Feasibility of Preoperative Tattooing of Percutaneously Biopsied Axillary Lymph Node: A Quasi Experimental Pilot Study
Conditions: Breast Neoplasm
Sponsor: Aga Khan University

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Feasibility of Preoperative 
Tattooing of Percutaneously 
Biopsied Axillary Lymph Node: 
A Quasi Experimental Pilot 
Study  
 
Last Modified: May 3, 2019  
Last Human Subjects Review: December 27, 2018 
 

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Feasibility of Preoperative Tattooing of Percutaneously Biopsied Axillary Lymph Node: A Quasi 
Experimental Pilot Study 
 
BMJ OPEN 
Feasibility of Preoperative Tattooing of Percutaneously Biopsied Axillary Lymph Node: A Quasi 
Experimental Pilot Study 
Wardah Khalid1, Basim Ali2,  Imrana Masroor3, Shaista Afzal4, Mohammad Usman Tariq5, Romana Idress6, 
Maseeh Uzzaman7, Abida K. Sattar8 
1   Senior Instructor, Department of Surgery, Aga Khan University, Karachi Pakistan 
2 Teaching Associate, Department of Biological and Biomedical Sciences, Aga Khan University, 
Karachi Pakistan 
3  Associate Professor, Department of Radiology, Aga Khan University, Karachi Pakistan 
4  Assistant Professor, Department of Radiology, Aga Khan University, Karachi Pakistan 
5  Assistant Professor, Department of Pathology, Aga Khan University, Karachi Pakistan 
6  Assistant Professor , Department of Pathology, Aga Khan University, Karachi Pakistan 
7 Professor, Department of Radiology, Aga Khan University, Karachi Pakistan 
8 Assistant Professor, Department of Surgery, Aga Khan University, Karachi Pakistan 
 
Email Addresses  
Name 
Email address 
Wardah Khalid 
Basim Ali                  
wardah.khalid@aku.edu 
basim.ali@aku.edu 
Imrana Masroor  
imrana.masroor@aku.edu 
Shaista Afzal 
shaista.afzal@aku.edu 
Mohammad Usman Tariq 
mohammad.usman@aku.edu 
Romana Idress 
romana.idress@aku.edu 
Maseeh Uzzaman 
maseeh.uzzaman@aku.edu 
Abida K. Sattar 
abida.sattar@aku.edu 
 
Corresponding Author 
 
Abida K. Sattar 
Assistant Professor Department of Surgery 

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Link Building, Stadium Road, Aga Khan University Karachi 74800 Pakistan 
Phone: 92-21-34864751  
Email: abida.sattar@aku.edu 
Abstract 
Introduction: Breast cancer is the leading cause of cancer mortality among females. Sentinel lymph node 
biopsy helps avoid axillary lymph node dissection and the associated morbidity. However, its utility is 
unclear in patients with biopsy proven axillary disease that has been treated with neoadjuvant 
chemotherapy. The objective of our study is to evaluate a) the feasibility of a novel technique of 
preoperatively tattooing suspicious lymph nodes with India ink, their intraoperative identification and 
retrieval (Phase I) and b) the application of this technique in patients who undergo neoadjuvant 
chemotherapy (Phase II).  
Methods and Analysis: A quasi-experimental pilot study will be conducted to evaluate the feasibility of 
tattooing suspicious lymph nodes with sterile black India ink. The study will be conducted in two phases, 
with the second phase conditional upon the successful results of the first. In Phase I, only 10 patients who 
are committed to undergo upfront surgery (without neoadjuvant chemotherapy) will be included. These 
patients will have the suspicious lymph node tattooed by injecting India ink at the time of core needle 
biopsy. Intraoperatively, the axilla will be inspected to determine whether India ink tattooed in the lymph 
nodes can be visualized by the surgeon. Microscopic inspection for the presence of the dye in nodes 
retrieved by sentinel biopsy and/or axillary dissection will also be done. In Phase II, this process will be 
repeated for patients who undergo surgery after neoadjuvant chemotherapy. 
Ethics and Dissemination: Ethical approval was obtained from the Aga Khan University Ethical Review 
Board (ERC# 2018-0345-1105). The process of sentinel node biopsy is a safe and routinely performed 
procedure at our institution. Intradermal injection of methylene blue can lead to skin necrosis; intra-
parenchymal injection can cause induration and erythema with associated pain. These side effects can be 
minimized by diluting methylene blue which will be done. The complications that can result from all 
additional steps of study procedures will be discussed with the patient and their full responsibility will be 
borne by the institution. Although sterile black India Ink is safe to administer but possible side effects can 
include mucosal inflammation due to spillage of the ink, abscess formation and others such as an allergic 
reaction. If any of these situations are encountered full responsibility to treat the side effects will be borne 
by the study team using departmental funds. No dissemination plan has been identified at the time of writing 
of this protocol.  
ClinicalTrials.gov Identifier:  

