Cellular Therapies Flashcards
What percentage of patients have an HLA-matched sibling?
30%
How many allo-transplants have ABO incompatibility?
50%
What are the consequences of major ABO incompatibility in allo-HSCT?
Hemolysis
Pure red cell aplasia
Delayed engraftment
What are the consequences of minor ABO incompatibility in allo-HSCT?
Increased GVHD (controversial) Delayed hemolysis (passenger lymphocyte syndrome)
When should ABO type be switched in HSCT?
No consensus, but maybe:
+100d
Donor cells present at 2+ typing
Based on presence of isohemagglutinins
What is the significance of RhD matching in stem cell transplant?
It is only of concern in young women. Even then, only 10% of D+ recipients getting D+ donors will develop anti-D.
Why is it important to minimize platelet transfusions in the peritransplant period?
Excessive platelet transfusion will stimulate alloimmunization; anti-HLA antibodies can cause graft failure.
What isohemagglutinin titers are recommended for ABO-incompatible SOLID ORGAN transplant?
Titers should be less than or equal to 16, at least in young children?
How can ABO-incompatible solid organ transplant cases be supported by the blood bank?
Try to give blood that is compatible for both organ and recipient (eg. O pt receiving A liver should get A plasma and O red cells). No need to irradiate. MINIMIZE pre-transplant transfusions.
How significant are A subgroups in transplant settings?
Not at all; can treat A2 as O and A2B as B.
What is passenger lymphocyte syndrome, and what drives its relative risk?
Transfer of established B-cell lineages that generate anti-host antibodies (usually A/B, but also D, K, Fya). The risk correlates to the degree of lymphoid tissue present in the transplanted tissue.
What disorders are best treated with autologous transplant?
Those with a high dose-treatment response curve, such as B-cell lymphomas and plasma cell myelomas.
What are the 8 required loci for HSCT matching?
HLA-A
HLA-B
HLA-C
HLA-DRB1
How do cord transplants compare to non-cord?
Lower-dose, slower engrafting. Best in children. High concentration of CD34+ cells.
What is the benefit of conditioning chemotherapy in transplant? What is used to condition?
Autologous: Eliminate as much tumor as possible
Allogeneic: Suppress native immunity (and eliminate tumor).
Bone marrow failure conditions do not require conditioning.
Condition with purine analogs and anti-T-cell therapy (formerly cyclophosphamide, busulfan, TBI)
What is the minimum dose of CD34+ cells required for stem cell transplant?
2.5 x 10^6 CD34+ cells per kilogram.
How do HPC(A) transplants compare to HPC(M)?
HPC(A) engrafts faster but is associated with more reactions and GVHD due to higher CD3+ load.
What markers allow HPCs to interact with their bone marrow niche?
VLA-4 MAC-1 CXCR44 CD44 CD62L
How does G-CSF promote mobilization? When is the peak?
It increases expression of MMP-9 and other proteolytic enzymes. 5d to peak.
How does Plerixafor promote mobilization? When is the peak?
Disrupts CXCR4-CXCL12 interactions. Peak is rapid; 6 hours
What are alternatives to G-CSF and plerixafor for mobilization?
Chemo
EPO, TPO, GM-CSF, PT? Lithium? Natalizumab…all meh.
What happens to circulating CD34+ count during harvest?
It does NOT fall, it actually holds steady or even increases as a response to harvest.
What happens to platelet count during harvest
It does fall, and patients can become transiently thrombocytopenic.
How are donors screened before stem cell transplant?
There is a DHQ-equivalent and medical records are reviewed. IDMs are performed up to 30d BEFORE harvest.
Can you collect from an ineligible donor?
Yes, if the medical need is great.
When should an HPC sample be drawn for culturing?
After collection but before processing & cryopreservation.
How can neoplastic cells in the product bag be reduced?
Harvest using chemo prime
Select/enrich for stem cells
Direct treatment in bag (doesn’t work well?)
What diseased states can have cause complications for stem cell harvesting?
