Fecal Calprotectin vs FIT for Mucosal Healing
Fecal Calprotectin vs FIT for Mucosal Healing
Since 2006, UC patients who regularly attend Okayama University Hospital have been requested to routinely prepare and bring fecal samples at each visit, including the day of colonoscopy, in order to evaluate the amount of fecal occult blood via FIT. In addition, all consecutive UC patients who underwent scheduled colonoscopy between October 2012 and February 2014 were requested to bring another fecal sample for the examination of Fcal. Both fecal samples were collected on the day or a few days before colonoscopy, and stored in a refrigerator until the day of colonoscopy. All of the patients had an established diagnosis of UC according to endoscopic and histologic assessments and had received adequate medical therapy. Mucosal status assessed via colonoscopy was compared with the results of FIT vs. Fcal.
Clinical disease activity was evaluated by using the Mayo score (Scoring Systems for Assessment of UC), consisting of the following four subscores: stool frequency (0, normal number of stools for this patient; 1, 1–2 stools more than normal; 2, 3–4 stools more than normal; and 3, ≥5 stools more than normal), rectal bleeding (0, no blood seen; 1, streaks of blood with stool less than half the time; 2, obvious blood with stool most of the time; and 3, blood alone passed), endoscopic findings (0, normal or inactive disease; 1, mild disease with erythema, decreased vascular pattern, mild friability; 2, moderate disease with marked erythema, absent vascular pattern, friability, erosions; and 3, severe disease with spontaneous bleeding, ulceration), and physician's global assessment (0, normal; 1, mild disease; 2, moderate disease; and 3, severe disease). Clinical remission was defined as a Mayo stool frequency subscore of 0 or 1 and a Mayo rectal bleeding subscore of 0. Patients who failed to fulfill the definition of clinical remission were considered to have clinically active disease.
Details of the method used for FIT have been described previously. In brief, patients collected fecal samples using an OC-Hemodia sampling probe (Eiken Chemical, Tokyo, Japan). Submitted stool samples were immediately processed and examined using OC-SENSOR neo (Eiken Chemical) that can accurately measure fecal hemoglobin concentrations from 50 to 1,000 ng/ml. Fecal specimens with a hemoglobin concentration of >1,000 ng/ml were measured by further dilution, whereas those with a hemoglobin concentration of <50 ng/ml were categorized as one (0–50 ng/ml) because FIT results are inaccurate when the hemoglobin concentration is <50 ng/ml.
Fecal samples collected by the patients for calprotectin analysis were stored at −70 °C until shipment to the laboratory. The samples were sent to the Institute of Applied Technology for Innate Immunity (Kagawa, Japan), where calprotectin in stools is measured by a Phical Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Benshem, Germany). The quantitative range of calprotectin was between 0.65 and 84,000 μg/g after the appropriate dilution of fecal samples ranging from 1:50 to 1:100,000.
Bowel preparation was performed with a polyethylene glycol-based or magnesium citrate-based electrolyte solution according to the standard protocol in our hospital. After the colonic lavage fluid was cleared, patients underwent colonoscopy. Patients were excluded from the study if the colonoscopic examination was incomplete because of problems with the bowel preparation or if the colonoscope could not be inserted into the cecum.
The mucosal status of UC patients was assessed via the MES classification. Evaluation was performed at each portion of the colorectum (cecum and ascending colon combined, transverse colon, descending colon, sigmoid colon, and rectum), and the maximum score in the colorectum of each patient was used for analysis. The total inflammation score was defined as the sum of MES in the five colonic portions, and ranged from 0 (no inflammation) to 15 (severe and extensive inflammation). MH was defined as an MES of 0, or 0 or 1 throughout the colorectum. Some patients underwent biopsy from the portion with maximum endoscopic inflammation for pathological examinations. All colonoscopic examinations were performed by experienced colonoscopists who were blind to the results of the FIT and Fcal. In addition, the MES and the fecal results of each patient were determined independently by investigators who did not know patient's status including symptoms.
Histologic studies were evaluated using Geboes scores by gastrointestinal pathologists. Geboes scores for assessment of UC histologic disease activity and MH are classified into 6 grades from grade 0 to grade 5: grade 0 (subgrades 0.0 to 0.3), structural (architectural changes); grade 1 (subgrades 1.0 to 1.3), chronic inflammatory infiltrate; grade 2, lamina propria neutrophils and eosinophils (subgrades 2A.0 to 2A.3 for eosinophils, subgrades 2B.0 to 2B.3 for neutrophils); grade 3 (subgrades 3.0 to 3.3), neutrophils in epithelium; grade 4 (subgrades 4.0 to 4.3), crypt destruction; grade 5 (subgrades 5.0 to 5.4), erosion or ulceration. When one or more biopsy specimens were evaluated on each patient, the highest score was used for analysis.
Patient characteristics were analyzed by the JMP program (version 11.0 for Windows, SAS Institute, Cary, NC). Sensitivity, specificity, positive predictive value and negative predictive value with 95% confidence intervals (CIs) for detecting mucosal status were determined based on the FIT and calprotectin results. To estimate appropriate cutoff values for FIT and calprotectin, receiver operating characteristic (ROC) curve analysis was performed, and the area under the curve was calculated. Spearman's rank correlation test was performed to determine the correlation coefficient between the FIT values or Fcallevels and the Mayo endoscopic scores. All P values were two sided and considered statistically significant when <0.05.
This study was approved by the institutional review board of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. Informed consent was obtained from each patient before bringing fecal samples.