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Keywords: Lower Middle Income Countries, Axillary Lymph Node Dissection, Sentinel Lymph Node 
Biopsy, Neoadjuvant Chemotherapy, and De-Escalating Surgery, Preoperative Tattooing of the Biopsied 
Lymph Node, Black or India Ink 
 
Strengths of Study 
 
Robust measures will be employed as trained and credentialed surgeon, radiologists and pathologists 
will perform the procedures and assess the outcomes.  
Limitations of Study 
 
This will be a single institutional study, so generalizability will be limited.  
 
Sample size is small, but we are doing a pilot study to evaluate the efficacy of sterile black India ink. 
 
This study will be conducted only on female patients; breast cancer is rare in males, we will not be able 
to report the efficacy among male patients.  
 
Background 
Based on GLOBOCAN estimates, 18.1 million new cancer cases and 9.6 million cancer deaths were 
reported worldwide in 2018. Female breast cancer constituted 11.6% of the cancer burden, second only 
to lung cancer (1). The current treatment of breast cancer involves various combinations of multiple 
treatment modalities including surgery, radiation and systemic therapy. Traditionally, regional lymph 
node status was thought to be the most important determinant of the extent of surgery and need for 
systemic and radiation therapy (2). Axillary lymph node dissection (ALND) is, however, associated 
with significant morbidity (lymphedema, pain and decreased range of motion postoperatively). Sentinel 
lymph node biopsy (SLNB) has become the standard of care in clinically node negative (cN0) early 
breast cancer (EBC) due to low false negative rates, without worsening of local control or survival (3) 
(4). This less extensive staging procedure has allowed safe omission of ALND in approximately 60% 
of patients with no pathological evidence of disease in the excised sentinel lymph node (SLN) (5).  

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Recent clinical trials have focused on further de-escalating surgery and have demonstrated that in 
patients with limited axillary disease (i.e. those with micro or macro-metastasis in <3 axillary lymph 
nodes) who undergo breast conservation and receive adjuvant systemic therapy and/or radiation, routine 
ALND may be safely omitted without impacting survival and maintaining the rate of local recurrence 
to <1% (6). 
In locally advanced breast cancer with cN0 axilla, performance of SLNB after neoadjuvant 
chemotherapy (NCT) is now the standard of care. However, for patients undergoing NCT who 
demonstrate pathological evidence of disease in the axilla before commencing treatment, an ALND after 
completion of NCT has been the standard of care despite its associated morbidity. Inspired by the 
possibility and success of less surgery on the axilla in EBC, several studies have assessed the application 
of SLNB after NCT to patients with pathologic evidence of disease in the axilla at initial presentation 
(7). However, management of the axilla in such patients remains controversial (8). A trial by American 
College of Surgeons Oncology Group (ACOSOG) Z1071 evaluating the efficacy of SLN biopsy 
compared to ALND in cN+ patients who underwent NAC reported a false negative rate of 12.6% for 
SLNB which was higher than the pre-set acceptable rate of 10%. Additionally, the false negative rate 
was dependent on the number of lymph nodes retrieved, with 3 nodes being the number leading to an 
acceptable FNR of <10%. However, the retrieval of 3 SLNs cannot be guaranteed in the procedure 
regardless of currently used single or dual tracer technique thus making it impractical. Thus the utility 
of SLNB remains limited after an axilla is rendered clinically node negative by NCT due to a high false 
negative rate, unless at least 3 SLNs are recovered (9) (10). 
In an attempt to lower the false negative rate of SLN in cN+ disease after NCT, preoperative tagging of 
the biopsied lymph node to facilitate retrieval has been evaluated (11). A study from MD Anderson 
evaluated the feasibility of marking a percutaneously biopsied lymph node with a clip at the time of core 
needle biopsy, followed by ultrasound guided placement of radioactive iodine-125 seed into this clipped 
node to facilitate identification and retrieval intra-operatively. The results showed a false negative rate 

