Вы находитесь на странице: 1из 7

Clin Chem Lab Med 2015; 53(1): 65–71

Els N. Dumoulin, Stephanie Van Biervliet, Michel R. Langlois and Joris R. Delanghe*

Proteolysis is a confounding factor in the


interpretation of faecal calprotectin
Abstract Keywords: α1-antitrypsin; calprotectin; faeces; inflamma-
tory bowel disease; proximal gut lesions; trypsin.
Background: Calprotectin is a 36  kDa calcium and zinc
binding protein. An increased level of calprotectin points DOI 10.1515/cclm-2014-0568
towards inflammatory bowel disease. However, the bio- Received May 28, 2014; accepted June 13, 2014; previously published
marker calprotectin shows 14 potential cleavages sites for online July 4, 2014
trypsin. Next to trypsin, also the presence of its inhibitor
α1-antitrypsin after a gastrointestinal bleeding may affect
calprotectin testing. In this study, effects of trypsin and α1- Introduction
antitrypsin as potential confounders for faecal calprotec-
tin testing are investigated. Calprotectin is a 36 kDa calcium and zinc binding protein,
Methods: An in vitro model was created. As calprotectin accounting for about 60% of soluble proteins in neutro-
source, leukocytes were isolated and subsequently lysed philic cytosol. It is secreted extracellularly by neutrophils
(1% Triton X-100) and diluted in faecal matrix. Trypsin upon stimulation or by cell disruption [1–3]. It can be
digestion was carried out by adding trypsin. Incubation measured in faeces and is believed to be resistant to enzy-
occurred for 24  h or 48  h (37 °C). To study the influence matic degradation. Calprotectin is reported to be stable
of α1-antitrypsin on trypsin, the same experiment was up to 1 week at room temperature and for several months
repeated after adding serum containing α1-antitrypsin. when stored at –20 °C [4]. Faecal calprotectin is regarded
Results: In vitro experiments enabled monitoring of the as a marker of inflammation in the gastrointestinal tract.
faecal calprotectin digestion, leading to loss of immuno- It is used to discern patients with an organic bowel disease
reactivity. Trypsin activity was a potential confounder in from the ones with functional complaints. In adults and
the interpretation of calprotectin, in particular for proxi- children above the age of 4, an increased value leads to
mal lesions, where exposure of calprotectin to trypsin is the identification of patients with suspicion of inflamma-
prolonged. Relative calprotectin loss was proportional to tory bowel disease (IBD) and in need of an endoscopy [1,
the amount of trypsin. Decrease of calprotectin was more 5, 6]. Calprotectin is also studied as surrogate biomarker
pronounced after 48 h of incubation in comparison to 24 h of mucosal improvement in patients with Crohn’s disease
of incubation. Analogue experiments also showed stable (CD). Endoscopic responders achieved a significant
calprotectin values after adding α1-antitrypsin. decrease of faecal calprotectin, whereas in the majority of
Conclusions: Transit time, trypsin activity and addition the endoscopic non- and partial responders these markers
of blood as a source of α1-antitrypsin may be regarded as remained abnormal [7]. Endoscopic score and histological
potential confounders in the interpretation of calprotectin findings correlated significantly with faecal calprotectin in
results. Age-related cut-off values depending on the ana- ileocolonic and colonic disease. However, ileal endoscopic
tomical localisation of the lesions could improve the diag- scores and histological findings failed to correlate with
nostic efficiency of calprotectin testing. faecal markers, such as calprotectin and lactoferrin. In the
study of Siponnen et al. 21 out of 87 patients were classified
as ileal disease. As the majority of these patients had a stric-
*Corresponding author: Joris R. Delanghe, Department of Clinical
Chemistry, Microbiology and Immunology, Ghent University turing disease type, larger studies of faecal markers’ behav-
Hospital, De Pintelaan 185, Ghent, Belgium, Phone: +32 9 3322956, iour in ileal and small bowel disease are necessary [8].
Fax: +32 9 3324985, E-mail: joris.delanghe@ugent.be Trypsin (EC 3.4.21.4) is an endopeptidase found in the
Els N. Dumoulin: Department of Clinical Chemistry, Microbiology intestinal lumen where it hydrolyses proteins. Trypsin is pro-
and Immunology, Ghent University Hospital, Ghent, Belgium
duced in large amounts by the human pancreas as the inac-
Stephanie Van Biervliet: Department of Pediatric Gastroenterology
and Nutrition, Ghent University Hospital, Ghent, Belgium
tive proenzyme trypsinogen. The reported enzyme activities
Michel R. Langlois: Department of Laboratory Medicine, AZ Sint-Jan, in duodenal juice vary from 20–50 U/mL between meals to
Bruges, Belgium 80–180 U/mL in the early postprandial and 60–150  U/mL

