1 2 , 3 4 5 – 16 99 m 2 3 17 17 26 18 11 19 , 20 21 22 22 , 23 The objective of this study was to develop a method for confirming that a FNB specimen was obtained from a true SLN by measuring the level of antimony (present in radiocolloid used during preoperative lymphoscintigraphy) in the FNB sample by ICP-MS. Materials and Methods Instrumentation 1 121 123 103 TABLE 1. Operating parameters for Agilent 7500ce Inductively Coupled Plasma–Mass Spectrometry Parameter Condition Radiofrequency power 1500 W Sample depth 8 mm Plasma gas flow  −1 Carrier gas flow −1 Peristaltic pump .15 rps Sample read delay 60 s Rinsing time 45 s Dwell time 100 ms Replicates 6 rps, revolution per second. Reagents and Chemicals All reagents used were of the highest purity available. Seventy percent double-distilled nitric acid, 37% hydrochloric acid (Arastar), and 30% hydrogen peroxide were obtained from Sigma-Aldrich, Australia. Ultra-high-purity water was produced by passing distilled water through a Milli-Q deionizing system (Millipore, Australia). Standards and Certified Reference Materials Certified reference material GBW 07601 Human Hair Powder (Langfang, China) was selected because of its certified antimony levels. Multiple samples of the reference material were also analyzed with each batch of samples and during method development to ensure the accuracy and precision of the analytic technique. SLN Biopsy Procedure 99 m 2 3 24 The SLN biopsy procedure was performed within 24 hours of the radiocolloid injection, so that residual radioactivity in lymph nodes could be measured intraoperatively with a handheld gamma probe (Navigator GPS, RMD Instruments, Watertown, MA). Fifteen minutes before the operative procedure, multiple intradermal injections of Patent Blue V dye (Guerbet, Roissy, France) were made around the primary cutaneous melanoma site. SLN identification was based on visualization of the nodal blue dye staining and results from the preoperative lymphoscintigraphy, with gamma probe confirmation. The experimental protocols of this study were approved by the University of Sydney Ethics Review Board in accordance with the precepts established by the Declaration of Helsinki. FNB Samples 2 4 2 22 Microwave Digestion Procedure 17 Statistical Analysis t P Results m b b m b b R 2 2 3 P 1 TABLE 2. Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from sentinel lymph nodes Sample No. Antimony concentration (ppb) 1 <LOD 2 <LOD 3 <LOD 4 <LOD 5 .0666 6 .0979 7 .1738 8 .2985 9 .3020 10 .3085 11 .3257 12 .3807 13 .3812 14 .3864 15 .3903 16 .4045 17 .4971 18 .6079 19 .6407 20 .7243 21 .8582 22 1.091 23 1.438 24 1.444 25 1.724 26 1.760 27 1.847 28 2.013 29 2.150 30 2.298 31 2.756 32 3.248 LOD, limit of detection (.048 ppb). TABLE 3. Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from nonsentinel lymph nodes Sample No. Antimony concentration (ppb) 1 <LOD 2 <LOD 3 <LOD 4 <LOD 5 <LOD 6 <LOD 7 <LOD 8 <LOD 9 <LOD 10 <LOD 11 <LOD 12 <LOD 13 <LOD 14 <LOD 15 .1159 LOD, limit of detection (.048 ppb). Fig. 1. Antimony concentration in digested fine-needle biopsy samples from sentinel lymph nodes and nonsentinel lymph nodes.  Discussion 25 6 , 8 – 10 , 12 , 14 26 sentinel 18 27 , 28 26 27 26 P 19 , 20 22 , 23 23 29 , 30 The results of our preliminary work provide evidence that determining antimony levels in FNB specimens by ICP-MS can confirm SLN identity and can differentiate SLNs from non-SLNs. This technique may be a useful adjunct to other techniques assessing tumor-harboring status of SLNs on FNB specimens such as MRS. Potentially, proton MRS analysis of a FNB from a node confirmed to be a true SLN by ICP-MS could provide a reliable method for determining the tumor-harboring status of SLNs. Although the results are promising and raise the possibility that SLN assessment may be performed on FNB specimens (rather than histologic specimens of excised SLNs) in the future, further validation studies are necessary before such techniques are used in widespread clinical practice. Furthermore, the highly specialized and expensive nature of the equipment used for ICP-MS will likely limit the technique to specialist centers.