Q J Med 2013; 106:117–131

doi:10.1093/qjmed/hcs186 Advance Access Publication 24 October 2012

Review

Indications, stains and techniques in chromoendoscopy

P.J. TRIVEDI1,2 and B. BRADEN2
From the 1Centre for Liver Research and NIHR Institute of Biomedical Research, 5th Floor IBR
Building, University of Birmingham, Birmingham B15 2TT, UK and 2Translational Gastroenterology
Unit, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK

Address correspondence to Prof. Barbara Braden, FEBG Translational Gastroenterology Unit, John Radcliffe
Hospital, Headley Way, Oxford OX3 9DU, UK. email: braden@em.uni-frankfurt.de

Summary
Early detection of malignancies within the gastro-
intestinal tract is essential to improve the prognosis
and outcome of affected patients. However, conven-
tional white light endoscopy has a miss rate of up to
25% for gastrointestinal pathology, specifically in
the context of small and flat lesions within the
colon. Chromoendoscopy and other advanced ima-
ging techniques aim at facilitating the visualization
and detection of neoplastic lesions and have
been applied throughout the gastrointestinal tract.
Chromoendoscopy, particularly in combination
with magnifying endoscopy has significantly
improved means to detect neoplastic lesions in the
gastrointestinal mucosa, particularly in ulcerative
colitis and Crohn’s colitis. In addition,
chromoendoscopy is beneficial in the upper gastro-
intestinal tract, especially when evaluating Barrett’s
oesophagus (BO) for the presence of dysplasia.
Furthermore, it also improves characterization, dif-
ferentiation and diagnosis of endoscopically
detected suspicious lesions, and helps to delineate
the extent of neoplastic lesions that may be amen-
able to endoscopic resection. This review discusses
the dyes, indications and advanced endoscopic
imaging methods used in various chromoendo-
scopic techniques, and presents a critical overview
of the existing evidence supporting their use in cur-
rent practice with a particular emphasis on the role
in inflammatory bowel disease and BO.

reducing the incidence of colon cancer by 76–

Introduction
Chromoendoscopy aims at a facilitated visualization
and detection of dysplastic and malignant lesions
in the gastrointestinal tract, which can be difficult to
distinguish from normal mucosa. Chromoendoscopy
is a diagnostic method in which, a chemical sub-
stance is sprayed onto the mucosal surface of the
gastrointestinal tract to highlight specific areas or
distinguish among different types of epithelia.
Screening colonoscopy for bowel cancer is an
example of how chromoendoscopy can increase
the diagnostic potential. Although standard defin-
ition ‘white light’ colonoscopy is efficient in
90%, it may miss significant numbers of smaller,
flat lesions, particularly on the right side. As a
result, anywhere between 10 and 24% of individ-
uals may still develop interval colorectal cancer.1,2
Polypoid adenomas are usually easy to detect
whereas only 20–50% of flat, intra-epithelial neo-
plasias may be picked up via conventional
colonoscopy.3,4
Chromoendoscopic techniques improve the recog-
nition of minute changes in the surface pattern by
enhancing the contrast of raised and deepened
areas. The stains used can be subdivided into ‘absorp-
tive’ or ‘vital’ stains, which, as the name suggests,

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118 P.J. Trivedi and B. Braden
are absorbed into tissue or into ‘non-absorptive’ con-
trast stains, which simply pool into mucosal tissue
allowing better visual definition. Other agents are
based on chemical reactions that indicate special
functions of the underlying tissue and are eponym-
ously named ‘reactive’ stains. Advanced imaging,
such as magnification endoscopy, allows brilliant
resolution of suspicious structures; although this
latter technique is not widely available outside spe-
cialist units. Nonetheless, most chromoendoscopic
methods can also be applied to standard endoscopes
in an effort to improve the detection rate of
pre-malignant lesions. Chromoendoscopy is particu-
larly helpful in surveillance programmes aiming to
detect dysplasia and pre-neoplastic lesions [e.g. in
Barrett’s oesophagus (BO) or inflammatory bowel dis-
ease (IBD)] with the diagnostic yield of targeted
‘smarter’ biopsies being superior to random biop-
sies,5,6 thus reducing the histopathologic workload
and potentially offsetting the costs for additional
procedure time.
In this article, we summarize the indications, dyes
and methodologies of various chromoendoscopic
techniques, providing a critical review of the exist-
ing evidence pertaining to IBD and BO, as well as a
comparison against digital, high-definition and vir-
tual endoscopic techniques. Articles have been
identified using PubMed, Medline and Ovid search
engines, alongside pre-existing clinical management
protocols and guidelines.
Equipments and general
chromoendoscopic techniques
The staining substances are generally inexpensive
and readily available, although not specifically mar-
keted for chromoendoscopy. Spraying catheters
allow the most controlled and precise application
of the dye as a fine mist onto the gastrointestinal
surface. They are disposable, flexible plastic sheaths
with a luer lock and metal nozzle tip. A good bowel
preparation is conditio-sine-qua-non for chromoen-
doscopy in the colon and up to 13% of patients may
not meet this requirement.4 Some staining tech-
niques also require pre-treatment with N-acetylcys-
teine as mycolyticum to clear excess mucus from
the mucosal surface. The use of n-butylscopolamine
is recommended to avoid bowel peristalsis and an
uneven distribution of the dye.
