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At present, fecal elastase-1 ELISA determination is the most sensitive and specific tubeless pancreatic function test available. However, the results are not available the same day in routine clinical practice. This prospective study aims at evaluating the sensitivity and specificity of the Elastase-1 Quick™ Test by comparing the results with the ELISA test.
Methods
The study was composed of three groups: the screening-diagnosed cystic fibrosis (CF) patients (n = 28), the screened, but non-CF subjects (n = 36) and non-screened CF patients (n = 62). Pancreatic status (normal vs abnormal) was evaluated using the Pancreas Elastase-1 Quick™ Test. Fecal elastase-1 concentration was determined with a commercially available ELISA kit, used as reference. The cut-off for abnormal results was set at <200 μg/g of stool.
Results
The Pancreatic Elastase-1 Quick Test™ showed the following sensitivities and specificities in the studied groups: 92.8% and 96.6% in all subjects, 90.5% and 100% in screening samples, and 92.8 and 90.5% in CF patients.
Conclusion
Pancreatic Elastase-1 Quick Test™ proves to be a rapid and reliable option to qualitatively evaluate pancreatic function for diagnostic purposes in a clinical setting of CF care.
Cystic fibrosis (CF) is a disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. As a result, the increased viscosity of mucus blocks the ducts of exocrine glands such as the lungs and pancreas. In the latter case, this leads to severe exocrine pancreatic insufficiency with maldigestion in about 85–90% of CF patients, called pancreatic insufficient (PI). Nevertheless, patients evaluated as being pancreatic sufficient (PS) do not present steatorrhea, but may demonstrate decreased pancreatic volume, abnormal bicarbonate concentration and altered enzyme secretion due to the fact that fat malabsorption may not appear until there is a 98% loss of pancreatic function [
]. PI is the cause of malnutrition, poor clinical status and survival, so it is crucial to rapidly and accurately determine pancreatic status in CF patients.
The diagnosis can be obtained by direct and indirect testing. Direct tests for estimation of pancreatic function such as the gold standard secretin-cholecystokinin test are unsuitable (time consuming, invasive, and expensive) for routine evaluation in children. Therefore several indirect tests, including fecal chymotrypsin, pancreolauryl test, mixed triglyceride breath test and fecal elastase-1 assessment, have been developed and evaluated for diagnosing pancreatic status [
]. Human pancreatic elastase-1 is an endoprotease and sterol binding protein that remains stable through the intestinal passage. Its concentrations in feces reach up to 5–6 times higher than in pancreatic juice, which indicates that it can serve as a diagnostic tool for evaluating exocrine pancreatic function. To date, the test has been shown to have the highest sensitivity among indirect tests [
Early delineation of PI subjects is crucial from a practical point of view. Moreover, the choice of the test should base on its sensitivity, specificity and noninvasiveness. Towards the need for rapid diagnosis of CF patients with pancreatic involvement, a novel test has been developed. This prospective study aims at evaluating the sensitivity and specificity of the Elastase-1 Quick Test by comparing the results with the ELISA test, the gold standard for non-invasive exocrine pancreatic function.
2. Methods
2.1 Subjects
The study was composed of three groups: the CF screened (28 subjects with CF aged from 1.5 to 3 months diagnosed in CF screening program), non-CF screened (36 subjects aged from 1.5 to 6.5 months in whom CF in the course of CF screening program was excluded) and CF non-screened (62 subjects diagnosed with CF in a classical way aged from 8 months to 25 years old) for a total of 126 subjects (53 females and 73 males) (Table 1). Participants were recruited at the Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan, Poland and the Lviv Cystic Fibrosis Centre, Lviv, Ukraine. The diagnosis of CF was based on history, clinical manifestation, increased sweat chloride concentrations, and CFTR gene examination. The genotypes of the CF patients were as follows: F508del/F508del (n = 30), F508del/2184insA (n = 11), F508del/unknown mutation (n = 8), F508del/N1303 K (n = 5), F508del/1898 + 1G-A (n = 4), F508del/2143delT (n = 4), F508del/G542X (n = 4), F508del/dele2,3(21 kb) (n = 3), F508del/3849 + 10kbC/T (n = 2), F508del/3272-26A > G (n = 2), F508del/2721del11 (n = 1), E585X/1524 + 1G > A (n = 1), F508del/R553X (n = 1), C524X/G542X (n = 1), F508del/L88IfsX22 (n = 1), F508del/185 + 1G > T (n = 1), F508del/R347H (n = 1), N1303K/2183AA-G (n = 1), F508del/W1282X (n = 1), 2183AA > G/E92K (n = 1), 3849 + 10kbC > T/F1074L (n = 1), F508del/G85E (n = 1), F508del/621-1G > T (n = 1), 621-1G > T/3849 + 10kbC > T (n = 1), F508del/V1001-I1005del (n = 1), D806G/unknown (n = 1), L257G/unknown (n = 1). An informed written consent from the parents of each patient was obtained.
