. 2023 Dec 15;4(4):102576.

doi: 10.1016/j.xpro.2023.102576. Epub 2023 Sep 20.

Ultrasound-guided lymph node fine-needle aspiration for evaluating post-vaccination germinal center responses in humans

Larissa L S Scholte¹, David J Leggat², Kristen W Cohen³, Lara Hoeweler⁴, Guacyara C Erwin⁴, Farhard Rahaman⁵, Angela Lombardo⁵, Vincent Philiponis⁵, Dagna S Laufer⁵, Heather Siefers⁵, Alexis M Ruppel⁶, Joshua Brand⁶, Janine Maenza⁷, Rhi Bronson⁶, Madhu Prabhakaran⁶, Jalen Jean-Baptiste⁶, Orpheus Kolokythas⁸, Aimee A Desrosiers⁴, Caroline K Thoreson⁴, Antje Heit³, Nadia J Khati⁴, Elissa Malkin⁴, M Juliana McElrath⁷, Adrian B McDermott⁹, William R Schief¹⁰, David Diemert⁴, Jeffrey M Bethony⁴

Affiliations

¹ Vaccine Research Unit, The George Washington University, Washington, DC 20037, USA. Electronic address: larissascholte@gwu.edu.
² The US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA.
⁴ Vaccine Research Unit, The George Washington University, Washington, DC 20037, USA.
⁵ IAVI, New York, NY 10004, USA.
⁶ Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
⁷ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA; Department of Medicine, Division of Allergy and Infectious Disease, University of Washington, Seattle, WA 98195, USA.
⁸ Department of Radiology, University of Washington, Seattle, WA 98195, USA.
⁹ Vaccines Research and Development, Sanofi, Swiftwater, PA 183709, USA.
¹⁰ IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Development, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA; The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02139, USA.

PMID: 37733596
PMCID: PMC10519838
DOI: 10.1016/j.xpro.2023.102576

Ultrasound-guided lymph node fine-needle aspiration for evaluating post-vaccination germinal center responses in humans

Larissa L S Scholte et al. STAR Protoc. 2023.

. 2023 Dec 15;4(4):102576.

doi: 10.1016/j.xpro.2023.102576. Epub 2023 Sep 20.

Authors

Affiliations

¹ Vaccine Research Unit, The George Washington University, Washington, DC 20037, USA. Electronic address: larissascholte@gwu.edu.
² The US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA.
⁴ Vaccine Research Unit, The George Washington University, Washington, DC 20037, USA.
⁵ IAVI, New York, NY 10004, USA.
⁶ Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
⁷ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA; Department of Medicine, Division of Allergy and Infectious Disease, University of Washington, Seattle, WA 98195, USA.
⁸ Department of Radiology, University of Washington, Seattle, WA 98195, USA.
⁹ Vaccines Research and Development, Sanofi, Swiftwater, PA 183709, USA.
¹⁰ IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Development, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA; The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02139, USA.

PMID: 37733596
PMCID: PMC10519838
DOI: 10.1016/j.xpro.2023.102576

Abstract

The lymph node (LN) is a critical biological site for immune maturation after vaccination as it includes several cell populations critical for priming the antibody response. Here, we present a protocol for sampling the LN and isolating cell populations to evaluate immunogens targeting germline cells. We describe steps for media and tube preparation and sample collection using an ultrasound-guided LN fine-needle aspiration procedure. This protocol is safe, quick, low-cost, and less invasive than excisional biopsy. For complete details on the use and execution of this protocol, please refer to Leggat et al. (2022).¹.

Keywords: Cell Isolation; Clinical Protocol; Immunology.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1**
Ultrasound-guided lymph node fine-needle aspiration (A) The ultrasound probe is positioned for optimal visualization over an enlarged axillary lymph node of the arm recently receiving an immunization of an investigational product (i.e., a “draining lymph node”). (B) Once puncture of the LN is visualized on US, negative pressure is applied to the syringe by withdrawing plunger to approximately the 2–3 mL mark on the syringe barrel using a gentle “to” and “fro” motion. (C) US image of a 23-gauge needle entering the cortex of an axillary lymph node. This procedure is repeated up to 4 times to gather a sufficient amount of sampling material.

**Figure 2**
50 mL conical tubes after FNA collection The tube containing the sample “For Collection” shows a darker reddish color than the tube containing media “For Flushing” only.

**Figure 3**
Workflow summarizing the LN-FNA pre-analytics, processing, and storage steps

**Figure 4**
US-LN-FNA cell pellet after centrifugation (A–D) Stages in processing the sample for GCC isolation.

**Figure 5**
US-LN-FNA sample images captured by the Cellometer Auto 2000 using the AOPI viability method (A and B) (A) shows a bright-field image within the expected cell concentration and highlights an area containing red blood cells (red arrow in the zoomed-inbox) that are not shown in the combined fluorescent panel (B) since only nucleated cells are counted using AOPI. (B) Shows combined fluorescent images with counted live cells circled in green and counted dead cells circled in red (highlighted in the zoomed-in box by an orange arrow).

**Figure 6**
Representative dataset showing the total live cell count and viability of sixty-two US-LN-FNA samples collected and processed following this protocol The y-axis shows a log10 transformation. The vast majority of samples show high viability (>80%) and a total live cell count >500,000 cells. Note: This figure shows the viability for fresh samples. US-LN-FNA aliquots properly cryopreserved and thawed following best practices should not result in viability decrease greater than 10% post-cryopreservation.

See this image and copyright information in PMC

References

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Ultrasound-guided lymph node fine-needle aspiration for evaluating post-vaccination germinal center responses in humans

Affiliations

Ultrasound-guided lymph node fine-needle aspiration for evaluating post-vaccination germinal center responses in humans

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources