Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 254–258
ORIGINAL ARTICLE
Fecal calprotectin in healthy children
TONJE OORD1 & NETE HORNUNG2
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Toronto on 10/02/14 For personal use only.
1University
of Århus, Århus and 2Department of Clinical Biochemistry, Randers Regional Hospital, Denmark
Abstract Calprotectin is a protein found in the cytosol of inflammatory cells and is a marker of the presence and the degree of inflammation in the bowel system. Calprotectin in feces has great diagnostic value in the matter of inflammatory bowel disease (IBD). In feces, the protein is stable up to seven days, and since the protein can easily be measured with an ELISA, the use of fecal calprotectin (FC) means no invasive measures. For adults and children over 4 years, a cut-off level of 50 mg/kg has been well established for diagnostic purposes. Because previous studies have proven that children under the age of four in general have higher FC values than older children and adults, there is a need for a cut-off level for this age group. In order to establish that, the normal values for FC in children from 0–4 years were investigated. Some 75 stool samples from healthy children were collected and the levels of FC were analyzed. The results were compared to 157 pediatric cases where FC analysis had been performed for diagnostic purposes. As a result, three cut-off levels were established based on the 97.5% percentiles of FC in different age groups: 538 mg/kg (1 ⬍ 6 months), 214 mg/kg (6 months ⬍ 3 years) and 75 mg/kg (3 ⬍ 4 years). Key Words: Calprotectin, inflammatory bowel disease, child, reference values, feces
Introduction In 1979, the calcium-binding protein, Calprotectin, was first described by Fagerhol et al. [1]. It is a protein which constitutes about 60% of the soluble proteins in neutrophile granulocytes and is also found in activated macrophages and monocytes, all derived from the myelomonocytic cell lineage [2,3]. Elevated concentrations of fecal calprotectin (FC) is described in Crohn’s disease (CD), ulcerative colitis (UC), cystic fibrosis, rheumatoid arthritis and bacterial infection, as well as in patients with colorectal cancer and Non-Steroidal AntiInflammatory Drugs (NSAID)-induced enteropathy [4–6]. Since 1992, an FC Enzyme-linked immunosorbent assay (ELISA) has been available [7]. The FC test is simple, non-invasive and inexpensive, with a high sensitivity and specificity, and it is established as a distinguished marker of disease activity in patients with inflammatory bowel disease (IBD), both in children and adults [7–11]. However, for children under 4 years, the use of the marker is limited by the lack of valid reference limits. There are several studies on pediatric patients investigating
FC as a marker for IBD. Few studies have specifically addressed FC levels in healthy children and studies including healthy children as controls have various limitations such as low number of subjects, no subjects below 4 years of age or no distinction of different age groups. This is summarized in a recent review by Kostakis et al. [11]. The aim of this study was to measure the concentrations of FC in healthy children from 0 to 4 years old in order to propose cut-off values for FC in this age group. We verify the cut-off values using existing FC data from sick children in the same age group and finally we hold the cut-off values towards established values for children more than 4 years old.
Materials and methods Collection of samples In order to collect feces from presumably healthy children from 0–4 years, over a 6-month period, four kindergartens in Aarhus, Denmark, were addressed. The parents were contacted either personally or by
Correspondence: Tonje Oord, University of Århus, Århus, Denmark. Tel: ⫹ 47 4054 7179. E-mail: [email protected] (Received 15 January 2013 ; accepted 29 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.879732
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Fecal calprotectin in healthy children from 0–4 years flyers. In addition, nurses visiting the youngest children, in order to engage their parents, were contacted. Staff at the Region Hospital of Randers, with children in the appropriate age group, was also recruited. All together, 75 samples of feces from healthy children (37 girls and 38 boys) under the age of four were collected. A healthy child was defined as no signs of a cold, flu, stomach problems or similar for the past two weeks, evaluated by their parents. Because the children were apparently healthy, no further tests were needed. In order to re-examine the results from the healthy children, FC data were withdrawn from the Laboratory Information System (LIS) from children in Denmark under the age of 5. The FC test was performed for clinical reasons in a one-year period from November 2010 to October 2011 and the FC analysis had been performed with the same assay in the same laboratory as the analysis of the healthy controls. Clinical data were obtained from the patients’ reports and the FC concentrations were compared to the patients’ final diagnosis. Clinical and laboratory results from 157 children from 0–4 years old were used to verify the proposed cut-off values. Measurement of calprotectin concentration Stool samples were delivered by mail and stored at ⫺20°C. Before analysis, the samples were thawed at room temperature. The FC was measured by a sandwich immunoassay manufactured by Bühlmann Laboratories AG (Schonenbuch, Schwitzerland). This analysis had a total coefficient of variation (CV) of 15% including the extraction procedure and the lower detection limit was 30 mg/kg. The analysis was performed according to the manufacturer’s instructions. Briefly, 50–100 mg of feces was homogenized with the extraction buffer (weight to volume ratio 1:49) and centrifuged. The supernatants from the extraction procedure were diluted 1:150 with incubation buffer for the Extended Range ELISA Procedure. 100 μL of samples, standards and controls were transferred to a micro titer plate coated with a monoclonal capture antibody highly specific to the calprotectin complexes and incubated at room temperature for 30 min. After a washing step, a detection antibody conjugated with horseradish peroxidase was added and after a second incubation and washing step, tetramethylbenzidine was added. Finally a stopping solution was added and the color absorbance was measured at 450 nm. Samples ⬎ 1800 mg/kg were diluted 1:1 and reanalyzed. The trueness of the method was ensured by participation in external quality program and standard operative procedures were established to monitor the accuracy over time.
