Cord blood banking brief presentation(1).pptx

Cord blood banking brief presentation

1. CORD BLOOD BANKING Dr. Jahnavi III Yr Resident KAMSRC

2. OVERVIEW Cord blood banking Benefits of CBB over BMT UCB procedure: a) collection b) processing c) freezing d) Thawing

3. Committed lineage- restricted progenitor cells- referred as Hematopoietic progenitor cells(HPCs) Pluripotent hematopoietic stem cells- capable of self renewal & differentiation When transplanted into conditioned recipients, reconstitute the marrow HPCs can be collected from: Marrow, peripheral blood, umbilical cord blood

4. Characteristics Umbilical cord blood Bone marrow Mobilised peripheral blood stem cells Ease of collection No safety risks for mother or child Needs to be anesthetised, Procedure in the OT, takes several hours Requires mobilization, discomfort to the donor Time to engraft Median time of 3 weeks Median time of 16-18 days Median time of 13-15 days Viral infections Rare common common GVHD Uncommon and less severe even if there is GVHD Common in mismatched grafts More risk of chronic GVHD than that of bone marrow HLA mismatch HLA mismatch well tolerated Requires a perfect HLA match Requires a perfect HLA match Repeat transplant Not possible Possible Possible HSC numbers Low and hence poor engraftment High, hence better engraftment High, hence better engraftment

5. Benefits of cord blood over BMT UCB is more easily accessed 6/6 V/s 8/8 match Research published in 2007 revealed that five-year leukemia-free survival rate in 503 children with ALL who received an HLA mismatched UCB transplant was similar to the survival rate of 282 children who received BMT

6. Results of the 2001 study showed that the median time of locating a bone marrow match was 49 days, compared to 13.5 days for a suitable UCB match Reduced numbers of mature T lymphocytes , which reduces the risk for GVHD.

7. Because any patient shares exactly one HLA haplotype with each biological parent or child and there is a 50% chance that a sibling will share one HLA haplotype, an eligible HLA-haploidentical donor can be identified rapidly in nearly all cases Clinical studies for COVID-19 treatments, the use of human umbilical cord mesenchymal stem cells ( hUCMSCs ) was shown to produce inflammatory remission in a patient with severe level symptoms

8. Cord Blood Bank Cord blood bank means a place or organization or unit for carrying out and responsible for operations of collection, processing, testing, banking, selection, and release of cord blood units 1988- First UCB as an alternative source of HSCs in Fanconi anaemia Only 25-30% of patients who require allogenic HSCT can find an HLA-matched sibling donor

9. The Indian rules define umbilical cord blood as “the whole blood including HPC collected from placental and/or Umbilical cord blood vessels after the umbilical cord has been clamped ”.

10. Umbilical cord blood Blood that remains in the placental blood vessels and in the attached umbilical cord, which connects the unborn baby to the mother's womb UCB is collected from the placenta when the expectant mother is in the third stage of labor (after delivery of baby) or after the delivery of the placenta

11. Rich in HSCs , which possess the properties of self-renewal as well as the ability to differentiate into myeloid and lymphoid cell lineages Rich in mesenchymal cells , are self-renewing and minimally immunogenic, play a key role in immune suppression in response to Graft-versus-host disease (GVHD) No documented benefits of banking the placental tissue or Wharton’s jelly

12. Criteria for choosing UCB unit Should be HLA typed to high-resolution for HLA-A, -B, -C, and DRB1 loci to select an unrelated UCB with ≤ 2 allele mismatches In a double UCB transplant , both the UCBs should be at least 4/6 matched to the patient using intermediate resolution (antigen level) for HLA loci A and B and high resolution (allelic) typing for HLA DRB1

13. Advantages and disadvantages of UCB Advantages: Ease of collection No risk for the mother or the child Less time needed for processing (more quickly available for use) More economical than bone marrow collection Decreased risk for transmission of TTI infection Lesser requirements for stringent HLA typing Relatively lesser possibility of GVHD

14. Disadvantages: Slower degree of engraftment Limited cell dose in the inoculum making its use limited to children. Small volume of the Unit Additional cell doses unavailable Storage issues at ultra-low temperatures and related costs Poor immune reconstitution

15. DONOR MANAGEMENT Maternal and infant donor evaluation: To assess the risk of disease transmission from the infant donor to the recipient To assess the health status of the infant To record the personal, family, medical and genetic history of the families of both parents before UCB collection In case of surrogate mother, genetic history is not required Not advisable in complicated deliveries like twin gestation and prematurity

16. Maternal screening and testing: Establish written criteria for maternal screening Detailed physical examination and medical history Information on the donor’s lifestyle Detailed medical and genetic history Travel history of mother Testing of maternal blood for infectious agent ( HIV, HBV,HCV, HTLV, CMV, Malaria and Treponema Pallidum) Within 7 days before or after delivery Consent form and process(Informed consent prior to delivery)

17. UCB collection procedure Aseptically using 16 gauze needle , allowing placental blood to be removed by gravity in a disposable PVC bag and containing an adequate quantity of sterile pyrogen-free anticoagulant like CPD / CPD-A  Pope  et al . suggested that the ratio of anticoagulant could cause decrease cell viability in CBUs with a volume lower than 60 ml. Further, it was suggested that lower WBC viability in low-volume CBUs could be due to a higher ratio of anticoagulant

18. Collection may be in-utero (common) or ex-utero. Volume collection- 50 to 200 ml Correlation between weight of the bag and TNC count. Some CBB establish thresholds for banking based on TNC count.

