PO Box 2345, Beijing 100023, China World J Gastroenterol 2007 March 14; 13(10): 1569-1574
`www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327
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`
` RAPID COMMUNICATION
`
`Usefulness of fecal lactoferrin and hemoglobin in diagnosis of
`colorectal diseases
`
`Ichiro Hirata, Masahiro Hoshimoto, Osamu Saito, Masanobu Kayazawa, Takashi Nishikawa, Mitsuyuki Murano,
`Ken Toshina, Fang-Yu Wang, Ryoichi Matsuse
`
`Ichiro Hirata, Department of Gastroenterology, Fujita Health
`University, Japan
`Masahiro Hoshimoto, Osamu Saito, Masanobu Kayazawa,
`Takashi Nishikawa, Mitsuyuki Murano, Ken Toshina, Second
`Department of Internal Medicine, Osaka Medical College, Japan
`Fang-Yu Wang, Department of Gastroenterology, Jinling
`Hospital, Nanjing University School of Medicine, China
`Ryoichi Matsuse, Kyoto Medical Science Laboratory, Japan
`Correspondence to: Dr. Ichiro Hirata, Department of
`Gastroenterology, Fujita Health University, 1-98 Dengakugakubo
`Katsukake-Cho, Toyoake, Aichi 470-1192,
`Japan. ihirata@fujita-hu.ac.jp
`Telephone: +81-562-932345 Fax: +81-562-938300
`Received: 2006-12-19 Accepted: 2007-03-05
`
`© 2007 The WJG Press. All rights reserved.
`
`Key words: Feces; Lactoferrin; Hemoglobin; Diagnosis;
`Colorectal disease
`
`Hirata I, Hoshimoto M, Saito O, Kayazawa M, Nishigkawa T,
`Murano M, Toshina K, Wang FY, Matsuse R. Usefulness of
`fecal lactoferrin and hemoglobin in diagnosis of colorectal
`diseases. World J Gastroenterol 2007; 13(10): 1569-1574
`
` http://www.wjgnet.com/1007-9327/13/1569.asp
`
`Abstract
`AIM: To evaluate prospectively usefulness of fecal
`lactoferrin (Lf ) and fecal hemoglobin (Hb) in the
`diagnosis of colorectal diseases.
`
`METHODS: Fecal Lf and Hb were measured using
`ELISA in 872 patients before they underwent colorectal
`endoscopy.
`
`RESULTS: Lf was positive in 18 (50%) of 36 patients
`with colorectal cancer, 25 (15.9%) of 157 with colorectal
`polyps, 29 (46.8%) of 62 with ulcerative colitis, and 25
`(62.5%) of 40 (62.5%) with Crohn’s disease. The Hb-
`positive rates were 50%, 12.1%, 41.9% and 32.5%,
`respectively. Of the 318 patients free of abnormalities
`by colorectal endoscopy, Lf was positive in 29 (9.1%)
`and Hb was positive in 15 (4.7%). Among patients with
`Crohn’s disease, the Lf-positive rate was significantly
`higher than the Hb-positive rate. If either high Lf or Hb
`levels were considered positive, the positive rates rose
`to 61.1%, 51.6%, and 67.5% in the colorectal cancer
`group, ulcerative colitis group, and Crohn’s disease
`group, respectively. If both high Lf and Hb levels were
`rated positive, the positive predictive values (PPV) were
`21% for colorectal cancer, 33% for ulcerative colitis, and
`17% for Crohn’s disease, and PPV of high Hb level alone
`was 18%, 25% and 13%, respectively.
`
`CONCLUSION: Fecal Lf and Hb were found useful in
`the detection of colorectal diseases, and the combination
`of the two measurements appears to increase the
`sensitivity and efficacy of diagnosis.
