TY - JOUR
AU1 - Wang,, Sen
AU2 - Wang,, Xian
AU3 - Tang,, Shengnan
AU4 - Chen,, Yuxin
AU5 - Jia,, Jia
AU6 - Chen,, Junhao
AU7 - Shen,, Han
AU8 - Bai,, Bing
AB - Abstract Background The sensitivity of the commonly used immunochromatographic fecal occult blood test (iFOBT) is critical in the routine screening of digestive tract cancers. Objective To determine whether the performance of the iFOBT might be improved by optimizing detergents and buffers in stool solution. Methods We tested different buffers and detergents in stool solution. The specificity of the new solution condition was confirmed by dot immunoblotting. A total of 1122 clinical stool specimens were collected to evaluate the clinical application of this refined stool solution. Results The different salts exerted minor effects; also, sodium dodecyl sulfate (SDS) prevented the test from working as designed. However, 1% Triton X-100 improved the sensitivity significantly, as confirmed by dot immunoblot testing. With 1% Triton X-100 in the stool solution, the rate of positive iFOBT results in patients with digestive tract cancers increased, from 40.4% to 49.4%. Conclusion Use of 1% Triton X-100 improves the sensitivity of the iFOBT and thus, this substance should be routinely included in iFOBT for screening of digestive tract cancers. FOB, Triton X-100, immunochromatography, hemoglobin, digestive tract cancer, detergent Digestive tract cancers are a leading cause of human death worldwide.1 A common assay performed in the routine screening of these cancers is the immunochromatographic fecal occult blood test (iFOBT).2 However, the sensitivity of this type of test might be limited.3 Fecal specimens might have different pH, salt, and water content; also, other microenvironmental conditions exist in feces that are largely different from those in red blood cells (RBCs) and plasma.4,5 These conditions might lead to denaturing of hemoglobin, especially when blood is secreted into the stool long before its excretion, making hemoglobin difficult to dissolve completely in the fecal solution during the iFOBT. Therefore, the salts, buffers, and detergents in the stool solution of the iFOBT kit could be optimized, to achieve higher iFOBT sensitivity for more-frequent, more-accurate discovery of digestive tract cancers.6 Sodium dodecyl sulfate (SDS) is widely known to be efficient in solubilizing proteins but affects antigen-antibody binding due to its denaturing power. For these reasons, we initially decided to use 0.1% SDS in fecal solution to facilitate hemoglobin dissolving and then to neutralize it using 1% Triton X-100 (The Dow Chemical Company) for the subsequent iFOBT.7-9 Meanwhile, we also wanted to test other, different salt and buffer conditions in the fecal solution that might enhance the sensitivity of this test. Materials and Methods Human Hemoglobin Specimen and Stool Solutions We centrifuged 100 µL of fresh human whole blood, collected using ethylenediaminetetraacetic acid (EDTA) anticoagulant, at 800 g for 1 minute to collect the cells and then washed the resulting material in saline 3 times. After that, we resuspended the RBCs in 1 mL 10 mM tris(hydroxymethyl)aminomethane (Tris)–HCl buffer, pH 7.4, to release the hemoglobin from the RBCs. After centrifugation at 20,000 g for 3 minutes, the supernatant was collected and diluted by experimental solutions, as indicated in the following paragraphs. We tested 9 different solution conditions, including the solution supplied by the iFOBT kit, saline, Tris buffer (50 mM; pH, 7.4), 0.1% SDS in Tris buffer, 1% Triton X-100 in Tris buffer, 0.1% Tween 20 in Tris buffer, 1% Tween 20 in Tris buffer, 0.1% SDS and 1% Triton X-100 in Tris buffer, and 0.1% SDS and 1% Tween 20 in Tris buffer. We purchased all the detergents (Triton X-100 [T9284], Tween 20 [P7949], SDS [L6026]) from Sigma-Aldrich Corporation. Immunochromatographic Fecal Occult Blood Tests We performed all procedures according to the instructions provided in the kit by the manufacturer, as is standard in iFOBTs. Briefly, for each stool specimen, the specimen-collection stick pierced the feces at multiple different positions. Then, the stick was inserted into the stool solution to dissolve thoroughly by gentle vortexing and left on a shaker for 30 minutes at room temperature. After that, the iFOBTs were performed using commercial strips (ABON Biopharm Co., Ltd.). The 3 