AllCells Leuko Pak Webinar 12-01-2011
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The biotechnology and pharmaceutical industry is continuing to implement more primary cell based assays in drug discovery due to their biological relevance and there is increased interest in cells of hematopoietic and immunological origin. This webinar will provide an introduction to leukapheresis, a process that can yield large quantities of primary cells from peripheral blood. In addition we will present data on immunological and hematopoietic cells derived from the leukapheresis process.
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AllCells Leuko Pak Webinar 12-01-2011
- 1. AllCells, LLC – Webinar Presentation Leuko Pak 101: How Leukapheresis Products are Best Utilized in the Lab Leading Provider of Human Hematopoietic Cells and Services
- 2. Corporate Background • Founded in 1998 • Centrally located within San Francisco Bay Area (Emeryville, CA) • Key products (>700) • Hematopoietic and Immunological Cells from diseased and healthy donors • Mesenchymal Stem Cells • Primate, rat and mouse tissue • AllCells has a dedicated onsite collection facility operating under IRB approved protocols • Contract Assay Services: • Hematopoietic & Immunological Assays • Flow Cytometry Services • Luminex Multianalyte Assays • Cell Based Assays for Drug Discovery • Diseased primary cells and cell lines • In vitro expansion and differentiation of primary hematopoietic cells Leading Provider of Human Hematopoietic Cells and Services
- 3. Apheresis • Derived from Greek, “to carry away” • A technique in which whole blood is taken and separated extracorporealy, separating the portion desired from the remaining blood • Performed using Cobe Spectra (Terumo BCT) Leading Provider of Human Hematopoietic Cells and Services
- 4. Apheresis Principle plasma Platelet collection bag RBC bag collection bag Vent bag Anticoagulant Leading Provider of Human Hematopoietic Cells and Services
- 5. Plasmapheresis • Therapeutic – Plasma: • Removal (replaced with albumin, plasma and saline solution) helps reduce circulating antibodies and immune complexes – Diseases include: Waldenstrom's macroglobulinemia, Myasthenia gravis and others • Donation – Plasma removed to supply blood components such as clotting factors Leading Provider of Human Hematopoietic Cells and Services
- 6. Plateletpheresis • Therapeutic – Myeloproliferative disorders • Thrombocytosis can occur – Removal of platelets can help to avoid complications of thrombosis and bleeding • Donation – Most common means for supplying HLA matched platelets to patients who have become HLA sensitized Leading Provider of Human Hematopoietic Cells and Services
- 7. Therapeutic Leukapheresis • Performed to decrease a very high white blood cell count • In hematological malignancies such as AML, WBC counts may be high enough to cause hemostasis and "sludging" in the capillaries – Affect retinal vasculature leading to vision changes – Pulmonary vasculature leading to shortness of breath from decreased efficiency in oxygen exchange – Neurological deterioration of a patient from cerebrovascular compromise • Stem Cell Harvesting for transplantation Leading Provider of Human Hematopoietic Cells and Services
- 8. Non-mobilized Leuko Pak • Donor criteria – Age: 18-55 years old – Weight: > 140 lbs – Complete Blood Count (CBC) lab test must meet protocol specifications – Donors cannot be pregnant – Donors are screened for HIV, Hepatitis B and C – Donors must be in good general health – Donors are required to sign a procedure-specific consent form prior to the collection procedure – All consent and protocols have been approved by an IRB – Donors must have adequate peripheral veins Leading Provider of Human Hematopoietic Cells and Services
- 9. Non-mobilized Leuko Pak Procedure – 3 total blood volumes processed – 4 hours – Minimum of 6 months between donations – Life time maximum of 6 donations – Average yield: 1.5x1010 MNCs/bag (1-3.5x1010) • ¼ Bag, ½ Bag and full Bag – Total volume 300 mL Leading Provider of Human Hematopoietic Cells and Services
- 10. Non Mobilized Leukapheresis Pre-apheresis Lymphocytes: 38.2% Monocytes : 6.9% Granulocytes : 54.9% WBC 5.17x106/mL Apheresis product Lymphocytes: 83.6% Monocytes : 15.3% Granulocytes : 1.1% WBC 9.8x107/mL Total cell #: 2.94x1010 Leading Provider of Human Hematopoietic Cells and Services
- 11. G-CSF (Neupogen™) Mobilized Leuko Pak • Granulocyte-Colony Stimulating Factor (G-CSF) also known as Neupogen® a recombinant form of human granulocyte colony stimulating factor (G-CSF) • Used to treat – Chemotherapy – Bone marrow transplantation – AML – Severe/chronic neutropenia • Used to increase the number of hematopoietic stem cells prior to leukapheresis Leading Provider of Human Hematopoietic Cells and Services
