2016, Volume 32, Number 2, Page(s) 112-118
Inking in Surgical Pathology: Does the Method Matter? A Procedural Analysis of a Spectrum of Colours
Divya PURSNANI, Swati ARORA, Palur KATYAYANI, Ambica C, Balasaheb RAMLING YELIKAR
B.L.D.E.U's Shri B.M Patil Medical College, Department of Pathology, VIJAYAPUR, INDIA
Keywords: Inks, Acrylic colours, India ink, Surgical pathology
Proper identification of surgical margins along with margin status holds utmost importance in histopathology. Inking margins is
one way. India ink has long been used but it can ink only one margin. On other hand acrylic colours, available in variety of colours can be used
for inking multiple margins along with many more advantages. The present study was undertaken to analyse acrylic colours and Indian ink for
inking surgical margins via three different methods for optimum results.
Material and Method: Thirteen acrylic colours along with India ink were evaluated via three different methods on radical specimens of breast
and colon after preliminary requisites of grossing were completed. In Method 1, coloured inks were applied to an overnight formalin fixed
specimen and representative sections were taken. In Method 2, the specimen was inked and kept for overnight fixation. The specimen was
sampled following day. In Method 3, the specimen was inked and kept for overnight fixation. The following day, it was re-coloured with the same
colours as of the previous day and subsequently sections were taken. Coloured inks were assessed on different parameters for their performance
as surgical ink and given scores.
Results: Acrylic shades #04, #22, #06, #01, #02 and India ink had very good to excellent score on microscopy in all three methods. Shades #64,
#18, #09, #23 had poor microscopic visibility in all three methods. Shade #09 showed loss of colour and the shade #23 showed penetration into
deeper tissues on microscopy. Results were best with Method 3 followed by Method 1 and 2, respectively.
Conclusion: Acrylic colours have more advantages compared to India ink. Method 3 is recommended for inking. Few acrylic shades meet the
criteria of surgical inks in all three methods.
Histopathological assessment of radical or wide excision
specimens requires proper orientation of the specimen
along with identification of all surgical margins (1-8).
Several methods have been utilised for margin assessment
like the marking of surgical margins with sutures and clips
or applying India ink (1,2). India ink has long been used in
the field of surgical pathology (1,2,5,7,9,10). However, when
multiple margins are to be assessed, there has been a lack of
use of varied coloured inks (1,2). The use of an assortment
of ink colours is useful in the following ways: 1) For margin
status, 2) For orientation of a specimen, 3) For the benefit
of post-grossing three-dimensional reconstruction, 4)
To reduce identification error when multiple sampling is
required from the same tissue (e.g. prostate needle biopsies)
or when obtaining similar specimens from different patients
(e.g. biopsies) (1,2,10,11).
Indeed, many times the critical decision of whether the
margin is positive for tumour cells depends entirely on the
presence or absence of ink (4).
Though tissue marking dyes (TMD) are available in
the market, they are expensive and not readily available
(2,3,7,8). Acrylic colours are easily available in numerous
colours, and are affordable, non-toxic, non-flammable and
stable (1,3,6,7,10) (Figure 1A-E). However, not all acrylic
colours withstand tissue processing and fulfill the criteria
of being used as surgical ink; plus though the inking has to
be done before grossing, whether application of ink after
overnight fixation is better or inking followed by fixation
and grossing is preferable is to be sought.
Click Here to Zoom
|Figure 1: A) Acrylic colours and paint brushes. B) Inking of gross
specimen by acrylic colours. C) Contamination of processing fluid
by acrylic yellow colour. D) Visibility of acrylic colours on blocks.
E) Visibility of acrylic colours on naked eye examination of slide.
The objective in this study was to analyze different
commercially available acrylic colours and India ink for
inking surgical margins; and also to analyze these coloured
inks by three different methods for optimum results and
convenience of use.
This was a cross-sectional comparative study. Radical
surgical specimens of breast and colon were included in the
study. After the preliminary requisites of gross specimens
were completed, the specimens were taken for grossing.
Thirteen Fevicryl® acrylic hobby idea colours (Table I)
along with India ink were used in the study. Inking was
done using half inch paint brushes. After each application,
brushes were thoroughly washed with water before the next
application. The colour inks and India ink were analysed
for their effectiveness as surgical inks via three different
methods. India ink was also analysed by the following three
methods which were used for analysis of coloured inks.