other iFOBT strips were from Deep Blue Medical Technology Co., Ltd.; Yiyou Biotech Co., Ltd.; and Boson Biotech Co., Ltd. All of these strips were coated with mouse monoclonal antibodies against human hemoglobin. Dot-Blot Assay We added 2 µL of serially diluted hemoglobin specimens from lysed human blood cells or fecal solution from stool specimens onto a nitrocellulose membrane. The membrane was dried in a 37°C incubator for 5 minutes and then blocked by 5% skim milk in PBST (phosphate-buffered saline with 0.05% Tween 20) buffer for 15 minutes. We added the rabbit monoclonal antibody (84934S, Cell Signaling Technology) that targets human hemoglobin to incubate with the membrane for 2 hours and then washed the product before further incubation with a horseradish peroxidase (HRP)–conjugated secondary antibody (H2014, Santa Cruz Biotechnology) for 30 minutes. Finally, the signal was developed by the enhanced chemiluminescent (ECL) substrate (32109; Thermo Fisher Scientific). Clinical Fecal Specimens Fecal specimens were unselectively and continuously collected in approximately 1 month, largely from inpatients with diseases such as lung cancer, heart failure, hepatitis, gallstones, colon inflammation, and neurological disorders. The FOB tests were generally completed within 1 hour after specimen arrival. Strip-Band Intensity Quantification, Statistical Analysis, and Ethics Committee Approval We quantified the intensities of the iFOBT bands and the immunoblotting dots using the software Image J (National Institutes of Health: https://imagej.nih.gov/ij/). On each strip, the intensity of the test band (which detects human hemoglobin) was divided by the intensity of the control band (which indicates the success of this test) for normalization. We performed statistical analyses using GraphPad (GraphPad Software, Inc.) or Microsoft Office Excel 2013 (Microsoft Corporation) software. This study was approved by the ethics committee of the Drum Tower Hospital, which is affiliated with Nanjing University Medical School, China. Results Triton X-100 and the Sensitivity of iFOBT Using human hemoglobin prepared from human blood cells, we tried 9 different solution conditions, including different combinations of detergents (SDS, Triton X-100, and Tween 20) and buffers (the kit solution, saline, and Tris) (Figure 1A). We discovered that the different buffers yielded similar results, whereas 0.1% SDS provided a much weaker test band. In contrast, 1% Triton X-100 yielded a stronger test band than any other results under different buffer and detergent conditions. Figure 1 View largeDownload slide Effects of different buffers and detergents on the immunochromatographic fecal occult blood test (iFOBT). A, iFOBT results with selected buffers and detergents in the specimen solution. The specimen used was hemoglobin (50 ng/mL) extracted from human red blood cells. The arrow indicates the test band. We used 1-way analysis of variance (ANOVA) (n = 5 for each solution); P <.01. B, Failed iFOBT using 0.1% sodium dodecyl sulfate (SDS) in the specimen solution. We added 0.1% to the fecal solution to assist dissolving of the hemoglobin in the stool specimen. Later, we added 1% Triton X-100 (The Dow Chemical Company) to neutralize the SDS before performing the iFOBT. Figure 1 View largeDownload slide Effects of different buffers and detergents on the immunochromatographic fecal occult blood test (iFOBT). A, iFOBT results with selected buffers and detergents in the specimen solution. The specimen used was hemoglobin (50 ng/mL) extracted from human red blood cells. The arrow indicates the test band. We used 1-way analysis of variance (ANOVA) (n = 5 for each solution); P <.01. B, Failed iFOBT using 0.1% sodium dodecyl sulfate (SDS) in the specimen solution. We added 0.1% to the fecal solution to assist dissolving of the hemoglobin in the stool specimen. Later, we added 1% Triton X-100 (The Dow Chemical Company) to neutralize the SDS before performing the iFOBT. The mixture of 0.1% SDS and 1% Triton X-100 did not affect our results considerably; therefore, we used 0.1% SDS to dissolve the stool specimen for 30 minutes at room temperature, according to our initial design, and then added 1% Triton X-100 to neutralize the SDS before the test. We were surprised to discover that this protocol almost completely abolished the test bands (Figure 1B), probably because the SDS