255
Ethical considerations The protocol of this study was approved by the Danish Data Protection Agency and the National Board of Health in Denmark. The Local Committee on Health Research Ethics was consulted and further ethical approval was not required, neither regarding the analysis of stool samples from healthy children nor regarding the register analysis. For the participation of healthy children, the parental approval was obtained when the parents sent in the fecal sample for measurements. Statistical analysis GraphPad Prism (Graph-Pad Software, CA, USA) statistical software package was used for graphic and statistical calculations. Grubb’s test was used to detect outliers. Nonparametric Kruskal-Wallis test was used to compare medians in different age groups. Results The numbers of stool samples in the various age groups are shown in Table I. There were no samples from neonates between 0 and 4 weeks. One FC result of 556 mg/kg in the age group 1 ⬍ 2 years and one result of 252 mg/kg in the age group 3 ⬍ 4 years are excluded since the results are significantly different from the groups (p ⬍⬍ 0.05). There is no statistical difference between the medians in group 6 ⬍ 12 months, 1 ⬍ 2 years and 2 ⬍ 3 years (p ⫽ 0.58). When combining data from these three age groups (6 months ⬍ 3 years) the results are n ⫽ 56, FC median ⫽ 47 mg/kg, 97.5% percentile ⫽ 214 mg/kg. The data are presented graphically in Figure 1. The median in the 3 ⬍ 4 years group is not significantly different from the groups from 6 months to 3 years. However, it is clear that the scattering of the results changes remarkably in the 3 ⬍ 4 years group and the 97.5% percentile approaches the established reference limit of 50 mg/kg for children 4 years old and adults [11–13]. A cut-off calculated from 6 months ⬍ 4 years group (median: 47 mg/kg, 97.5% percentile: 207 mg/kg) may therefore cause false negative results and consequently a decrease in the sensitivity of the test for children between 3 and 4 years old. Children from 1 ⬍ 6 months have the highest concentrations of FC and the median in this group is significantly different from the medians in Table I. FC levels (mg/kg) in different age groups of healthy children. Age n Median 97.5% percentile
1⬍6 months
6 ⬍ 12 months
1⬍2 years
2⬍3 years
3⬍4 years
9 192 538
12 72 162
20 47 210
24 31 197
8 36 75
256
T. Oord & N. Hornung 600 550 500
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Faecal calprotectin (mg/kg)
450 400 350 300 250 200 150 100 50 0
0 < 6 mo
6 mo < 3 years
3 < 4 years
Figure 1. Diagram of FC levels in healthy children. Dotted lines are 97.5% percentiles, even lines are medians. Two outliers, one in age group 1 ⬍ 2 years and one in age group 3 ⬍ 4 years, have been removed, see text.
the 6 months ⬍ 3 years group and the 3 ⬍ 4 year group (p ⫽ 0.01). The results clearly indicate that more than one cut-off level is necessary for children less than 4 years old. The cut-off levels based on the 97.5% percentiles of FC values are: 538 mg/kg for 1 ⬍ 6 months, 214 mg/kg for children 6 months to ⬍ 3 years old and 75 mg/kg for the 3–4 years old children. In order to re-examine the cut-off levels a total of 157 patient reports were subsequently scrutinized regarding diagnosis and FC concentration (Table II). The patients were categorized in three groups according to their final diagnosis. Category 1 consisted of patients with milder diseases such as diarrhea without definite cause, abdominalia, Toddler’s diarrhea, constipation, poor well-being and other non-specific diagnosis.