`
`INTRODUCTION
`In the United States, colorectal cancer is the second
`leading cause of death[1]. In Japan, the prevalence of
`colorectal cancer and the percentage of this cancer among
`all cancer deaths have recently been rising as well. Because
`the prognosis of patients with colorectal cancer depends
`on the stage of cancer at the time of detection, screening
`for early diagnosis of colorectal cancer is considered
`essential[2,3]. In the United States, fecal occult blood
`testing by the guaiac method has been used as a screening
`method for colorectal cancer. A randomized study found
`that this method reduced the mortality rate of colorectal
`cancer[4,5]. In Japan, immunological fecal occult blood
`testing with a target of hemoglobin (Hb) has often been
`used in screening for colorectal cancer[6]. Fecal occult
`blood testing is also useful in the diagnosis and evaluation
`of various colorectal diseases other than colorectal
`cancer[7-9]. However, Hb in feces is unstable, which can
`be a cause of false-negative cases. Furthermore, Hb is
`not useful in the detection of lesions not accompanied
`by bleeding. For these reasons, a fecal marker with a high
`sensitivity and specificity has to be developed. Lactoferrin
`(Lf), which is released from neutrophil-specific granules,
`is stable in feces and is an excellent marker of activity of
`inflammatory bowel diseases such as ulcerative colitis and
`Crohn’s disease[10]. It has also been reported that fecal Lf
`level is higher in the patients with not only inflammatory
`bowel diseases but also colorectal tumors than in healthy
`individuals[11]. In a pilot study comparing fecal Lf level
`with fecal occult blood testing (immunological qualitative
`method) in 351 patients, Lf was found as useful as
`fecal occult blood testing in the diagnosis of colorectal
`diseases[12].
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`1570 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 14, 2007 Volume 13 Number 10
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`This prospective study involved a larger number of
`subjects than in previous studies to compare the usefulness
`of fecal Lf with that of fecal Hb as a quantitative
`parameter for the diagnosis of colorectal diseases and
`to evaluate the combination of fecal Lf and Hb using
`fecal samples collected from patients on the day before
`colorectal endoscopy.
`
`MATERIALS AND METHODS
`Subjects and fecal sampling method
`The subjects of this study were 872 patients scheduled to
`undergo colorectal endoscopy at the Second Department
`of Internal Medicine, Osaka Medical College University
`Hospital, who provided written informed consent to
`participate in the study. Their ages ranged from 12 to 90
`years (14 > 20 years; 34, 20-29 years; 51, 30-39 years; 116,
`40-49 years; 244, 50-59 years; 274, 60-69 years; 130, 70-79
`years; and 9 over 80 years). Examination for colorectal
`cancers and polyps was also performed in 657 subjects
`over 50 years of age. Patients with severe diseases of
`liver, gallbladder or pancreas were excluded from this
`study. Feces were collected from each patient on the day
`before colorectal endoscopy. The container used for fecal
`sampling had a stick type, designed to allow collection of
`about 10 mg feces into 1 mL buffer solution by thrusting
`the stick into feces at several points. On the day of the
`test, each subject was pretreated with intestinal lavage in
`the morning and underwent colorectal endoscopy in the
`afternoon. During endoscopy, the entire large intestine was
`examined, with biopsy performed as needed.
`
`Measurement of Lf and Hb
`Lf and Hb levels in feces were measured by sandwich
`ELISA, using 96-well microplates. For measurement
`of Lf, rabbit anti-human Lf antibody (Dakopatts,
`Glostrup, Denmark) and peroxidase-labeled anti-human
`Lf antibody were used, with tetramethylbendizine as a
`color developer[13]. For Hb, anti-human Hb antibody and
`alkaline phosphatase-labeled anti-human Hb antibody were
`used by the Kind-King method for color development[7].
`Concentrations were calculated, referring to the standard
`curves prepared from Lf originating from human
`colostrum (Cappel Co. Durham, NC) and Hb derived from
`the blood of healthy adults. All samples were subjected to
`measurement in blind fashion.
`
`Statistical analysis
`McNemar’s test was used for statistical analysis. P < 0.05
`was considered significant. For a given disease, sensitivity
`was defined as the positive rate, while specificity was
`defined as (number of true-negative cases)/(number of
`true-negative cases + number of false-positive cases). To
`compare the usefulness of fecal Hb and Lf, a operating
`characteristic analysis (ROC) was conducted. The ROC
`analysis involved preparing a sensitivity-false-positive
`rate curve and comparing areas under the curve (AUC),
`with an AUC of 0.5 uninformative and an AUC of 1
`perfect[13].
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`Table 1 Comparison of fecal Lf and Hb between normal and
`abnormal groups
`
`Group
`
`Normal group
`Fecal Hb
`
`+
`-
`Total
`Abnormal group
`Fecal Hb
`+
`-
`Total
`
` Fecal Lf
` + -
`
` Sum
`
`Specificity % (No.)
