Processing & Utilization of Cord Blood for Transplant

Processing & Utilization of Cord Blood for Transplant 2010-Jan Jan-15 Nicole L. Prokopishyn, PhD HPC Processing Laboratory Director Calgary Laboratory Services

Overview Cord Blood Processing Pre-Freeze Post-Thaw Cord Blood Potency Cell Recovery Cell Potency Engraftment Ability ISCT & AABB Meeting Updates Concerns New Trends Future Plans

Pluripotential Adult Stem Cells HPC Cancers Immunodeficienies Sickle Cell Anemia GVHD Crohn s Disease Muscular Dystrophy MSC

Adult Stem Cells in the Bone Marrow Bone Natural kill (NK) cell Lymphoid Progenitor cell T lymphocytes Neutrophil Basophil B lymphocyte Eosinophil Hematopoietic Stem cell Multipotential Blood stem cell Myeloid Progenitor cell Monocyte/macrophage Platelets Bone matrix Blood vessel Stromal cell Mesenchymal Stem cell Osteoblast Osteocyte Red blood cells Hematopoietic Supportive stroma Marrow Adipocyte Osteoclast Adipocyte Skeletal muscle stem cell? Hepatocyte stem cell? Hematopoietic Stem cell

Number of Blood Stem Cells Needed for Transplant HPC, Apheresis Blood Stem Cell Immature Undifferentiated CD34 + CD38 - Thy-1 lo ~1 x 10 4 /kg CD34 + CD38 - ~1 x 10 5 /kg Mononuclear Cells ~2 x 10 8 /kg CD34 + Cells ~3 x 10 6 /kg Mature Differentiated

Cord Blood Collection Collected at both vaginal delivery and Caesarean section Collected once the baby has been delivered Collected from the remains of the umbilical cord Old Method: - Collected by gravity by squeezing blood out of the cord. ~50-150 ml of blood is collected

Cord Blood Collection New Method: - Collected with a syringe by puncturing the umbilical vein immediately after the cord has been clamped, cut, and cleaned ~50-150 ml of blood is collected

Cord Blood Processing Pre- Freeze Buffy Coat Majority of Banks Older Method -just add DMSO & Freeze StemCyte Processing Plasma Depletion NO Red Cell Removal

Stem Cell Processing at a cord bank..\desktop http://www.cordlife.com/sg/whybankcordblood/whybankcordblood.php? target=processingandstorage

Cord Blood Processing Old Methods Pre-Freeze Cord + DMSO -> Freeze Volumes 100-800 ml Mononuclear cell Buffy Coat isolation of the past

Cord Blood Processing Newer Methods Pre-Freeze http://www.biosafe. ch/en/international /Library/Sepax+ani mation.html Average Volumes 20-50 ml

Storage & Shipping Cord Blood Units are stored in liquid nitrogen for >15 years

Global cord blood banks and registries (Courtesy of Mary Halet, NMDP)

What happens on transplant day? Dilution Post Thaw Sample Thawed Cord Unit Original Cord Bag Washed 2x with ReCon Solution Rapid Thaw (37 0 C) 2-3 minutes ReCon Solution 10% LMD40: 5% HSA) ~15 minutes Transported at 4 0 C to unit for infusion Expiry ~ 4 hrs from thaw Diluted Cord Product (1 part cord, 3 parts ReCon) Post Dilution Sample QC sample taken for Hematology TNC testing Flow Cytometry CD34 & TNC

What happens on transplant day? Washing Post Thaw Sample Thawed Cord Unit Original Cord Bag Washed 2x with ReCon Solution Rapid Thaw (37 0 C) 2-3 minutes ReCon Solution 10% LMD40: 5% HSA) Diluted Cord Product (1 part cord, 3 parts ReCon) 60-90 minutes Washed Cord Product (volume similar to starting volume) Re-spin Supernatant Transported at 4 0 C to unit for infusion Expiry ~ 8 hrs from thaw Post Wash Sample Re-suspend in fresh ReCon

Cord Blood Infusion Thawed @ beside Thawed in the lab Neutrophil Recovery 21-35 days Diluted/Washed in Osmotic Solution Hung to Infuse Donor Cells Engraft HPCs home to the bone marrow

Pre-Freeze Cord Bank Values Post-Thaw Values Calculated Recovery

AABB Oct 2009 Majority of centres doing dilution protocol, some using washing protocol All use Dextran-40 & Human Serum Albumin Percents and dilution factor vary All protocols based on NY Blood Centre protocol Washing Protocol with Sepax System being tested by Duke and MD Anderson Concerns regarding non-rbc depleted (e.g. StemCyte) cords Cord Blood Bank & Unit Qualification

AABB Meeting Oct 2009 % Recovery: Post Thaw/Dilution or Wash vs Original Pre-Freeze Values from Banks Most groups only reporting Hematology TNC values Average ~70% recovery; 40% is not uncommon Some reporting CD34+ cell recovery; Average 60-70% Dependent on Flow method used: Single Platform Lower TNC & CD34 values Double Platform Higher TNC & CD34 values? What is one platform picking up that the other isn t?? Which is more indicative of actual stem cell present?