Method 1: An overnight formalin-fixed specimen was
patted dry with a paper towel and all surgical margins were
inked with different colours. The inks were allowed to air
dry and representative sections from the coloured margins
Method 2: The specimen was patted dry with a paper towel
and all surgical margins were inked with different colours.
After air drying, the specimen was kept for overnight
fixation. Following day, representative sections from the
coloured margins were taken.
Method 3: The specimen was patted dry with a paper towel
and all surgical margins were inked with different colours.
After air drying, the specimen was kept for overnight fixation. Following day, the specimen was re-coloured with
the same colours as on the previous day and representative
sections from the coloured margins were taken.
Sections were cut at 4 μm thickness and stained with
haematoxylin and eosin (H&E).
The coloured inks were assessed on the following
1) Ease of application,
2) Drying time of ink,
3) Contamination of other tissues and processing fluids,
4) Visibility on paraffin blocks,
5) Visibility on slide,
6) Visibility on microscopy,
7) Penetration into deeper tissue and interference with
nuclear and cytoplasmic morphology.
Visibility on microscopy of the coloured inks was scored
between 1 and 4 by individual observations. Score 1 was
excellent, 2 was very good, 3 was good and 4 was poor. For
each colour in the three respective methods an average score
was calculated and the results were tabulated (Table I).
Click Here to Zoom
|Table I: Analysis of Acrylic Colours and India Ink via Methods 1, 2 and 3 on microscopy
Statistical analysis in the study was by mean and standard
In method 1, three acrylic colours (black, burnt sienna,
turquoise blue) and India ink gave excellent results on
microscopic examination. In method 2, crimson red (acrylic
colour) gave excellent result on microscopic examination.
In method 3, four acrylic colours (dark green, orange,
turquoise blue, crimson red) gave excellent results on
microscopic examination. Some acrylic colours had a shade
lighter on gross specimen during method 2, e. g orange
and burnt sienna. These colours gave better performance
with method 3. The performance of other colours on
microscopic examination is enlisted in Table I. Mean + SD
of all colours for method 1 was 2.2±1.2, for method 2 was
2.4±1.1 and for method 3 was 2.1±1.2. Least value of mean
was observed for method 3 followed by method 1 and 2,
respectively. Thus, method 3 showed best results followed
by method 1 and 2, respectively.
Analysis of coloured inks on the above mentioned
parameters is interpreted (Table II) as follows:
Click Here to Zoom
|Table II: Analysis of Acrylic Colours and India Ink on different parameters
Visibility on paraffin block: All the acrylic colours and
India ink had good visibility on the paraffin blocks.
Visibility on naked eye examination of slides: Out of 1260
slides, 29 slides did not show visibility on the slide.
Visibility on microscopy: Out of 1260 sections, 24
sections showed near total loss of colour on microscopic
examination. 120 sections had interrupted staining on
microscopy (turquoise blue, ultramarine blue, Prussian
blue, light green).
Penetration into deeper tissues and interference with
nuclear and cytoplasmic details: Out of 1260 slides, 117
slides (ultramarine blue) showed penetration of the colour
into the underlying tissue interfering with the nuclear and
Drying time: Time required for drying of acrylic colours
was significantly lower than that of India ink.
Ease of application: Radical specimens were painted by 13
different commercially available acrylic colours and India
ink with different brushes. All the colours applied had
equal ease of application.
Contamination of other tissues and processing fluids:
Processing fluids and other solutions showed contamination
by yellow colour.
Presence or absence of tumour cells in the surgical margins
holds important therapeutic and prognostic implications.
Till date, morphological assessment of margin status is
considered the gold standard (1,2,5,6). Also, orientation
or laterality of the excised specimen is desirable for both
post-grossing specimen examination and microscopic
identification of resected margins (1,2,7). Inking the
margins is more reliable than sectioning techniques and
suture markers, for identification of surgical margins
before and after tissue processing (2,10). India ink is the
traditional marking ink in the field of surgical pathology
(1,2). Other marking techniques include painting with
Alcian blue, plain gelatine, gelatine with added colours,
TMD, erythrocytes and surface coating with starch (3,6,7).
Currently, margins are marked by TMD, gelatine, Alcian
blue, inorganic artist’s pigment suspended in acetone,
India ink and acrylic colours (3-5,7-10). There are some
known disadvantages such as inorganic pigments tend to
be radio dense and mimic or obscure micro calcification
on sliced specimen mammogram; gelatine requires
reheating and maintaining its liquid state is laborious and
time consuming; TMDs are expensive, hazardous, have a
longer drying time and can cause false positive results with
immunohistochemical stains (2,7,8).