`
` 7
`22
`29
`
` 59
`105
`164
`
` 8
`281
`289
`
` 27
`363
`390
`
` 15
`303
`318
`
` 86
`468
`554
`
`Lf
` Hb
`P = 0.0176
`
` Lf
` Hb
`P < 0.0001
`
`90.9 (289/318)
`95.3 (303/318)
`
`29.6 (164/554)
`15.5 (86/554)
`
`Table 2 Sensitivity of fecal Lf and Hb in various diseases
`
`Diseases
`
`Colorectal cancer
`Colorectal polyp
`Ulcerative colitis
`Crohn’s desease
`Colorectal diverticulum
`Internal hemorrhoids
`Nonspecific colitis
`Other diseases1
`
` n
`
` 36
`157
` 62
` 40
` 73
`142
` 20
` 24
`
` Sensitivity
`Hb > 100
`% (No.)
`50.0 (18/36)
`12.1 (19/157)
`40.3 (25/62)
`32.5 (13/40)
` 1.4 (1/73)
` 4.2 (6/142)
`15.0 (3/20)
` 4.2 (1/24)
`
`Lf > 65
`% (No.)
`50 (18/36)
`15.9 (25/157)
`46.8 (29/62)
`62.5b (25/40)
`19.2b (14/73)
`27.5b (39/142)
`20 (4/20)
`41.7b (10/24)
`
`bP < 0.01 vs Hb. 1Including 4 cases of previous intestinal tuberculosis, 4 of
`submucosal tumor, 2 of ischemic colitis, 2 of rectal carcinoid, 2 of Behcet
`disease, 2 of rectal mucosal prolapse syndrome, 2 of Cronkhite-Canada
`syndrome, 2 of Cowden disease, 1 of mucosa-associated lymphoma, and 1 of
`periappendiceal abscess.
`
`RESULTS
`Diagnosis
`Of the 872 subjects, 554 were found to have abnormalities
`on colorectal endoscopy, while 318 were rated as free of
`abnormalities (Tables 1 and 2). Polyps were defined as
`adenomas over 5 mm in size. Individuals with colorectal
`polyps were classified as having colorectal polyps even
`accompanied with internal hemorrhoids and/or colorectal
`diverticula. Individuals with both internal hemorrhoids and
`colorectal diverticula were classified as cases of internal
`hemorrhoids.
`
`Establishment of cut-off levels for fecal Lf and Hb
`For the 25 healthy individuals, the mean + 2SD (the upper
`limit of the normal range) was 5.96 μg/g for fecal Lf and
`9.18 μg/g for fecal Hb[10]. In this study, feces were diluted
`1:100 with the buffer solution in the container, the mean +
`2SD for healthy individuals was equivalent to 59.6 ng/mL
`for Lf and 91.8 ng/mL for Hb. We therefore set the cut-
`off levels for this study at 65 ng/mL (Lf) and 100 ng/mL
`(Hb).
`The AUC of the ROC curve was greater for Lf (0.600)
`than for Hb (0.556), although this difference was not
`statistically significant.
`
`Analysis of groups with and without abnormalities
`In the abnormality-free group (n = 318), fecal Lf was
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`Hirata I et al. Fecal lactoferrin in colorectal diseases 1571
`
`Table 3 Diagnosis value using combination of fecal Lf and Hb
`
`Diseases
`
` n
`
` 36
`Colorectal cancer
`157
`Colorectal polyp
` 62
`Ulcerative colitis
` 40
`Crohn’s desease
`Colorectal diverticulum 73
`Internal hemorrhoids
`142
`Nonspecific colitis
` 20
`Other diseases
` 24
`
` Sensitivity
`Hb>100 or
`Hb>100 and
`Lf>65 % (No.)
`Lf>65 % (No.)
