TITLE: SVF Isolation and Immunophenotyping using Flow Cytometry in Leprosy Patients
AUTHORS: Sondang P. Sirait, Kusmarinah Bramono, Sri Linuwih Menaldi, Jeanne Adiwinata Pawitan, Wresti Indriatmi, Tiara Aninditha

[DESCRIPTION]
Adipose derived stromal vascular fraction (SVF) contains a heterogeneous population of mononuclear cells, progenitor cells and about 1-10% are mesenchymal stromal cells. These cells are an ideal candidate for regenerative medicine for peripheral neuropathy. Leprosy is a disabling disorder with neuropathy, usually with consequences of permanent disability of the extremities. Before we could inject the SVF, we need to learn whether the SVF is appropriate to be used for neuropathy treatment using the flow cytometry characterization. Liposuction was done to four leprosy patients and adipose tissue was processed into SVF with a closed system.  Following the process, the SVF were characterized and counted the CD73, CD90, CD34, CD45, Lin-Negatif, and CD105 cells using flow cytometry, cell counting, sterility and presence of mycobacteria. The process was done by ProStem using a traditional flow cytometer, without blocking and negative isotypes. The expected results from these protocols are the appropriate amount of mesenchymal cells needed to meet the IFATS and ISCT standards for SVF.

[STEPS]
SECTION: Isolation of Abdominal Adipose Tissue
2. The area was marked by using skin marker. After aseptic and antiseptic procedures the area was covered with a sterile surgical drape.
SECTION: Isolation of Abdominal Adipose Tissue
1. The following procedure is performed after patient identification and obtaining patient consent.
SECTION: Isolation of Abdominal Adipose Tissue
3. The local anesthesia was done with pehacain 2% and a small skin incision (1–3 mm) was made with number 11 blade afterwards.
SECTION: Isolation of Abdominal Adipose Tissue
4. The tumescent solution (TLA) was injected using a blunt tip cannula in the form of a fan-shaped. The tumescent solution is made based on Lilis formula with 1.000 mL NaCl 0.9%, 50 mL lidocaine 2%, 10 mL sodium bicarbonate 8.4%, and 1 mL epinephrine 1:1000.
SECTION: Isolation of Abdominal Adipose Tissue
5. The adipose tissue was collected using Mercedes aspiration cannula that connected with the canister and suction machine. The cannula, which is 3 mm in diameter, was directed in a radial, fan-shaped pattern. The fat above Scarpa's fascia was sucked out through the cannula with criss cross technique. Up to 100 mL of adipose tissue was collected.
SECTION: Isolation of Abdominal Adipose Tissue
6. The remaining tumescent solution is pushed caudally by pressing the operation area while the patient is standing.
SECTION: Isolation of Abdominal Adipose Tissue
7. The incision area was sutured and covered with sofratulle, gauze, sanitary pad, and Hypafix®. A compression bandage was applied using a large sized Hypafix®.
SECTION: Isolation of Stromal Vascular Fraction
7. 
SECTION: Isolation of Stromal Vascular Fraction
8. The tissue collected was processed into SVF using Sepax2® method. 

SECTION: Flow cytometry and cell counting
13. Cell count and viability are analyzed with Automated Cell Counter Countess II for enumerating the cells and cell counting.
SECTION: Flow cytometry and cell counting
14. After counting, 100.000 cells in a 100 μL medium were exported into the test tube.
SECTION: Flow cytometry and cell counting
15. The cells were added 20 μL positive antibody cocktail (contains CD90-FITC, CD73-APC, CD105-PerCP.Cy5.5, and 20 μL negative antibody cocktails (contains CD45-PE, CD34-PE, CD11b-PE, CD19-PE, and HLA-DR-PE.
SECTION: Flow cytometry and cell counting
16. Samples were incubated for 30 minutes and washed with Phosphate Buffer Saline (PBS) afterwards.
SECTION: Flow cytometry and cell counting
17. Samples in PBS were analyzed in Flow cytometer BD FACSCanto II using negative isotypes for the beads and positive isotypes for the CDs with enumeration control methods.
SECTION: Flow cytometry and cell counting
18. CD73+, CD90+, CD105+, Lineage Negative, CD34+, CD34+/CD45+ are counted and plotted into graphs using the methods.
SECTION: Flow cytometry and cell counting
19. The process was done by 1 clinical laboratory scientist, verified by another clinical laboratory scientist, and authorized by 1 clinical pathologist.
SECTION: Isolation of Stromal Vascular Fraction
9. The liposuction sample was transferred to a sterile collection bag without contact with air using a luer lock to luer lock connector. Then 0.15% collagenase solution (Serva) was added and incubated at 37 ºC for 60 minutes.
SECTION: Isolation of Stromal Vascular Fraction
10. Using CS-900.2 kit the incubated adipose tissue was connected to a Sepax2® device. Press “Start Procedure” to start a fully automated cellular separation.
SECTION: Isolation of Stromal Vascular Fraction
11. For cell collection, the system diluted the residual pellets and extracted the content into the final bag, and automatically rinsed the chamber. After the procedure is completed, a text “Remove Bags and Air Filter” will appear on the device. About twelve mL of SVF was collected in a sterile 20 mL syringe.
SECTION: Isolation of Stromal Vascular Fraction
12. Samples were taken for count of live cells using Trypan Blue method, flow cytometric analysis, endotoxin testing using Lonza® kit, contamination assessments using Mycoplasma kit, and blood agar for bacterial culture.