Sickle cell: G-CSF promotes sickling.
Myeloma: Paraproteins affect the interface.
Autoimmune disorders: G-CSF can cause flares.
What are the adverse effects of G-CSF?
Bone pain, headache, nausea.
Splenomegaly is common, ruptures are rare.
Neutrophil activation syndromes are rare.
How do pediatric harvests differ from adult?
Kids have fewer complications overall, but are more likely to need CVC access and experience more vasovagal reactions and citrate toxicity.
What is TC(A)?
Therapeutic cells (usually CD3+) from apheresis, often collected in tandem with stem cells for later DLI.
What are the acceptance criteria for a cell processing lab to take HPCs?
Need IDMs, ABO/Rh, an adequate label (donor, recipient, facility, product, DIN). HPC(A) should be >60% MNCs and have >90% viability.
How are HPCs processed to minimize major/minor ABO incompatibility issues?
Spin to remove red cells, wash to remove plasma. Do both in bidirectional incompatibility.
What is the benefit of cellular selection and depletion in stem cell processing?
Can enrich for CD34+ cells to make a more product and select against tumor. Can also select for or against T-cells, Dendritic cells, and B-cell lineages.
What is stem cell expansion?
Ex vivo culturing of stem cells with cytokines and growth factors. Can expand HPC (CB) but not HPC (A)?
What is dendritic cell generation?
An ex vivo process of enriching MNCs and exposing them to IL-4 or IL-13 to generate dendritic cells, which enhances graft vs tumor effect.
How do reactions to DMSO generaly present?
Very similarly to allergic transfusion reactions.
What immunophenotypic features distinguishes a plasmacytoid dendritic cell from a conventional/myeloid dendritic cell?
Strong expression of CD123 and no expression of CD11c.
What is the concept of dendritic cell vaccines?
Dendritic cells can be loaded with antigen and activated to generate a recipient immune response against viral or tumor antigens. In practice, this has not been very promising.
Describe the structure of the CAR.
Rather than a modified TCRa/b, it consists of a zeta intracytosolic region as well as a CD28 costimulatory region affixed to an extracytosolic single-chain variable fragment which bears the target specificity.
What are the transcription factors needed to induce pluripotency in fibroblasts?
c-Myc
Sox-2
Oct-4
Klf-4
What is a real example of cellular transdifferentiation?
Pancreatic exocrine cells can be induced to transform into beta-endocrine cells.
What can influence an injured tissue’s ability to undergo regeneration (rather than repair)?
Age of immunity (fetal tissue more able to regenerate), relative pluripotency of niche stem cells.
What is the difference between pluripotency and multipotency?
Pluripotency means the ability to generate cells from all 3 germ layer lineages. Multipotency can only generate from one.
What cellular interactions drive hematopoietic progenitor cell homing?
CXCL12/CXCR4, E-selectin, VLA-4/5, VCAM, and others.
What are the four transcription factors that can induce pluripotency in fibroblasts?
c-Myc, Klf4, Oct4, Sox2
What is tissue transdifferentiation?
Conversion of one committed cell lineage to another (eg. Pancreatic exocrine to beta-endocrine)
What is the driving principle behind DC vaccines?
Dendritic cells can be prepared with an antigen to promote a robust T-cell response.
How can an antigen presenting cell be loaded with antigen for the purpose of cellular therapy?
Can expose to cellular lysates, proteins, and nucleic acids. Can transfect with a viral vector.
What is the relative occurrence rate of complications arising from mismatching of non-HLA blood groups in HSCT?
1-8% have complications. These are almost always to ABO, though rare complications with RhD, Kell, Kidd, and Lewis have been described.
At what isohemagglutinin titer is hemolysis a concern in ABO incompatible transplant?
> 256
What defines delayed engraftment and/or PRCA?
Normal engraftment should take 3-4mo to reach >2.5 x 10^9 retics per liter. If less than 1% retics after 28d, PRCA.
What is transplant associated TMA?