`61.1 (22/36)
`38.9 (14/36)
`25.5 (40/157)
` 2.5 (4/157)
`51.6 (32/62)
`35.5 (22/62)
`67.5 (27/40)
`27.5 (11/40)
`19.2 (14/73)
` 1.4 (1/73)
`28.9 (41/142)
` 2.8 (4/142)
`20.0 (4/20)
`15.0 (3/20)
`45.8 (11/24)
` 0.0 (0/24)
`
`Table 4 Positive predictive value (PPV) of fecal Lf and Hb in
`colorectal diseases
`
`Diseases
`
`
`
` 36
`Colorectal cancer
`157
`Colorectal polyp
` 62
`Ulcerative colitis
` 40
`Crohn’s desease
`Colorectal diverticulum 73
`Internal hemorrhoids
`142
`Nonspecific colitis
` 20
`Other diseases
` 24
`
` PPV
` n Hb > 100
`Hb > 100 and Lf>65
` % (No.)
` % (No.)
`17.8 (18/101)
` 21.2 (14/66)
`18.8 (19/101)
` 6.1 (4/66)
`24.8 (25/101)
` 33.3 (22/66)
`12.9 (13/101)
` 16.7 (11/66)
` 1.0 (1/101)
` 1.5 (1/66)
` 5.9 (6/101)
` 6.1 (4/66)
` 3.0 (3/101)
` 4.5 (3/66)
` 1.0 (1/101)
` 0.0 (0/66)
`
`Table 5 Comparison of fecal Lf and Hb in subjects aged over
`50 yr
`
`Table 6 Sensitivity of fecal Lf and Hb in colorectal cancer
`
`Normal group
`Fecal Hb +
`-
`Total
`Abnormal group
`Fecal Hb +
`-
`Total
`
` Fecal Lf
` + -
`
`Sum
`
` 7
` 18
` 25
`
` 32
` 76
`108
`
` 8
`209
`217
`
` 20
`287
`307
`
` 15
`227
`242
`
` 52
`363
`415
`
`Specificity
`
` % (No.)
`
`Lf
`Hb
`P = 0.0776
`
`Lf
`Hb
`P < 0.0001
`
`89.7 (217/242)
`93.8 (227/242)
`
`26.0 (108/415)
`12.5 (52/415)
`
`Colorectal cancer
`
`Total
`Early stage
` Right side
` Left side
`Advanced stage
` Right side
` Left side
`
`n
`36
`17
` 5
`12
`19
` 6
`13
`
`Note: n. number of cases.
`
` Sensitivity
`Hb % (No.)
`Lf % (No.)
`50.00 (18/36)
`50.00 (18/36)
`11.80 (2/17)
`29.40 (5/17)
`20 (1/5)
`40.00 (2/5)
` 8.30 (1/12)
`25.00 (3/12)
`84.20 (16/19)
`68.40 (13/19)
`66.70 (4/6)
`83.30 (5/6)
`92.30 (12/13)
`61.50 (8/13)
`
`positive in 29 cases (specificity: 90.9%) and fecal Hb was
`positive in 15 cases (specificity: 95.3%). Specificity was
`thus significantly higher for Hb than for Lf. In the group
`with abnormalities (n = 554), fecal Lf was positive in 164
`cases (sensitivity: 29.6%) and fecal Hb was positive in 86
`cases (sensitivity: 15.5%). Sensitivity was thus significantly
`higher for Lf than for Hb.
`
`Analysis in each disease group
`Sensitivities were compared between fecal Lf and Hb in
`each disease group (Table 2). The sensitivity of fecal Lf
`was significantly higher than that of fecal Hb in the Crohn’
`s disease, internal hemorrhoid, and colorectal diverticulum.
`There was no significant difference in sensitivity between
`Lf and Hb in the colorectal cancer, colorectal polyp, and
`ulcerative colitis.
`
`Diagnosis using a combination of fecal Lf and Hb
`When either high Hb or high Lf (Hb > 100 or Lf > 65)
`was rated positive, sensitivity in disease detection rose to
`61.1% for colorectal cancer, 25.5% for colorectal polyps,
`51.6% for ulcerative colitis, 67.5% for Crohn’s disease,
`19.2% for colorectal diverticulum, 28.9% for internal
`hemorrhoids, 20.0% for nonspecific colitis, and 45.8% for
`other diseases (Table 3). When both high Hb and high Lf
`(Hb > 100 and Lf > 65) were rated positive, the number
`of individuals rated positive among the 318 abnormality-
`free individuals decreased to 7 (Table 1). The positive rate
`by each disease group also decreased (Table 3), though
`the magnitude of the decrease was relatively mild in the
`colorectal cancer, Crohn’s disease, and ulcerative colitis.