Table 1Basic demographic data of the study subjects (n = 126).
Two small samples of feces were collected from the same stool sample of each subject. Fecal elastase-1 concentration was determined with a commercially available ELISA kit (ScheBo® Biotech AG, Giessen, Germany) [
]. The values of elastase-1 were rounded to the nearest integer and the cut-off value for abnormal results of the test was set at <200 μg/g in the stool. Evaluation of the pancreatic status was also done with the use of the Pancreas Elastase-1 Quick™ Test (ScheBo® Biotech AG, Giessen, Germany). The qualitative results of the test would present as two stripes for a positive (normal) result and one stripe for a negative (abnormal) result. The commercially available kits were kindly provided by ScheBo® Biotech AG.
The ScheBo Pancreas Elastase-1 Quick stool test is a visual immunochromatographic rapid test for the detection of pancreatic elastase-1 in stool samples. It is based on the same monoclonal antibodies as the Pancreatic Elastase-1 ELISA stool test. Elastase-1 in the stool sample reacts with a monoclonal antibody bound to gold particles. This complex migrates along the test membrane and reaches the test line (T) which has a second monoclonal antibody against elastase-1 attached. If the exocrine pancreatic function is normal (=high elastase-1 concentration) the gold labeled antibody + pancreatic elastase-1 complex binds to the test line (T) and a pink color develops. In the event of an exocrine pancreatic insufficiency (=low elastase 1 concentration) the sample does not contain antibody + elastase-1 complex that can bind to the test band (T) so no color becomes visible. Development of a pink control line (C) guarantees that sample application and migration have taken place correctly and that the test was properly performed. The appearance of two lines corresponds with fecal elastase-1 concentration >200 μg/g.
The test results are available within 5 min after stool sample extraction. Pancreas Elastase 1 Quick stool test is a convenient point-of-care test which can be performed at the bedside. The test can be stored at room temperature. The stool sample can be stored at room temperature for 1 week. The frozen stool sample is stable for 1 year at −20 °C.
The flow diagram describing the accuracy of the test was set according to STARD guidelines. The study was carried out in accordance with the Declaration of Helsinki. The protocol was approved by the Bioethical Committee of Poznań University of Medical Sciences, Poland (decision 181/15). The study was funded by the Poznań University of Medical Sciences, Poland.
3. Results
Fecal concentrations of elastase-1 measured using ELISA in subjects with normal and abnormal Elastase-1 Quick Test results are shown in Table 2. The discordant values of elastase-1 concentration in patients defined as abnormal by Elastase-1 Quick Test were 479; 492 [μg/g]. The discordant values of elastase-1 concentration in patients identified as having normal pancreatic function were 45; 112; 132; 184; 191 [μg/g]. The sensitivity and the specificity of Pancreas Elastase-1 Quick Test in comparison to classical elastase-1 ELISA test are summarized in Table 3.
Table 2Fecal elastase-1 concentrations (ELISA) in patients with normal and abnormal results of Elastase-1 Quick Test.
Table 3Sensitivity and specificity of Pancreas Elastase-1 Quick Test in the determination of normal/abnormal pancreatic function in comparison to the ELISA Elastase-1 test.
The flow diagrams describing the accuracy of the Pancreas Elastase-1 Quick Test are presented in Fig. 1. The frequency distribution of elastase-1 concentrations measured by the ELISA Elastase-1 test is shown in Fig. 2. The comparative characteristic of the Pancreas Elastase-1 Quick Test is given in Table 4.