A rare thrombotic complication of hematopoietic stem cell transplant. Diagnosed by renal biopsy, but not really well treated by plasma exchange.
What is the role of monitoring isohemagglutinin titers pre-transplant and peri-transplant?
Pre-transplant: Can identify high titers that require exchange or adsorption before transplant
Peri-transplant: Can be used as a marker of native immune decline / myeloablation
How should ABO matching be done in a major incompatible graft? Minor incompatible? Bidirectional?
Major: Give red cells of recipient type, plasma products of donor type.
Minor: Try to match both directions (eg AB patient getting A cells»_space; Give A cells, AB plasma)
Bidirectional: Fuck it, O cells and AB plasma.
What non-HSC cells contribute to the HSC niche?
Osteogenic progenitors, osteoclast, osteoblasts, adipocytes, and endothelial cells
What infectious disease testing is required of allogeneic HPCs? Auto?
Allogeneic: HIV-1/2, HTLV-1/2, HBV, HCV, Syphilis, CMV
Autologous: All except CMV
What is the maximum harvest goal in HPC-M?
20ml/kg.
How are HPCs stored?
In vapor phase nitrogen at < -150C.
What quality assays should be done on an HPC product upon receipt by a cell therapy lab?
CBC/Diff CD34+ flow Sterility studies CFU (clonogenic assay) Viability (Trypan, 7-AAD, acridine)
What are the features of a DMSO transfusion reaction?
Nausea, vomiting, cough, headache, redness
What federal regulations oversee the handling and production of minimally manipulated HPCs? Specially altered products?
Minimally manipulated: Section 31 of CBER’s PHSA
Altered: Title 21, CFR pt 1271
How does AABB recommend tissue allografts be handled?
By a central transfusion medicine service.
What are the aspects of donor assessment in tissue allograft harvest?
Consent/authorization, review of medical history, a physical assessment, perhaps autopsy reports. IDM testing is the same as with blood…
What types of tissue grafts does the FDA regulate?
Allografts, xenografts, xenotransplants. Not autografts.
Compare and contrast freezing of tissue allografts with lyophilization.
Cryopreservation can preserve cellular viability if a cryoprotectant solution is used. Lyophilization does not.
Do tissue allografts require ABO matching, HLA matching, or both?
Neither!
What infectious agents can be transmitted in allografts?
Virtually all; viruses, bacteria, fungi, prions, and even sometimes neoplasms.
What are the responsibilities of hospital tissue services?
Must handle tissue, trace/detect/report adverse events. They must handle inspection, consenting, and record-keeping.
What is the most common tissue auto-graft?
Craniotomy specimens
What are typical storage condition of most simple tissue grafts?
Most are stored either in fridge at 1-10C or frozen at ***
What disease states get the most benefit from donor lymphocyte infusion (DLI)?
Most promising early data was in the setting of CML. AML derives some benefit, while ALL, myeloma, and lymphomas derive very little benefit.
What are the benefits and consequences of donor lymphocyte infusions?
Can induce remission during relapse, can also fight off opportuistic infections. Associated with worse GVHD and can cause marrow aplasias.
In cellular gene therapy, what are some possible methods of genetic transduction?
Non-viral: Injection, liposomes, electroporation.
Viral: Lentiviruses, retroviruses.
What are the strengths and weaknesses of using a retrovirus as compared to other viral vectors?
Retroviruses randomly insert into the genome, causing insertional mutagenesis and requiring the cells to be actively dividing. But, response is more durable.
What are some criteria for a disease to be targetable by cellular gene therapies?
The disease should have high morbidity/mortality, should not have good treatment otherwise, and should have a specific and reversible phenotype. Usually these are monogenic disorders driven by cells that are easy to harvest, modify, and administer.
Tocilizumab
Anti-IL-6 antibody used to mitigate cytokine-storm-like inflammatory response in CAR-T treatment.
Tsagenlecleucel / Kymriah
Novartis-based CD19 CAR-T therapy
Axicabtagene / Yescarta
Gilead-based CD19 CAR-T therapy
