TITLE: BIDMC TMC - SOP for collection of peripheral lymphatic vessels
AUTHORS: nkalavro, Ioannis Vlachos, Dhruv Singhal

[DESCRIPTION]
BIDMC Lymphatic TMC SOP for peripheral lymphatic vessel collection

[STEPS]
SECTION: Standard Operating Procedure Overview
1. 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.
SECTION: Donor Criteria
2. 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.

SECTION: Consenting
4. 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)
SECTION: Donor metadata
3. The following information is recorded per study participant: Age, Sex, BMI, Hospitalization Time, Comorbidities, and Clinical history from the OMR.
SECTION: Collection of Peripheral Lymphatic Vessel
5. 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.
SECTION: Collection of Peripheral Lymphatic Vessel
6. The sites are massaged for 1 minute.
SECTION: Collection of Peripheral Lymphatic Vessel
7. Identified lymphatic vessels are harvested (2x5mm, or longer) with microsurgical scissors and microclips applied to free lymphatic ends.
SECTION: Collection of Peripheral Lymphatic Vessel
8. 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.
SECTION: Collection of Peripheral Lymphatic Vessel
9. The distance from thevessel to the edge of the axillary vein is recorded.
SECTION: Collection of Peripheral Lymphatic Vessel
10. The peri-lymphatic adipose tissue of the lymphatic vessel is removed as closely to the vessel as possible.

    

SECTION: Sample Processing
11. Prepare an isopentane and liquid nitrogen bath.
SECTION: Sample Processing
12. 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.
SECTION: Sample Processing
13. Cover the tissue completely with OCT. Label the cryomold to mark the orientation of the tissue.
SECTION: Sample Processing
14. Using forceps, lower the embedded tissue into the isopentane without fully submerging.
SECTION: Sample Processing
15. Keep cryomold in contact with isopentane until the OCT has solidified and turned white. Once frozen, place the cryomold on powdered dry ice.
SECTION: Sample Processing
16. Store frozen embedded tissue in a sealed container at -80C or liquid nitrogen for long-term storage.