The amount of staining solution required, de-
pends on the surface area to be stained (e.g. pan-
colonic staining; or further characterization of a
single lesion), but the smallest amount necessary
should be applied to avoid dye pooling. For pan-
colonic staining, the endoscope is slowly
withdrawn, while the endoscope tip with a 1–2 cm
protruding spraying catheter is directed in spiral
movements onto the mucosa and simultaneously,
the dye is constantly sprayed. Step-by-step, seg-
ments of 20 cm are stained and then carefully
inspected.
The additional time needed for chromoendo-
scopy, including preparation of the tissue surface,
dye spraying, washing of excessive dye, inspection
and interpretation, varies from 2 to 20 min. This is
dependent upon staining objectives; a targeted
lesion may need further, local characterization
(e.g. colonic polyp), vs. an entire organ needing to
be stained for detection of dysplastic areas (e.g. the
colon in IBD).
Vital stains
Acetic acid
Acetic acid (vinegar) is a weak acid that breaks up
the disulphide bridges of glycoproteins that build the
mucus layer and results in a reversible denaturation
of proteins. Acetic acid is not a colouring agent, but
when sprayed onto the tissue surface, it can en-
hance the structural surface pattern similar to a con-
trast agent. It has been used in colposcopy for a
number of years as it brings out dysplastic squamous
lesions of the cervix.7
Procedure. Pre-treatment of the surface with
mucolytic agents is not required. Concentrations of
1.5–3% (v/v) acetic acid are usually sprayed in
20 ml aliquots onto the oesophageal mucosa.
Within a few seconds, a whitish discolouration of
the epithelia is noted. This staining method has
been introduced to predict the presence of specia-
lized columnar-lined epithelium in the oesophagus
using magnification endoscopy.
After acetic acid application onto suspected
Barrett’s epithelium, different mucosal surface pat-
terns can be observed (Table 1). When oesophageal
biopsies are taken from type III/IV pit pattern areas
(villous and cerebriform appearance), the diagnostic
yield for specialized columnar-lined epithelium is
>87%, whereas it is <11% when taken from type I
or II areas (regular round pits or circular and oval
pits).8
Methylene blue
Chromoendoscopy using methylene blue as a vital
stain involves active mucosal absorption of the dye
by small intestinal and colonic epithelium. The stain
is not absorbed by non-absorptive mucosa such as
squamous or gastric epithelium. Targeted biopsies
should be aimed at heterogeneously stained or
unstained areas, as high grade dysplasia (HGD)
 Indications, stains and techniques in chromoendoscopy 119

Table 1 Definition of oesophageal mucosa pit patterns following application of acetic acid in BO

Classification Pattern description

Pattern 1 Round pits with a characteristic pattern with regularly and orderly arranged dots
Pattern 2 Reticular, circular or oval pits which are regular in shape and arrangement
Pattern 3 Villous pattern with no discernible pits present, rather a fine villiform appearance with regular shape
and arrangement
Pattern 4 Ridged with no pits but instead, a thick villous convoluted cerebriform appearance with regular
shape and arrangement

and early cancers absorb the dye to a lower degree
due to loss in goblet cells and decreased cytoplasm.
Procedure. Methylene blue chromoendoscopy
requires prior mucus removal from the mucosal sur-
face to ensure homogenous uptake of dye by epi-
thelial cells in the upper gastrointestinal tract. This
can be obtained by spraying 10% solution of
N-acetylcysteine as a mucolytic onto the mucosal
surface prior to the application of 0.5% methylene
blue. Excess dye is carefully washed off with water
until the staining pattern is stable.
With pan-colonic dye staining, segments of
20–30 cm of colon are sprayed and evaluated at a
time. A slightly lower concentration (0.1%) of
methylene blue is applied, using a spraying catheter
onto the colonic mucosa. Excessive dye is removed
by suction after a staining time of 1 min.
Side effects. Generally, chromoendoscopy with
methylene blue is safe. However, methylene blue
might induce oxidative damage to DNA in the epi-
thelium in combination with photosensitization by
white light endoscopy.9 Up till now, no increased
risk of cancer development has been proven in
patients undergoing methylene blue-based chro-
moendoscopy, although many centres prefer using
indigo carmine (see below) for this reason, to avoid
any potential DNA damage in patients who already
have a pre-malignant condition. Chromoendoscopy
using methylene blue may also cause a transient,
harmless, blue discolouration of urine and faeces.
Cresyl violet—cytoendoscopy
Cresyl violet (gentian violet) solution is preferentially
taken up in the crypts of Lieberkühn, which appear
as dots or pits, providing very clear deEnition of
patterns having histological correlates. Cresyl violet
staining can be combined with methylene blue and
indigo carmine staining to detect small, early malig-
nant changes in the colon. Cresyl violet has also
been used as an intra-vital stain for the detection
and characterization of early upper gastrointestinal
cancers.10
Procedure. Cresyl violet (0.05–0.2%) is usually
applied in small amounts (1–2 ml) to avoid exces-
sive darkening of stained surfaces. Combined with
confocal laser endomicroscopy (CLE), cresyl violet
may be applied topically to allow simultaneous
chromoendoscopy and endomicroscopy, thereby
providing accurate prediction of histology, as well
as visualization of nuclear morphology.11
Lugol
Lugol is an iodine-based solution, which is used to
demarcate dysplasia and cancer in squamous epi-
thelium (Figure 1). The iodine is incorporated in the
glycogen, which is abundant within non-keratinized
squamous epithelium. This results in a typical ‘rep-
tile skin’-like endoscopic appearance after staining.