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of 4.2% (95% CI, 1.4 to 9.5) when the clipped node was retrieved along with sentinel lymph nodes and 
10.1% when SLNs alone were retrieved without the clipped node (95% CI, 4.2 to 19.8). Furthermore, 
25% of the time the clipped node was not a sentinel node and would have been missed if targeted 
retrieval had not been performed (8). While the study showed promising results the technique requires 
a sonographically visible bio-resorbable polymer (HydroMark T3) and a separate procedure for 
placement of radioactive iodine-125 seeds limiting application (2) (12) (13) (14) (15).  
Thus, alternative methods need to be identified for marking the percutaneously biopsied lymph node at 
the time of core needle biopsy. One recent study that evaluated the feasibility of tattooing axillary lymph 
nodes with black ink before NAC identified that the black ink was visible in 93% of the patients 
intraoperatively, and lasted for almost 7 months in cases where lymph node retrieval was performed 
after NCT (14, 16). This concept is adopted based on the successful experience in gastroenterology 
where tattooing is widely used for marking lesions or tumors biopsied during endoscopy as the tattoo 
ink can remains identifiable over a long period of time (17).   
There is thus a need to identify and test techniques that are practical, limit the number of procedures 
performed and could help limit the extent of surgery without negatively impacting outcomes. For the 
patient undergoing neoadjuvant chemotherapy with biopsy proven disease in the axilla, this could be 
achieved by developing a technique that allows marking, identification and retrieval of the biopsied 
lymph node, after completion of NCT at the time of SLNB. Retrieval of this previously biopsied lymph 
node along with SLNs, if found to be representative of the status of the remainder of the axilla, could 
potentially eliminate the need for routine ALND and thus limit morbidity.  
To the best of our knowledge this will be the first study conducted to evaluate the feasibility of tattooing 
and retrieval of suspicious lymph nodes using India ink. The primary objective of our study is to evaluate 
whether sterile black India ink, injected percutaneously into the biopsied lymph node, can be visualized 
intraoperatively (Phase I). The secondary objective is to determine whether the injected India ink remains 

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visible at the time of surgery even after neoadjuvant chemotherapy (lag time) and is the status of sentinel 
and tattooed lymph nodes predictive of the axillary nodal status. 
Study Objectives 
Primary Outcomes 
• 
Intra-operative identification of tattooed lymph node(s) in upfront surgery group (Phase I)  
• 
Intra-operative identification of tattooed lymph node(s) in the post-neoadjuvant surgery group 
(Phase II) 
Secondary Outcome  
• 
Concordance rate between identified sentinel node(s) and tattooed lymph node(s) 
 
Methodology 
Study Design 
A quasi-experimental pilot study will be conducted to evaluate the feasibility of tattooing suspicious 
lymph nodes with sterile black India Ink in breast cancer patients. Patients with suspected (who have not 
had a breast biopsy yet but clinically are suspected to have breast cancer) or biopsy proven breast cancer, 
with clinically suspicious axillary nodes, who have not had axillary lymph node biopsy yet, will be 
considered for this study. Such patients will then undergo an ultrasound guided core needle biopsy of the 
breast (if not already done) as well as of the suspicious axillary lymph node, in the radiology department 
by study radiologists as per our current institutional protocol. For the purpose of the study, at the time of 
the lymph node core biopsy, the biopsied node will be tattooed by injecting 0.1-0.5 ml (larger lymph 
nodes will be injected with more ink) of sterile black India ink.   
In the first phase, 10 patients planned for upfront surgery will be included. This part of the study will help 
to evaluate whether black India ink (injected percutaneously into the lymph node at the time of 
percutaneous axillary biopsy) can be identified intraoperatively by the surgeon and does it help in 
retrieval of this percutaneously biopsied lymph node. All lymph nodes excised including sentinel, 
tattooed (if recovered) and non-sentinel will be submitted in separately labelled containers for further 

