Comparison of Fine Needle Aspiration Techniques

Abstract

Background:

Fine needle aspiration (FNA) has been reported since 1912 beginning with the use of trocars and other specialized instruments that were impractical. Since then, FNA has proven to be a successful alternative technique to excisional biopsy for some assays despite a few limitations.

Methods:

In this study we compared four different techniques for FNA in rhesus macaques by evaluating total live cells recovered and cell viability using a standard 6 ml syringe and 1.5-inch 22-gauge needle.

Results:

Technique B, which was the only technique in which the needle was removed from the syringe after collection of the sample to allow forced air through the needle to expel the contents into media followed by flushing of the syringe and needle resulted in the highest total cell count and second highest cell viability in recovered cells.

Conclusion:

Based on our results, Technique B appears to be the superior method.

Introduction

Fine needle aspiration (FNA) has been documented for use in obtaining specimens for analysis since 1912, when Ward suggested its use in diagnosing lymphoblastomas. Others, such as Goeller in 1920, Guthrie in 1921 and Forkner in 1927, described its use for studying prostate tissue, Hodgkin’s disease, and general use FNA, respectively. The early reports required specialized instruments which were not readily available, not practical, or caused significant tissue trauma.¹⁶ However, in 1926, Hayes, Martin, and Ellis at Memorial Hospital in New York began collecting aspirations with an 18 gauge needle and 20 ml syringe, both of which were readily available at clinics and hospitals.^7,16 The advantages to their technique were no hospitalization or extensive patient preparation and minimal discomfort to the patient during the rapid procedure.¹⁶

FNA has proven to be an atraumatic, economical, and fairly rapid technique for obtaining biopsy results.^1,7,17,18,24 There are limitations with FNA compared to excisional biopsies, mainly with the cell yield; however, most studies report a range of 1×10⁵⁻⁶ cells/collection, which is normally sufficient for immunophenotyping.^{2,7,10,11,14,15,17–20,24} The aim of this study was to compare the total live cells yield and viability of four different techniques for FNA in rhesus macaques.

Materials and Methods

This study was conducted at the Tulane National Primate Research Center (TNPRC), which is fully AAALAC-accredited. All procedures were IACUC-approved and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The animal housing rooms were maintained on a 12:12-light: dark cycle, with a relative humidity of 30% to 70% and a temperature of 64 to 84 °F(18–29°C). All animals were fed a standard, commercially formulated primate diet with fruit offered at least 3 times weekly as part of the enrichment program. Fifteen animals were enrolled in this study, 6 females and 9 males. The average age was 9.74 years, with a range of 4.5 to 21.7 years. The average weight was 8.2 kg, with a range of 4.8 – 12.4 kg.

Four FNA techniques, A-D, were evaluated as part of the study design and each animal served as its own control as all four techniques were performed on each animal (Table 1). FNA for technique A and C were performed on the left inguinal lymph nodes and techniques B and D were performed on the right inguinal lymph node. Sampling was consistent for each animal with technique A performed first, then technique C on the left inguinal lymph node and technique B performed first, then technique D on the right inguinal lymph node. All lymph node FNA were collected during necropsy with a 6 ml syringe and a 22-gauge, 1.5-inch needle (Exelint International, Redondo Beach, CA). Debakey forceps were used to isolate the lymph nodes for FNA for all techniques (Figure 1A, B). Techniques A and B were performed with a syringe and needle under negative pressure and the needle was directed several times throughout the lymph node during aspiration. Negative pressure was then discontinued prior to removal of the needle from the lymph node. Technique A, the needle and syringe remained attached after sample collection to flush contents into media. Technique B, the needle was removed after collection of the sample and the syringe was filled with air and then reconnected to the needle to force air through the needle to expel contents into media. Technique C utilized a needle only, no syringe for a needle core biopsy. The needle was directed several times throughout the lymph node. Following the core biopsy, the needle was attached to a syringe to flush contents into media. Technique D utilized the INRAD Aspiration Biopsy Syringe Gun (Inrad Inc., Kentwood, MI) for sample collection (Figure 2) with the same 6 ml syringe and 22-gauge, 1.5-inch needle. The FNA was collected under negative pressure by directing the needle several times throughout the lymph node during aspiration. Technique D maintained the same procedure post FNA as technique A in which the needle and syringe remained attached after sample collection to flush contents into media. For each technique, contents of the aspirate or needle core biopsy were flushed with media three times and then expelled into the collection tube.

Table 1.

Description of FNA collection procedures for techniques A-D.