`Positive predictive value (PPV) (number of positive cases
`in a given disease group divided by the number of all
`
`positive cases) rose to 21.2% (14/66) for colorectal cancer,
`33.3% (22/66) for ulcerative colitis, and 16.7% (11/66) for
`Crohn’s disease (Table 4).
`
`Analysis of subjects over ��� years of ageof subjects over ��� years of age subjects over ��� years of age
`
`
`Because examinations for colorectal cancer are usually
`performed in individuals aged 50 years or older, we
`performed an analysis confined to the 657 individuals
`aged over 50 years[14]. Among the 242 subjects free of
`abnormalities, Lf was positive in 25 (specificity, 89.7%)
`and Hb was positive in 15 (specificity, 93.8%), as shown
`in Table 5. Among the 415 patients with abnormalities, Lf
`was positive in 108 (sensitivity, 26.0%) and Hb was positive
`in 52 (sensitivity, 12.5%) (Table 5). We then examined the
`sensitivity in detection of colorectal cancer and polyps. In
`the 33 patients with colorectal cancer aged over 50 years,
`Hb was positive in 16 (sensitivity, 48.5%) and Lf was
`positive in 17 patients (sensitivity, 51.5%). When either
`high Lf or high Hb was rated positive, sensitivity rose
`to 60.6% for colorectal cancer and 25.2% for colorectal
`polyps. When both high Lf and high Hb were rated
`positive, PPV rose to 33.3% in the colorectal cancer group.
`
`Analysis of patients with colorectal cancer
`Of 36 patients aged 36-81 years with colorectal caner,
`14 had both Lf and Hb levels below the cut-off levels.
`Twelve of the 14 patients had colorectal cancer at an early
`stage confined to the submucosal layer. When analyzed
`by location (left or right side of the colon) and stage of
`cancer (early and advanced), the Hb-positive rate (12/13)
`was higher than Lf-positive rate (8/13) in patients with
`advanced left side colon cancer (Table 6). Among patients
`with right side colon cancer, however, the Lf-positive
`rate was slightly higher than the Hb-positive rate in both
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`1572 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 14, 2007 Volume 13 Number 10
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`early and advanced patients(2/5 vs 1/5 and 5/6 vs 4/6,
`respectively). Macroscopically, bloody stool was noted in
`10 of the 36 patients with colorectal cancer. Therefore,
`excluding these 10 cases, routine fecal test was enough for
`screening of colorectal cancer. Among the 26 cases, the
`Lf-positive rate (46.2%) was higher than the Hb-positive
`rate (38.5%).
`
`Analysis of patients with ulcerative colitis or Crohn’s
`disease
`Ulcerative colitis was considered in active stage if active
`lesions were revealed by colorectal endoscopy. Among
`the 38 cases of active ulcerative colitis, Lf and Hb were
`positive in 26 and 22 cases, respectively. Among the 24
`cases of inactive ulcerative colitis, Lf and Hb were positive
`in 3 and 3 cases, respectively.
`Crohn’s disease was considered active if the Crohn’s
`disease activity index (CDAI) was over 150 or if active
`lesions were detected by colorectal endoscopy[15]. Among
`the 26 cases of active Crohn’s disease, Lf and Hb were
`positive in 18 and 12 cases while in the 12 cases of inactive
`Crohn’s disease, Lf and Hb were positive in 8 cases and 1
`case, respectively. In patients with Crohn’s disease, minor
`active lesions of the small intestine often persist, but the
`CDAI is likely to be lower than 150 during treatment,
`often leading to the judgment that the patient has inactive
`disease clinically.
`
`DISCUSSION
`Lf is an ironbound protein with a molecular weight of
`about 80 000. It is found not only in neutrophil-specific
`granules but also in milk, tears, saliva, etc.[16,17]. The
`presence of Lf in the cytoplasm of colorectal cancer and
`adenoma cells has also been reported[18,19]. The high Lf
`level in the feces of patients with intestinal inflammation
`is believed to originate from the neutrophils which have
`infiltrated the intestinal mucosa[20-22]. The reason for
`elevation of fecal Lf level in patients with colorectal
`cancer has not yet been fully determined. It has been
`reported that some colorectal cancers are accompanied
`by local inflammatory reaction, and that leukocyte
`scintigraphy is sometimes positive in patients with colorectal
`cancer[23-25]. Neutrophil elastase and calprotectin, which are
`neutrophilic granular proteins, are absent in tumor cells,
`though their levels in feces of patients with colorectal
`cancer are high[26, 27]. Neutrophils thus appear to be a more
`important source of fecal Lf than exfoliated tumor cells in
`patients with colorectal cancer. In this study, the usefulness
`of fecal Lf in the diagnosis of various colorectal diseases
`was prospectively evaluated.
