DOI 10.1515/cclm-2014-0017 Clin Chem Lab Med 2014; 52(7): e143–e145
Letter to the Editor Nacer Adam Benahmed, Dieudonné Manéné, Laurence Barbot-Trystram and Nathalie Kapel*
Evaluation of Calfast® immunochromatographic quantitative assay for the measurement of calprotectin in faeces Keywords: ELISA; faecal calprotectin; point-of-care testing. *Corresponding author: Nathalie Kapel, Laboratoire de Coprologie Fonctionnelle, Groupe Hospitalier Pitié-Salpêtrière, 47-83 Boulevard de l’Hôpital, 75013 Paris, France, Phone: +33 1 42162652, Fax: +33 1 42162654, E-mail: [email protected] Nacer Adam Benahmed, Dieudonné Manéné and Laurence BarbotTrystram: Coprologie fonctionnelle, AP-HP, Groupe Hospitalier PitiéSalpêtrière, Paris, France Nathalie Kapel: Coprologie fonctionnelle, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; and Ecosystéme intestinal, probiotiques, antibiotiques (EA 4065), Université; Paris Descartes, Sorbonne Paris Cité, France
To the Editor, Calprotectin is a calcium and zinc binding protein which belongs to the S100 protein family. It is secreted extracellularly from stimulated neutrophils and, to a lesser extent, from monocytes and reactive macrophages. It can also be released by cell disruption or death as a result of mucosal inflammation [1]. Faecal calprotectin has been shown to be remarkably resistant to proteolysis and colonic bacterial degradation when bound to calcium, thus allowing its measurement in stools [2]. Various data including recent meta-analyses have shown the relevance of faecal calprotectin assay in the differential diagnosis of inflammatory bowel disease (IBD) from irritable bowel syndrome, both in adults and paediatric populations [3, 4]. According to their meta-analysis, van Rheenen et al. concluded that measurement of faecal calprotectin is a useful screening tool for identifying patients who are most likely to need rapid endoscopy for suspected IBD [3]. Using calprotectin as a screening factor, the number of colonoscopies could thus be reduced substantially [5]. ELISA is the reference method for calprotectin measurement in faeces and the most accepted cut-off is 50 µg/g of stools for healthy adults and children over 5–7 years of age [2]. ELISA is both a time-consuming and costly technique which
requires incubations for up to 2 h, so laboratories often run samples in batches to reduce costs at the expense of long turnaround times. However, in the meantime most patients have already undergone colonoscopy. By reducing this delay, it would thus be possible to improve upon the selection of patients requiring complementary clinical investigations. Since 2008, rapid semi-quantitative and quantitative immunochromatographic tests have been developed for faecal calprotectin that offer the opportunity for better turnaround times with global satisfactory results [6–9]. In this study, we have evaluated a new point-of-care testing (POCT) (CalFast®, Eurospital, Italy) which is based on a combination of a mixture of anti-calprotectin polyclonal and monoclonal antibodies and compared it to ELISA (Calprest®, Eurospital, Italy) routinely used in our laboratory. The comparative study used faecal samples sent to us for routine analyses. Calprotectin was measured within 2 weeks on an aliquot of native stools stored at −80 °C for ELISA, while for POCT, a second aliquot of native stools was stored at −80 °C for a maximum of 2 months. The Calprest ELISA method was carried out on the BEP2000® Advance system (Siemens, Germany). Fifty to 90 mg of faeces was weighed (avoiding any undigested particulate material in the sample) and diluted 1/50 (weight/volume) with extraction buffer. After centrifugation, the supernatant was used for analysis performed at two different dilutions (1:2500 and 1:25000 final dilutions). Using this method, the range of measurement was 15–5000 µg/g of stools and 50 µg/g of stools was considered as the cut-off normal limit as recommended by the manufacturer. For the Calfast POCT, faecal sampling was performed using the device provided in the kit. The supernatant was diluted 1/50 and 100 µL of the dilution was loaded onto the coated test cartridge. Signal intensity (from gold particles) was measured after 20 min by the dedicated reader using a lot-specific calibration curve to calculate calprotectin concentration. Using this method, the range of measurement was 50–300 µg/g of stools and 70 µg/g of stools was considered as the cut-off
Brought to you by | provisional account Unauthenticated | 128.103.149.52 Download Date | 6/27/14 6:31 PM
e144 Benahmed et al.: Evaluation of CalFast rapid test for faecal calprotectin
>300 250 POCT, µg/g stool
normal limit as recommended by the manufacturer. The intra-assay (n = 10) and the inter-assay (n = 6) variations were evaluated by assays of three stools samples selected to have calprotectin concentrations under the cut-off value (
