TITLE: Case Processing SOP for Lymph Nodes 
AUTHORS: Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this Standard Operating Procedure is to outline procedures for processing and storing lymph node tissue received for HuBMAP consortium assay and analysis. </div></div>

[STEPS]
?. The tissues received will be identified as follows: Lymph Node (LN)
?. Sample prepared from tissues will be identified using the following nomenclature, abbreviations, and formats: Formalin Fixed Paraffin Embedded---FFPE (paraffin block)Formalin Fixed Frozen Embedded---Fixed OCT BlockFresh Frozen Embedded---Fresh OCT Block Fresh Tissue piece cut to size---Tissue (in 4% PFA or complete medium in 50 cc conical)
?. Case Number Assignment: HuBMAP donor tissues will be assigned case numbers adhering to the following template: two digit year identifier, hyphen, sequential 3 digit number beginning with 001.
?. Labeling Samples:     a. Cassettes for paraffin embedding and Fixed OCT block fixation step        i. Generate label using cassette printer or print legibly in pencil        ii. Line #1: Case ID + Sample type abbreviation         iii. Line #2: fixative used    b. Cryomolds for fixed or fresh frozen OCT blocks        i. Print on mold legibly using permanent ink, apply printed label to bottom        ii. Line #1 as for cassettes        iii. Line #2 as for cassettes + FX if prepared to receive fixed frozen tissue    c. Dishes, foil wraps and containers (culture, petri or conical)        i. Print legibly using permanent ink or apply printed label        ii. Case ID + Sample type abbreviation (Table 1)
?. Processing Records:     a. Tissue handling data will be recorded on the Case Worksheet form.    b. Required fields during case processing include case identification, date received, any shipment or packaging anomalies, processing date and time (start and end), staff, tissues received, aliquot types and numbers, measurements and tissue quality notations.    c. Photo documentation should be employed to verify harvests are in keeping with guidelines.
?. Tissue Dissection:     a. Using sterile instruments, collect one lymph node of roughly 5 mm in size.        i. Place in individual RNAse free, 1.5 ml cryotubes and flash freeze in liquid nitrogen. Store at -80C for Quality Control analysis.    b. Dissect individual LNs away from fat. Place each in a single well of a 24 well cluster dish containing ice cold 1X D-PBS  until collection of all lymph nodes is completed.         i. A minimum of twelve lymph nodes (or pieces) are required for a complete harvest.         ii. D-PBS volume should completely cover the lymph nodes    c. Remove any residual fat or connective tissues from each LN. Measure circumference using grid provided on dissection board and record size. Lymph nodes larger than 7 mm can be bisected if needed.         i. Photo document.     d. Assign lymph nodes to the various applications/fixations and record on worksheet.    e. Cassette tissue for three paraffin blocks using two medium LNs or three small LNs. Tissue must fit in a maximum area of 1 cm x 1 cm.     i. Place two cassettes into a container of NBF, the other into 4% PFA, as designated on the cassette labels. The NBF cassettes are reserved for Imaging Mass Cytometry and CODEX modalities.         Use a fixative volume of at least 20 mls per cassette added.         *cassettes are moved to 70% ethanol at 20-24 hours of fixation, and will be processed within 3 days.    f. Fix tissue for one fixed frozen OCT block using one to two medium LNs or three small LNs . Place (in a labeled cassette) into a container of 4% PFA. Use a fixative volume of at least 20 mls per cassette added.     i. Complete processing and embedding following SOP:https://dx.doi.org/10.17504/protocols.io.basniede   g. Make one fresh frozen OCT block from one medium LN or several small LNs. Quickly blot excess moisture on a clean kimwipe or gauze and submerge tissue into an OCT filled labeled cryomold.     ii. Complete processing and embedding following SOP: https://dx.doi.org/10.17504/protocols.io.bcwsixee        Hold and transport prepared blocks on dry ice.     h. Place 3 three LNs into labeled cassettes and emmerse in 4% PFA for use in CLARITY analysis. Use a ratio of at least 20 volumes of fixative to volume of tissue. Fix tissue for 20-24 hours at room temperature with gentle agitation.         * Tissue will be transferred into hydrogel solution and incubated 5 days at 4C on a rocking platform.    i. Obtain three LN to be used for 10x analysis.         i. If 8mm or larger in size, mince the tissue into small (5 x 5 mm) pieces. Transfer each lymph node into a 50 ml tubes containing 25 ml complete DMEM/F12 media. Store tissue at 4C until released for use or further isolation.    j. If the tissue is selected to be used for collaboration projects indicate protocol B on the worksheet.         i. Perform protocols as provided by the requesting lab(s).
?. Sample Archiving:     a. All materials obtained for this program will be inventoried by the University of Florida Tissue Mapping Center (UF TMC) and archived in the Organ Processing and Pathology Core (OPPC) until use.    b. Samples will be released for use or shipped to collaborator(s) upon request by the Principle Investigator.
?. Processing Worksheet for Lymph Nodes
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