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
66      Dumoulin et al.: Confounders of calprotectin interpretation

in the late postprandial period [9]. Trypsin cleaves peptide manufacturer’s instruction with the Faecal Sample Preparation Kit
chains mainly at the carboxyl side of the amino acids lysine or (Roche diagnostics, Mannheim, Germany). After adding a standard-
ised amount of faeces to a tube, 5  mL of EliA Calprotectin Extrac-
arginine, except when either is followed by proline. Trypsin
tion Buffer (Thermo Scientific, Uppsala, Sweden) was added and the
activity has been reported to be stable 7 h after faecal sample sample was vortexed. After centrifugation the supernatant was used
collection between 0 and 4 °C [10, 11]. Following release of as a faecal matrix in our experiments. Faecal samples from outpa-
trypsinogen, the enzyme reaches its highest activity in the tients consulting the Department of Gastro-Enterology were used.
duodenum. During transit, activity of proteases decreases, Calprotectin was prepared from white blood cells (WBC) obtained
resulting in a residual activity of only 20%–30% in the ter- from fresh ethylenediaminetetraacetic acid (EDTA) blood samples.
After isolation of WBC with erythrocyte lysis buffer (Qiagen, Hilden,
minal ileum. For trypsin, its enzymatic activity is preserved
Germany) from a sample with a high neutrophil count (45.5×103 per
better than its immunoreactivity. Structural integrity may not μL), a 1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) lysis buffer
be essential for the proteolytic activity [12, 13]. Since calpro- (TRIS 50 mM, Sigma Aldrich; NaCl 150 mM, Merck Chemicals, Darm-
tectin contains 14 residues which may act as potential cleav- stadt, Germany) adjusted to pH 7.5 was used to release the cytosolic
age sites, (partial) digestion of calprotectin by trypsin can be calprotectin. Aliquots of 1 mL were frozen.
An optimal dilution in faecal matrix of 1:100 was chosen by
postulated (Figure 1).
serial dilutions (calprotectin levels of 102–103 mg/kg). Trypsin diges-
α1-Antitrypsin (AAT) is a 52  kDa plasma glycoprotein tion of calprotectin was carried out by adding trypsin (Sigma Aldrich)
that inhibits a variety of serine proteinases, such as trypsin. solution (1 mg/mL, 1 mM HCl). Incubation occurred at 37 °C. Samples
Normal plasma levels range from 1 g/L to 2 g/L. A common were analysed for calprotectin after 24  h or 48  h of incubation. To
method used for establishing a protein-losing enteropathy evaluate the influence of AAT, routine serum samples were selected
is the clearance of AAT from plasma. It requires a blood in the normal range. First, incubation of trypsin with a serum sam-
ple was performed at room temperature for 30 min. Second, 100 μL
sample for AAT analysis and a 24  h stool collection to
serum or saline was added to the mixture of trypsin and calprotec-
determine stool volume and stool AAT level. Normal AAT tin in faecal matrix and incubated under the same conditions as
clearance is   ≤  27 mL/24 h. Blood or protein loss in stool will described above.
thus be accompanied by a variable release of AAT. As AAT
does not undergo proteolysis or degradation in the gas-
trointestinal lumen, it can be regarded as a confounder of Methods and analysis
residual trypsin activity in stool samples [14, 15].
Calprotectin was assayed on the Phadia ImmunoCAP 250 analyser
Clinical efficiency of calprotectin testing is lower for
(Thermo Scientific) according to the manufacturer’s instructions.
proximal lesions [8]. The reasons for these findings are Results were expressed in mg/kg [16]. Trypsin activity was measured
poorly understood. Therefore, we postulate a potential using a colourimetric method with Nα-Benzoyl-L-arginine 4-nitroan-
role for trypsin and AAT as confounders in calprotectin ilide hydrochloride (BAPNA, Sigma Aldrich) as a substrate for trypsin
test interpretation. In the present study, we have studied (Sigma Aldrich). The amount of 4-nitroaniline released per minute
was measured by increase in absorbance 405 nm at 25 °C. A trietha-
in detail the effects of trypsin and its inhibitor protein AAT
nolamine solution (TEA 0.2 mol/L, Sigma Aldrich; CaCl2 0.02 mol/L,
on faecal calprotectin analysis using an in vitro model. Sarstedt, Nümbrecht, Germany) adjusted to pH 7.8 was used as buffer.
Results were expressed in BAPNA U/L [10]. AAT concentration was
measured on the Behring Nephelometer Analyzer II (Siemens, Mar-
Materials and methods burg, Germany) according to the manufacturer’s instructions [17].

