TITLE: Case Processing SOP (Spleen)
AUTHORS: Marda Jorgensen

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

[STEPS]
?. The tissues received will be identified as follows: Spleen (SP)
?. 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 50cc 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: Block number. Where applicable, block # is determined by common coordinate region followed by        position moving lateral (hilar capsule) to medial (central)     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) + common coordinate location if relevant
?. 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, and tissue quality notations.    c. Photo documentation should be employed to verify harvests are in keeping with common coordinate guidelines.
?. Tissue Dissection:    a. Photo document intact organ from all angles. Orient with hilum to left, inferior end at top.     b. Place a 0.5 cm cube of tissue for RNA QC purposes into a sterile vial and flash freeze in liquid nitrogen. Use RNAse free conditions and collect from an interior, non-Common Coordinate site.    c. Harvest Common Coordinate tissue regions (Appendix). Cut 1 cm thick cross sectional slabs and photo document location of origin by sliding slabs slightly to right of residual organ. Use ruler or grid to map locations.    d. Place slabs with superior pole surface facing up and rotate until hilum is located at 12 o’clock. Cut a 1 cm wide strip through center of each slab starting at middle of hilum facing side. Photo document position prior to harvesting strip.    e. Cut strips into individual blocks of 1 cm3 tissue. A 1 cm X 1 cm tissue matrix may be used to assist. Photo document cuts. Maintain orientation and order.     f. Place the Worksheet (Appendix) designated 1cm3  , bisected block into the two cassettes labeled for NBF (p), maintaining orientation. Immerse in NBF, room temperature with mild agitation, at a 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.    g. Place the Diagram Worksheet (Appendix) designated 1cm3 cut block into the cassettes labeled for PFA (p) maintaining orientation. Immerse in 4% PAF at a 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.    h. PPlace the Diagram Worksheet (Appendix) designated 1 cm3 cut block into the cassette labeled for FX frozen OCT, maintaining orientation. Immerse in 4% PFA at a volume of at least 20 mls per cassette added. Complete processing and embedding following  SOP:https://dx.doi.org/10.17504/protocols.io.basniede    i. Place the Diagram Worksheet (Appendix) designated 1 cm3 cut block for Fresh OCT into a labeled, OCT filled cryomold maintaining orientation. Complete processing and embedding following  SOP: https://dx.doi.org/10.17504/protocols.io.bcwsixee  Hold and transport prepared blocks on dry ice.      j. Place the Diagram Worksheet (Appendix) designated 1cm3 block of tissue into 4% PFA for 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 is then transferred into hydrogel solution and incubated 5 days at 4C.    k. Prepare the Diagram Worksheet (Appendix) designated 1 cm3 cut block of spleen tissue to be used for 10X analysis.        i. Place the tissue into a 50 ml tubes containing 25ml complete DMEM/F12 media. Store in the refrigerator until released for further isolation.    i. If the tissue is selected to be used for collaboration projects identify protocol B on the work sheet and follow 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.
?. Worksheet /Appendix for Spleen Processing
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