`Among the subjects free of abnormalities, the LF-
`positive rate was 9.1% (29/318) and the Hb-positive
`rate was 4.7% (15/318), both of which were higher than
`those in the general screening. In patients with symptoms
`such as diarrhea and abdominal pain, fecal Lf may be
`rated positive due to transient or minor inflammation not
`detectable with colorectal endoscopy, even when they are
`considered free of abnormalities by colorectal endoscopy.
`In patients who have been found positive for fecal occult
`blood before admission to the hospital, the percentage
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`of the patients who are fecal Hb-positive without any
`abnormality is high.
`Many of our patients were positive in fecal occult
`blood prior to visiting the hospital or visited the
`hospital complaining of macroscopically bloody stool.
`This suggests that patients with colorectal lesions of
`hemorrhagic subtype accounted for a high percentage
`of the subjects of this study. For example, colorectal
`cancers which likely cause bleeding or positive findings
`for fecal occult blood were found in a high percentage of
`patients with colorectal cancer in this study. Thus, based
`on the fact that Hb is expected to be more useful than
`Lf, the sensitivity of Lf was significantly higher than that
`of Hb in patients with abnormalities. Because this study
`involved quantitative measurement of both Lf and Hb, we
`compared the usefulness of Lf and Hb in the diagnosis
`of colorectal diseases by means of ROC analysis. A
`greater AUC was found in Lf than in Hb, although this
`difference was not significant. These findings suggest that
`Lf is comparable to or more useful than Hb in detecting
`colorectal diseases.
`By type of disease, the sensitivity of testing for Lf was
`comparable to that for Hb for colorectal cancer, colorectal
`polyps, ulcerative colitis, and nonspecific colitis. For Crohn’
`s disease, internal hemorrhoids, colorectal diverticulum,
`and other colorectal diseases, the sensitivity of Lf testing
`was significantly higher than that of Hb testing.
`In patients with Crohn’s disease, the Lf-positive rate
`was high even the disease was rated as inactive. It seems
`more rational to interpret this result as that inflammation
`of the intestine persists in patients with Crohn’s disease
`with high fecal Lf levels, rather than as a false-positive
`result for the following reasons: (1) in Crohn’s disease,
`the intestine often remains inflamed even after the CDAI
`decreases to below 150 after treatment; and (2) complete
`evaluation of residual inflammation of the intestine is
`difficult even with colorectal endoscopy. The “other
`diseases” mentioned above include important diseases such
`as old intestinal tuberculosis, submucosal tumor, rectal
`carcinoid, Behcet disease, Cronkhite-Canada syndrome,
`Cowden disease, and mucosa-associated lymphoid tissue
`lymphoma (MALT). Significantly higher sensitivity of
`testing for Lf than that for Hb in the “other diseases”
`group is clinically important.
`The results of this study suggest that it is possible
`to increase the accuracy of screening based on the
`conventional fecal occult blood test with a target set
`at Hb by combining Hb with Lf. For example, when
`immunological fecal occult blood testing is used for
`screening of colorectal cancer, feces are often sampled
`repeatedly to increase the sensitivity of detection.
`However, if a combination of Hb and Lf is used and
`results are considered positive in cases with high Lf or Hb,
`it is possible to perform a highly sensitive screening that
`requires only one fecal sampling. In addition, if results
`were considered positive in cases with both high Lf and
`Hb, PPV rose in the colorectal cancer, ulcerative colitis,
`and Crohn’s disease. If PPV is high, it will be easier to
`convince patients to take further examinations such as
`colorectal endoscopy, etc. because of the likelihood of the
`presence of colorectal disease.