Neoplastic tissue usually has low glycogen storages
and therefore appears unstained. However, other
conditions which result in depleted glycogen stor-
age in the cells, such as inflammatory diseases
(e.g. reflux oesophagitis) or BO might show a
decreased or missing stain uptake. Lugol voiding
lesions in patients with squamous cell cancer of
the head and neck are a strong predictor for syn-
chronous or metachronous oesophageal squamous
cancer and identifies patients who should be under
close endoscopic surveillance.12
Procedure. Following initial inspection, 20–30 ml
of 1–2% Lugol’s iodine solution (e.g. 12 g iod-
ine + 24 g potassium iodide in 1000 ml water) is
sprayed from the gastro-oesophageal junction to the
upper oesophageal sphincter using a spray catheter.
Side effects. The application of iodine can cause
thyrotoxicosis in patients with underlying thyroid
disease. Severe allergic reactions to iodine have
been reported, and it should not be administered
to patients with a history of iodine hypersensitivity.
Retro-sternal discomfort induced by the mucosal
irritation of iodine has been reported in up to 30%
of patients. This side effect can be reduced by spray-
ing 20 ml of 5% sodium thiosulphate solution after
chromoendoscopy.13
120 P.J. Trivedi and B. Braden
Figure 1. (a) Conventional white light endoscopy and (b) chromoendoscopy using Lugol’s solution of a patch of high grade
dysplastic squamous epithelium in the mid-oesophagus. The dysplastic area remained unstained, whereas glycogen deposits
within the normal surrounding squamous epithelium show a darker, more intense colouration.
In 1998, 225 adults with balloon cytologic evi-
dence of oesophageal dysplasia or carcinoma
underwent conventional, standard definition,
white-light endoscopy followed by staining with
1.2% Lugol’s iodine solution. Before staining, the
sensitivity of visible lesions for identifying HGD or
cancer was only 62% and the speciEcity, 79%;
whereas after staining, the sensitivity of unstained
lesions for identifying HGD or cancer, rose to
96%. Although no additional patients with invasive
carcinoma were detected with staining alone, dys-
plastic lesions in 17 of 31 patients with moderate
dysplasia (55%) and 8 of 35 patients with severe
dysplasia (23%) were identiEed only after iodine
staining.14 Chromoendoscopy with Lugol’s iodine
may also prove useful for reducing misclassification
in patients with reflux oesophagitis.15
Non-absorptive contrast stains
Indigo carmine
Indigo carmine (E132; often used as food dye) is a
contrast dye that neither reacts with nor is absorbed
by the mucosa, but simply pools in the mucosal
grooves and crevices, allowing better topographic
definition (Figure 2).
Procedure. During continuous extubation, indigo
carmine (0.4%) is gently applied to achieve diffuse
coverage of the entire mucosal surface. Only a small
volume of dye is applied to avoid excess dye accu-
mulation. This continuous one-step low volume
technique is much simpler than previously
described multiple-step techniques involving seg-
mental staining, followed by re-examination after
excess dye has been aspirated. Indigo carmine is
easily applied using a special dye-spray catheter.4
Prior application of acetic acid has also been used in
the upper gastrointestinal tract in some studies.16
In contrast to methylene blue, patients receiving
indigo carmine dye spraying do not seem to have
increased DNA damage induced to colonocytes.17
Indigo carmine appears to be photostable and poses
little potential for damage to genetic material
in vitro.
The different colonic staining patterns are categor-
ized according to the Kudo pit pattern classification
(type I: round pits; type II: reticular pattern, stellar or
papillary pits; type IIIl: tubular, large pits and IIIs:
rounded, compact, smaller pits; type IV: elongated,
branched and sulcus-like pits and type V: irregular,
non-structural pits),18 which predicts histology
with good accuracy. This classification is based on
an early Japanese study where, 2050 colorectal
tumour lesions were assessed by endomicroscopy,
stereomicroscopy and histopathology. Based on
stereomicroscopy, lesions with a regular type I or II
pit pattern were non-tumours, whereas lesions with
types IIIs, IIIL, IV and/or V pit patterns were neoplas-
tic tumours. When the diagnosis by magnifying
endoscopy was compared with the stereomicro-
scopic diagnosis, there was agreement in 1130 of
1387 lesions (81.5%).19
Indigo carmine has also proven useful in delineat-
ing the extent of gastric lesions. In 2009, Lee et al.
performed both conventional endoscopy and acetic
acid–indigo carmine chromoendoscopy in 141 pa-
tients with early gastric cancer to clearly identify the
extension of tumour. The latter technique clarified
the border in a significantly higher percentage of
differentiated adenocarcinomas (89.8 vs. 68.5%;
P < 0.001). However, the technique had no add-
itional benefit in demarcating undifferentiated
adenocarcinomas.16
 Indications, stains and techniques in chromoendoscopy
Figure 2. Indigo carmine chromoendoscopy delineating mucosal alteration in the sigmoid: (a) before staining and (b) after
staining.
Reactive stains
Congo red
Congo red is a pH indicator that changes colour
from red to dark blue or black when exposed to
acidic environments (pH < 3). It has been used to
map ectopic sites of excessive acid production and
is useful in the evaluation of post-vagotomy patients.