In vitro model Statistics


To create an in vitro model and study the influence of possible All calculations were made in Excel 2010 and MedCalc (version
inhibitors in faeces, faecal extracts were prepared according to the 11.6.1.0, Mariakerke, Belgium). Results were given as median and

Figure 1 The molecular structure of calprotectin.


Trypsin cleaves peptide chains at the carboxyl side of the amino acids lysine (K) or arginine (R). Fourteen potential cleavage sites are
present, as shown by the arrows.

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
Dumoulin et al.: Confounders of calprotectin interpretation      67

inter-quartile ranges between brackets. Comparisons between condi- decrease of –28.0 [(–42.0)–(0.5)]%. It is, however, clear
tions were made by using a Wilcoxon test. A value of p < 0.05 was con- that decreases after 48 h of incubation were higher, as also
sidered statistically significant. Regression analysis was performed
seen in Figure 3. Differences between decreases after 24 h
and box-and-whisker plots were made.
and 48 h of incubation were evaluated using a Wilcoxon
test. Both differences, absolute and relative decrease, were
statistically significant (respectively, p < 0.01 and p = 0.02).
Results We also studied the effect of AAT addition on trypsin
activity. First, 1 mg/mL trypsin solution was added to a
At first effects of trypsin digestion on calprotectin were routine serum sample with an AAT concentration of 2 g/L.
evaluated by incubating a 1:100 dilution of calprotectin 50 μL trypsin solution was added to, respectively, 25 μL, 50
in faecal matrix with different added trypsin concentra- μL and 100 μL samples. Incubation at room temperature
tions dissolved in 1 mM HCl (a serial dilution with trypsin was performed for 30 min. Baseline trypsin activity was 90
levels between 12 and 61 U/L). Differences were evaluated U/L, after adding AAT activity decreased to, respectively,
after 24 h and 48 h of incubation at 37 °C. Figure 2 depicts 37 U/L, 49 U/L, and 48 U/L. Figure 4 shows the decrease
the effects of these different trypsin concentrations on in trypsin activity. Second, analogue incubation experi-
calprotectin immunoreactivity in faecal matrix after 24 h ments were performed to evaluate the influence of AAT in
and 48  h of incubation. The more trypsin was added to our in vitro model. To the mixture of 500 μL trypsin solu-
the mixture the higher the percentage of calprotectin pro- tion and 500 μL 1:100 diluted calprotectin in faecal matrix,
teolysis after 24 h. After 48 h of incubation the effect was 100 μL saline (blank) or 100 μL serum (AAT concentration
still present but the trend towards more proteolysis when of 1.47 g/L) was added. After 24 h and 48 h of incubation
more trypsin was added to the mixture was less clear. at 37 °C calprotectin values remained stable in the mixture
For further evaluation an ideal ratio of 500 μL trypsin with AAT, whereas in the blank mixture values decreased
solution (1 mg/mL) and 500 μL 1:100 diluted calprotectin 63% after 24 h and even more after 48 h (value below the
in faecal matrix was chosen. Table 1 represents the indi- limit of detection,  < 15 mg/kg).
vidual results. A median baseline trypsin activity in the
used faecal matrix of 6 [0–19] U/L was found. The median
trypsin activity after adding trypsin was 68.5 [62.0–
73.5]  U/L. Following trypsin treatment, relative decrease Discussion
of c­alprotectin immunoreactivity showed a broad
­variation. A median absolute decrease of –38.5 [(–68.5)– Calprotectin present in a faecal extract matrix is subject
(–5.5)] mg/kg is seen after 24 h of incubation, correspond- to trypsin digestion in vitro, leading to loss of immu-
ing to a relative decrease of –14.0 [(–28.5)–( –14.0)]%. After noreactivity. Human calprotectin shows 14 potential
48  h of incubation a median absolute decrease of –57.0 cleavages sites for trypsin. In the present study, trypsin
[(–80.0)–(–2.5)] mg/kg was found, equivalent to a relative activities in the range of values typically observed in the