Table II. Data from children who have had an FC analysis performed for diagnostic purposes compared to cut-offs established in healthy children. FC (mg/kg) N Minimum Median Maximum
Category 1
Category 2
Category 3
101 ⬍ 30 78 ⬎ 3600
52 ⬍ 30 423 ⬎ 3600
4 550 1556 ⬎ 3600
Number of patients with FC values above 97.5% percentile (total) 0 – ⬍ 0.5 years 0.5 – ⬍ 3 years 3–4 years
3 (6) 12 (72) 6 (23)
2 (7) 19 (31) 10 (14)
0 4 (4) 0
The patients in category 2 were children with final diagnosis such as gastroenteritis with verified infective agent (viruses, bacteria, amoebas), colorectal cancer or polyps, anal fissures or hemorrhoids. The third category included one case of CD and three cases of UC and they were categorized in a separate group. The FC levels in IBD patients were 3600, 550, 2082 and 1030 mg/kg at the time of diagnosis, and the main symptoms were rectal bleeding and diarrhea. No data existed of children diagnosed with irritable bowel syndrome. Table II also presents the number of patients with FC values above the 97.5% percentile of the healthy children and the distribution of patients in the different age groups. It is important that the four IBD patients have FC values above the 97.5% percentile and the high number of patients in category 1 below the 97.5% percentile is also worth noticing. Applying the suggested cut-offs from healthy children on these patients, the sensitivity of the test is 100% and the specificity of the test is 0.66. Table III presents category 1 patients according to age and FC levels compared to healthy children with the same age. It also shows that the youngest children in category 1 have higher FC levels and that a clear decrease in median FC is observed with increased age. This is in accordance with the FC levels seen in healthy children. FC concentrations in patients with mild disease are in general higher than FC levels in healthy children but almost 80% proved to have FC values below the 97.5% percentile of the healthy children.
Fecal calprotectin in healthy children from 0–4 years Table III. Data from healthy children and patients in category 1 divided into different age groups. Healthy children
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n Median 97.5% percentile Patients in category 1 N Minimum Median Maximum
1⬍6 months
6 months ⬍ 3 years
3⬍4 years
9 192 538
56 47 214
8 36 75
6 102 582 1373
72 ⬍ 30 73 1036
23 ⬍ 30 72 3600
Discussion According to the guideline How to define and determine reference intervals in the clinical laboratory; approved guideline – 3rd ed by Clinical and Laboratory Standards Institute, the optimal method to define a reference value for medical diagnosis is a direct sampling technique used on healthy children [14]. They suggest that a minimum of 120 samples for analysis should be collected and when dividing into subclasses, 120 should be in each class. Ideally, far more tests from healthy children are desired, but in general this is difficult to accomplish. But it is also stated that as an alternative, the laboratory can verify an already established reference interval, by collecting and measuring as few as 20 samples [14]. We collected a total of 75 samples and because of significant different medians in the various age groups the data were combined into three subclasses: 1 ⬍ 6 months, 6 months ⬍ 3 years and 3 ⬍ 4 years. The cut-off values were estimated by calculating the 97.5% percentile in each subclass but rounded figures are suggested for patient safety reasons and practical use in the clinic. The proposal for cut-offs values to be used for the different subclasses are 1 ⬍ 6 months: 530 mg/kg, 6 months ⬍ 3 years: 220 mg/kg and 3 ⬍ 4 years: 75 mg/kg. In this context it is important to stress that decision limits different from the established cut-offs might be useful in specific clinical settings. In 2002, Rugtveit and Fagerhol measured FC concentrations in 115 Norwegian children visiting two local community health centers in Oslo for routine examinations [15]. The children were from 6 weeks to 5 years of age. They found high values in children less than one year comparable to FC concentrations seen in older children and adults with IBD. They also found large variation within the various age groups and our study on Danish children are highly comparable to their findings. Another study by Olafsdottir et al. [16] showed that 27 healthy infants from 2–10 weeks had mean FC concentration of 278 ⫾ 105 mg/kg which is also in accordance with the 1 ⬍ 6 months group in our study. High levels of FC have also been shown in
257