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pathologic evaluation. Determination of concordance between sentinel and tattooed nodes, will be 
primarily done visually by the surgeon and confirmed by 2 independent pathologists, blinded to each 
other’s findings. Pathologic evaluation will determine concordance between sentinel/tattooed and non-
sentinel nodes.  
In Phase 2, we will  perform the same procedure as detailed above (percutaneous lymph node biopsy and 
tattooing of lymph node with black India Ink) on 30 patients who are planned to undergo neoadjuvant 
chemotherapy. Chemotherapy typically is administered over 4-6 months, which leads to a lag time 
between percutaneous lymph node biopsy/tattooing and surgery. We will need to determine whether the 
tattoo on the lymph node remains intraoperatively visible and retrievable despite the lag of several months 
(while undergoing neoadjuvant chemotherapy).  Pathologic evaluation will determine concordance 
between sentinel/tattooed and non-sentinel nodes.  
Clinical stage and evaluation by the surgeon as well as patient preference will determine the timing of 
surgery, upfront or after NCT. In all cases, whether pathologically negative or positive on biopsy, a SLNB 
will be performed at the time of definitive surgery, with dual tracer technique using technitium-99 sulfur 
colloid and methylene blue as per current institutional protocol. Intraoperatively, the axilla will be 
inspected to determine whether the black India ink tattooed in the lymph nodes can be visualized or not, is 
it easily distinguishable from methylene blue and is the tattooed lymph node also the SLN. In cases where 
biopsy of lymph node was negative, an intraoperative frozen section will be performed on the retrieved 
lymph nodes (SLN and tattooed) and need for ALND will be determined according to current institutional 
protocol. All those with evidence of metastatic disease on previous core biopsy or on frozen section of 
SLN/tattooed LN will then proceed to have an ALND.  All lymph nodes i.e. sentinel, tattooed and non-
sentinel (retrieved in ALND) submitted will be processed and evaluated as per routine protocol by a 
single study pathologist, who will issue the final pathology report. The additional study part will require 
evaluation of methylene blue and India Ink tattoo by the second independent study pathologist, to see if 
the two are grossly and/or microscopically distinguishable. Both pathologists will be blinded to each 

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other’s findings. This additional evaluation will not affect the pathologic stage or further treatment 
decisions. This step/part of the study could however impact future surgical technique. 
 
Study Setting  
The study will be conducted at Department of Surgery in collaboration with Department of Pathology and 
Radiology, the Aga Khan University Hospital Karachi-Pakistan (AKUH). AKUH is a Joint Commission 
International Accreditation (JCIA) accredited hospital and is one of the biggest private tertiary care 
hospitals in Karachi. The multidisciplinary AKUH breast surgery team consists of well qualified breast 
surgeons, radiologists, medical oncologists and pathologists who provide evidence based standard care to 
breast cancer patients following strict adherence to institutional guidelines. The reason for conducting 
single center study is to limit variability in management of breast cancer patients at different centers as 
AKUH provides a non-biased, controlled environment to evaluate the feasibility of injecting sterile black 
India ink and subsequently retrieval of the SLN using dual tracer technique, with attempted identification 
and retrieval of the marked/tattooed lymph node. 
Study Participants 
Women greater than 18 years old with histologically confirmed diagnosis of breast cancer or clinically 
suspected breast cancer and clinically suspicious ipsilateral axillary lymph nodes (on clinical exam or 
ultrasound) presenting to AKUH fulfilling our eligibility criteria and willing to give written informed 
consent for the whole duration of study period will be recruited.  
 
Eligibility Criteria 
Inclusion Criteria  
 
Female greater than 18 year of age 

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Breast cancer patient (biopsy proven or clinically suspected) with clinically suspicious (palpable on 
clinical exam or abnormal by ultrasound criteria but not biopsied yet) ipsilateral axillary lymph node(s). 
The ultrasonographic examination will be considered suspicious for metastasis if one or more of 
following criteria are present: 1. Eccentric cortical enlargement (>3 mm) or lobulation with 
displacement of hilum 2. Absent hilum and irregular border and hypoechoic echotexture 3. Spherical 
node 4. Perinodal vascularity.  (18-20) 
 
Participants willing to undergo axillary lymph node percutaneous biopsy with marking/tattooing of the 
biopsied lymph node at Aga Khan University Hospital  
 
Participants intending to have definitive surgery at Aga Khan University Hospital  
 