First used in the 20th century, acrylic paints are coloured
particulate pigments suspended in acrylic polymer
solutions as a thick emulsion. They are increasingly popular,
cost-effective, non-hazardous, easy to apply, dry quickly,
insoluble in water and solvent once dry thus being long
lasting (1-3,6,10) (Table III). As they are available in varied
colours, multiple margins can be marked (2,10). It allows
multiple usages as acrylic paint brushes and other equipment
can be washed with soap and water. Acrylic colours can be
painted in layers and as they are not transparent previous
colour is not seen through (2). India ink on the other hand
is a colloidal suspension of inert carbon black used since
fourth century B. C under the name of “Mashi” in India (1).
All shades of acrylic colours do not fulfil the criteria to be used
as surgical ink. 1 Few shades that fix on fresh and formalin
fixed tissue, withstand tissue processing and routine H&E
staining and are visible on both block and microscopic
examination, without contaminating processing fluids
and not obscuring nuclear and cytoplasmic morphological
details suffice the criteria of surgical ink (1). Acrylic colours
have an added advantage that they do not interfere with
immunohistochemical staining (1,6,10). They are easily
available in stationary shops and can be bought as single
units at nominal prices (1,2).
In our study acrylic shades numbered 04 (crimson), 22
(turquoise blue), 06 (dark green), 01 (burnt sienna) and
02 (black) had very good to excellent score on microscopic examination in all three methods. (Figure 2A-F) Shade
17 (orange) had excellent visibility when used by method
3 whereas it had good to very good visibility through
methods 1 and 2. Shade 15 (mauve) had very good visibility
in methods 1 and 3 but good in method 2. Shades 64
(deep brilliant purple), 18 (pink), 09 (golden yellow), 23
(ultramarine blue) had poor microscopic visibility in all
methods. Golden yellow in every method and in all slides
showed loss of colour and presented as black colour with
foci of yellow colour. (Figure 2A-F) Shade 12 (light green)
and 19 (Prussian blue) had good visibility on microscopy.
The results were in accordance with the study done by
Tampi et al. and Chiam et al. The authors also stated that
acrylic colours were easily visible both macroscopically and
Click Here to Zoom
|Figure 2: Micrographs showing excellent visibility of acrylic colours. A) Black. B) Light green. C) Dark green. D) Turquoise blue on
microscopy (H&E; x100). E) Micrograph showing penetration of acrylic colour ultramarine blue into deeper tissue (H&E; x100).
F) Micrograph showing loss of colour by acrylic yellow colour (H&E; x100).
We observed that the outcome, out of the three methods
was best by method 3 followed by 1 and 2 respectively.
method 2 had least performance because the colours
painted on the surgical margins were a shade lighter after
overnight formalin fixation. Method 3 gave best results as
the specimens were re-coloured after overnight formalin
fixation thus compensating for any loss of colour. The
outcome of method 1 was mediocre as the specimens
were directly sent for tissue processing without overnight
A similar observation was made with regards to visibility
of colours on the paraffin blocks and on naked eye
examination of the slides. Method 2 had slightly lighter
colour on blocks compared to blocks made by methods 1
and 3, the latter again having the best colour visibility. Few
colours were discernible only on microscopic examination
of the section though not identified specifically externally
on the paraffin blocks or slides. This was especially seen
for mauve colour (shade 15). Also in a few cases scanner
(x40) identification of mauve colour was difficult and
confirmation was obtained only on low (x100) and high
power (x400). A similar observation was made by Williams
et al. in their study (6).
Shade 23 (ultramarine blue) showed penetration into
deeper tissues leading to obscuration of cellular and nuclear
morphology (Figure 2A-F) All acrylic colours showed
equal ease of application. In a similar study done by Sarode
et al.., acrylic colours had faster drying time compared to India ink, thus making them better suited for routine use
(2). Tissue marking dyes also take a longer time to dry and
thus are cumbersome to use (7). It was observed in our
study that yellow colour caused contamination of tissue
processing solutions contrary to India ink in the study
done by Sarode et al. (2) (Figure 1A-E).