other studies on healthy term and preterm infants [17,18] and reviewed by Kapel et al. [19]. Since there are no infants from 0 ⬍ 4 weeks in our study we cannot compare the specific results directly but it clearly indicates that neonates also need cut-off values higher than adults. Healthy children older than 4 weeks have also been investigated by others and the results from Danish children are also in agreement with these findings [11,16,20]. None of these studies have, however, suggested cut-off levels and subsequently re-examined the cut-off levels for clinical use. The practical collection of fecal samples shows that FC concentrations may increase up to 30% when the sample is collected from a diaper which may be explained by the water absorption into the diaper [16]. In our study, the parents were encouraged to collect the fecal sample from the morning stool, but there was no follow-up on this matter in our study. It is not stated in the other studies how samples were collected but it stresses the importance of maintaining a strict quality assurance program that includes preanalytical matters and clear recommendations for the patients/parents of how to collect the stool sample. Our study confirms the great value of the FC marker as a diagnostic tool when suspecting IBD in children. A recent review and meta-analysis by Henderson et al. [21] showed a pooled sensitivity as high as 0.978 for the diagnosis of suspected IBD in pediatric patients and the pooled specificity was 0.682. 100% of the patients with IBD had FC concentrations above the suggested cut-offs in our study. On the other hand, enhanced FC results are not specific for IBD and category 2 (Table II) presents several different diagnoses resulting in an elevated FC level in agreement with the relatively low specificity of the test [21] also confirmed in our study. Clearly the diseases are more serious than category 1 and many of the cases are expected to have FC levels above the reference limit according to literature [22]. Indeed, almost 60% of these patients have FC levels higher than healthy children. A recent study has shown that FC is elevated in bacterial gastrointestinal diseases and that it may be a valuable marker for infectious disease in children [22]. This underlines that elevated FC has several causes but also that low values help the clinician to exclude IBD. This is the first study of FC measured in a Danish cohort of healthy infants and small children. The results are highly comparable to previous findings and clearly show that healthy, younger children have higher FC concentrations than adults and older children. To our knowledge this is also first study to suggest specific cut-off levels and verifying the clinical performance of the proposed cut-off values by subsequent comparison with FC results and final diagnosis of sick children. The latter was
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T. Oord & N. Hornung
possible since the FC analysis were performed in the same laboratory using the same assay. There were four cases of children diagnosed with IBD in the age group 7 months ⬍ 3 years. Because four is a small number, the verification cannot be assessed as standards for IBD, but most importantly all four cases had FC levels highly above the suggested cutoff level of 220 mg/kg. In addition, almost 80% of the patients with milder diseases had FC values below the proposed cut-off. This may assure the clinician that a search for a more serious disease may not be relevant. Finally, most patients with FC levels above cut-off were diagnosed with specific gastroenteritis caused by various microorganisms which indicate that FC results above the cut-off levels should increase the suspicion and further investigations should be initiated.
[8]
[9]
[10]
[11]
[12]
Acknowledgements
[13]
The expert technical assistance of Joan Povlsen is appreciated.
[14]
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
[15]
[16]
References [1] Fagerhol MK, Dale I, Andersson T. Release and quantitiation of a leucocyte derived protein (L1). Scan J Haematol 1980; 24:393–8. [2] Fagerhol MK, Andersson KB, Naess-Andresen CF, Brandtzaeg P, Dale I. Calprotectin (the L1 leucoyte protein). In: Smith VL, Dedman JR, eds. stimulus response coupling: the role of intracellular calcium binding proteins. Boca Raton, FL: CRC Press; 1990;187–210. [3] Dale I, Brandtzaeg P, Fagerhol MK, Scott H. Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes. Immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin. Am J Clin Pathol 1985;84:24–34. [4] Johne B, Fagerhol MK, Lyberg T, Prydz H, Brandtzaeg P, Naess-Andresen CF, Dale I. Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol 1997;50:113–23. [5] Roseth AG, Kristinsson J, Fagerhol MK, Schjonsby H, Aadland E, Nygaard K, Roald B. Faecal calprotectin: a novel test for the diagnosis of colorectal cancer? Scand J Gastroenterol 1993;28:1073–6. [6] Tibble JA, Sigthorsson G, Foster R, Scott D, Fagerhol MK, Roseth A, Bjarnason I. High prevalence of NSAID enteropathy as shown by a simple faecal test. Gut 1999;45:362–6. [7] Roseth AG, Fagerhol MK, Aadland E, Schjonsby H. Assessment of the neutrophil dominating protein calprotectin
[17]
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[19]
[20]
[21]
[22]