denatured the hemoglobin in the solution and because the antibody on the strip only recognizes hemoglobin in its native form. Evaluated iFOBT Sensitivity with Use of Triton X-100 To determine the sensitivity of the assay in the presence of Triton X-100, we prepared 400 ng per mL human hemoglobin from RBCs and serially diluted it by 2-fold (Figure 2A). In the results, without Triton X-100 in the stool solution, the test bands were observable across the dilutions down to 12.5 ng per mL. In contrast, when 1% Triton X-100 was added to the solution, the test bands could be further observed until 6.3 ng per mL of hemoglobin. More experiments with statistical analysis confirmed the increased sensitivity in the presence of Triton X-100 (Figure 2B). Figure 2 View largeDownload slide Increased sensitivity of immunochromatographic fecal occult blood test (iFOBT) using Triton X-100 (The Dow Chemical Company). A, iFOBT sensitivity in the presence or absence of 1% Triton X-100 in the specimen solution. B, The results of further sensitivity experiments and statistical analysis; we used 2-way analysis of variance (ANOVA) (n = 5); P <.05. C, The Triton X-100 effect occurred in strips from 3 other commercial iFOBTs. D, Analysis of the results from all iFOBT strips. Paired Student t testing was performed for each commercial strip; P <.01 (indicated by *) in all 4 tests. Figure 2 View largeDownload slide Increased sensitivity of immunochromatographic fecal occult blood test (iFOBT) using Triton X-100 (The Dow Chemical Company). A, iFOBT sensitivity in the presence or absence of 1% Triton X-100 in the specimen solution. B, The results of further sensitivity experiments and statistical analysis; we used 2-way analysis of variance (ANOVA) (n = 5); P <.05. C, The Triton X-100 effect occurred in strips from 3 other commercial iFOBTs. D, Analysis of the results from all iFOBT strips. Paired Student t testing was performed for each commercial strip; P <.01 (indicated by *) in all 4 tests. Next, we further investigated this effect of Triton X-100 by using 3 more commercial kits to test the extracted human hemoglobin specimen with or without 1% Triton X-100 in the specimen solution. The strips from all 3 kits consistently showed stronger test bands in the presence of Triton X-100 (Figure 2C). Statistical analysis of the results from more specimens yielded a significant difference, confirming the enhancing effect of Triton X-100 (Figure 2D). Validated Effect of Triton X-100 in Testing Clinical Specimens We further investigated whether the effect of Triton X-100 on human stool specimens would persist, so that we could determine whether to recommend adding Triton X-100 to specimen solution for future clinical application. Indeed, 1% Triton X-100 in the stool solution enhanced sensitivity by approximately 2-fold (Figure 3A), which is consistent with our previous results (Figure 2A). We tested more clinical stool specimens for comparison, which yielded similar results, supporting the practical use of Triton X-100 in clinical specimens (Figure 3B). Figure 3 View largeDownload slide Validated effect of Triton X-100 (The Dow Chemical Company) on clinical fecal specimens. A, 1% Triton X-100 increased the sensitivity of the immunochromatographic fecal occult blood test (iFOBT) when testing a clinical stool specimen. The stool specimen was dissolved in the kit solution and then serially diluted by 2-fold in the solution with or without 1% Triton X-100. B, More clinical stool specimens were tested in the presence (+) or absence (−) of 1% Triton X-100 in the fecal solution. Figure 3 View largeDownload slide Validated effect of Triton X-100 (The Dow Chemical Company) on clinical fecal specimens. A, 1% Triton X-100 increased the sensitivity of the immunochromatographic fecal occult blood test (iFOBT) when testing a clinical stool specimen. The stool specimen was dissolved in the kit solution and then serially diluted by 2-fold in the solution with or without 1% Triton X-100. B, More clinical stool specimens were tested in the presence (+) or absence (−) of 1% Triton X-100 in the fecal solution. Confirmed Specificity of the Effect of Triton X-100 in Dot Immunoblotting To determine whether the iFOBT results in the presence of Triton X-100 were specific, we initially performed Western blot. No signal was detected, despite that highly concentrated human hemoglobin specimens