Congo red has been used for many years in screen-
ing early gastric cancers, and in combination with
methylene blue, which stains gastric intestinal
metaplasia.20
Procedure. This technique involves stimulation
of acid production with 250 mg of pentagastrin
given orally. During the endoscopy, 0.5% sodium
bicarbonate solution is sprayed prior to a 0.3–0.5%
Congo red solution. A positive reaction (black
colour change) results within minutes that delineate
acid secreting areas (blue/black) from non-acid-
secreting areas (red).
A double staining technique using methylene
blue and Congo red has been used to identify
early gastric cancers as ‘bleached’ areas of mucosa
that fail to stain with either methylene blue or Congo
red. This is in contrast to the red or blue–red col-
oured mucosa of non-cancerous areas. A very early
study by Iishi et al. demonstrated that the detection
of synchronous early gastric cancers increased from
28% under standard imaging to 89% after methy-
lene blue–congo red combination staining. The
technique also facilitates the detection of carcin-
omatous foci, 4–10 mm in size that are not visible
with conventional endoscopy.21,22
Phenol red
Phenol red changes colour from yellow to red in the
presence of an alkaline environment and has been
121
used to detect and map the distribution of
Helicobacter pylori infection within the stomach.
Procedure. Prior to the endoscopy, the patient is
given acid suppression therapy (either via a proton
pump inhibitor orally the day before, or via intra-
venous therapy 30–60 min before the procedure), an
oral anti-foaming mucolytic agent and an anti-
cholinergic drug to suppress gastric motility. The
entire surface of the stomach is sprayed over with
0.1% phenol red containing 5% urea. Positive stain-
ing of yellow to red usually occurs within 2–3 min.
The sensitivity of this method in detecting
H. pylori approaches 100%, and specificity
84.6%.23,24 However, the clinical relevance of the
phenol red technique is limited.
Chromoendoscopy in primary bowel
cancer screening
Screening for colorectal cancer using faecal occult
blood testing, sigmoidoscopy or colonoscopy is rec-
ommended in several countries, mostly in those pa-
tients aged >50 years with an average risk, and
earlier in people with a strong family history or
other risk factors. Adenomatous polyps are deemed
to be precursors of colorectal cancer and removal of
polyps and post-polypectomy surveillance decreases
the incidence of colorectal cancer.25,26 Colonoscopy
is considered to be the reference standard against
which the accuracy and efficacy of other colorectal
cancer screening tests is compared with; however,
the miss rate for polyps is >20%.27
Several trials have evaluated the ability of chro-
moendoscopy to increase detection of adenomatous
lesions during primary bowel cancer screening pro-
grammes in the general population. One of the first,
large prospective trials used vital staining with
122 P.J. Trivedi and B. Braden
indigo carmine on all visible lesions in 100 consecu-
tive patients without visible inflammatory changes.
If findings on macroscopic examinations were unre-
markable, the sigmoid colon and rectum were
stained with indigo carmine over a defined segment
(0–30 cm ab ano) and inspected for lesions visible
only after staining. Using this technique, 178 add-
itional lesions in the sigmoid colon were identified
as being detectable only after dye spray. This
study failed to control for withdrawal time, which
may explain in part, the increased detection with
indigo carmine.28 A second randomized trial
of 260 patients comparing indigo carmine pan-
chromoendoscopy vs. a control group of targeted
biopsy colonoscopy (without dye spray) reported
a higher number of adenomas in the pan-
chromoendoscopy group (112 vs. 57; P < 0.05).
Flat lesions (82 vs. 54; P < 0.05), right-sided lesions
(79 vs. 31; P < 0.05) and the number of patients
with more than three adenomas (13 vs. 4; P < 0.01)
were also significantly higher in the pan-
chromoendoscopy group.29
A recent study from Germany evaluated 1008 pa-
tients aged >45 years, who presented for primary
bowel cancer screening over an 18-month period.
Patients were randomized to receive standard col-
onoscopy or colonoscopy with pan-colonic dye
spray using indigo carmine. There was a significant
increase in the overall detection rate for adenomas
in the pan-colonic dye-spray arm in comparison
with the control group (46.2 vs. 36.3%; P = 0.002).
In the pan-colonic dye-spray group, the detection
rate for both flat (0.56 vs. 0.28 per patient) and ser-
rated (1.19 vs. 0.49 per patient) adenomas was
nearly double that of the conventional colonoscopy
arm (P < 0.001). However, in the subgroup of
advanced adenomas >1 cm, despite a trend towards
greater detection in the dye-spray arm, this did not
reach statistical significance, echoing the results of
previous studies.4
Although the introduction of chromoendoscopy,
both in isolation and in combination with magnify-
ing endoscopy, has significantly improved the
means to detect both pre-neoplastic and neoplastic
changes, not all studies in primary bowel cancer
screening have demonstrated clear benefits. One
such randomized controlled trial of pan-colonic
chromoendoscopy (indigo carmine) vs. standard
colonoscopy included 259 patients, and although
a statistically significant increase in detection rate
was found for both <5 mm adenomas (89 vs. 36,
P = 0.026), as well as number of patients with 53
adenomas (15 vs. 3; P = 0.002), no significant differ-
ence in the detection rate of adenomas overall was
observed.30 The prospective trial by Le Rhun et al.