Pre-Freeze Cord Bank Values Post-Thaw Values Calculated Recovery

Post Thaw Recovery of Cord Blood Cells 2008 & 2009 Hematology TNC Results: - Reported by most centres - Average ~70% - <40% non-conformance - Non-specific Flow Cytometry TNC Results: - Rarely reported - Distinguished Live/Dead, clumps excluded Flow Cytometry CD34+ Results: - Reported by Duke, MD Anderson, St. Louis, Minn. -Very Specific -Average ~60-70% recovery % Recovery (PostDilution or Wash/PreFreeze) Component # Hematology Flow Cytometry Notes Cord Age TNC TNC CD34+ Year 2008 08-157 P nd 18% 64% Diluted 9.5 08-175 P nd 14% 39% Washed 3.3 08-183 P nd 36% 50% Diluted 2.6 08-225 P nd 18% 48% Diluted 9.3 08-270 A nd 46% 80% Diluted 3.6 08-005 A nd nd nd Diluted 3.5 08-184 P nd 22% 38% Diluted 10.6 08-246 P nd 7% 6% Diluted 0.6 08-302 P nd 27% 86% Diluted 2.9 08-193 A nd 48% 102% Diluted 5.3 08-186 A nd 30% 53% Diluted 8.2 Yr 2008 Average 27% 57% Year 2009 09-084 P 77% 40% 66% Diluted 7.0 09-289 A 69% 21% 135% Diluted 1.3 09-005 P 76% 43% 74% Diluted 2.5 09-248 A 94% 53% 77% Diluted 4.1 09-113 P 67% 25% 119% Diluted 8.9 09-194 P 58% 21% 18% Washed 3.2 09-180 A 86% 34% 75% Diluted 1.5 09-232 P 83% 36% 80% Diluted 2.3 09-022 P nd 20% 50% Diluted 2.9 09-025 A nd 16% 14% Diluted 11.9 09-114 P 94% 41% 82% Diluted 1.1 09-001 P nd 22% 94% Diluted 1.3 Yr 2009 Average 78% 31% 74%

What influences recovery? Age of Cord? Collection Procedure? Time between collection & processing? Processing Method Pre-Freeze? Storage Conditions? Processing Method Post-Thaw? Inherent nature of the cord & cells?

Does Age of Cord Blood Influence Recovery of Cells? Cord Age and % Recovery (Diluted Products Only) 160% CD34 140% Flow Cytometry TNC Hematology TNC % Recovery 120% 100% 80% 60% R 2 = 0.1972 Linear (CD34 ) Linear (Hematology TNC) Linear (Flow Cytometry TNC) 40% R 2 = 0.266 20% R 2 = 0.1804 0% 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Age of Cord

What influences recovery? Age of Cord? Collection Procedure? Gravity method vs syringe system Time between collection & processing? Short the better Processing Method Pre-Freeze? Buffy coat preparation vs plasma depletion vs none Storage Conditions? Processing Method Post-Thaw? Inherent nature of the cord & cells? Do some cords freeze and thaw better than others?

What about pre-freeze processing? Does it influence recovery of cells??

Pre-Freeze Processing and Adverse Events during infusion Adverse Events in patients receiving double cord transplants -the patients received at least one cord that was NOT red cell depleted (e.g. Stemcyte processing method) -Cord was not washed prior to infusion -suspected cause: red cells & red cell debris? Recommendations: -wash cords that have not been red cell depleted or dilute greater than 1:10

Updates from ISCT Somatic Meeting Cord Blood Expansion MD Anderson studies Typically in culture only 2 fold expansion of CD34 cells in CB Addition of MSC and extracellular matrix and get better expansion Addition of angioblast. Angioblast MPC 15 fold TNC & 47 fold CD34 cells (9.6e7/kg TNC, 9e5/kg CD34+) Cord #1 Thawed, split into flasks & cultured with MSC and angioblast. On transplant Day: Cord #2 unmanipulated infused into patient Cord #1 expanded cells, media, MSC infused into patient Engraftment Day 16 for neutrophils and Day 14 for platelets 10 patients so far

Updates from ISCT Somatic Meeting Cord Blood Expansion cont d Notch Ligand based cord blood expansion Double cord transplants Day -16 thaw and cd34 select smaller unit and expand in culture with notch ligand (41-471 fold expansion) Day 0 Unmanipulated larger cord given Wait 4 hrs and give expanded 2 nd cord. Neutrophil engraftment Day 14.5 average (nonexpanded day 26) Neutrophils initially from expanded unit. Usually only 1 unit survives in bone marrow

Updates from ISCT Somatic Meeting Cord blood & non-hematopoietic therapeutic indications Joanne Kurtzberg Duke Genetic & acquired brain injuries Enzyme replacement for lysosomal storage diseases Post thaw CD34 good predictor of engraftment ALDH Bright cells isolated from 20% fraction of CB infused directly or after cytokine stimulation 80% CB fraction infused & then ALDH sort 4 hrs later Neutrophils engrafted more quickly Trial of fetal transplants Auto CB transplant for Cerebral Palsy Infusion of ALDHbr cells

Cord Blood Unit Qualification How do we know if is the correct cord blood unit when it comes to the lab? Do we get a segment for HLA testing or is it a QC vial? ABO/Rh at time of thaw? CD34/TNC hematology & flow Quick HLA (serologicial)?

Cord Blood Bank Qualification What standards does the bank follow? How do they test? And how consistent is the testing? TNC Viability CD34 content CFU activity How do they process the units? Buffy Coat enrichment traditional method vs Biosafe system Plasma reduction only How do they store the units?