0
24 h
-10 48 h

-20
∆rel calprotectin, %

-30

-40

-50

-60

-70

-80
0 10 20 30 40 50 60 70
Trypsin concentration, U/L

Figure 2 Effects of in vitro trypsin digestion (U/L) on calprotectin levels (mg/kg) in faecal matrix.
Relative differences are expressed as compared to the baseline calprotectin levels (%). The upper graph shows the effect after 48 h and the
lower graph after 24 h of incubation at 37 °C. h, hours; Δrel, relative difference.

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
68      Dumoulin et al.: Confounders of calprotectin interpretation

Table 1 Evaluation of in vitro trypsin digestion [median activity 68.5 (62.0–73.5) U/L] on calprotectin levels (expressed as mg/kg) in faecal
matrix. Relative and absolute differences are expressed as compared to the baseline calprotectin levels (n = 20).

Trypsin activity,  Faecal trypsin  ΔAbs calprotectin,  ΔAbs calprotectin,  ΔRel calprotectin,  ΔRel calprotectin,
U/L activity baseline, U/L mg/kg; 24 h mg/kg; 48 h %; 24 h %; 48 h

74  23  –72  –70  –24  –24


69  30  50  28  12  7
87  30  –12  –38  –8  –26
84  17  40  8  18  4
77  23  –65  –57  –43  –38
73  21  –12  10  –8  7
50  UD  –51  –83  –26  –43
59  UD  –139  –563  –10  –41
68  11  –8  –32  –4  –14
65  5  –33  –61  –18  –34
65  4  –49  –57  –30  –35
65  UD  14  8  13  7
75  6  –19  –9  –6  –3
72  UD  0  4  0  4
71  3  –63  –77  –25  –30
52  7  –128  –156  –36  –56
57  UD  –158  –150  –27  –74
69  UD  –103  –113  –72  –21
66  6  –44  –58  –41  –46
58  UD  –3  –19  –5  –71

Δabs, absolute difference; h, hours; Δrel, relative difference; UD, undetectable.

20 median trypsin activity of 68.5 (62.0–73.5) U/L and incuba-


tion times of 24 h/48 h] were in the same order as the ones
observed in vivo [18]. Our experiments confirmed an effect
0
of proteolytic enzymes, such as trypsin on the faecal bio-
marker calprotectin. The results proof trypsin activity to
-20 be a potential confounder in the interpretation of calpro-
tectin results, in particular for proximal lesions, where the
-40 exposure of calprotectin to trypsin is prolonged.
We also showed a statistically significant difference
between absolute and relative proteolysis of calprotectin
-60
after 24  h and 48  h of incubation, confirming that transit
time is also an important confounder in calprotectin results.
-80 It is described that children younger than 4 years have higher
∆rel calprotectin, %; 24 h ∆rel calprotectin, %; 48 h values of faecal calprotectin, the reasons still remain unclear
[19]. Most studies do not include subjects below 4 years of
Figure 3 Distribution of relative decreases of calprotectin after
adding a median trypsin concentration of 68.5 (62–73.5) U/L. age or make no distinction of different age groups [20]. Oord
All results are shown separately in Table 1, n = 20. h, hours; et  al. were the first to establish age-specific cut-off values
Δrel, relative difference. using healthy controls (n = 75): 538  mg/kg (1–6 months),
214 mg/kg (6 months–3 years) and 75 mg/kg (3–4 years).
In contrast, a cut-off value of 50 mg/kg is used for children
gastrointestinal lumen were used. In the proximal part of older than 4 years and adults [21]. Possible explanations for
the intestinal tract, trypsin activities are maximal as the these differences are the migration of neutrophils through
pancreas enzymes enter the gastrointestinal tract in the the mucosal membrane during regulation of the microbial
duodenum. In the more distal segments, trypsin activities flora [22]. However, transit time could also be a possible
drop rapidly. The experimental conditions of our study [a explanation for this phenomenon.