Fig. 1Flow diagram of diagnostic accuracy of the Pancreas Elastase-1 Quick Test of screened (A), non-screened (B) and all samples (C).
Table 4The description of utility of the Pancreas Elastase-1 Quick Test in the determination of normal/abnormal pancreatic function in comparison to the ELISA Elastase-1 test for the whole CF group studied (n = 126).
Rapid evaluation of exocrine pancreatic function in CF patients allows for adequate intervention and should result in better disease outcomes. To date, ELISA fecal elastase-1 estimation shows superior value to other indirect pancreatic function tests. The present study is the first to evaluate the clinical utility of a novel Pancreas Elastase-1 Quick Test. The test assesses the pancreatic status with 92.8% sensitivity and 96.6% specificity in comparison to classical ELISA fecal elastase-1 test for all subjects studied. The obtained values prove high clinical applicability of Pancreas Elastase-1 Quick Test as a bedside screening tool.
Thus far, several indirect tests have been developed to assess pancreatic status including breath tests, acid startocrit, fecal elastase-1 and immunoreactive trypsinogen (IRT). However, the study by Durie et al. shows that IRT fails to determine pancreatic function in patients under 7 years old [
Age-related alterations of immunoreactive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic insufficiency.
]. The gold standard for the assessment of fecal fat loses among indirect tests is the quantitative 72-hour fecal fat excretion test, although the procedure is labor-intensive [
]. These tests are highly sensitive and specific, but only in evident steatorrhea. In cases of borderline values the use of aforementioned tests is vastly limited. Pancreatic Elastase-1 ELISA stool test has been shown to be the most reliable indirect test for the assessment of pancreatic function. The functional pancreatic status established by elastase-1 values early in life may not correspond to the patient's diagnosis later on, as the pancreatic function may deteriorate with age [
]. This is especially true for CF babies diagnosed in CF neonatal screening program for whom we should have an immediate answer. In many CF centers the samples are analyzed for fecal elastase-1 once/twice per year or even less frequently. Therefore, a simple and rapid test to evaluate the function on a regular basis would be helpful in the proper assessment of pancreatic status.
Although high sensitivity and specificity values were obtained in the present study, two of the patients showed high elastase-1 values (479; 492 [μg/g]) still being defined as an abnormal result by the Elastase-1 Quick Test. Likewise, some patients identified as having normal pancreatic function were below the cutoff value of 200 μg/g in stool (45; 112; 132; 184; 191 [μg/g]). It is worth noting that two of these values fall within the range of the 10% margin of the test error and four of them were higher than the cutoff level (100 μg/g) for PI status [
]. Since improper assessment of stool sample with low fecal elastase-1 concentration as defined with Pancreas Elastase-1 ELISA Test was surprising for us we repeated the Pancreas Elastase-1 Quick Test from the second available stool sample. Abnormal pancreatic function was documented twice.
The principle of the Elastase-1 Quick Test is based on chromatographic separation and requires only a small sample of the stool to obtain the results. Moreover, there is no need to discontinue the enzyme replacement therapy for the patient as it does not affect the test result. The indisputable advantage of the test is the qualitative result that can be obtained within minutes.
In conclusion, the present study proves that the Elastase-1 Quick Test can be a useful tool in a bedside detection of abnormal pancreatic status and can therefore serve in a clinical setting for rapid identification of CF patients requiring pancreatic enzyme replacement therapy.
Competing financial interests
The authors declare no competing financial interests.
Author contributions
Jarosław Walkowiak and Aleksandra Lisowska have made substantial contributions to the conception and design of the study, all authors have contributed to the data acquisition, analysis and interpretation, Aleksandra Glapa and Jarosław Walkowiak drafted the article, Jan Krzysztof Nowak and Aleksandra Lisowska revised it critically for important intellectual content, all authors finally approved the version to be submitted.
Acknowledgements
The study was supported by Poznań University of Medical Sciences, Poland (grant number 502010110311507588). The commercially available kits were kindly provided by ScheBo® Biotech AG.
Age-related alterations of immunoreactive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic insufficiency.
Months for screening, years in non-screening patients.
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