were used, probably because the antibody could not recognize the denatured hemoglobin. Therefore, we directly added the specimen material to the solution, with 1% Triton X-100, placed it onto a nitrocellulose membrane, and conducted the dot immunoblot testing. Correlation of the dot intensities and the test band intensities for the extracted human hemoglobin specimens yielded a high correlation coefficient (R2 = 0.90) (Figure 4A). Also, consistently, we achieved correlative results using iFOBTs and dot immunoblots on patient fecal specimens (Figure 4B). Figure 4 View largeDownload slide Evaluated specificity of the effect of Triton X-100 (The Dow Chemical Company). A, Dot-blot immunoassay to confirm the immunochromatographic fecal occult blood test (iFOBT) specificity in the presence of Triton X-100; the specimen was extracted hemoglobin from human red blood cells. B, Dot-blot immunoassay and iFOBT of clinical stool specimens showed that those specimens dissolved in the presence of 1% Triton X-100. C, iFOBTs of animal stool specimens with or without human hemoglobin (50 ng/mL) added, and with or without 1% Triton X-100 in the stool solution. Figure 4 View largeDownload slide Evaluated specificity of the effect of Triton X-100 (The Dow Chemical Company). A, Dot-blot immunoassay to confirm the immunochromatographic fecal occult blood test (iFOBT) specificity in the presence of Triton X-100; the specimen was extracted hemoglobin from human red blood cells. B, Dot-blot immunoassay and iFOBT of clinical stool specimens showed that those specimens dissolved in the presence of 1% Triton X-100. C, iFOBTs of animal stool specimens with or without human hemoglobin (50 ng/mL) added, and with or without 1% Triton X-100 in the stool solution. It is difficult to obtain human stool specimens with absolutely no hemoglobin. Therefore, we obtained stools from mice, rats, and rabbits from our animal core facility as human hemoglobin–free specimens, for evaluation of the iFOBT result specificity in the presence of Triton X-100. None of those specimens showed any positive iFOBT results, even in the presence of Triton X-100 (Figure 4C). However, when 50 ng per mL human hemoglobin was added to those specimens, the results were positive and were stronger in the presence of 1% Triton X-100. Application of Triton X-100 in Large-Scale Clinical iFOBTs We unselectively recruited 1122 clinical stool specimens from inpatients with different types of diseases and tested each of the specimens in the presence or absence of 1% Triton X-100 in iFOBTs (Figure 5A). Without Triton X-100, 293 specimens tested positive. In contrast, when Triton X-100 was added to the stool solution, 121 additional specimens yielded positive results, increasing the overall positivity rate from 26.1% to 36.9% (Figure 5B). We consider it notable that none of the specimens that had tested positive via the regular kit solution, without Triton X-100, showed a negative result when Triton X-100 was added. This finding strongly suggests the specificity of the effect of Triton X-100. Results of χ2 testing showed a statistical difference between the stool solutions with and without Triton X-100 (Figure 5B). Figure 5 View largeDownload slide Application of Triton X-100 (The Dow Chemical Company) in large-scale (n = 1122) clinical immunochromatographic fecal occult blood tests (iFOBTs). A, Workflow of the experimental design. B, Comparison of the iFOBT results in the presence or absence of 1% Triton X-100. We performed χ2 testing; P <.01. C, Positive rates of iFOBT results with 1% Triton X-100 or no Triton X-100. Each dot represents patients with diseases at a same bodily location (such as digestive tract, heart, lung, brain, kidney, nervous system, blood, spine, thyroid, throat, bladder, uterus, knee, prostate, leg, breast, ovary, cervix, spermatic cord, ureter, and chest); or with a same systemic disease (hypertension, diabetes); or individuals who received routine physical examinations. Only the bodily locations at which more than 10 case individuals were affected were selected for inclusion. D, The positive iFOBT rates of different diseases in the digestive tract. Figure 5 View largeDownload slide Application of Triton X-100 (The Dow Chemical Company) in large-scale (n = 1122) clinical immunochromatographic fecal occult blood tests (iFOBTs). A, Workflow of the experimental design. B, Comparison