TECHNIQUE	FNA PROCEDURE	POST FNA COLLECTION PROCEDURE
A	NEEDLE ATTACHED TO SYRINGE, NEGATIVE PRESSURE APPLIED	SYRINGE AND NEEDLE REMAIN ATTACHED AFTER SAMPLE COLLECTION TO FLUSH SYRINGE AND NEEDLE WITH MEDIA 3X
B	NEEDLE ATTACHED TO SYRINGE, NEGATIVE PRESSURE APPLIED, NEEDLE IS REMOVED AFTER COLLECTION OF SAMPLE	NEEDLE IS REMOVED AFTER COLLECTION OF SAMPLE, AIR IS FORCED THROUGH NEEDLE TO EXPEL CONTENTS INTO MEDIA, SYRINGE AND NEEDLE FLUSHED WITH MEDIA 3X
C	NEEDLE WITHOUT SYRINGE IS INSERTED INTO LYMPH NODE	AFTER COLLECTION OF SAMPLE, NEEDLE IS ATTACHED TO SYRINGE FILLED WITH AIR TO EXPEL CONTENTS INTO MEDIA, SYRINGE AND NEEDLE FLUSHED WITH MEDIA 3X
D	INRAD ASPIRATION BIOPSY SYRINGE GUN, NEEDLE ATTACHED TO SYRINGE, NEGATIVE PRESSURE IS APPLIED	SYRINGE AND NEEDLE REMAIN ATTACHED AFTER SAMPLE COLLECTION TO FLUSH SYRINGE AND NEEDLE WITH MEDIA 3X

Figure 1.

(A)Debakey forceps isolating the inguinal lymphnode. (B) Fine needle aspiration of the lymph node with a 22 gauge needle.

Figure 2.

INRAD Aspiration Biopsy Syringe Gun

All FNA samples were passed through a 70 μm cell strainer (SureStrain^™, Premium Cell Strainers, MTC BIO Incorporated, Metuchen, NJ) to remove fat and tissue debris, the collection tubes and cell strainers were washed with 2 ml cold 2% fetal bovine serum (FBS) in PBS. The cells were then centrifuged at 4^° C for 10 minutes at 300xg. The supernatant was completely discarded. If red blood cell contamination was still present, the cells were resuspended in 1 ml ammonium chloride potassium and incubated for 5 minutes at room temperature. Three mls of cold 2% FBS was added to the cells and centrifuged at 4^° C for 10 minutes at 300 xg. The supernatant was completely discarded and the cells were resuspended in 0.5 ml cold 2% FBS.

Samples were evaluated for viability and total live cells by acridine orange propidium iodide staining and counting on Cellometer Auto 2000 (Nexcelom Bioscience LLC, Lawrence, MA). Data were analyzed using the nonparametric Friedman Test with Dunn’s multiple comparison test to determine significant differences between groups, p<0.05.

Results

Technique A had a viability of 58% and an average of 3.8×10⁶ live cells. Technique B had a viability of 71% and 5.2×10⁶ total live cells. Technique C had 76% viability and 2.2×10⁶ total live cells and technique D had 69% viability and 4.7×10⁶ total live cells. (Table 2) With regards to cell viability, the only significant difference was between techniques A and C, with technique C demonstrating the higher percentage of viable cells obtained. There were no other significant differences between techniques. (Figure 3A,B) Total live cell measurements revealed the only significant difference was between techniques B and C with technique B had a higher number of live cells obtained. There were no other significant differences between techniques. (Figure 4A,B)

Table 2.

Total live cells recovered and percent viability by FNA techniques A-D.

TECHNIQUE	A	B	C	D
VIABILITY	58.04%	70.98%	76.46%	68.79%
TOTAL LIVE CELLS	3.8×10^6	5.2×10^6	2.2 × 10^6	4.7×10^6

Figure 3 A and B.

Median and 95% confidence intervals for the percent viability of cells recovered by FNA techniques A-D. There was an overall significant difference among techniques (Friedman statistic = 9.69, p = 0.0214). Technique A had a significantly lower percent of viable cells compared to Technique C (Dunn’s multiple comparison Z = 2.83, p = 0.0281). No other significant differences were found.

Figure 4 A and B.

Median and 95% confidence intervals for the total live cells recovered by FNA techniques A-D. There was an overall significant difference among techniques (Friedman statistic = 10.04, p = 0.0182). Technique B had a significantly more live cells compared to Technique C (Dunn’s multiple comparison Z = 3.11, p = 0.0112). No other significant differences were found.

