Comparison of Three Different Tissue Acquisition Techniques During Endoscopic Ultrasound-guide Fine Needle Biopsies of Solid Tumors: A Randomized Single Blind Clinical Trial.
Phase: NA
Conditions: Solid Tumor
Sponsor: Texas Tech University Health Sciences Center, El Paso

5/7/2020
1
Comparison of three different tissue acquisition techniques during endoscopic ultrasound-
guided fine needle biopsies of solid lesions: A randomized single blind clinical trial. 
ABSTRACT
a. Endoscopic ultrasound guided fine-needle aspiration (EUS-FNA) has been used since 
1990’s for the diagnosis and staging of esophageal, gastric, duodenal, pancreatobiliary, 
rectal mediastinal lesions and intra-abdominal lymphadenopathy. Studies have shown a 
variable range of specimen adequacy when performing pancreatic biopsies with the 
standard fine needle aspiration (FNA) needles with this modality. There are several 
factors that affect the overall diagnostic yield of this procedure, such as endosonographer 
experience, presence of cytopathologist during the procedure, the needle diameter and the 
number of passes. In this study we will compare the yield of recently available fine 
biopsy needles (FNB) using three different techniques to obtain samples from solid 
lesions. All solid lesions will be studied with the endoscopic ultrasound.    If during the 
procedure the characteristics of the lesion are comfortably determined and it is decided 
that it does not need a biopsy, then it won’t be done (i.e. lipomas). These lesions are very 
well determined on EUS and do not need a biopsy. The three techniques to be compared 
in this study are: stylet slow pull (SP) vs dry suction (DS) vs wet suction (WS). 
OBJECTIVES
The primary objective of this study is to compare the rate of adequacy for cytologic and/or 
histologic diagnosis of specimens obtained using three different techniques of tissue acquisition 
when performing EUS guided sampling of solid lesions with FNB needles. The three techniques 
to be compared are: SP, DS and WS.  The secondary objectives are to compare the rate of blood 
contamination of the specimen obtained by each technique 
BACKGROUND
Endoscopic ultrasound guided fine-needle aspiration (EUS-FNA) has been used since 1990’s for 
the diagnosis and staging of esophageal, gastric, duodenal, pancreatobiliary, rectal and 
mediastinal lesions and intra-abdominal lymphadenopathy (1). Studies have shown a variable 
range of specimen adequacy when performing pancreatic biopsies with the standard fine needle 
aspiration (FNA) needles with this modality (2,3).  When sampling the lesions, the use of a stylet 
and/or suction during FNA of solid lesions has been used to increase the accuracy, efficiency and 
quality of the specimen. Theoretically, the use of a stylet prevents obstruction of the needle 
lumen as it penetrates the gastrointestinal wall tissue, resulting in less contamination from GI 
wall cells (4).
In the “suction technique” the stylet of the needle can be left in place or removed before 
puncturing the lesion.  Once the needle is inside the target, negative pressure is applied through a 
10 or 20 cc syringe connected to the needle.  This technique has yielded adequate specimens in 
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
2
approximately 70% of cases in some studies, with no difference seen between the stylet vs no 
stylet modality (5, 6).   Some alterations of this technique have been used. In cases of 
neuroendocrine tumors especially, no suction techniques have been tried to avoid the effect of 
dilution by too much bleeding in such highly vascular tumors (7).  In a recent study on 52 patients 
(8), the accuracy was higher utilizing the suction technique compared to no suction.  The 
bloodiness of the samples was not different in each arm.  The lesions were located in the 
mediastinum, pancreas, adrenals and others. Another study on 100 patients in 2014 did not report 
any difference in the adequacy of samples obtained with or without suction (9). The samples were 
more contaminated with blood in the suction group though. It has been concluded that at the 
expense of a bloodier sample, suction method yields a high cellular material (10).   The wet 
suction technique consists of flushing of the needle with 5 ml of saline solution to replace the 
column of air within the lumen of needle with saline solution before needle aspiration. Once the 
needle is flushed, negative pressure is applied with a 10 or 20 cc syringe connected to the needle. 