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
Dumoulin et al.: Confounders of calprotectin interpretation      69

0.7
Baseline activitty 25 µL sample 50 µL sample 100 µL sample
0.6
Absorbance, 405 nm

0.5

0.4

0.3

0.2

0.1

0
0 1 2 3 4 5 6 7 8 9 10
Time, min

Figure 4 Influence of α1-antitrypsin (AAT) on trypsin activity after 30 min of incubation at room temperature and adding 25 μL, 50 μL or
100 μL of serum sample (with an AAT concentration of 2 g/L).
Absorbance is monitored in function of time to calculate trypsin activity (a decrease is seen from 90 U/L to respectively, 37 U/L, 49 U/L, and
48 U/L).

Kaiser et al. also reported higher levels of calprotec- counteracted by a variety of protease inhibitors in the intes-
tin in distal inflammation compared with more proximal tinal lumen. Our experiments demonstrate that effects of
inflammation but these were not significant [23]. However, trypsin digestion are not always straightforward, which
Siponnen et  al. reported that ileal endoscopic score can be explained by a variable stability of trypsin or the
and histological findings failed to correlate with faecal presence of inhibitors. The occurrence of the endogenous
markers, such as calprotectin and lactoferrin [8]. They protease inhibitor protein, AAT, may play a role. Human
also showed that although the good specificity, sensitiv- plasma contains a variable amount of AAT. This variation
ity remained too low to exclude small bowel CD. Despite is partly due to the existence of genetic polymorphisms.
small bowel active CD faecal calprotectin values remained Currently, several variants are known, leading to deficient
low [24]. These findings may be explained by a major effect or non-detectable plasma levels with an increased risk of
of pancreatic trypsin on the investigated biomarkers. In a lung emphysema. The highest prevalence of the Z variant
serial dilution experiment we were also able to prove that peaks is found in southern Scandinavia, Denmark, the
the more trypsin is added to calprotectin, the higher the Netherlands, the UK and northern France. The distribu-
percentage of calprotectin proteolysis is seen. Proteolysis tion of the S variant differs and the highest frequency is
is also more pronounced when incubation times are pro- found in southern Europe [28]. AAT phenotypes are char-
longed, as demonstrated with our experiments. acterised by their own reference range, PiS shows about
Patients with cystic fibrosis (CF) have a marked defi- 60% of the normal level, PiW 80%, PiZ only 15%, and Pi
ciency of trypsin due to pancreatic exocrine insufficiency. null 0% [29]. The proposed interim reference range for
This insufficiency leads to maldigestion and therefore Caucasians is 0.9–2 g/L [30]. The modulation of AAT syn-
malabsorption and gastrointestinal complaints. Bruzz- thesis by the acute phase response is another important
ese et  al. reported that faecal calprotectin levels are sig- factor. AAT could be present in the gastrointestinal lumen
nificantly higher in children with CF compared to healthy after a minor or major bleeding at a proximal lesion or in
controls. Despite the increasing evidence of intestinal case of a protein-loosing enteropathy [15]. In these cases,
inflammation explaining these findings, the exact under- AAT is normally present in the excreted stool, mostly
lying pathologic mechanism is still unknown. Probiotic complexed to pancreatic trypsin and elastase. This could
administration reduces faecal calprotectin values and gas- lead to inactivation of the present trypsin, at least when
trointestinal complaints, supporting the presence of intes- patients are not deficient for AAT. Our first experiments
tinal inflammation. As trypsin activities of CF patients are demonstrated the inactivation of trypsin by AAT in serum.
lower in the gastrointestinal lumen due to pancreas insuf- However, also when testing the effect of AAT in our in
ficiency, calprotectin proteolysis is impaired and levels vitro model, we proved that calprotectin values remained
in stool are higher. In this way, our data are in agreement stable after inactivating trypsin by addition of AAT.
with clinical observations [25–27]. As trypsin shows a limited stability, our findings do
Next to the biological variation of trypsin concen- not imply major pre-analytical precautions following sam-
trations in the faecal matrix, proteolytic effects are pling [10, 11]. In most cases, residual trypsin activity in