included 203 patients and compared adenoma
detection of standard resolution colonoscopy
against high resolution pan-colonic chromoendo-
scopy (indigo carmine). Once again, although
more polyps were detected by the latter, the total
number of polyps detected per patient was not sig-
nificantly different between groups.31
Chromoendoscopy in IBD
The increased risk of colorectal cancer in ulcerative
colitis (UC) has long been recognized.32,33 However,
the previously estimated lifetime risk of 30% is
probably greater than estimated, when applied to
the present patient population. Recent population-
based studies estimate the 30-year cumulative risk
to be between 2.1 and 10.8%.34–36 The risk of
cancer in Crohn’s disease with colonic involvement
is less clear. Longstanding disease, extensive colonic
involvement, a family history of colorectal cancer,
the degree of inflammatory activity and the co-exist-
ence of primary sclerosing cholangitis37 are add-
itional factors that increase the risk of colonic
cancer in patients with pre-existing IBD. This led to
the development of colonoscopy surveillance pro-
grams which centred upon multiple non-targeted
random biopsies at pre-defined intervals based on
the presence or absence of the aforementioned risk
factors.38,39
Older guidelines recommended 2–4 biopsies be
taken every 10 cm in the colorectum, rendering
20–50 biopsies per examination. This approach
was time-consuming, expensive and associated
with significant miss rates of pre-neoplastic lesions.
In 2003, Kiesslich et al. randomized 165 patients
with longstanding ulcerative colitis to receive
either conventional colonoscopy or colonoscopy
with chromoendoscopy using methylene blue. In
the chromoendoscopy group, there was significantly
better correlation between the endoscopic assess-
ment of degree and extent of colonic inflammation
and the histopathologic findings, compared with the
conventional colonoscopy group (89 vs. 52%;
P < 0.0001). More targeted biopsies were possible
and significantly more intra-epithelial neoplasia
was detected in the chromoendoscopy arm (32 vs.
10; P = 0.003). Using the modified pit pattern clas-
sification, both the sensitivity and specificity for dif-
ferentiation between non-neoplastic and neoplastic
lesions were 93%.40 A Japanese study published in
the same year followed up 57 patients with pan-
colitis between 5 and 7 years and found higher
diagnostic accuracy for dysplasia in biopsies tar-
geted by chromoendoscopy vs. standard definition
colonoscopy (sensitivity 85.7 vs. 38.1%).41
 Indications, stains and techniques in chromoendoscopy
In 2004, Rutter et al. conducted back-to-back
colonoscopies on 100 patients with long standing,
extensive ulcerative colitis. During the first examin-
ation, both visible abnormalities and quadrantic,
non-targeted areas (every 10 cm) were biopsied.
Pan-colonic indigo carmine dye spraying was per-
formed during the second examination and any
additional visible abnormalities biopsied. The
non-targeted biopsy protocol detected no dysplasia
in 2904 biopsies. About 43 mucosal abnormalities
(20 patients) were detected during the pre-dye-spray
colonoscopy, of which, 2 (2 patients) were dysplas-
tic. A total of 114 additional mucosal abnormalities
(55 patients) were detected only following dye
spraying, of which seven (5 patients) were dysplastic
on histological analysis. Although there was a strong
trend towards increased dysplasia detection follow-
ing dye spraying, this did not quite reach statistical
significance (P = 0.06).42 More recently, Ratiu et al.
conducted a study (n = 55; mean age 60 years) to see
whether indigo carmine provides a greater adenoma
detection rate in the distal colon during examination
with flexible sigmoidoscopy. Chromoendoscopy
with indigo carmine significantly improved the
adenoma detection rate for lesions <5 mm (15 vs.
7 adenomas; P < 0.01); however, no significant dif-
ference was detectable for lesions >5 mm between
chromoendoscopy and conventional sigmoidos-
copy.43 A larger study from Mt. Sinai Hospital,
New York prospectively investigated 102 patients
with either ulcerative colitis or Crohn’s colitis.
Patients underwent standard surveillance colonos-
copy with quadrantic random biopsies every
10 cm (minimum 32 samples) or colonoscopy
using a targeted biopsy protocol or finally, methy-
lene blue (0.01%) dye-spray chromoendoscopy and
Table 2 Indications and preferred dyes in surveillance chromoendoscopy
Clinical Preferred staining Sensitivity (%)
indication technique
Dysplasia Acetic acid 1.5–3% 95.5–100
detection
in BO Indigo carmine 83
0.4–0.8%
Dysplasia Indigo carmine 0.4% 93
detection Overall: 83
in IBD Methylene blue 0.1% 72
123
targeted biopsy. Each patient had a single examin-
ation that included two passes of the colonoscope.
Targeted biopsies with dye spray revealed signifi-
cantly more dysplastic lesions than random biopsies
(17 vs. 3; P = 0.001) although the benefit over the
targeted non-dye-spray approach did not reach stat-
istical significance (P = 0.057).6
A recent meta-analysis evaluating the efficacy
of methylene blue or indigo carmine chromoendo-
scopy for detecting dysplasia in patients with ulcera-
tive colitis demonstrated a pooled sensitivity of
83%, specificity of 91.3% and a diagnostic odds
ratio of 17.5.44 A second meta-analysis found
that the incremental yield of dysplasia detection
between chromoendoscopy and white light endos-
copy was 7% [95% confidence interval: 3.2–11.3]
on a per-patient analysis, with a number needed
to treat of 14.3 to detect one extra patient with dys-
plasia or cancer (Table 2). The difference in pro-
portion of flat lesions detected by targeted biopsies
was 44 vs. 27% in favour of chromoendoscopy.45
This evidence on the superiority of chromo-
endoscopy with targeted biopsies compared with
standard colonoscopy has led to the recommenda-
tion of pan-colonic dye spray with targeted
biopsies (where available), rather than random
biopsies in the new guidelines for surveillance
colonoscopy by the British Society of Gastro-
enterology and American Gastroenterological
Association.38,39
Chromoendoscopy in BO
Longstanding gastro-oesophageal reflux can lead to
the development of BO which affects 2% of adults
Specificity (%) Comments
80 Neoplasia detection rate increased
by 2.5-fold in one study.53
88 High sensitivity and specificity
restricted to long (53 cm) Barrett’s
intestinal metaplasia segments having
an irregular, distorted pattern
(pit-pattern III/IV).