of the iFOBT results in the presence or absence of 1% Triton X-100. We performed χ2 testing; P <.01. C, Positive rates of iFOBT results with 1% Triton X-100 or no Triton X-100. Each dot represents patients with diseases at a same bodily location (such as digestive tract, heart, lung, brain, kidney, nervous system, blood, spine, thyroid, throat, bladder, uterus, knee, prostate, leg, breast, ovary, cervix, spermatic cord, ureter, and chest); or with a same systemic disease (hypertension, diabetes); or individuals who received routine physical examinations. Only the bodily locations at which more than 10 case individuals were affected were selected for inclusion. D, The positive iFOBT rates of different diseases in the digestive tract. If increased sensitivity occurs purely because of the enhanced detection limit of hemoglobin, the increase in positive rates should depend solely on the amount of hemoglobin in the stool, with no regard for any particular types of diseases. Therefore, we categorized all cases based on the locations of the lesions on/in the body (such as the digestive tract, heart, lungs, and brain) and selected the category that included more than 10 cases for the analysis. As we expected, the rates of positive results in all categories demonstrated a nearly parallel shift, namely, an approximately 10% increase (Figure 5C). Next, we further examined the major diseases in the digestive tract and discovered that the positive rates increased from 40.4% to 49.4% for cancer and 47.4% to 52.6% for tumors to be pathologically characterized. This finding suggests an increased positive predictive value of this test in the presence of Triton X-100 (Figure 5D). Discussion In this study, we examined the effects of salts, buffers, and detergents in the stool solution on the performance of the iFOBT. Although the salts and buffers had minor effects, the detergents exerted greater effects—for instance, 1% Triton X-100 increased the sensitivity of the assay significantly. This effect of Triton X-100 might be related to its influence on antigen stabilization and protein residual on the membrane during chromatography. It is known that Triton X-100 surrounds proteins and maintains them in their native forms,10 to preserve the conformational epitope. As a detergent, Triton X-100 also reduces the viscosity of the specimen solution, as well as the nonspecific binding between the proteins and the nitrocellulose membrane. This makes the chromatography on the strip much more efficient, leaving less protein residual and, thus, carrying more antigens in the flow for testing. Indeed, in our preliminary study, we examined the strips after the iFOBT by Western blotting and found less residual hemoglobin on the membrane when Triton X-100 was present in the specimen solution. Although a positive iFOBT result might be attributed to many factors,11-15 any improved sensitivity of this test with no compromised specificity will certainly increase its ability to detect digestive tract cancers. Therefore, we recommend that laboratory technicians routinely add 1% Triton X-100 to stool solution when performing clinical iFOBTs. Abbreviations iFOBT immunochromatographic fecal occult blood test RBCs red blood cells SDS sodium dodecyl sulfate EDTA ethylenediaminetetraacetic acid Tris tris(hydroxymethyl)aminomethane; HRP horseradish peroxidase ECL enhanced chemiluminescent Acknowledgments We thank all the laboratory members for their helpful discussions with us. This work was partially supported by the Chinese Fundamental Research Funds for the Central Universities (021414380299, to B.B.) and the Nanjing Drum Tower Hospital Research Initiation (RE445, to B.B.). References 1. Siegel RL , Miller KD , Jemal A . Cancer statistics, 2018 . 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Non-neoplastic findings at colonoscopy after positive faecal occult blood testing: data from the English Bowel Cancer Screening Programme . J Med Screen . 2014 ; 21 ( 2 ): 89 – 94 . Google Scholar Crossref Search ADS PubMed © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Study of Immunochromatographic Fecal Occult Blood Test Methods
JF - Laboratory Medicine
DO - 10.1093/labmed/lmy057
DA - 2019-04-08
UR - https://www.deepdyve.com/lp/oxford-university-press/a-study-of-immunochromatographic-fecal-occult-blood-test-methods-mHJG7Ehg7O
SP - 130
VL - 50
IS - 2
DP - DeepDyve
ER -