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
70      Dumoulin et al.: Confounders of calprotectin interpretation

faeces is very low as demonstrated in our results, which is 7. Sipponen T, Bjorkesten CG, Farkkila M, Nuutinen H, Savilahti E,
almost negligible versus the huge concentrations observed Kolho KL. Faecal calprotectin and lactoferrin are reliable sur-
rogate markers of endoscopic response during Crohn’s disease
in the duodenum [9]. Addition of protease inhibitors to the
treatment. Scand J Gastroenterol 2010;45:325–31.
sample will therefore only have a marginal effect on test 8. Sipponen T, Karkkainen P, Savilahti E, Kolho KL, Nuutinen H,
results. Post-sampling stability of calprotectin has also Turunen U, et al. Correlation of faecal calprotectin and lactofer-
been confirmed in other pre-analytical studies dealing rin with an endoscopic score for Crohn’s disease and histologi-
with calprotectin [1]. Taking these different facts into cal findings. Aliment Pharmacol Ther 2008;28:1221–9.
9. Keller J, Layer P. Human pancreatic exocrine response to nutri-
account, numerous caveats should be taken into account
ents in health and disease. Gut 2005;54(Suppl 6):vi1–28.
when interpreting calprotectin test results. Transit time, 10. Geiger R, Fritz H. Trypsin. In: Bergmeyer HU, Bergmeyer J,
trypsin activity and addition of blood as a source of AAT Grassl M, editors. Methods of enzymatic analysis. Volume V.
may be regarded as potential confounders. Age-related Enzymes 3: peptidases, proteinases and their inhibitors, 3rd
cut-off values depending on the anatomical localisation ed. Weinheim, Deerfield Beach, Florida; Basel: Verlag Chemie,
of the lesions could therefore improve the diagnostic effi- 1984;119–29.
11. Smith JS, Ediss I, Mullinger MA, Bogoch A. Fecal chymotrypsin
ciency of calprotectin testing.
and trypsin determinations. Can Med Assoc J 1971;104:691–4.
12. Layer P, Go VL, DiMagno EP. Fate of pancreatic enzymes dur-
Acknowledgments: The authors would like to thank ing small intestinal aboral transit in humans. Am J Physiol
Thermo Scientific (Uppsala, Sweden) for providing the 1986;251:G475–80.
necessary calprotectin reagents. We also wish to thank 13. Layer P, Keller J. Pancreatic enzymes: secretion and luminal
nutrient digestion in health and disease. J Clin Gastroenterol
Elke Lecocq, Jonas Himpe and Mieke Hutsebaut for their
1999;28:3–10.
technical support. 14. Strygler B, Nicar MJ, Santangelo WC, Porter JL, Fordtran JS.
Author contributions: All the authors have accepted Alpha 1-antitrypsin excretion in stool in normal subjects and
responsibility for the entire content of this submitted in patients with gastrointestinal disorders. Gastroenterology
manuscript and approved submission. 1990;99:1380–7.
Financial support: None declared. 15. Umar SB, DiBaise JK. Protein-losing enteropathy: case illustra-
tions and clinical review. Am J Gastroenterol 2010;105:43–9.
Employment or leadership: None declared.
16. Oyaert M, Trouve C, Baert F, De Smet D, Langlois M,
Honorarium: None declared. Vanpoucke H. Comparison of two immunoassays for measure-
Role of sponsor: The funding organisation(s) played no ment of faecal calprotectin in detection of inflammatory bowel
role in the study design; in the collection, analysis, and disease: (pre)-analytical and diagnostic performance character-
interpretation of data; in the writing of the report; or in the istics. Clin Chem Lab Med 2014;52:391–7.
17. Fink PC, Romer M, Haeckel R, Fateh-Moghadam A, Delanghe J,
decision to submit the report for publication.
Gressner AM, et al. Measurement of proteins with the Behring
Nephelometer. A multicentre evaluation. J Clin Chem Clin Bio-
chem 1989;27:261–76.
18. Kim SK. Small intestine transit time in the normal small
References bowel study. Am J Roentgenol Radium Ther Nucl Med
1968;104:522–4.
1. Basso D, Zambon CF, Plebani M. Inflammatory bowel diseases: 19. Henderson P, Anderson NH, Wilson DC. The diagnostic accuracy
from pathogenesis to laboratory testing. Clin Chem Lab Med of fecal calprotectin during the investigation of suspected
2014;52:471–81. pediatric inflammatory bowel disease: a systematic review and
2. Fagerhol MK, Dale I, Andersson T. A radioimmunoassay for a meta-analysis. Am J Gastroenterol 2014;109:637–45.
granulocyte protein as a marker in studies on the turnover of 20. Kostakis ID, Cholidou KG, Vaiopoulos AG, Vlachos IS, Perrea D,
such cells. Bull Eur Physiopathol Respir 1980;16(Suppl):273–82. Vaos G. Fecal calprotectin in pediatric inflammatory bowel
3. Poullis A, Foster R, Mendall MA, Fagerhol MK. Emerging role disease: a systematic review. Dig Dis Sci 2013;58:309–19.
of calprotectin in gastroenterology. J Gastroenterol Hepatol 21. Oord T, Hornung N. Fecal calprotectin in healthy children.
2003;18:756–62. Scand J Clin Lab Invest 2014;74:254–8.
4. Roseth AG, Fagerhol MK, Aadland E, Schjonsby H. Assessment of 22. Rugtveit J, Fagerhol MK. Age-dependent variations in fecal
the neutrophil dominating protein calprotectin in feces. calprotectin concentrations in children. J Pediatr Gastroenterol
A methodologic study. Scand J Gastroenterol 1992;27:793–8. Nutr 2002;34:323–4.
5. van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin 23. Kaiser T, Langhorst J, Wittkowski H, Becker K, Friedrich AW,
for screening of patients with suspected inflammatory bowel Ruefferet A, et al. Faecal S100A12 as a non-invasive marker
disease: diagnostic meta-analysis. Br Med J 2010;341:c3369. distinguishing inflammatory bowel disease from irritable bowel
6. Van de Vijver E, Schreuder AB, Cnossen WR, Muller Kobold AC, syndrome. Gut 2007;56:1706–13.
van Rheenen PF. Safely ruling out inflammatory bowel disease in 24. Sipponen T, Haapamaki J, Savilahti E, Alfthan H, Hämäläinen E,
children and teenagers without referral for endoscopy. Arch Dis Rautiainen H, et al. Fecal calprotectin and S100A12 have low
Child 2012;97:1014–8. utility in prediction of small bowel Crohn’s disease detected