Unable to distinguish low-grade
dysplasia from non-dysplastic
intestinal metaplasia.
91 Data extracted from recent
Overall: 91 meta-analysis with an
92 AUROC of 89%.44
124 P.J. Trivedi and B. Braden
in the West, and is more common in men aged
>50 years. The lifetime oesophageal adenocarcin-
oma risk in this pre-malignant condition is in the
order of 3–5%.47 This is 30–100 times higher than
in the general population. In view of this, endo-
scopic surveillance programs have been instituted
with an aim of detecting dysplasia or neoplasia at
an early stage.48,49 Most guidelines recommend sur-
veillance endoscopy every 2–5 years in patients
with BO to detect early signs of HGD, which
poses a risk of progressing to adenocarcinoma of
6–19% per year,49 as well as early, treatable neo-
plastic lesions.
There has been a longstanding practice for sur-
veillance endoscopies to take random four quadrant
biopsies every 1–2 cm unless obvious nodules or
mucosal irregularities are present (‘Seattle protocol’).
However, high grade dysplasia and early cancer
often present as flat lesions that are difficult to iden-
tify. The Seattle protocol is time consuming, expen-
sive and may still miss 41–66% of high grade
dysplasia.5 Therefore, great effort has been under-
taken to improve the visualization of subtle mucosal
changes, which indicate the histological presence of
early neoplasia. Although methylene blue has
proven useful at diagnosing gastric lesions,50 its
use in Barrett’s surveillance has produced discrepant
results. A meta-analysis by Ngamruengphong et al.
concluded that the diagnostic yield for the detection
of specialized intra-epithelial metaplasia and dys-
plasia is only comparable, but not superior to
taking random biopsies.51 As methylene blue stain-
ing is cumbersome, time consuming and bears at
least a potential risk of DNA damage, it cannot
widely be recommended for routine Barrett’s sur-
veillance. However, it might be helpful to delineate
dysplastic or malignant lesions for further endo-
scopic therapy (endoscopic mucosal resection or
endoscopic submucosal dissection).
Whereas methylene blue chromoendoscopy
does not seem to increase the diagnostic yield in
the context of BO, highlighting irregularities in the
surface pattern by acetic acid spraying (Figure 3)
and taking targeted biopsies from said areas does
result in a higher detection rate of dysplasia and
cancer.52,53 Although many studies apply acetic
acid staining in combination with magnification
endoscopy, it is also helpful when applied to
conventional, standard definition ‘white light’
endoscopes.46 Staining with the naturally brown-
ish-coloured balsamic vinegar combines the advan-
tage of chromoendoscopy and surface structure
enhancement by the acetic acid.54 Pech et al. pro-
spectively evaluated the diagnostic value of staining
with balsamic vinegar, and in combination with
high resolution video-endoscopes without
magnification, balsamic vinegar staining obtained
an accuracy of 90%, sensitivity of 100% and speci-
ficity of 82% in predicting the presence of Barrett’s
epithelium compared with histology.54 A recent
study from Portsmouth (UK) collected data on 190
patients undergoing upper gastrointestinal endos-
copy and found acetic acid chromoendoscopy to
have a sensitivity of 95.5% and specificity of 80%
for the detection of neoplasia. In this study, visible
neoplasia was observed during 43 procedures with
white-light endoscopy and in 102 following dye
spray with acetic acid (P = 0.001), yielding an im-
proved neoplastic-lesion detection rate of 2.5-fold
when compared with conventional white light en-
doscopy. Moreover, a high degree of correlation be-
tween lesions predicted to be neoplastic by acetic
acid chromoendoscopy and those diagnosed by
histological analysis was found (r-value = 0.99).46
However, the results of a recent randomized study
(n = 137) were more sobering, with targeted biopsies
using enhanced magnification acetic acid chro-
moendoscopy, yielding specialized intestinal meta-
plasia at the same frequency as standard
endoscopy, calling into question, the diagnostic util-
ity of this technique.55 Nonetheless, as advanced
endoscopic imaging techniques using acetic acid
identify the vast majority of early neoplasia, targeted
biopsies using acetic acid chromoendoscopy and
high resolution endoscopes may yet replace
random quadrant biopsies as part of Barrett’s sur-
veillance programmes. Moreover, acetic acid
localizes neoplastic lesions in the majority of pa-
tients and could potentially represent significant
cost savings in high-risk patients with BO and sus-
pected neoplasia.56
Chromoendoscopy using indigo carmine in BO
may also be helpful. In 2003, Sharma et al. per-
formed indigo carmine dye spray and magnification
chromoendoscopy in 80 patients with suspected
>3 cm BO. The yield of intestinal metaplasia was
97% when a ridged, villous pattern was observed
and 100% for high grade dysplasia when an irregu-
lar, distorted pattern could be seen. Although all
patients with long segment BO were identified
using this technique, the value dropped to 82%
when evaluating for short segment diseases.57
A more recent, multi-centre study prospectively
evaluated 56 patients with BO using indigo carmine
magnification chromoendoscopy. This study also
found that the presence of an irregular, distorted
pattern throughout the Barrett’s segment was
highly sensitive (83%) and specific (88%) for high
grade dysplasia.58 However, indigo carmine is
not able to accurately distinguish low-grade dyspla-
sia from non-dysplastic intestinal metaplasia.57
 Indications, stains and techniques in chromoendoscopy
Figure 3. Acetic acid application and narrow band imaging in BO. (a) Standard white light endoscopy; (b) after acetic acid
staining; (c) combination of acetic acid and narrow band imaging.
