Dec 30, 2022
BIDMC TMC - SOP for collection of peripheral lymphatic vessels
- Nikolaos Kalavros1,
- Ioannis Vlachos1,
- Dhruv Singhal1
- 1BIDMC
Protocol Citation: Nikolaos Kalavros, Ioannis Vlachos, Dhruv Singhal 2022. BIDMC TMC - SOP for collection of peripheral lymphatic vessels. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjeb7lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 27, 2022
Last Modified: December 30, 2022
Protocol Integer ID: 74514
Funders Acknowledgement:
NIH
Grant ID: U54-165440
Abstract
BIDMC Lymphatic TMC SOP for peripheral lymphatic vessel collection
Standard Operating Procedure Overview
Standard Operating Procedure Overview
Standard Operating Procedure to outline the process for obtaining, processing, and storing peripheral lymphatic vessel samples collected for the HuBMAP Lymphatic TMC.
The vessels are collected through a permanent removal of right axillary lymphatic vessel during lymphedema surgery from patients with malignant neoplasm of right female breast.
Donor Criteria
Donor Criteria
Patients with age >18 years from any race (White, Black, Hispanic, and Asian) are included.
Patients are excluded if they have one of the following characteristic:
- Have a known underlying lymphatic disease.
- Suffer from a chronic inflammatory condition (e.g. RA).
- Have ahistory of filarial infection.
- Have a history of allergy to lymphazurin blue.
- Are actively breastfeeding.
- Have a history of sensitivity to triphenylmethane.
- Have a history of systemic therapeutic treatment (e.g. chemotherapy).
- Have a history of pathology at anticipated site of lymphatic vessel harvest.
Donor metadata
Donor metadata
The following information is recorded per study participant: Age, Sex, BMI, Hospitalization Time, Comorbidities, and Clinical history from the OMR.
Consenting
Consenting
Following confirmation of the above inclusion and exclusion criteria by a study coordinator, the patient is informed about the study and provides informed consent according to the BIDMC IRB criteria.
Donors accepting to participate into the study are assigned an ascending 3-digit number (beginning with 001)
Collection of Peripheral Lymphatic Vessel
Collection of Peripheral Lymphatic Vessel
During the planned radial forearm free flap, 3 injections of 0.1cc of ICGare injected into the fascia 2cm apart proximal to the radial styloid.
The sites are massaged for 1 minute.
Identified lymphatic vessels are harvested (2x5mm, or longer) with microsurgical scissors and microclips applied to free lymphatic ends.
A 9-0 suture (peri-adventitial) is placed at the 12 o’clock position (i.e. the side of the vessel facing the axillary vein) – on the side farthest from the heart, to mark orientation.
The distance from thevessel to the edge of the axillary vein is recorded.
The peri-lymphatic adipose tissue of the lymphatic vessel is removed as closely to the vessel as possible.
Fig 1a: Marking of lymphatic vessel with the use of a 9-0 suture (peri-adventitial) at the 12 o’clock position, on the side furthest from the heart.
Fig 1b: Marking the distance below the axillary vein for orientation.
Sample Processing
Sample Processing
Prepare an isopentane and liquid nitrogen bath.
In a petri dish, carefully coat fresh tissue sample with room temperature OCT. Using a spatula, place the OCT-coated tissue into an appropriately sized cryomold.
Cover the tissue completely with OCT. Label the cryomold to mark the orientation of the tissue.
Using forceps, lower the embedded tissue into the isopentane without fully submerging.
Keep cryomold in contact with isopentane until the OCT has solidified and turned white. Once frozen, place the cryomold on powdered dry ice.
Store frozen embedded tissue in a sealed container at -80C or liquid nitrogen for long-term storage.