in feces. A methodologic study. Scand J Gastroenterol 1992;27:793–8. Roseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of indium-111-labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol 1999;34:50–4. Roseth AG, Aadland E, Jahnsen J, Raknerud N. Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein. Digestion 1997;58:176–80. Bunn SK, Bisset WM, Main MJ, Golden BE. Fecal calprotectin as a measure of disease activity in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2001;32:171–7. Kostakis ID, Cholidou KG, Vaiopoulos AG, Vlachos IS, Perrea D, Vaos G. Fecal calprotectin in pediatric inflammatory bowel disease: a systematic review. Dig Dis Sci 2013;58:309–19. Fagerberg UL, Loof L, Myrdal U, Hansson LO, Finkel Y. Colorectal inflammation is well predicted by fecal calprotectin in children with gastrointestinal symptoms. J Pediatr Gastroenterol Nutr 2005;40:450–5. Ton H, Brandsnes, Dale S, Holtlund J, Skuibina E, Schjonsby H, Johne B. Improved assay for fecal calprotectin. Clin Chim Acta 2000;292:41–54. Horowitz GL, Altaie S, Boyd JC, Ceriotti F, Garg U, Horn P, Pesce A, Sine HE, Zakowski J. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – 3rd ed. 2008;28. Rugtveit J, Fagerhol MK. Age-dependent variations in fecal calprotectin concentrations in children. J Pediatr Gastroenterol Nutr 2002;34:323,4; author reply 324–5. Olafsdottir E, Aksnes L, Fluge G, Berstad A. Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children. Acta Paediatr 2002;91:45–50. Rhoads JM, Fatheree NY, Norori J, Liu Y, Lucke JF, Tyson JE, Ferris MJ. Altered fecal microflora and increased fecal calprotectin in infants with colic. J Pediatr 2009; 155:823–8. Nissen AC, van Gils CE, Menheere PP, Van den Neucker AM, van der Hoeven MA, Forget PP. Fecal calprotectin in healthy term and preterm infants. J Pediatr Gastroenterol Nutr 2004;381:107–8. Kapel N, Campeotto F, Kalach N, Baldassare M, Butel MJ, Dupont C. Faecal calprotectin in term and preterm neonates. J Pediatr Gastroenterol Nutr 2010; 51:542–7. Fagerberg UL, Loof L, Merzoug RD, Hansson LO, Finkel Y. Fecal calprotectin levels in healthy children studied with an improved assay. J Pediatr Gastroenterol Nutr 2003;37:468–72. Henderson P, Anderson NH, Wilson DC. The diagnostic accuracy of fecal calprotectin during the investigation of suspected pediatric inflammatory bowel disease: a systematic review and meta-analysis. Am J Gastroenterol 2012; 107:941–9. Chen CC, Huang JL, Chang CJ, Kong MS. Fecal calprotectin as a correlative marker in clinical severity of infectious diarrhea and usefulness in evaluating bacterial or viral pathogens in children. J Pediatr Gastroenterol Nutr 2012; 55:541–7.
ORIGINAL ARTICLE
Fecal calprotectin in healthy children
TONJE OORD1 & NETE HORNUNG2
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Toronto on 10/02/14 For personal use only.
1University
of Århus, Århus and 2Department of Clinical Biochemistry, Randers Regional Hospital, Denmark
Abstract Calprotectin is a protein found in the cytosol of inflammatory cells and is a marker of the presence and the degree of inflammation in the bowel system. Calprotectin in feces has great diagnostic value in the matter of inflammatory bowel disease (IBD). In feces, the protein is stable up to seven days, and since the protein can easily be measured with an ELISA, the use of fecal calprotectin (FC) means no invasive measures. For adults and children over 4 years, a cut-off level of 50 mg/kg has been well established for diagnostic purposes. Because previous studies have proven that children under the age of four in general have higher FC values than older children and adults, there is a need for a cut-off level for this age group. In order to establish that, the normal values for FC in children from 0–4 years were investigated. Some 75 stool samples from healthy children were collected and the levels of FC were analyzed. The results were compared to 157 pediatric cases where FC analysis had been performed for diagnostic purposes. As a result, three cut-off levels were established based on the 97.5% percentiles of FC in different age groups: 538 mg/kg (1 ⬍ 6 months), 214 mg/kg (6 months ⬍ 3 years) and 75 mg/kg (3 ⬍ 4 years). Key Words: Calprotectin, inflammatory bowel disease, child, reference values, feces
Introduction In 1979, the calcium-binding protein, Calprotectin, was first described by Fagerhol et al. [1]. It is a protein which constitutes about 60% of the soluble proteins in neutrophile granulocytes and is also found in activated macrophages and monocytes, all derived from the myelomonocytic cell lineage [2,3]. Elevated concentrations of fecal calprotectin (FC) is described in Crohn’s disease (CD), ulcerative colitis (UC), cystic fibrosis, rheumatoid arthritis and bacterial infection, as well as in patients with colorectal cancer and Non-Steroidal AntiInflammatory Drugs (NSAID)-induced enteropathy [4–6]. Since 1992, an FC Enzyme-linked immunosorbent assay (ELISA) has been available [7]. The FC test is simple, non-invasive and inexpensive, with a high sensitivity and specificity, and it is established as a distinguished marker of disease activity in patients with inflammatory bowel disease (IBD), both in children and adults [7–11]. However, for children under 4 years, the use of the marker is limited by the lack of valid reference limits. There are several studies on pediatric patients investigating
FC as a marker for IBD. Few studies have specifically addressed FC levels in healthy children and studies including healthy children as controls have various limitations such as low number of subjects, no subjects below 4 years of age or no distinction of different age groups. This is summarized in a recent review by Kostakis et al. [11]. The aim of this study was to measure the concentrations of FC in healthy children from 0 to 4 years old in order to propose cut-off values for FC in this age group. We verify the cut-off values using existing FC data from sick children in the same age group and finally we hold the cut-off values towards established values for children more than 4 years old.