This novel technique was compared with the conventional FNA technique by using 22-gauge 
needles (11).  A total of 117 samples from 95 patients and 72 lesions were obtained in an 
alternating pattern between the two techniques. If the first pass was done by wet suction then the 
second one was done by the FNA conventional method. Higher cellularity scores and adequacy 
of specimen were achieved with wet suction (68% vs 44%, respectively) and (85% vs 75, 
respectively). However, the diagnostic yield difference was not specified. 
In the slow pull technique, the stylet is left in place in the needle and is slightly retracted prior to 
puncturing the lesion. Once the needle is inside the target, the stylet is pushed completely into 
the needle to remove any contaminant cells and several back and forth movements are done 
while slowly withdrawing the stylet.  One study compared slow pull technique versus suction (12).  
Although scores of cellularity were lower in the slow pull group (A score of more than or equal 2 
in 37.5% vs 76.7%), the contamination of samples observed was lower (25% vs 66.7%). The 
accuracy and sensitivity of the slow-pull technique were significantly higher than those of the 
suction techniques for solid pancreatic lesions (13).  Specifically for malignancy, the slow pull 
technique has demonstrated sensitivity for diagnosis of 90% compared to 68% with the suction 
technique. (12) Moreover, this technique showed less false negatives results than suction (11 vs 
26%) (14 ABSTRACT).
Recently new fine needle biopsy (FNB) needles have become widely available in the US.  These 
needles differ from FNA needles in its design and allow for long cores of tissue to obtained. 
Recent studies have already suggested that samples obtained with these needles are superior to 
the ones obtained with standard FNA needles (15).  Given the novelty of these FNB needles, 
literature regarding different techniques of tissue acquisition with them is scarce.  Therefore, in 
this study we aim to compare the cellularity of specimens obtained with these needles using the 
three different techniques described above in a randomized fashion. 
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
3
RESEARCH PLAN
Study Design                                                                                                                               
This study is planned as a single–blind, parallel group randomized clinical trial (RCT).  After receiving 
consent from the eligible subjects, the subjects will be randomly assigned to one of the three techniques 
and one of two needles, stratified by the three (3) endoscopists performing EUS-FNB.    
Primary endpoint
1. Compare the rate of adequate specimens obtained by slow pull vs dry suction vs wet 
suction techniques using the new fine needle biopsy (FNB) needles in solid lesions.
Secondary outcome
1. Compare the degree of blood contamination obtained by all three techniques.
Inclusion criteria
b. Ages 18 years and older
c.
Male or female
d. Patients who require EUS and tissue sampling of solid lesions (size >1 cm) anywhere in 
the body: lymph nodes, stomach, esophagus, colon, small intestine, pancreas, liver, 
spleen or kidney. 
e.
Patients who are able to give consent
Exclusion criteria 
a) Pregnant female
b) Coagulation disorders (platelets < 50,000/mm3, INR > 2)
c) Patients with acute pancreatitis in the immediate 2 weeks prior to the procedure (if the 
lesion to be biopsied is in the pancreas). .
d) Cardiorespiratory dysfunction that precludes sedation. 
e) Unable to provide informed consent
f)
Previous chemotherapy or radiotherapy for pancreatic neoplasm
Patient Enrollment: Patients will be prospectively enrolled until the target sample size has been 
reached. This study is planned as a single-blind, parallel group randomized trial. There will be 
three parallel groups.  The first group will be assigned to the slow pull technique, the second one 
to the dry suction technique and the third one to the wet suction technique.  Our calculated 
sample size is 300 patients. In our unit, we perform approximately 10 – 15 EUS guided 
pancreatic mass biopsies per month. Therefore, we expect to enroll the desired number of 
patients in approximately 60 months. 