Chromoendoscopy in combination
with high definition and magnifying
endoscopy
Good resolution and appropriate magnification are
essential for achieving high quality visualization
during any endoscopic examination. Video reso-
lution is defined as the ability to optically distinguish
two closely approximated points or objects.59
High-resolution imaging improves the ability to
discriminate detail, whereas magnification enlarges
the image. Several studies have argued that high-
resolution imaging endoscopy may be enough to
detect BO,60,61 with no additional benefits being
obtained with acetic acid,60 or in detecting HGD
or early cancer with indigo carmine chromoendo-
scopy.61 However, even with high-resolution endo-
scopes, four-quadrant biopsies are still necessary,
and acetic acid spraying may yet improve visualiza-
tion of suspicious lesions.46,62
Recent advancements in endoscopic technology
have produced high magnification endoscopes that
allow real time visualization of mucosal morph-
ology in greater detail. The clinical utility of this
125
modality had been limited by the size of the endo-
scope in the past. However, improvement in the
design of the charged-couple device, an electronic
light-sensing apparatus located at the tip of the
endoscope, has given rise to less bulky and more
manageable apparatus. Magnification endoscopy
has been used in combination with indigo carmine
to diagnose BO with a sensitivity of 97%, and HGD
with a sensitivity of 100%.57 As already discussed,
enhanced magnification endoscopy with acetic acid
allows clear visualization of the epithelial pit pat-
terns within BO; however, despite the increasing
availability of high resolution magnification endo-
scopes, there is a lack of diagnostic criteria for mag-
nified endoscopic images.
There are also large cases’ series that report the
utility of using magnification colonoscopy and
pit-pattern analysis to differentiate neoplastic and
non-neoplastic lesions. One prospective trial rando-
mized patients (n = 660) to either magnification
chromocolonoscopy with indigo carmine or conven-
tional (non-magnified) chromocolonoscopy and
found that the accuracy of the former in distinguish-
ing neoplastic from non-neoplastic lesions <10 mm
in size (92%) was significantly higher compared with
126 P.J. Trivedi and B. Braden
the latter (68%).63 The higher accuracy of magnifica-
tion chromocolonoscopy has been validated in
other reports.64,65 In the colon at least, it may be
that the mucosal magnification in combination with
tissue staining is more important than high-resolution
imaging, as the number of lesions detected between
high-resolution colonoscopy without tissue staining
and standard colonoscopy does not appear to be dif-
ferent between modalities.31
Magnification chromoendoscopy in combination
with methylene blue dye spray has also been stu-
died in ulcerative colitis, and is significantly better
than magnification endoscopy alone at identifying
intra-epithelial neoplasia.40 Prospective trials with
targeted chromocolonoscopy (indigo carmine) con-
firmed these findings and demonstrated improved
detection of intra-epithelial neoplasia compared
with random quadrantic biopsies.66,67 In small stu-
dies, magnification chromocolonoscopy was also
used to assess colitis disease severity and may
even predict disease relapse.68–70
How does chromoendoscopy
compare with other endoscopic
advancements?
All chromoendoscopic methods have to compete
with newer, quicker and neater imaging techniques,
which offer on-demand imaging of the gastrointes-
tinal mucosa during the endoscopic examination
whenever further characterization of a suspicious
area is required. For many of these novel techniques
the clinical value over conventional white light and
high-definition endoscopy in the detection of aden-
omatous, dysplastic and neoplastic lesions of the
gastrointestinal tract has yet to be rigorously
evaluated. Moreover, few studies provide a
‘head-to-head’ comparison of these newer endo-
scopic enhancements vs. chromoendoscopy.
Narrow-band imaging (NBI; Olympus, Tokyo,
Japan) offers better visualization of the superficial
mucosal detail and vasculature by pressing a
button on the hand control of the endoscope with-
out the need for any additional dyes. The NBI
system has special red–green–blue filters in which
the band-pass ranges have been narrowed and
the relative contribution of blue light has been
increased. NBI has become increasingly popular in
the upper gastrointestinal tract, as it assists in un-
masking pre-malignant and neoplastic lesions in pa-
tients with BO.71–73 However, despite initial
enthusiasm and promise, well-designed prospective
randomized controlled trials and meta-analysis have
failed to prove superiority of narrow-band imaging
in screening or surveillance colonoscopy, com-
pared with standard definition white light endos-
copy.74–78 Nonetheless, improved further
characterization of already detected colonic polyps
by NBI may allow prediction of the histology with
acceptable accuracy, thereby allowing a ‘resect and
discard’ strategy, which might be cost-effective and
safe for diminutive colonic polyps.79
Newer generation endoscopy systems include
Fuji intelligent chromoendoscopy (Fujinon) or
i-Scan (Pentax); both also from Japan. These modal-
ities utilize the light reflected from the intestinal
mucosa, which is then modified by ‘post-processor
computer algorithms’ that allow different forms of
enhancements leading to accentuation of the vascu-
lature, surface architecture or tissue pattern visua-
lization. Although these techniques provide
accurate classification and differentiation of colo-
rectal polyps and neoplastic lesions,80 a clear
advantage in adenoma detection rate over chromo-
colonoscopy or high resolution white light endos-
copy has not yet been shown.81
Autofluorescence imaging (AFI) endoscopy uses
short wavelengths of light to stimulate endogenous
substances, so called ‘fluorophores’ such as nicoti-
namide adenine dinucleotide (NADH), collagen,
aromatic aminoacids and porphyrines in the tissue
to emit fluorescent light of a longer wavelength.