Materials and methods Collection of samples In order to collect feces from presumably healthy children from 0–4 years, over a 6-month period, four kindergartens in Aarhus, Denmark, were addressed. The parents were contacted either personally or by
Correspondence: Tonje Oord, University of Århus, Århus, Denmark. Tel: ⫹ 47 4054 7179. E-mail: [email protected] (Received 15 January 2013 ; accepted 29 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.879732
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Toronto on 10/02/14 For personal use only.
Fecal calprotectin in healthy children from 0–4 years flyers. In addition, nurses visiting the youngest children, in order to engage their parents, were contacted. Staff at the Region Hospital of Randers, with children in the appropriate age group, was also recruited. All together, 75 samples of feces from healthy children (37 girls and 38 boys) under the age of four were collected. A healthy child was defined as no signs of a cold, flu, stomach problems or similar for the past two weeks, evaluated by their parents. Because the children were apparently healthy, no further tests were needed. In order to re-examine the results from the healthy children, FC data were withdrawn from the Laboratory Information System (LIS) from children in Denmark under the age of 5. The FC test was performed for clinical reasons in a one-year period from November 2010 to October 2011 and the FC analysis had been performed with the same assay in the same laboratory as the analysis of the healthy controls. Clinical data were obtained from the patients’ reports and the FC concentrations were compared to the patients’ final diagnosis. Clinical and laboratory results from 157 children from 0–4 years old were used to verify the proposed cut-off values. Measurement of calprotectin concentration Stool samples were delivered by mail and stored at ⫺20°C. Before analysis, the samples were thawed at room temperature. The FC was measured by a sandwich immunoassay manufactured by Bühlmann Laboratories AG (Schonenbuch, Schwitzerland). This analysis had a total coefficient of variation (CV) of 15% including the extraction procedure and the lower detection limit was 30 mg/kg. The analysis was performed according to the manufacturer’s instructions. Briefly, 50–100 mg of feces was homogenized with the extraction buffer (weight to volume ratio 1:49) and centrifuged. The supernatants from the extraction procedure were diluted 1:150 with incubation buffer for the Extended Range ELISA Procedure. 100 μL of samples, standards and controls were transferred to a micro titer plate coated with a monoclonal capture antibody highly specific to the calprotectin complexes and incubated at room temperature for 30 min. After a washing step, a detection antibody conjugated with horseradish peroxidase was added and after a second incubation and washing step, tetramethylbenzidine was added. Finally a stopping solution was added and the color absorbance was measured at 450 nm. Samples ⬎ 1800 mg/kg were diluted 1:1 and reanalyzed. The trueness of the method was ensured by participation in external quality program and standard operative procedures were established to monitor the accuracy over time.