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
4
Data Collection:  
For each patient, the following data will be collected:
1. Age
2. Gender
3. Ethnicity
4. EUS characteristics of the lesion (number, location, size, echogenic pattern, invasion into 
other structures)
5. Number of needle passes
6. Other maneuvers if  3 passes  yields an inadequate specimen
7. Immediate complications (bleeding, perforation, hemodynamic instability)
Statistical analysis
Sample size:  The sample size computation was done on the basis of published data (5, 6, 11, 13).  A 
previous study [11] showed that Wet suction technique produced moderate to high cellularity 
(67.5%) compared to conventional FNA technique (44.4%). Another manuscript demonstrated 
that slow-pull technique had higher moderate to high cellularity (71%) as compared to suction 
technique with 5-mL (57.9%) (13). Other studies (5, 6) reported cellularity between 46% and 
73.6%. Based on this prior information, we assumed that one of the techniques will yield at least 
25% higher cellularity compared to the baseline 45%. Based on this, a sample size of 95 per 
group will be sufficient to detect significant difference in at least one pair of techniques with 
more than 80% power and at 1% level of significance using a two-sided Z-test with pooled 
variance. Further, we also validated sample size computation for comparing average cellularity 
score across three groups. A study reported average cellularity score by dry technique as 1.85 
(SD: 0.79) and by WET as 1.70 (SD:0.72) compared to average 1.45 (SD: 0.78) (11).   Using this 
information, a sample size of 100 per group will be sufficient to detect significant difference in 
average cellularity scores among groups using a one way analysis of variance (F-test) with 80% 
power and at 1% level of significance.  The level of significance was adjusted due to multiple 
comparisons in both calculations. This sample size seems to be appropriate for detecting 
significant differences in other outcomes (such as diagnostic yield) as well. The sample size 
computation was carried out using PASS 14 (PASS 14 Power Analysis and Sample Size 
Software (2015). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/pass.). 
Statistical Analysis:  All categorical variables will be described using count and percentage 
whereas the quantitative variables will be described using mean and standard deviation(SD). The 
baseline characteristics will be compared among three groups using either one way analysis of 
variance (for quantitative data) or  Fisher’s exact tests (for categorical data).  The primary 
outcome (cellularity score: quantitative and categorical forms and percentage positive diagnostic 
yield) will be compared between groups using an F-test or Fisher’s exact tests. The proportion 
differences between two groups will be compared using two sided Z-test between two groups 
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
5
while quantitative scores will be compared using t-tests with Bonferroni corrected method. The 
effect size will be summarized using proportion difference along with 95% confidence interval 
(CI) estimated using Binomial distribution. Further, relative risk regression analysis (16) will be 
performed for determining adjusted differences in cellularity and diagnostic yields between 
groups. The regression analysis will be carried out separately for each outcome. This analysis 
will also help to identify any factors associated with increased percentage of cellularity and 
diagnostic yield.  The secondary outcomes will also be compared between groups using a 
Fisher’s exact test or an F-test depending on the type of outcome. The results of relative risk 
regression analysis will be summarized using relative risk along with 95%CI and p-value. P-
values less than 5% will be considered statistically significant results. All the statistical analyses 
will be processed using STATA 14 and SAS 9.4. 
METHODS
Patients will be enrolled in a prospective manner at the University Medical Center of El Paso 
endoscopy unit.  At the time of the pre-op appointment the nurse will perform the usual 
evaluation of the patient and will explain the EUS/FNB procedure. The day of the procedure, the 
endoscopist performing the procedure explain the EUS/FNB procedure and the risks of the 
procedure, explain the study and will consent the patients.  He will randomize them using a 
random number list generated by a simple block randomization scheme by our study statistician, 
to one of three possible groups: SP, DS or WS.  The randomization number will be kept in sealed 
opaque envelopes in numerical sequence. The random number sequence will be generated using 
SAS software.  The patients will be asked to sign the consent only after the study has been 
explained to them and all their questions have been answered by the performing physician. The 
randomization will be via sealed envelopes that will contain cards with the technique and the 
needle to be used.  Following randomization each patient will be assigned a code corresponding 
to one of the three technique arms. This code will be noted and placed in a master spreadsheet 
that only the study coordinator and/or assigned research personnel will handle. 