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`Hirata I et al. Fecal lactoferrin in colorectal diseases 1573
`
`Hb is likely to be degraded by bacteria or enzymes
`contained in feces, resulting in loss of antigenicity. In
`buffer fluid, both fecal Lf and fecal Hb remained stable
`for 3 days, retaining 75% or greater activity compared
`to that recorded immediately after sampling[12]. In this
`study, feces were collected into a buffer fluid. For this
`reason, we believe that stability after sampling was not
`different between Lf and Hb in this study. In the intestine,
`however, Lf is believed to be more stable than Hb. We
`therefore, analyzed Lf- and Hb-positive rates by location
`of colorectal cancer, and found that Hb was highly
`sensitive in detecting advanced cancer of the left side of
`the colon, i.e., the sensitivity of Hb was lower in detection
`of colorectal cancer than in advanced cancer of the left
`side of the colon. This finding appears to be related to the
`following factors: (1) relatively low stability of Hb within
`the intestine, as noted above; and (2) the feces in the right
`portion of the colon are still too soft to cause bleeding.
`Although the sensitivity of Lf in detecting cancer of the
`left side of the colon was lower than that of Hb, it was
`higher in detecting cancers affecting other portions of
`the colon compared to Hb. The low sensitivity of fecal
`occult blood testing in the detection of early colorectal
`cancer has been noted in previous reports. In this study,
`only 2 of the 17 cases of early colorectal cancer were
`positive by this method. However, of these 17 patients
`with early colorectal cancer, 14 had been found in other
`clinics to be positive for fecal occult blood and visited our
`hospital for this reason. This suggests that in cases of early
`colorectal cancer, bleeding is often intermittent rather than
`continuous. On the other hand, Lf was positive in 5 of
`the 17 cases of early colorectal cancer, and its sensitivity
`in detecting early colorectal cancer was higher than that
`of Hb. Thus, the findings of the analyses by location and
`stage of colorectal cancer also suggest that a combination
`of Lf and Hb can compensate the shortcomings of
`these two parameters used individually for detection of
`colorectal cancer.
`Examinations for colorectal cancer are usually
`performed in individuals over 50 years of age. When
`analysis was confined to this age group, specificity of Lf
`was slightly lower (89.7%) than that of Hb (93.8%), while
`sensitivity of Lf was slightly higher (51.5%) than that
`of Hb (48.5%). When results were considered positive
`if both Lf and Hb were high, PPV rose to 33.3%. The
`combination of Lf and Hb is thus useful in judging
`whether an individual aged over 50 years has a high risk
`for colorectal cancer.
`Numerous studies have been published concerning
`noninvasive screening methods, which may replace the
`fecal occult blood test used for screening of colorectal
`caner [28,29]. A multi-target assay, designed to detect
`abnormal DNA in feces, exhibited a high sensitivity in
`detecting colorectal cancer, although no comparison of
`this assay had been made with fecal Hb measurement.
`Furthermore, this assay is too expensive for general use in
`clinics or health checkups[30]. Measurement of calprotectin,
`one of the neutrophil-specific granules, is highly sensitive
`in detecting colorectal cancer, though its specificity is low.
`Furthermore, no comparison of calprotectin with fecal Hb
`in detection of colorectal cancer has been performed[27,31].
`
`In conclusion, our results suggest that the usefulness
`of fecal Lf measurement appears comparable to that
`of fecal Hb measurement in detection of colorectal
`diseases. Furthermore, the combination of Lf and Hb
`measurement appears to increase the sensitivity and
`efficacy of diagnosis.
`
` COMMENTS
`Background
`The high Lactoferrin (Lf) level in the feces of patients with intestinal inflammation
`is believed to originate from the neutrophils which have infiltrated the intestinal
`mucosa. The presence of Lf in the cytoplasm of colorectal cancer and adenoma
`cells has also been reported. The reason for elevation of fecal Lf level in patients
`with colorectal cancer has not yet been fully determined. It has been found that
`some colorectal cancers are accompanied by local inflammatory reaction, and that
`leukocyte scintigraphy is sometimes positive in patients with colorectal cancer.
`Neutrophil elastase and calprotectin, which are neutrophilic granular proteins,
`are absent in tumor cells, although their levels are high in feces from patients
`with colorectal cancer. Neutrophils thus appear to be a more important source of
`fecal Lf than exfoliated tumor cells in patients with colorectal cancer. Therefore,
`the usefulness of fecal Lf in the diagnosis of various colorectal diseases was
`prospectively evaluated in this study.