Letter to the Editor Nacer Adam Benahmed, Dieudonné Manéné, Laurence Barbot-Trystram and Nathalie Kapel*
Evaluation of Calfast® immunochromatographic quantitative assay for the measurement of calprotectin in faeces Keywords: ELISA; faecal calprotectin; point-of-care testing. *Corresponding author: Nathalie Kapel, Laboratoire de Coprologie Fonctionnelle, Groupe Hospitalier Pitié-Salpêtrière, 47-83 Boulevard de l’Hôpital, 75013 Paris, France, Phone: +33 1 42162652, Fax: +33 1 42162654, E-mail: [email protected] Nacer Adam Benahmed, Dieudonné Manéné and Laurence BarbotTrystram: Coprologie fonctionnelle, AP-HP, Groupe Hospitalier PitiéSalpêtrière, Paris, France Nathalie Kapel: Coprologie fonctionnelle, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; and Ecosystéme intestinal, probiotiques, antibiotiques (EA 4065), Université; Paris Descartes, Sorbonne Paris Cité, France
To the Editor, Calprotectin is a calcium and zinc binding protein which belongs to the S100 protein family. It is secreted extracellularly from stimulated neutrophils and, to a lesser extent, from monocytes and reactive macrophages. It can also be released by cell disruption or death as a result of mucosal inflammation [1]. Faecal calprotectin has been shown to be remarkably resistant to proteolysis and colonic bacterial degradation when bound to calcium, thus allowing its measurement in stools [2]. Various data including recent meta-analyses have shown the relevance of faecal calprotectin assay in the differential diagnosis of inflammatory bowel disease (IBD) from irritable bowel syndrome, both in adults and paediatric populations [3, 4]. According to their meta-analysis, van Rheenen et al. concluded that measurement of faecal calprotectin is a useful screening tool for identifying patients who are most likely to need rapid endoscopy for suspected IBD [3]. Using calprotectin as a screening factor, the number of colonoscopies could thus be reduced substantially [5]. ELISA is the reference method for calprotectin measurement in faeces and the most accepted cut-off is 50 µg/g of stools for healthy adults and children over 5–7 years of age [2]. ELISA is both a time-consuming and costly technique which
requires incubations for up to 2 h, so laboratories often run samples in batches to reduce costs at the expense of long turnaround times. However, in the meantime most patients have already undergone colonoscopy. By reducing this delay, it would thus be possible to improve upon the selection of patients requiring complementary clinical investigations. Since 2008, rapid semi-quantitative and quantitative immunochromatographic tests have been developed for faecal calprotectin that offer the opportunity for better turnaround times with global satisfactory results [6–9]. In this study, we have evaluated a new point-of-care testing (POCT) (CalFast®, Eurospital, Italy) which is based on a combination of a mixture of anti-calprotectin polyclonal and monoclonal antibodies and compared it to ELISA (Calprest®, Eurospital, Italy) routinely used in our laboratory. The comparative study used faecal samples sent to us for routine analyses. Calprotectin was measured within 2 weeks on an aliquot of native stools stored at −80 °C for ELISA, while for POCT, a second aliquot of native stools was stored at −80 °C for a maximum of 2 months. The Calprest ELISA method was carried out on the BEP2000® Advance system (Siemens, Germany). Fifty to 90 mg of faeces was weighed (avoiding any undigested particulate material in the sample) and diluted 1/50 (weight/volume) with extraction buffer. After centrifugation, the supernatant was used for analysis performed at two different dilutions (1:2500 and 1:25000 final dilutions). Using this method, the range of measurement was 15–5000 µg/g of stools and 50 µg/g of stools was considered as the cut-off normal limit as recommended by the manufacturer. For the Calfast POCT, faecal sampling was performed using the device provided in the kit. The supernatant was diluted 1/50 and 100 µL of the dilution was loaded onto the coated test cartridge. Signal intensity (from gold particles) was measured after 20 min by the dedicated reader using a lot-specific calibration curve to calculate calprotectin concentration. Using this method, the range of measurement was 50–300 µg/g of stools and 70 µg/g of stools was considered as the cut-off
Brought to you by | provisional account Unauthenticated | 128.103.149.52 Download Date | 6/27/14 6:31 PM
e144 Benahmed et al.: Evaluation of CalFast rapid test for faecal calprotectin
>300 250 POCT, µg/g stool
normal limit as recommended by the manufacturer. The intra-assay (n = 10) and the inter-assay (n = 6) variations were evaluated by assays of three stools samples selected to have calprotectin concentrations under the cut-off value (