All the procedures will be performed by three experienced endoscopists (Cesar Garcia, M.D., 
Sherif Elhanafi, M.D. and Antonio Mendoza-Ladd, M.D.) using 22 gauge SharkCore™ 
(MEDTRONIC) or AcquireTM (BOSTON SCIENTIFIC) FNB needles.  The techniques to be 
used are described here:
DS: the stylet will be left in place in the needle and once the lesion has been punctured, the stylet 
will be withdrawn. After this, negative pressure with a 10 cc syringe will be applied to the 
needle.  Once the lesion has been punctured, the valve between the syringe and the needle will be 
opened and the endoscopist will obtain the sample with multiple back and forth movements. 
SP: the stylet will be left inside the needle and retracted slightly prior to puncturing the lesion in 
order to sharpen the needle.  Once the lesion has been punctured, the stylet will be pushed 
completely into the needle to expel any debris from the gastrointestinal wall, then it will be 
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
6
slowly withdrawn from the needle as the endoscopist is making the back and forth movements to 
create negative pressure in the system. 
WS: Prior to puncturing the needle, the stylet will be completely withdrawn and the needle will 
be flushed with 10 cc of normal saline until the entire needle lumen is completely primed with 
fluid.  After this, negative pressure with a 10 cc syringe containing 1 cc of NS will be applied in 
a similar manner as in the DS technique.  The procedures will be performed under deep sedation 
according to the principles of monitored anesthesia care. The patients will be anesthetized with 
intravenous administration of propofol by trained anesthesists. Once the lesion is evaluated by 
EUS, the endoscopist will select the shortest pathway, while avoiding blood vessels, to reach the 
pancreatic lesion. Under real-time visualization, each lesion will be punctured with 3 needle 
passes. 
If the tissue sample is deemed suboptimal after these 3 passes, then the endoscopist will be 
allowed to use a different needle and/or technique according to his preference.  The specimen 
obtained will be assessed preliminarily by the cytology technician who will determine if more 
tissue is needed.  Once the tissue is deemed to be sufficient, the pathologist evaluating the 
specimen will dictate the official cellularity and blood contamination scores according to scales 
previously described by Gerke et al (17) and Wee et al (18).  These will be the scores used for 
analysis.  A score of ≥2 and ≥4 will be considered adequate for cytological and histological 
diagnosis respectively. The pathologists evaluating the sample will be blinded to the type of 
needle and technique used.
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
7
REFERENCES
1) Yamao K, Sawaki A, Mizuno N, et al. Endoscopic ultrasound-guided-fine-needle 
aspiration biopsy (EUS-FNAB): past, present, and future. J Gastroenterol. 2005; 40: 
1013-23.
2) Weston BR, Bhutani MS. Optimizing diagnostic yield for EUS-guided sampling of solid 
pancreatic lesions: A technical review. Gastroenterol Hepatol. 2013;9: 352-63
3) Hewitt MJ, McPhail, Possamai L, Dhar A, et al. EUS-guided FNA for diagnosis of solid 
pancreatic neoplasms: A meta-analysis. Gastrointest Endosc. 2012; 75:319-31.
4) Rastogi A, Wani A, Gupta N, et al. A prospective, single-blind, randomized, controlled 
trial of EUS-guided FNA with and without stylet. Gastrointest Endosc. 2011; 74: 58-64.
5) Wani S, Early D, Kunkel J, et al. Diagnostic yield of malignancy during EUS-guided 
FNA of solid lesions with and without stylet: a prospective, single blind, randomized, 
controlled trial. Gastrointest Endosc. 2012; 76:328-35. 