`
`Research frontiers
`In the United States, fecal occult blood testing (FOBT) by the guaiac method has
`been used as a screening for colorectal cancer. A randomized study found that this
`method lowered the mortality rate of colorectal cancer. In Japan, immunological
`FOBT with a target of hemoglobin (Hb) has often been used in screening for
`colorectal cancer. FOBT is also useful in the diagnosis and evaluation of various
`colorectal diseases other than colorectal cancer. However, since Hb in feces
`is unstable, it may result in false-negative cases. Furthermore, Hb is not useful
`in the detection of lesions not accompanied by bleeding. For these reasons,
`development of a fecal marker with high sensitivity and specificity is needed.
`Lf, which is released from neutrophil-specific granules, is stable in feces and
`is an excellent marker of activity of IBD. Fecal Lf level has been reported to be
`higher in the patients with not only IBD but also colorectal tumors than in healthy
`individuals. In a pilot study comparing fecal Lf level with fecal occult blood testing
`in 351 individuals, Lf was found to be as useful as fecal occult blood testing in the
`diagnosis of colorectal diseases.
`
`Innovations and breakthroughs
`The measurement of fecal Lf and Hb was found to be useful in the detection of
`colorectal diseases, and the combination of the two measurements appears to
`increase the sensitivity and efficacy of diagnosis.
`
`Applications
`The combined measurement of fecal Lf and Hb should be useful for screening of
`colorectal diseases including colorectal cancer.
`
`Terminology
`Lf is an ironbound protein with a molecular weight of about 80 000. It is found not
`only in neutrophil-specific granules but also in milk, tears, saliva, etc. The high Lf
`level in the feces of patients with intestinal inflammation is believed to originate
`from the neutrophils which have infiltrated the intestinal mucosa.
`
`Peer review
`Hirata et al demonstrated the usefulness of fecal Lf and Hb levels for screening of
`colorectal cancer and IBD. This study might be clinically relevant. The reliability of
`their data is dependent on the presentation of determination method. More precise
`methods of their ELISA for lactoferrin and hemoglobin should be described.
`
`REFERENCES
`1 Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer
`statistics, 2001. CA Cancer J Clin 2001; 51: 15-36
`Rex DK, Johnson DA, Lieberman DA, Burt RW, Sonnenberg A.
`Colorectal cancer prevention 2000: screening recommendations
`
`2
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`www.wjgnet.com
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`
`8
`
`6
`
`of the American College of Gastroenterology. American
`College of Gastroenterology. Am J Gastroenterol 2000; 95:
`868-877
`3 McLoughlin RM, O'Morain CA. Colorectal cancer screening.
`World J Gastroenterol 2006; 12: 6747-6750
`4 Kronborg O, Fenger C, Olsen J, Jørgensen OD, Søndergaard
`O. Randomised study of screening for colorectal cancer with
`faecal-occult-blood test. Lancet 1996; 348: 1467-1471
`5 Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM,
`Schuman LM, Ederer F. Reducing mortality from colorectal
`cancer by screening for fecal occult blood. Minnesota Colon
`Cancer Control Study. N Engl J Med 1993; 328: 1365-1371
`Saito H. Screening for colorectal cancer: current status in
`Japan. Dis Colon Rectum 2000; 43: S78-S84
`7 Uchida K, Matsuse R, Miyachi N, Okuda S, Tomita S, Miyoshi
`H, Hirata I, Tsumoto S, Ohshiba S. Immunochemical detection
`of human blood in feces. Clin Chim Acta 1990; 189: 267-274
`Saitoh O, Matsumoto H, Sugimori K, Sugi K, Nakagawa
`K, Miyoshi H, Hirata I, Matsuse R, Uchida K, Ohshiba
`S. Intestinal protein loss and bleeding assessed by fecal
`hemoglobin, transferrin, albumin, and alpha-1-antitrypsin
`levels in patients with colorectal diseases. Digestion 1995; 56:
`67-75
`9 Howarth GF, Robinson MH, Jenkins D, Hardcastle JD, Logan
`RF. High prevalence of undetected ulcerative colitis: data
`from the Nottingham fecal occult blood screening trial. Am J
`Gastroenterol 2002; 97: 690-694
`10 Uchida K, Matsuse R, Tomita S, Sugi K, Saitoh O, Ohshiba S.
`Immunochem