Multi-site Ultrasound-guided Fine Needle Aspiration to Study Cells and Soluble Factors From Human Lymph Nodes

Abstract

Lymph nodes (LNs) are specialized secondary lymphoid tissues essential to the priming and maintenance of adaptive immune responses, including the B cell germinal center response; thus, they are central to immunity. However, the anatomically restricted and time-resolved nature of immune priming means that sampling disease-relevant human LNs requires specialized techniques. This article describes the application of ultrasound-guided fine-needle aspiration (FNA) to sample LNs, using cervical LNs of the head and neck as an exemplar. This minimally invasive technique allows collection of both immune cells and cell-free material that are relevant to both neuroimmune diseases and basic lymphatic functions. Downstream use of cellular material can include multiplexed flow cytometry, single-cell transcriptome sequencing (RNA-seq), and B cell cultures. The cell-free supernatant can be used for proteomics or other similar 'omics approaches. This unit describes collection of samples by FNA as well as processing and storage of samples for downstream assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sampling of human cervical lymph nodes by ultrasound-guided fine-needle aspiration Alternate Protocol: Sampling of human lymph nodes by ultrasound-guided fine-needle aspiration with negative pressure Basic Protocol 2: Processing and storage of human lymph node samples.

Keywords: cervical lymph nodes; fine needle aspiration; lymph; lymph node.

Figure 1

Participant satisfaction survey. Participant survey data (n = 19) on experience of CLN FNA. Survey participant age range from 26 to 75 years.

Figure 2

Example informal participant information. Example text used in the informal participant information document as part of ANIMATE study (23/LO/0459) to discuss LN FNA.

Figure 3

Cervical LN sampling. (A) Anatomic localization of the cervical LNs within the neck. Our prior sampling has been primarily from level 1 and 5 LNs, highlighted in green. Reproduced from Al‐Diwani et al. (2022). (B) Locating an accessible cervical LN. (C) Ultrasound image of the neck with detailed anatomic annotation of key major structures. (D) Sampling of the cervical LN under ultrasound guidance. (E) Ultrasound image of the neck with the LN and the FNA needle highlighted.

Figure 4

Underlay procedure to remove red blood cells. (A) Orientation of sample collection tube from Basic Protocol 1 in the microcentrifuge to aid cell isolation. (B) Pipetting technique to remove LN FNA supernatant without disturbing the cell LN cell pellet. (C) Filling the Pasteur pipet with Ficoll. (D, E) Making a seal on the Pasteur pipet to allow insertion of the pipet into the sample tube. (F) Gravity‐aided flow of Ficoll into the bottom of the sample tube, creating the interface. (G) Removal of the Pasteur pipet once all Ficoll has drained. (H) After centrifugation, pipetting at the medium/Ficoll interface to collect the LN cells.

Figure 5

Protein and cell recovery considerations. (A) Median protein concentration recovered from LN FNA supernatants (n = 6) diluted to different volumes to model recovery of cells into different amounts of PBS. (B) Median protein concentration (n = 4; except needle 2 wash no. 1, n = 3) recovered from each needle wash of each of two FNA needle passes. Data in A and B were generated using the Olink assay as described in (Provine et al., 2024). (C) Number of cells recovered from CLN FNA by three different radiologists (n = 26). Median and interquartile range are shown in (B) and (C).