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM
Dumoulin et al.: Confounders of calprotectin interpretation      71

by wireless capsule endoscopy. Scand J Gastroenterol 28. Luisetti M, Seersholm N. Alpha1-antitrypsin deficiency. 1: epide-
2012;47:778–84. miology of alpha1-antitrypsin deficiency. Thorax 2004;59:164–9.
25. Fallahi G, Motamed F, Yousefi A, Shafieyoun A, Najafi M, 29. Tietz NW. Section I: general clinical tests. In: Clinical guide to
Khodadad A, et al. The effect of probiotics on fecal cal- laboratory tests. Section I: general clinical tests, 3rd ed. Phila-
protectin in patients with cystic fibrosis. Turk J Pediatr delphia: W.B. Saunders Company, 1995;66–7.
2013;55:475–8. 30. Dati F, Schumann G, Thomas L, Aguzzi F, Baudner S, Bienvenu J,
26. Lee JM, Leach ST, Katz T, Day AS, Jaffe A, Ooi CY. Update of faecal et al. Consensus of a group of professional societies and diag-
markers of inflammation in children with cystic fibrosis. Media- nostic companies on guidelines for interim reference ranges for
tors Inflamm 2012;2012:948367. 14 proteins in serum based on the standardization against the
27. Bruzzese E, Raia V, Gaudiello G, Polito G, Buccigrossi V, IFCC/BCR/CAP Reference Material (CRM 470). International Fed-
Formicola V, et al. Intestinal inflammation is a frequent feature eration of Clinical Chemistry. Community Bureau of Reference of
of cystic fibrosis and is reduced by probiotic administration. the Commission of the European Communities. College of Ameri-
Aliment Pharmacol Ther 2004;20:813–9. can Pathologists. Eur J Clin Chem Clin Biochem 1996;34:517–20.

Brought to you by | SUNY Stony Brook University Libraries


Authenticated
Download Date | 2/8/15 12:21 AM