 
Exclusion Criteria  
 
 
Participants with terminal disease like renal failure will be excluded because these conditions can have 
profound effect on their course of treatment 
 
Participants with distant metastases 
 
Participants with prior breast or axillary surgery 
 
Participants with bilateral breast cancer, and those who have  already had an axillary core needle biopsy 
prior to inclusion (to avoid a second procedure for tattooing) 
 
Participants with recurrent breast malignancy because their course of treatment might be different 
 
Participants that were initially suspected to have breast cancer, but pathology results did not confirm 
the diagnosis 
 
Pregnant and lactating women 
 
Men with breast cancer  
 
 
Sampling Strategy 

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The study participants will be recruited through purposive sampling technique from a population of female 
patients, presenting to AKUH with biopsy proven or suspected breast cancer with a clinically suspicious 
lymph node in the ipsilateral axilla.  
Sample Size Assumption 
The sample size assumptions were based on reported proportion of correctly identified marked/tattooed 
lymph of 93% (14), with confidence level of 95% and design effect of 1, the minimum sample size 
calculated is 100 breast cancer patients. Since we are conducting a small feasibility pilot study, it will be 
conducted on 40% of the calculated sample size, i.e. on 40 breasts cancer patients with clinically suspicious, 
percutaneously biopsied and tattooed ipsilateral axillary lymph node.  The sample size was calculated 
through Open-EPI version 3.01.  
The first 10 participants will be those with clinically suspicious nodes having undergone a core needle 
biopsy with simultaneous injection of India Ink and now planned for upfront surgery. The next 30 patients 
enrolled will be those who are planned for NCT, regardless of the pathologic results of the axillary core 
needle biopsy. Since surgery in these 30 patients will be deferred for approximately 6 months, it will allow 
us to know if delayed retrieval of the tattooed lymph node is possible after NCT.   
Study Procedure 
Female patients with histologically confirmed diagnosis of breast cancer or clinically suspected breast 
cancer with clinically suspicious ipsilateral lymph nodes (not biopsied yet) will be approached to participate 
in our study by the research team. Consent from patients will be obtained by those who meet the inclusion 
criteria and intend to have surgical treatment at AKUH.  
Participants as per the institutional guidelines will undergo the routine clinical breast examination by trained 
specialist breast surgeon and radiological investigation. Those with suspected or histologically confirmed 
breast cancer and suspicious (but not biopsied yet) ipsilateral axillary lymph nodes on clinical exam or 
ultrasound will undergo an ultrasound guided core needle biopsy of the breast as well as the single, most 

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suspicious axillary lymph node either concurrently with core biopsy of the breast or in addition to 
previously performed core needle biopsy of breast, in the radiology department by study radiologists. 
The biopsied lymph nodes will be tattooed with sterile black India ink immediately following sampling of 
the suspicious lymph node.  Those planned for upfront surgery (Phase I) will be prepared and consented 
for surgery. Those planned for NCT (Phase II) will receive NCT followed by definitive surgery after 
completion of chemotherapy.  
All study patients will have a sentinel lymph node biopsy and frozen section on retrieved SLN as part of 
axillary surgery. For this purpose after injection of TC99  and lymphatic mapping preoperatively, diluted 
methylene blue will be injected into the subareolar plexus intraoperatively. The axillary hot spot will be 
assessed and documented for presence or absence, using a gamma probe. Intraoperatively axilla will be 
inspected to determine whether sterile black India ink tattooed in the lymph nodes is visible or not. Sentinel 
lymph node biopsy will be performed in usual fashion including identification of palpable lymph nodes. 
Total number of sentinel lymph nodes retrieved, number of hot, number of blue and number of hot and blue 
will be documented. If the sentinel node is also the marked/tattooed node it will be assessed and 
documented.   
Subsequently, those with a biopsy proven axillary metastatic disease on axillary core needle biopsy 
(regardless of frozen section results), will proceed to have the planned breast procedure and axillary lymph 
node dissection levels I and II in usual fashion.  Management of axilla in those participants who did not 
have biopsy proven  metastatic disease on axillary core needle biopsy (but had a suspicious node leading 
to core biopsy), will be dictated by results of  frozen section analysis. If the frozen section results show 
absence of metastatic disease in the submitted lymph nodes, no additional axillary surgery will be 
performed. While the frozen section is in process and results are awaited, the surgeon may proceed with 
the planned breast procedure. ALND will be followed by placement of a drain in the axilla as per routine.   
For post NCT patients, extent of fibrosis (mild, moderate or severe) will be documented.  All submitted 
lymph nodes will be evaluated by two independent pathologists to ascertain the visualization of sterile black 

