; CD, Crohn’s disease

V. Kristensen et al.

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Figure 1. (a) Comparison plot of the Roche device and the EasyExtract. The continuous line is the best fitted linear line, and the dotted line is y ⫽ x. (b) Difference plot of the two extraction methods, demonstrating that the mean difference is close to zero.

Figure 1a demonstrates a comparison plot of the Roche device and the EasyExtract. The difference plot in Figure 1b demonstrates that the difference between the two methods increases with increasing f-calprotectin, but is evenly distributed and the mean difference is close to zero. Using the 100 μg/g cut-off value as indication of probable mucosal healing, a concordance analysis was performed, shown in Table IV. As demonstrated, we found the concordance rate between the Roche device and the EasyExtract to be 94%. The disagreements displayed no specific pattern and were evenly distributed.

The extraction process with the Roche device was more time consuming than with the EasyExtract. Due to a more cumbersome measurement process and also two additional steps of pipetting, we estimated that each sample extraction made with the Roche device required one extra minute compared to the EasyExtract. Method comparison – weighing vs. Roche device A subgroup of nine samples was extracted through a manual weighing process, and a comparison plot of

Validation of calprotectin extraction Table IV. Concordance analysis between the two extraction methods using f-calprotectin ⬍ 100 μg/g as a cut-off for probable mucosal healing. (a) all patients, (b) patients with UC, and (c) patients with CD are displayed. The shaded areas indicate the number of samples that fell into the same groups of decision limits.

Easy Extract

(a) Roche device All N ⫽ 135 ⬍ 100 μg/g ⱖ 100 μg/g

⬍ 100 μg/g 72 3

ⱖ 100 μg/g 6 54

Concordance ⫽ 94%

Easy Extract

UC n ⫽ 64

⬍ 100 μg/g

ⱖ 100 μg/g

⬍ 100 μg/g ⱖ 100 μg/g

37 1

1 25

Concordance ⫽ 97%

(c) Roche device Easy Extract

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(b) Roche device

CD n ⫽ 71 ⬍ 100 μg/g

⬍ 100 μg/g 35

ⱖ 100 μg/g 5

ⱖ 100 μg/g

2

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Concordance ⫽ 90%

the weighed samples and samples extracted with the Roche device is presented in Figure 2. Spearman rank correlation coefficient in this subgroup was 0.96.

Discussion In the present study we found that the two extraction devices, EasyExtract and the Roche device, had a very good overall correlation. When subdividing into

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groups according to f-calprotectin levels, the interval essential for considering treatment alterations, i.e. the lower subgroup, showed best correlation. A slight decrease in correlation with increasing f-calprotectin levels has no important clinical implications, as high levels confirm intestinal inflammation regardless of exact value [21]. Subdividing the samples according to faeces consistency did not alter the correlation, demonstrating that the grooves on the rod in the EasyExtract supply satisfactory surface tension to contain and maintain liquid faeces samples also. This is in contrast to previous studies demonstrating that extraction devices with dosing tips are unable to capture fluid faeces samples and therefore underestimate f-calprotectin compared to the Roche device [22,23]. Faeces is a heterogeneous material, and f-calprotectin is routinely measured from faeces spot samples without prior homogenization. The slight decrease in correlation with increasing f-calprotectin levels may be due to larger variation within the sample when mucosal inflammation is extensive. Our imprecision data could not support this hypothesis though, as the CV from multiple extractions of a high sample was found to be lower than from a low sample for both extraction methods. We have chosen a level of 100 μg/g f-calprotectin as a cut-off for IBD in remission. This choice was based on previous studies evaluating the coherence between f-calprotectin and mucosal healing. As it has previously been demonstrated that different f-calprotectin assays can conclude with different f-calprotectin cut-off levels for mucosal healing in IBD [21], we only considered studies using

Figure 2. Comparison plot of the weighing method and the Roche device. The continuous line is the best fitted linear line, and the dotted line is y ⫽ x.

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f-calprotectin ELISA assays from Calpro AS. Af Bjørkesten et al. [24] found 94 μg/g f-calprotectin the optimal cut-off level in CD patients, and Labaere et al. [21] found 89 μg/g in CD and 86 μg/g in UC. Using f-calprotectin ⱖ 100 μg/g as an indicator of inflamed mucosa therefore stands out as a practical clinical decision limit. The chosen cut-off level demonstrated an overall concordance rate between the Roche device and the EasyExtract at 94%, which we consider very good. In total, nine patients had f-calprotectin values where the two extraction methods placed the result on each side of the 100 μg/g cut-off. For all these patients, these values were close to 100 μg/g. This illustrates that when values are close to cut-off, re-evaluation with a second faeces sample can be essential for correct management of the disease. When evaluating UC and CD separately, both subgroups showed equally good method correlation and concordance. Hence, we were unable to demonstrate a greater variation among the CD patients, as we might have expected. In the presented project we have used the Roche device as the reference method, in contrast to the more conventional weighing used in the original method description [19]. A recent study has demonstrated that using this device for extraction may lead to an under-recovery of calprotectin of 28.1% compared to the manual weighing [23]. In our subgroup of samples where manual weighing was applied, the correlation between this and the Roche device was excellent, supporting further use of this device as a reference method. Involving fewer steps in the extraction process, EasyExtract was faster and easier to use and required significant fewer human resources than the Roche device. We estimated approximately 1 minute saved for each sample extracted. In a laboratory analyzing a considerable amount of samples, hours of labour may be saved weekly. In conclusion, comparing the extraction with EasyExtract with established routine method gives reliable results when managed by laboratory personnel. Stool sampling by EasyExtract is also intended for use by the patient at home. The patient can submit an already extracted faeces sample, and the laboratory is then able to save the time-consuming extraction process. There are however several possible sources of error and the use of EasyExtract for homebased extraction must be validated compared to laboratory-based extraction.

Acknowledgements The authors would like to thank the following persons for the collection of data: Tore Bjørn Grimstad, Stavanger University Hospital; Randi Opheim, Oslo University Hospital; Magne Henriksen, Østfold

Hospital Trust; Roald Torp, Innlandet Hospital Hamar; Øistein Hovde, Innlandet Hospital Gjøvik; Tomm Bernklev, Telemark Hospital Trust; Jørgen Jahnsen, Akershus University Hospital; Ingrid Prytz Berset, Ålesund Hospital; and Svein Oskar Frigstad, Vestre Viken Hospital Bærum. We would also like to thank Calpro AS for providing the ELISA kits and extraction devices used in this study. VK participated in the design of the study, carried out the studies and data analyses and drafted the manuscript. TL carried out the samples analyses, performed the statistical analyses and participated in drafting the manuscript. LPJJ participated in the design of the study, and supervised inclusion of study participants and data collection. BM designed the study, participated in coordination and participated in drafting the manuscript. All authors revised the manuscript for intellectual content, and read and approved the final manuscript. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Validation of a new extraction device for measuring faecal calprotectin in inflammatory bowel disease, and comparison to established extraction methods.

Faecal (f-) calprotectin is a widely used surrogate marker of intestinal inflammation. F-calprotectin analysis is labour demanding partly due to a tim...
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