255
Ethical considerations The protocol of this study was approved by the Danish Data Protection Agency and the National Board of Health in Denmark. The Local Committee on Health Research Ethics was consulted and further ethical approval was not required, neither regarding the analysis of stool samples from healthy children nor regarding the register analysis. For the participation of healthy children, the parental approval was obtained when the parents sent in the fecal sample for measurements. Statistical analysis GraphPad Prism (Graph-Pad Software, CA, USA) statistical software package was used for graphic and statistical calculations. Grubb’s test was used to detect outliers. Nonparametric Kruskal-Wallis test was used to compare medians in different age groups. Results The numbers of stool samples in the various age groups are shown in Table I. There were no samples from neonates between 0 and 4 weeks. One FC result of 556 mg/kg in the age group 1 ⬍ 2 years and one result of 252 mg/kg in the age group 3 ⬍ 4 years are excluded since the results are significantly different from the groups (p ⬍⬍ 0.05). There is no statistical difference between the medians in group 6 ⬍ 12 months, 1 ⬍ 2 years and 2 ⬍ 3 years (p ⫽ 0.58). When combining data from these three age groups (6 months ⬍ 3 years) the results are n ⫽ 56, FC median ⫽ 47 mg/kg, 97.5% percentile ⫽ 214 mg/kg. The data are presented graphically in Figure 1. The median in the 3 ⬍ 4 years group is not significantly different from the groups from 6 months to 3 years. However, it is clear that the scattering of the results changes remarkably in the 3 ⬍ 4 years group and the 97.5% percentile approaches the established reference limit of 50 mg/kg for children 4 years old and adults [11–13]. A cut-off calculated from 6 months ⬍ 4 years group (median: 47 mg/kg, 97.5% percentile: 207 mg/kg) may therefore cause false negative results and consequently a decrease in the sensitivity of the test for children between 3 and 4 years old. Children from 1 ⬍ 6 months have the highest concentrations of FC and the median in this group is significantly different from the medians in Table I. FC levels (mg/kg) in different age groups of healthy children. Age n Median 97.5% percentile
1⬍6 months
6 ⬍ 12 months
1⬍2 years
2⬍3 years
3⬍4 years
9 192 538
12 72 162
20 47 210
24 31 197
8 36 75
256
T. Oord & N. Hornung 600 550 500
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Faecal calprotectin (mg/kg)
450 400 350 300 250 200 150 100 50 0
0 < 6 mo
6 mo < 3 years
3 < 4 years
Figure 1. Diagram of FC levels in healthy children. Dotted lines are 97.5% percentiles, even lines are medians. Two outliers, one in age group 1 ⬍ 2 years and one in age group 3 ⬍ 4 years, have been removed, see text.
the 6 months ⬍ 3 years group and the 3 ⬍ 4 year group (p ⫽ 0.01). The results clearly indicate that more than one cut-off level is necessary for children less than 4 years old. The cut-off levels based on the 97.5% percentiles of FC values are: 538 mg/kg for 1 ⬍ 6 months, 214 mg/kg for children 6 months to ⬍ 3 years old and 75 mg/kg for the 3–4 years old children. In order to re-examine the cut-off levels a total of 157 patient reports were subsequently scrutinized regarding diagnosis and FC concentration (Table II). The patients were categorized in three groups according to their final diagnosis. Category 1 consisted of patients with milder diseases such as diarrhea without definite cause, abdominalia, Toddler’s diarrhea, constipation, poor well-being and other non-specific diagnosis.
Table II. Data from children who have had an FC analysis performed for diagnostic purposes compared to cut-offs established in healthy children. FC (mg/kg) N Minimum Median Maximum
Category 1
Category 2
Category 3
101 ⬍ 30 78 ⬎ 3600
52 ⬍ 30 423 ⬎ 3600
4 550 1556 ⬎ 3600
Number of patients with FC values above 97.5% percentile (total) 0 – ⬍ 0.5 years 0.5 – ⬍ 3 years 3–4 years
3 (6) 12 (72) 6 (23)
2 (7) 19 (31) 10 (14)
0 4 (4) 0
The patients in category 2 were children with final diagnosis such as gastroenteritis with verified infective agent (viruses, bacteria, amoebas), colorectal cancer or polyps, anal fissures or hemorrhoids. The third category included one case of CD and three cases of UC and they were categorized in a separate group. The FC levels in IBD patients were 3600, 550, 2082 and 1030 mg/kg at the time of diagnosis, and the main symptoms were rectal bleeding and diarrhea. No data existed of children diagnosed with irritable bowel syndrome. Table II also presents the number of patients with FC values above the 97.5% percentile of the healthy children and the distribution of patients in the different age groups. It is important that the four IBD patients have FC values above the 97.5% percentile and the high number of patients in category 1 below the 97.5% percentile is also worth noticing. Applying the suggested cut-offs from healthy children on these patients, the sensitivity of the test is 100% and the specificity of the test is 0.66. Table III presents category 1 patients according to age and FC levels compared to healthy children with the same age. It also shows that the youngest children in category 1 have higher FC levels and that a clear decrease in median FC is observed with increased age. This is in accordance with the FC levels seen in healthy children. FC concentrations in patients with mild disease are in general higher than FC levels in healthy children but almost 80% proved to have FC values below the 97.5% percentile of the healthy children.