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India ink within the nodes, and usual histologic assessment of all submitted specimens. Correlation 
between, marked/tattooed and sentinel nodes (including palpable) will be determined. Tumor pathology, 
including type, grade, receptors (ER, PR and Her-2/neu) will be reported. Credentialed surgeon, radiologists 
and pathologists according to institutional guidelines will participate in the study as this will lead to 
minimization of experimenter/assessor bias. (Study Flow Diagram) 
Statistical Analysis 
Analysis will be performed in Stata version 12. Descriptive analysis will be performed. For continuous 
variables either Mean± S.D or Median with IQR will be reported depending on the normality assumption 
of the variables. For categorical variables frequency with percentages will be reported. Proportion of lymph 
node visualized intraoperatively with Black India Ink will be reported. Concordance between the sentinel 
lymph nodes and tattooed biopsied lymph nodes will be reported. False negative rate (FNR) will be 
determined to assess the findings of preoperative pathological assessment against the final histological 
findings of the resected nodes and 95% Confidence interval (CIs) for FNRs will be calculated by exact 
method for the binomial proportion.  
Ethical Considerations  
Ethical approval is obtained Aga Khan University Ethical Review Committee no 2018-0345-1105. Written 
informed consent will be taken from the participants by properly trained data collectors. They will be 
explained the study procedures in detail along with the risks and benefits associated in taking part in the 
study. Data collectors will only ask to sign or stamp the consent form by the participants after they have 
understood the entire procedure. Data collectors will be trained by PI/Co-PI. Data collectors will be 
reinforced to recruit only those participants who will be willing to participate in the study. Participants will 
be allowed to leave the study at any point in time and they will continue to receive quality standard treatment 
services at AKUH. In addition all study materials containing personal identifiers will be kept in a locked 

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file cabinet. A unique study identification number will be assigned to each participant. After that data will 
be entered from hard copy into the electronic database that will be password protected and only accessed 
by the research staff of the study.  Participants will be explained in detail regarding the risks associated with 
the procedure. Complications that can result due to additional steps of study procedures, their full 
responsibility will be borne by the institution. Although sterile black India ink is safe to administer but 
possible side effects can include mucosal inflammation, due to spillage of the ink, abscess formation and 
allergic reaction (17). 
List of Abbreviations 
Aga Khan University Hospital 
AKUH 
Lower Middle Income Countries 
 
LMIC 
Axillary Lymph Node Dissection  
ALND 
Sentinel Lymph Node Biopsy  
SLNB 
Clinically Node Negative  
cN0 
Early Breast Cancer  
EBC 
Sentinel Lymph Node  
SLN 
Neoadjuvant chemotherapy  
NCT 
American College of Surgeons Oncology Group  
ACOSOG 
False Negative Rate  
FNR 
Joint Commission International Accreditation  
JCIA 
Standard Deviation 
S.D 
Interquartile Range 
IQR 
Confidence interval  
CI 
Principal Investigator/Co-PI 
PI/Co-PI 
 

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Declarations Section 
Ethics approval and consent to participate 
Study protocol was approved by the Aga Khan University Hospital Ethics Review Committee (ERC) with 
ERC Ref 2018-0345-1105. 
Consent for publication 
Not applicable 
Availability of data and material 
As this is a study protocol, we do not any study related data or material to share. 
Competing interests 
All authors declare no competing interest  
Funding 
The study is funded by Department of Surgery, Aga Khan University Faculty Research funds  
Authors' contributions 
AKS conceived and designed the study and wrote the manuscript. WK and BA directly overlooked all 
aspects of study design and wrote the manuscript with AKS. IM, SA, MUT, RI, MU assisted as local experts 
and informed aspects of development of the study intellectually. All authors have contributed intellectually 
to this manuscript. All authors read and approved the final manuscript. 
Acknowledgements 
We would also like to acknowledge Dr. Monica Morrow for her critical feedback on the study design and 
protocol. 
 
 
 
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