Due to different content of such fluorophores,
normal and neoplastic tissues differ in their autofluor-
escence spectra. Whereas normal mucosa appears
green, neoplastic areas are ‘flagged up’ in violet.
Modern endoscopic techniques combine white-light
endoscopy, AFI and NBI (trimodal endoscopic ima-
ging). However, studies have not consistently demo-
nstrated the superiority of this method compared
with high resolution endoscopy alone, for detection
of colonic or oesophageal dysplasia.82,83 Thus, AFI-
guided biopsies are not currently able to replace
random biopsies during surveillance endoscopies.
CLE is a technique that offers in vivo imaging of the
mucosal layer at cellular and subcellular resolutions
without the fixation artefacts one experiences with
histological specimens. In this system, fluorescent
dyes are applied either locally or systemically, and
subsequently excited by a low-power laser. The in-
tensity of the fluorescent energy is then captured as
an image. Commonly used agents are fluroscein
sodium and acriflavine. Studies have shown the high
accuracy (96.7%) with which CLE allows differenti-
ation from low-grade to high-grade intra-epithelial
colorectal neoplasia.84 Moreover, chromoendo-
scopy together with targeted CLE and endomiscro-
scopy is able to increase the diagnostic yield of
intra-epithelial neoplasia by 4.75-fold (P = 0.005) in
patients with chronic ulcerative colitis.85
 Indications, stains and techniques in chromoendoscopy
Other emerging in vivo endoscopic techniques
include light-scattered spectroscopy (LSS), a type of
reflectance spectroscopy which determines tissue
structure by determining elastic light scattering86,87
and optical coherence tomography, a probe-based
technique using infrared light which allows deeper
tissue penetration than CLE.88,89 However, the
clinical benefit of these novel modalities has not
yet been fully appreciated and further technological
improvements for gastrointestinal endoscopy are
warranted before they enter widespread usage.
Tumour markers and
chromoendoscopy
Biomarkers of malignancy or pre-malignancy in stool
or blood are a potentially attractive means of detect-
ing colorectal dysplasia and neoplasia, particularly
for high-risk populations. Unfortunately, many con-
ventional tumour markers such as CEA, CA19-9
and EpCAM reflect inflammation, rather than pre-
malignant change; therefore do not have an estab-
lished place in cancer, or pre-cancer detection.
However, specific molecular alterations (p53 muta-
tions, DNA aneuploidy, chromosomal instability,
K-ras mutations, p14 and p16 hypermethylation,
microsatellite instability, age-related methylation,
telomere length shortening) have been identified at
a higher frequency in the non-neoplastic epithelium
of UC patients with neoplasia, compared with
non-neoplastic epithelium from UC patients without
neoplasia.90 These emerging tools may assist in the
risk-stratification of future endoscopic surveillance
strategies and therapeutic intervention to patients at
highest risk of developing cancer. Although these
novel biomarkers do not improve absolute lesion de-
tection rates during endoscopy, emerging molecular
imaging methods could revolutionize the detection
of dysplasia in conjunction with chromoendoscopy
by providing a wide field of view while simultan-
eously highlighting molecular abnormalities in
‘real time.’ One such example is the use of specific
lectin probes that recognize coordinated changes in
glycan expression that accompany the transition
from BO through dysplasia to adenocarcinoma.91,92
Such advancements may not only help delineate the
extent of disease involvement, but also inform the
future choice of treatment if they can be validated
as an endoscopic tool in vivo.
Conclusions
Chromoendoscopy enables targeted biopsies and
thus improves the diagnostic yield of dysplastic
127
alterations. It also improves further characterization,
differentiation and diagnosis of endoscopically
detected suspicious lesions. Chromoendoscopy is
considered a safe, relatively inexpensive procedure
aside from the additional endoscopy time required.
The chromoendoscopic staining methods are not
technically demanding and easy to learn, but
require experience in the interpretation of the
observed staining pattern. The accessories needed
to perform chromoendoscopy, the dye agents
and spraying catheters, are readily available.
Standardization of staining protocols, a consensus
in terminology and classification of staining patterns
are still awaited. Virtual chromoendoscopy allows
further characterization of detected lesions and
can predict their histology with accuracy.
Therefore, an optical triage approach to ‘diagnose,
resect, discard’ or ‘diagnose and leave behind’
might reduce the costs of polypectomy. Newer evol-
ving technologies are likely to definitively change
the clinical algorithm of gastrointestinal endoscopy
in which hopefully the proportion of missed aden-
omas, dysplastic lesions and carcinomas will sub-
stantially decrease.
Funding
No funding was provided for the production of
this manuscript. P.J.T is independently funded by
the Wellcome Trust Clinical Research Fellowship
program.
Conflict of interest: None declared.
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