VCUHS Stem Cell Processing Lab

1. Bone Marrow Transplant Program VCUHS Stem Cell Processing Lab Rick Edwards, PA Sharon Whitlock, MT Christy McLaughlin, BS Sam Berhane, BS Olin Thomas, MT

2. Lab Accreditations • Foundation for the Accreditation of Cellular Therapy (FACT) MISSION: The Foundation for the Accreditation of Cellular Therapy promotes quality medical and laboratory practice of cellular therapy through its peer-developed standards and voluntary inspection and accreditation program. • Clinical Laboratory Improvement Amendments, Centers for Medicare and Medicaid Services (CLIA) (CLIA) of 1988 [Public Law 100-578] – Established quality standards to ensure the accuracy, reliability and timeliness of patient test results regardless of where the test was performed.

3. Sources of Hematopoetic Progenitor Cells (HPC) HPC,Aphersis HPC,Marrow HPC,Cord • Most common • Patient or donor is mobilized before collection • May take several days of collection to reach goal dose • Patient or donor not mobilized • Out-patient surgery • General anesthesia • Umbilical cord blood collected at birth for public or private use. • Stored cryopreserved at Cord Blood Banks • Adults will often require 2 different cord units

4. Receipt of Apheresis Product • Chain of Custody • Check product information • Sign upon receipt of product • Transported in room temperature cooler • Record time of arrival in lab

5. Product Manipulations • HPC,Apheresis • Volume Reduction: Reduces the volume of plasma by centrifugation. • High total nucleated cell recovery- low risk of cell dose loss. • Reduces volume of DMSO used • Reduces total infusion volume, particularly important to patients with multiple days of collections • Adoptive Cellular Immunotherapy (ACI): • Goal is to induce remission of cancer by graft-versus-tumor • Doses calculated prior to cryopreservation based on CD3 concentration • Cryopreserved in specific cell doses for each infusion

6. Product Manipulations, cont. • HPC,Marrow • Plasma Reduction: Reduces the volume of plasma by centrifugation. • High total nucleated cell recovery- low risk of cell dose loss. • Reduces volume of DMSO used, if cryopreserved, and total infusion volume

7. DMSO Side Effects • Unpleasant smell and taste • Oyster or garlic-like • Nausea • Headache • Sensitivity to light • Histamine-like reaction

8. Product Manipulations, cont. • HPC,Marrow, cont. • Red Blood Cell Reduction: Required when donor is ABO mismatch • Removes incompatible red cells and plasma • Possibility of 30% nucleated cell loss • Reduces volume of DMSO used, if cryopreserved, and total infusion volume

9. Product Manipulations, cont. • HPC, Cord • Wash • Removes DMSO • High risk of cell loss, approximately 30% due to centrifugation • Must be infused within 4 hours of initial thaw • Reconstitution • Does not involve centrifugation • Reduces the concentration of DMSO • Must be infused within 4 hours of initial thaw

10. Product Manipulations, cont. • Cryopreservation of HPC,A and HPC,M • 10% Dimethyl Sulfoxide (DMSO) used as the cryoprotectant in a freeze mix with plasmalyte and Human Albumin • Protects the cell from the lethal effects of the freezing process • Cryoprotectants work as an antifreeze to prevent formation of ice crystals and aid in the process of vitrification, a method of converting biological tissue into a strange kind of low-temperature glass that is totally free of ice crystals. • Preserves the ability to restore hematopoiesis • Cryocyte bags • Maintains integrity at ultra low temperatures • Impact and puncture resistant

11. Freezing Process • Controlled Rate Freezer • Slow freeze enhances cell viability. • The controlled rate freezer rapidly drops the temperature and then subsequently warms the freezer temperature which shortens the phase change period. This combined with the controlled rate cooling of the liquid and solid phases will provide maximum recovery of viable cells while minimizing freezing time. • Records temperature, freeze and end points as well as heat of fusion on graph printout.

12. Study Protocols • Osirus • Mesenchymal cells • Lab is unblinded • Cells are thawed and reconstituted • Standard dose determined by study • Delivered to unit for infusion • Bag retrieved after infusion

13. Study Protocols • Osirus • Patients randomized to placebo or investigational agent • Unit is blinded • Bags and tubing covered with amber wrap

14. Storage • Storage in LN2 Vapor • Stored in LN2 Vapor. • Monitor records temperature every 4 hours will alarm if temp. warmer than -145o C • Alarm calls four numbers, the last of which is the N10 nursing station where someone mans the phone 24 hours a day.

15. Product Evaluation • CD 34 analysis • CD3 analysis • WBC count • Weight • Culture- FDA approved method • Cellular Viability

16. Release of Products • Release to an internal destination • Paperwork • Check labels • Inspect for cracks or breaks • Requires two technicians • Remove from inventory • Record transplant date in stem lab • Begin following for date of engraftment

17. Internal Transporter (R2D2)

18. External Transport • Release to an external destination • Charge LN2 shipper 24 hours prior • Courier • Temperature monitored • Maintains temperature for 10 days

19. Record Keeping • Quality Assurance • Operational Self Assessment Record • Annual/Quarterly data: hematocrit, days to collect, mobilization type, viability, etc • Reagent validations and QC • Equipment maintenance and validation • Peer and supervisor review • Proficiency testing through CAP • Engraftment out through 45 days post transplant

20. Outcome Engraftment Data

21. Outcome

22. Record Keeping • Patient Records • StemLab software • Paper copy • Stored for a minimum of ten years • Records lot numbers of supplies used • Product evaluations

23. End of the week