Method 3 overall had highest degree of precision in both
block and microscopic findings followed by method 1 and
2 respectively. A slight difference between method 1 and
3 was observed. Keeping this in mind, it is best if we use
method 3 but if convenience and less time for grossing are
desirable, even method 1 is more than sufficient and gives
approximately same results. However, consideration should
be kept in mind that the colours which gave similar results
for both methods 1 and 3 should be used (e. g. shades
numbers 01,02,04,06,22). There are some colours where
significant difference was observed between methods 1 and
3 and with such colours it is preferable to follow method 3
for better outcome.
Every laboratory should have a protocol for inking margins
and should standardize different colours for different
margins (10). Some studies report that few marking
inks mimic microcalcification on specimen radiography
of breast lumpectomies and should be avoided, where
microcalcification is required to be identified after excision
by prior testing radio-opacity of ink (1,11).
A wide range of acrylic colours is available in market, but
trial and error is needed before they can be used in surgical
pathology. Acrylic black colour (No. 02) gave excellent
performance on all study parameters and therefore can be
used instead of India ink which is comparatively expensive,
takes longer time to dry and is toxic (2). Acrylic colours
require less time to dry compared to tissue marking dyes
and are therefore preferable in a busy surgical laboratory
The present study had certain limitations. The study
included limited set of acrylic colours and only one
manufacturer colours were analysed. Also the assessment in
the study was on the subjective interpretation of the authors.
Analyses of multiple colours from multiple manufactures
need to be tried. Image analysis has been used in some
studies. Though it provides quantitative interpretation, it
does not include subjective observation of human colour
perception and its use is not practical for routine surgical
In conclusion, acrylic colours are easily available, more
affordable and easy to use compared to India ink with an
added advantage of multiple colours for different margins.
Inking by method 3 is recommended. Acrylic shades of 04
(crimson), 22 (turquoise blue), 06 (dark green), 01 (burnt
sienna) and 02 (black) had very good to excellent visibility
on microscopic examination in all the three methods.
The authors express their gratitude to the staff of
Department of Pathology for their constant encouragement
and guidance. We are thankful to Mr. Mohd Shannawaz for
his help in statistical analysis of the study. We are grateful
to Mrs Jessy Joseph, Mr. N B Mathad and Mrs. V P Pawar
for their co-operation and technical assistance. The authors
would also like to thank Dr. Abhey Chawla, Dr. Lynda
Rodrigues and Dr. Jyotsna W for their help during the
CONFLICT OF INTEREST
Authors have no conflict of interest to declare.
1) Tampi C. In search of the rainbow: Colored inks in surgical
pathology. Indian J Pathol Microbiol. 2012;55:154-7.
2) Sarode SC, Sarode GS, Patil S, Mahajan P, Anand R, Patil A.
Comparative Study of Acrylic Color and India Ink for their use as
a surgical margin inks in oral squamous cell carcinoma. World J
3) Williams AS, Dakin Haché K. Variable fidelity of tissue-marking
dyes in surgical pathology. Histopathology. 2014;64:896–900.
4) Westra WH, Hruban RH, Phelps TH, Isacson C. Surgical
pathology dissection: An illustrated guide. 2nd ed. New York:
Springer; 2003. Chapter 1, General approach to surgical pathology
specimens; p. 2-13.
5) Shinde V, Phelan C, Gater W, Thomas J. Inking a specimen
without the mess. J Clin Pathol. 2008;61:783.
6) Williams AS, Hache KD. Recognition and discrimination of tissuemarking
dye color by surgical pathologists recommendations
to avoid errors in margin assessment. Am J Clin Pathol.
7) Chiam HW, Maslen PG, Hoffman GJ. Marking the surgical
margins of specimens: Commercial acrylic pigments are reliable,
rapid and safe. Pathology. 2003;35:204-6.
8) Wong JW, Bai H, Abdul-Karim FW, Maclennan GT. Simulation
of microcalcifications on specimen radiographs of breast biopsies
by inks used in marking the surgical resection margins. Breast
9) Paterson DA, Davies JD. Marking planes of surgical excision on
breast biopsy specimens: Use of artists’ pigments suspended in
acetone. J Clin Pathol. 1988;41:1013-6.
10) R anjan R, Singh L, Arava SK, Singh MK. Margins in skin
excision biopsies: Principles and guidelines. Indian J Dermatol.
11) Parkinson AB, Cannon CR, Hayne ST. Colour coding surgical
margins with the Davidson marking system. J Histotechnol.