Stability of Thawed Hematopoietic Progenitor Cells (HPCs) in Dimethyl Sulfoxide (DMSO) in Filtered and Unfiltered Products

Transcription

1 Stability of Thawed Hematopoietic Progenitor Cells (HPCs) in Dimethyl Sulfoxide (DMSO) in Filtered and Unfiltered Products Mehraboon S. Irani, MD, Robert Endres, PhD, Robin Medis, CHS(ABHI), Melissa Marlowe, MT(ASCP), CHS(ABHI), Stephen Hunt, BS, Roberta Bruhn, MS, PhD and Frank Nizzi, DO

2 Blood Systems Cellular Therapy Activity Blood Systems Laboratories, Stem Cell Processing Laboratory located in Tempe, AZ provides Cellular Therapy services both locally and to UBS customers in other states (Nevada, Texas, New Mexico) Local clients include Mayo Clinic Arizona, Phoenix Children s Hospital and Scottsdale Healthcare 2 Cellular Therapy

3 A study was undertaken to address two issues: after thawing and loss of HPCs after filtration DMSO is considered toxic to HPCs after thawing, so infusion of cryopreserved cells is typically accomplished by bedside thawing and immediate infusion 3

4 Blood Systems Stem Cell Processing Laboratory is located in Tempe, and is about 30 minutes away by car, from the nearest client hospital After the infusion there is a delay from the time the product is thawed to the technologist returning to the laboratory to perform post-thaw viability assays 4

5 Filtration of thawed HPCs prior to infusion is not commonly practiced because of fear of loss of cells Recent publication(letter): Post thaw filtration of umbilical cord blood does not affect product quality or likelihood of engraftment may prevent infusion-related adverse side effects, such as infusion of foreign bodies and pulmonary or paradoxical embolism Totoe et al Transfusion 51(10): ,

6 A standard blood filter ( µm) will capture bone spicules, clots and other material such as plastic fragments while allowing white blood cells (generally 8-20 µm in diameter) to pass into the patient Totoe et al Transfusion 51(10): ,

7 Three neurological events associated with HPC product infusion at the Dana Farber Cancer Institute Emboli documented by radiological studies All three patients had PFO Berg A, and Kao GS: Impact of Filtering Thawed Hematopoietic Progenitor Cells(HPC) with Routine Blood Filter on CD34+ Cell Number. AABB abstr 2006 Vol 46 Supplement SP71 7

8 The study was carried out on samples from 10 cryovials and 11 bags, thawed in a 37 C waterbath. The samples were diluted in an equal volume of 5% albumin and left at 1-10 C for 3 hours. Assays performed were CD45+ cell viability by 7-amino-actinomycin D (7AAD), CD34+ cell concentration, and colony forming unit (CFU) assessment 8

9 The samples were filtered at 0 and 3 hours from time of thaw, using a standard blood filter (SBF), with a pore size of µm The data for cryovials, and bags both unfiltered and filtered were analyzed using the Wilcoxon signed-rank test 9

10 Time after thawing 0h 3h Mean (SD) Median (Range) Mean (SD) Median (Range) R p* Cryovial (n=10) CD45 (7AAD%) CD34 cells/ul CFU /10 5 cells 59.5 (13.9) 59.3 ( ) (484.4) ( ) 97 (93.2) 73.5 (3 292) 56.8 (13.3) 61.1 ( ) (439.6) ( ) 92 (104.2) 50.5 (4 333) Bag, Unfiltered (n=11) CD45 (7AAD%) CD34 cells/ul CFU /10 5 cells 69.8 (12.3) 69.2 ( ) (692.7) ( ) 127 (83.5) 115 (2 336) 69.3 (10.3) 69.4 ( ) (771.4) ( ) 143 (70.0) 133 (9 279) Bag, Filtered (n=11) CD45 (7AAD%) CD34 cells/ul CFU /10 5 cells 70.7 (12.6) 70.7 ( ) (709.4) ( ) 156 (110.7) 157 (4 441) 68.9 (10.6) 68.5 ( ) (683.1) ( ) 151 (74.9) 165 (6 303) Stability of cells in DMSO

11 Cryovial (n=10)* Bag Unfiltered (n=11)* Bag Filtered (n=11)* 0h vs 3h 0h vs 3h 0h vs 3h CD45 (7AAD%) CD34 cells/ul CFU /10 5 cells *p-values for Wilcoxon signed-rank test 11

12 Bag Unfiltered vs Filtered (n=11) 0h 3h CD45 (7AAD%) Mean values p 69.8 vs * 69.3 vs * CD34 cells/µl values p Mean vs * vs * CFU /10 5 cells Mean values p 127 vs * 143 vs * *p-values for Wilcoxon signed-rank test 12

13 Results show good correlation with respect to CD45 viabilities, CD34+ cell concentration and CFU at 0 and 3 hours The Wilcoxon signed-rank test shows no significant difference in the values obtained at 0 and 3 hours No significant difference between the filtered and unfiltered samples with the exception of CFU at 0 h which was increased after filtration! 13

14 We conclude that HPCs are stable when thawed and refrigerated in DMSO for at least 3 hours This allows us to perform viability and CFU assays within 3 hours of thawing Also filtration using a SBF does not decrease the number of CD34+ cells or CFU in the product 14

15 Co-authors: Robert Endres, Robin Medis, Melissa Marlowe, Stephen Hunt, Roberta Bruhn, Frank Nizzi 15

16 Acknowledgements: HPC Transplant teams at Mayo Clinic Arizona and Phoenix Children s Hospital, Dr. James Slack, and Dr. Roberta Adams 16

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