6) Abe Y, Kawakami H, Oba K, et al. Effect of a stylet on a histological specimen in EUS-
guided fine-needle tissue acquisition by using 22-gauge needles: a multicenter, 
prospective, randomized, controlled trial. Gastrointest Endosc. 2015; 82:837-44.
7) Matsubayashi, Hiroyuki, et al. Endoscopic ultrasonography guided-fine needle aspiration 
for the diagnosis of solid pancreaticobiliary lesions: Clinical aspects to improve the 
diagnosis.World journal of gastroenterology 22.2 (2016): 628.
8) Puri, Rajesh, et al. Randomized controlled trial of endoscopic ultrasound-guided fine-
needle sampling with or without suction for better cytological diagnosis. Scandinavian 
journal of gastroenterology 44.4 (2009): 499-504.
9) Alizadeh, Amir, et al. Comparison of two techniques for endoscopic ultrasonography 
fine-needle aspiration in solid pancreatic mass. Endoscopic ultrasound 3.3 (2014): 174. 
10) Storm, Andrew C., and Linda S. Lee. Endoscopic ultrasound-guided techniques for 
diagnosing pancreatic mass lesions: Can we do better? World Journal of 
Gastroenterology 22.39 (2016): 8658.
11) Attam, Rajeev, et al. “Wet suction technique (WEST)”: a novel way to enhance the 
quality of EUS-FNA aspirate. Results of a prospective, single-blind, randomized, 
controlled trial using a 22-gauge needle for EUS-FNA of solid lesions. Gastrointest 
Endosc. (2015): 1401-1407.
12) Nakai, Yousuke, et al. Slow pull versus suction in endoscopic ultrasound-guided fine-
needle aspiration of pancreatic solid masses. Digestive diseases and sciences 59.7 (2014): 
1578-1585.
13) Chen JY, Ding QY, Lv Y. Slow-pull and different conventional suction techniques in 
endoscopic ultrasound-guided fine-needle aspiration of pancreatic solid lesions using 22-
gauge needles. World J Gastroenterol. 2016; 22(39): 8790-8797. 
14) Lee JM, Lee H, Lee SY, et al. The usefulness of slow-pull back technique of Eus-fna in 
patients with pancreatic masses; a prospective comparative study. Gastrointest Endosc. 
2017; 85 (5): AB 357.
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
8
15) Kandel P, Tranesh G, Nassar A, et al. EUS-guided fine needle biopsy sampling using a 
novel fork-tip needle: a case-control study. Gastrointest Endosc. 2016; 84(6):1034-1039
16) Dwivedi AK, Mallawaarachchi I, Lee S, Tarwater P. Methods for estimating relative risk 
in studies of common binary outcomes. J Appl Stat 2013;41(August 2014):484–500
17) Gerke H, Rizk MK, Vanderheyden AD, et al. Randomized study comparing endoscopic 
ultrasound-guided Trucut biopsy and fine needle aspiration with high suction. 
Cytopathology. 2010 Feb;21(1):44-51
18) Wee E, Lakhtakia S, Gupta R, et al. Endoscopic ultrasound guided fine-needle aspiration 
of lymph nodes and solid masses: factors influencing the cellularity and adequacy of the 
aspirate. J Clin Gastroenterol. 2012 Jul;46(6):487-93.
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020

5/7/2020
9
APPENDIX
score
Explanation
0
Insufficient material for interpretation
Cytology 1-2
1
Sufficient material for limited cytological interpretation; probably not representative
2
Sufficient material for adequate cytological interpretation
Histology 3-5
3
Sufficient material for limited histological interpretation
4
Sufficient material for adequate histological interpretation, low quality (total material < 
1 10× power field in length)
5
Sufficient material for adequate histological interpretation, high quality (> 1 10× power 
field in length)
IRB NUMBER: E17118
IRB APPROVAL DATE: 05/08/2020
IRB EXPIRATION DATE: 07/01/2020