- 12. G-CSF (Neupogen™) Mobilized Leuko Pak Procedure – Donor criteria: 18-40 year old – 3 total blood volumes processed – 4 hours – Minimum of 12 months between donations – Twice in a lifetime maximum – Average yield: 2.5x1010 MNCs/bag (0.9-5.6x1010) – Total volume: 300 mL – Three mobilization regimes Leading Provider of Human Hematopoietic Cells and Services
- 13. G-CSF (Neupogen™) Mobilized Leuko Pak Pre apheresis Viability: 99.05% WBC: 3.19 x107/mL CD34+:3.02x104/mL Apheresis Product Viability: 99.16% WBC: 1.90 x108 /mL CD34+: 8.14x105/mL 6 fold increase in WBC 27 fold increase in CD34+ cells Leading Provider of Human Hematopoietic Cells and Services
- 14. Dual Half Pak-Leukapheresis • For each donation a half Leuko Pak is collected • Shorter collection time (< 2 hrs) • Healthy donor undergoes leukapheresis twice within 6-8 weeks – Reduced variability between experiments • Potential studies – T cell activation/expansion – NK cell cytotoxicity Leading Provider of Human Hematopoietic Cells and Services
- 15. Dual Half Pak-Leukapheresis Donor 3501 1st donation Donor 3501 2nd donation WBC: 7.25x106/mL WBC: 7.36x106/mL % Lymphs: 36.7% % Lymphs: 34.1% %Monos: 9.8% %Monos : 11.3% %Grans: 53.5% %Grans : 54.6% WBC: 1.37x108/mL WBC: 1.13x108/mL % Lymphs: 53.2% % Lymphs: 66.1% %Monos : 15.4% %Monos : 20.9% %Grans : 31.4% %Grans : 13.0% Total Cell number: 2.2x1010 Total Cell number: 1.8x1010 Leading Provider of Human Hematopoietic Cells and Services
- 16. Application using Dual Half Pak • NK effector function has traditionally been examined by Cr51 release assays • We investigated a non radioactive FACS based assay for cytotoxicity using CD56+CD16+ NK cells and K562 as the target cell type Leading Provider of Human Hematopoietic Cells and Services
- 17. NK Cytotoxicity Assay Protocol 1. NK cells were isolated by immunomagnetic “untouched” selection using Leuko Paks from a healthy donor 2. K562 cells were labeled with 5 M CMFDA (CellTracker Green) for 1h at 37°C 3. NK cells were then co-incubated with K562 cells at a E:T ratio of 50:1 and 25:1 for 4 hours at 37°C 4. 7-AAD was then added and the cells were analyzed by flow cytometry 5. Experiment was repeated for 2nd leukapheresis on the same donor 6 weeks after the initial donation Leading Provider of Human Hematopoietic Cells and Services
- 18. NK Cytotoxicity Assay • Collections were performed 6 weeks apart • Consistent yield and purity between donations Donor 3501 Donor 3501 1st collection 2nd collection Leading Provider of Human Hematopoietic Cells and Services
- 19. NK Cytotoxicity Assay K562 only CellTracker™ Green CMFDA NK cells + K562 CellTracker™ Green CMFDA Leading Provider of Human Hematopoietic Cells and Services
- 20. Results: First Donation E:T=50 E:T=25 Leading Provider of Human Hematopoietic Cells and Services
- 21. Results: Second Donation E:T=50 E:T=25 Leading Provider of Human Hematopoietic Cells and Services
- 22. Summary • AllCells is a leading provider of non-mobilized and G-CSF mobilized leukapheresis products • We recruit donors directly based on your specific needs • Leuko Paks can be used as a source for large quantities of primary cells • Our new Dual Half Pak leukapheresis product – Allows for the same individual to donate twice within 6-8 weeks – Reduce variability – Illicit similar responses Leading Provider of Human Hematopoietic Cells and Services
- 23. Thank You – Q & A Leuko Pak 101: How Leukapheresis Products are Best Utilized in the Lab • Survey to follow • Discuss your project needs with us: www.allcells.com/technical_services.php • Sign-up for our free monthly eNewsletter: www.allcells.com • Presentation will be uploaded in ‘Support’ section Leading Provider of Human Hematopoietic Cells and Services
Notas do Editor
- This allows the desired portion (e.g., leukocytes) to be removed and the remainder returned to the donor
- In the cartoon on the left, an IV is inserted in one arm of the donor, then whole blood is then introduced into the chamber or centrifuge and the blood separates into components by gravity along the wall of the chamber. The component to be removed can be selected by moving the level of the aspiration device at the right. In this example, plasma is being removed. In this example, plasma is being removed. The remaining components are then returned to the donor through an Iv in the other arm.
- Reg A 8 ug/kg/d for 3 days;D4 apheresisRegE 5ug/kg/d for 5 days, apheresis on D5Reg C 5-7 ug/kg/d for 5 days and apheresis on D5 and D6
- CellTracker reagents are fluorescent chloromethyl derivatives that freely diffuse through the membranes of live cells. Once inside the cell, these mildly thiol-reactive probes react with intracellular components to produce cells that are both fluorescent and viable for at least 24 hours after loading. CellTracker Green CMFDA has a relatively low pKa, which ensures that it will exhibit bright, green fluorescence in the cytoplasm at all physiological pH levels.