Fecal calprotectin in healthy children from 0–4 years Table III. Data from healthy children and patients in category 1 divided into different age groups. Healthy children
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n Median 97.5% percentile Patients in category 1 N Minimum Median Maximum
1⬍6 months
6 months ⬍ 3 years
3⬍4 years
9 192 538
56 47 214
8 36 75
6 102 582 1373
72 ⬍ 30 73 1036
23 ⬍ 30 72 3600
Discussion According to the guideline How to define and determine reference intervals in the clinical laboratory; approved guideline – 3rd ed by Clinical and Laboratory Standards Institute, the optimal method to define a reference value for medical diagnosis is a direct sampling technique used on healthy children [14]. They suggest that a minimum of 120 samples for analysis should be collected and when dividing into subclasses, 120 should be in each class. Ideally, far more tests from healthy children are desired, but in general this is difficult to accomplish. But it is also stated that as an alternative, the laboratory can verify an already established reference interval, by collecting and measuring as few as 20 samples [14]. We collected a total of 75 samples and because of significant different medians in the various age groups the data were combined into three subclasses: 1 ⬍ 6 months, 6 months ⬍ 3 years and 3 ⬍ 4 years. The cut-off values were estimated by calculating the 97.5% percentile in each subclass but rounded figures are suggested for patient safety reasons and practical use in the clinic. The proposal for cut-offs values to be used for the different subclasses are 1 ⬍ 6 months: 530 mg/kg, 6 months ⬍ 3 years: 220 mg/kg and 3 ⬍ 4 years: 75 mg/kg. In this context it is important to stress that decision limits different from the established cut-offs might be useful in specific clinical settings. In 2002, Rugtveit and Fagerhol measured FC concentrations in 115 Norwegian children visiting two local community health centers in Oslo for routine examinations [15]. The children were from 6 weeks to 5 years of age. They found high values in children less than one year comparable to FC concentrations seen in older children and adults with IBD. They also found large variation within the various age groups and our study on Danish children are highly comparable to their findings. Another study by Olafsdottir et al. [16] showed that 27 healthy infants from 2–10 weeks had mean FC concentration of 278 ⫾ 105 mg/kg which is also in accordance with the 1 ⬍ 6 months group in our study. High levels of FC have also been shown in
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other studies on healthy term and preterm infants [17,18] and reviewed by Kapel et al. [19]. Since there are no infants from 0 ⬍ 4 weeks in our study we cannot compare the specific results directly but it clearly indicates that neonates also need cut-off values higher than adults. Healthy children older than 4 weeks have also been investigated by others and the results from Danish children are also in agreement with these findings [11,16,20]. None of these studies have, however, suggested cut-off levels and subsequently re-examined the cut-off levels for clinical use. The practical collection of fecal samples shows that FC concentrations may increase up to 30% when the sample is collected from a diaper which may be explained by the water absorption into the diaper [16]. In our study, the parents were encouraged to collect the fecal sample from the morning stool, but there was no follow-up on this matter in our study. It is not stated in the other studies how samples were collected but it stresses the importance of maintaining a strict quality assurance program that includes preanalytical matters and clear recommendations for the patients/parents of how to collect the stool sample. Our study confirms the great value of the FC marker as a diagnostic tool when suspecting IBD in children. A recent review and meta-analysis by Henderson et al. [21] showed a pooled sensitivity as high as 0.978 for the diagnosis of suspected IBD in pediatric patients and the pooled specificity was 0.682. 100% of the patients with IBD had FC concentrations above the suggested cut-offs in our study. On the other hand, enhanced FC results are not specific for IBD and category 2 (Table II) presents several different diagnoses resulting in an elevated FC level in agreement with the relatively low specificity of the test [21] also confirmed in our study. Clearly the diseases are more serious than category 1 and many of the cases are expected to have FC levels above the reference limit according to literature [22]. Indeed, almost 60% of these patients have FC levels higher than healthy children. A recent study has shown that FC is elevated in bacterial gastrointestinal diseases and that it may be a valuable marker for infectious disease in children [22]. This underlines that elevated FC has several causes but also that low values help the clinician to exclude IBD. This is the first study of FC measured in a Danish cohort of healthy infants and small children. The results are highly comparable to previous findings and clearly show that healthy, younger children have higher FC concentrations than adults and older children. To our knowledge this is also first study to suggest specific cut-off levels and verifying the clinical performance of the proposed cut-off values by subsequent comparison with FC results and final diagnosis of sick children. The latter was
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possible since the FC analysis were performed in the same laboratory using the same assay. There were four cases of children diagnosed with IBD in the age group 7 months ⬍ 3 years. Because four is a small number, the verification cannot be assessed as standards for IBD, but most importantly all four cases had FC levels highly above the suggested cutoff level of 220 mg/kg. In addition, almost 80% of the patients with milder diseases had FC values below the proposed cut-off. This may assure the clinician that a search for a more serious disease may not be relevant. Finally, most patients with FC levels above cut-off were diagnosed with specific gastroenteritis caused by various microorganisms which indicate that FC results above the cut-off levels should increase the suspicion and further investigations should be initiated.
[8]
[9]
[10]
[11]
[12]
Acknowledgements
[13]
The expert technical assistance of Joan Povlsen is appreciated.
[14]
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
[15]
[16]
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