Methods
Patients
Since 2006, UC patients who regularly attend Okayama University Hospital have been requested to routinely prepare and bring fecal samples at each visit, including the day of colonoscopy, in order to evaluate the amount of fecal occult blood via FIT. In addition, all consecutive UC patients who underwent scheduled colonoscopy between October 2012 and February 2014 were requested to bring another fecal sample for the examination of Fcal. Both fecal samples were collected on the day or a few days before colonoscopy, and stored in a refrigerator until the day of colonoscopy. All of the patients had an established diagnosis of UC according to endoscopic and histologic assessments and had received adequate medical therapy. Mucosal status assessed via colonoscopy was compared with the results of FIT vs. Fcal.
Clinical disease activity was evaluated by using the Mayo score (Scoring Systems for Assessment of UC), consisting of the following four subscores: stool frequency (0, normal number of stools for this patient; 1, 1–2 stools more than normal; 2, 3–4 stools more than normal; and 3, ≥5 stools more than normal), rectal bleeding (0, no blood seen; 1, streaks of blood with stool less than half the time; 2, obvious blood with stool most of the time; and 3, blood alone passed), endoscopic findings (0, normal or inactive disease; 1, mild disease with erythema, decreased vascular pattern, mild friability; 2, moderate disease with marked erythema, absent vascular pattern, friability, erosions; and 3, severe disease with spontaneous bleeding, ulceration), and physician's global assessment (0, normal; 1, mild disease; 2, moderate disease; and 3, severe disease). Clinical remission was defined as a Mayo stool frequency subscore of 0 or 1 and a Mayo rectal bleeding subscore of 0. Patients who failed to fulfill the definition of clinical remission were considered to have clinically active disease.
FIT Analysis
Details of the method used for FIT have been described previously. In brief, patients collected fecal samples using an OC-Hemodia sampling probe (Eiken Chemical, Tokyo, Japan). Submitted stool samples were immediately processed and examined using OC-SENSOR neo (Eiken Chemical) that can accurately measure fecal hemoglobin concentrations from 50 to 1,000 ng/ml. Fecal specimens with a hemoglobin concentration of >1,000 ng/ml were measured by further dilution, whereas those with a hemoglobin concentration of <50 ng/ml were categorized as one (0–50 ng/ml) because FIT results are inaccurate when the hemoglobin concentration is <50 ng/ml.
Fcal Analysis
Fecal samples collected by the patients for calprotectin analysis were stored at −70 °C until shipment to the laboratory. The samples were sent to the Institute of Applied Technology for Innate Immunity (Kagawa, Japan), where calprotectin in stools is measured by a Phical Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Benshem, Germany). The quantitative range of calprotectin was between 0.65 and 84,000 μg/g after the appropriate dilution of fecal samples ranging from 1:50 to 1:100,000.
Colonoscopy
Bowel preparation was performed with a polyethylene glycol-based or magnesium citrate-based electrolyte solution according to the standard protocol in our hospital. After the colonic lavage fluid was cleared, patients underwent colonoscopy. Patients were excluded from the study if the colonoscopic examination was incomplete because of problems with the bowel preparation or if the colonoscope could not be inserted into the cecum.
The mucosal status of UC patients was assessed via the MES classification. Evaluation was performed at each portion of the colorectum (cecum and ascending colon combined, transverse colon, descending colon, sigmoid colon, and rectum), and the maximum score in the colorectum of each patient was used for analysis. The total inflammation score was defined as the sum of MES in the five colonic portions, and ranged from 0 (no inflammation) to 15 (severe and extensive inflammation). MH was defined as an MES of 0, or 0 or 1 throughout the colorectum. Some patients underwent biopsy from the portion with maximum endoscopic inflammation for pathological examinations. All colonoscopic examinations were performed by experienced colonoscopists who were blind to the results of the FIT and Fcal. In addition, the MES and the fecal results of each patient were determined independently by investigators who did not know patient's status including symptoms.
Pathologic Findings
Histologic studies were evaluated using Geboes scores by gastrointestinal pathologists. Geboes scores for assessment of UC histologic disease activity and MH are classified into 6 grades from grade 0 to grade 5: grade 0 (subgrades 0.0 to 0.3), structural (architectural changes); grade 1 (subgrades 1.0 to 1.3), chronic inflammatory infiltrate; grade 2, lamina propria neutrophils and eosinophils (subgrades 2A.0 to 2A.3 for eosinophils, subgrades 2B.0 to 2B.3 for neutrophils); grade 3 (subgrades 3.0 to 3.3), neutrophils in epithelium; grade 4 (subgrades 4.0 to 4.3), crypt destruction; grade 5 (subgrades 5.0 to 5.4), erosion or ulceration. When one or more biopsy specimens were evaluated on each patient, the highest score was used for analysis.
Statistical Analysis
Patient characteristics were analyzed by the JMP program (version 11.0 for Windows, SAS Institute, Cary, NC). Sensitivity, specificity, positive predictive value and negative predictive value with 95% confidence intervals (CIs) for detecting mucosal status were determined based on the FIT and calprotectin results. To estimate appropriate cutoff values for FIT and calprotectin, receiver operating characteristic (ROC) curve analysis was performed, and the area under the curve was calculated. Spearman's rank correlation test was performed to determine the correlation coefficient between the FIT values or Fcallevels and the Mayo endoscopic scores. All P values were two sided and considered statistically significant when <0.05.
Ethical Considerations
This study was approved by the institutional review board of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. Informed consent was obtained from each patient before bringing fecal samples.