The lymph nodes were also identified as either normal size, small, or surrounded by adipose tissue. Small lymph nodes were barely palpable through the skin and lymph nodes defined as surrounded by adipose tissue were lymph nodes in which the margins of the lymph node was difficult to detect due to the surrounding adipose tissue. Four of the fifteen animals had moderate amount of adipose surrounding the lymph node, which can increase the difficulty in isolating the lymph node. Three animals included in the study had a sample with no cells obtained, all of which had adipose tissue surrounding the lymph node. Four of the fifteen animals had small lymph nodes. We examined the effects of lymph node grade (normal, small, or surrounded by adipose) on the results of cell viability and total live cells. There were no statistically significant difference for the main effects of technique or lymph node type or the interaction of the two on the outcome variables regarding cell viability or total live cells. (Figures 5 and 6)

Figure 5.

Median and 95% confidence intervals for the percent viability of cells recovered by FNA techniques A-D grouped by lymph node type. No statistically significant differences were found

Figure 6.

Median and 95% confidence intervals for the total live cells recovered by FNA techniques A-D grouped by lymph node type. No statistically significant differences were found due to the significant variability within subjects.

Discussion

It has been previously established that lymph node FNA has been proven as a viable alternative to excisional biopsy as a minimally invasive technique to obtain lymphocytes. FNA of the lymph nodes allows for longitudinal analysis of different cell subsets and, in some cases, lymphoid viral load with increased frequency.^{2,3,5,6,10,14,15,18–20,24} It has also been established that in order to allow for repeated sampling of lymphoid tissue to measure viral load and cell populations to assess treatment efficacy, less invasive sampling techniques are essential.^2,14 Previous publications have demonstrated that the percentages of particular T lymphocytes correlated significantly or strongly, depending on the cell subset, between lymph node FNA and lymph node excisional biopsy.^2,14,18,24 It has also been demonstrated that serial sampling of lymph nodes by FNA doesn’t affect the animals immune response, architecture of the lymph node, or interfere with future histological examination.^10,11,18,21

The technique for FNA of the lymph nodes has already been standardized. Different volume syringes have been used and most studies utilize 22–25-gauge needles, with the smaller needle diameter minimizing blood contamination. The skin over the lymph node is surgically prepared and the lymph node is isolated. The needle is directed into the lymph node and multiple passes of the needle through the lymph node are performed. Studies have reported collection of the aspirate both with and without vacuum of the syringe. If a vacuum is engaged, the pressure in the syringe is released prior to withdrawing the needle from the lymph node. The aspirate is then expelled into the media and the needle and syringe are normally flushed with the media.^{1,2,7,8,9,10,14,16,20,25}

Our results are consistent with other published findings with our total live cell counts (2.2×10⁶ – 5.2×10⁶). Our cell viability ranged from 58–76% and other reports describe a wide range from 30–90%.^15,18,22 Technique B had the highest total number of live cells and the second highest percentage viability. It was the only technique with a vacuum and air forced through the needle to expel the contents into media prior to flushing the needle with media. This technique also appears to be the standard and most common technique utilized in literature. Technique C had the highest percentage viability and was the only technique in which no vacuum was applied while collecting the sample and the pressure applied to generate a vacuum in the syringe for aspiration has been previously reported to damage cells.^12,25 Technique C also had the lowest total live cells obtained. Technique C, which has been described as Fine Needle Non-Aspiration (FNNA), core biopsy, or capillary technique has demonstrated lower cell yields than aspiration techniques, which provides less sufficient material for diagnosis. FNNA also performed worse than aspiration techniques with regards to architecture of the sample and has provided inadequate tissue in benign and fibrous lesions. FNNA has been reported to cause less blood contamination however it was not statistically significant.^4,9,12,13,15

We had three FNA samples in which no cells were obtained, all of which were sampled from lymph nodes surrounded by adipose tissue. It has been proposed that ultrasound guidance should be employed when the lymph nodes are not easily palpated due to surrounding fat to help ensure successful sample collection.²⁴

Several syringe guns have been tested including the Inrad Aspiration Biopsy Syringe Gun used here, and no differences were found in samples obtained compared to standard manual syringe and needle aspiration.¹² There are several disadvantages to some of the syringe guns commercially available. They can be cumbersome and difficult to control during the procedure, which can cause increased pain and hemorrhage.,^9,12,23 The cost is also significantly increased with the use of a syringe gun compared to the standard syringe and needle.

Based on our results, Technique B appears to be the superior method for lymph node FNA. Technique B had the highest total live cell count and the second highest cell viability and provided an adequate number of cells for phenotyping. Technique B was the only technique to utilize a standard syringe and needle in which the needle is removed from the syringe after collection of the sample to allow forcing air through the needle to expel the contents into media, followed by flushing the syringe and needle with the media.