19. In-utero Trained obstetricians after the delivery of the infant ( min of 34 weeks) Advantages Avoids the possibility of failed collection resulting from damage to placenta during delivery Reduces delay in collection An increase in volume collected Reduces incidence of clotted collections

20. Ex-utero Trained UCB bank staff after the infant and placenta are delivered Following delivery, placenta is passed to UCB bank staff to harvest the blood following cleaning the umbilical cord and aspiration of blood from placental vein

21. Higher UCB volumes yields greater nucleated cells, CFUs and CD34+ cells on engraftment, transplant-related events and survival A large CIBMTR study demonstrated that double HPC(CB) transplants had similar outcomes to single HPC(CB) transplants in patients with AML Minimum TNC count of 3×10 7 /kg at cryopreservation obtained for engraftment

22. For double HPC(CB) transplants, each HPC(CB) unit has a minimum TNC count of 1.5×10 7 /kg at cryopreservation A cryopreserved dose of 2.5 ×10 7 /kg accepted as the minimum cell dose for successful engraftment in single HPC(CB) transplants In double HPC(CB) transplants, pre cryopreservation CD34+ cell dose of ≥1×10 5 /kg used by several transplantation centres as the minimum cell doses for each unit

23. Askari  et al . showed that, collection within not greater than 5 minutes of placental delivery produced higher volume and TNC count. Increased TNC count also seen in Caucasian women, primi gravidae, female newborns, and collection duration of more than 5 minutes.

24. A time between delivery of newborn and placenta of not greater than 10 minutes predicted better volume and CD34+ count Ballin   et al . showed that CBUs collected during 10 min after cord clamping had lower thrombin activation within time

25. Elchalal   et al . showed that the CB collection by syringe causes higher number of TNC and volume than collecting by bag. However, there was no statistical difference in the percentage of CD34+ cell Using active syringe/flush/syringe method, for collecting UCB before placental delivery, results in greater volumes and lower discard rate

26. The collection of UCB in within 10 min after cord clamping in the closed system and without manipulation has higher volume, higher TNC, lower coagulation activity, and less contamination

27. The best time and temperature in different steps of umbilical cord blood collection until transplantation

28. Collection, processing and banking

29. Requirements for processing, testing and storage areas for UCB Cord blood reception- air-conditioned area of at least 10.00 sq. meters shall be provided Cord blood processing area- house Class 100 biological safety cabinets for UCB processing. Room temperature shall be maintained from 20ºC to 25ºC with a positive differential pressure of 10-15 Pascal and relative humidity of 50-60% . The minimum area shall be 10.00 sq. meters for the activity. Entry to this area shall be through the airlock

30. Haematology and Serology Laboratory- Testing for ABO grouping and Rh typing, total nucleated cell count, progenitor cell count, and viability test Transfusion transmissible infectious disease screening laboratory- HIV I & II; Hepatitis B & C virus, syphilis, malaria, CMV, and HTLV

31. Sterility testing laboratory- Area for media preparation, sterilization, incubation and decontamination HLA typing laboratory Sterilization-cum-washing Records and store rooms Cryogenic storage room- A minimum space of 20.00 sq. meters shall be provided. Temperature monitoring of storage vessels, liquid nitrogen level in storage vessels, and oxygen meter. space between each liquid nitrogen storage vessel, supply cylinders, and connecting hose should be a minimum of 1.00 sq. meters

32. Transportation From the birthing center to the designated laboratory Transportation procedure shall be validated to ensure the optimum survival of the stem cells Temperature should be between 18ºC to 28ºC Time period between collection and processing shall not exceed 72 hours Wada  et al . served a 1% decrease in viability for every 4-h increase in transport time for newly collected UCB units that were shipped at ambient temperature

33. Processing of UCB Centrifugation, sedimentation, for reducing red cell content, plasma volume S edimenting agents such as hydroxyethyl starch (HES), gelatin and dextran R ecovery of TNC and HCT of final products is the most important indicator of CBB processing quality Pablo Rubinstein method is a good procedure to reduce red cell count using HES and depletes the units of plasma and anticoagulant to minimize the final product and cryoprotectant volumes

34. Freezing technique DMSO is used by most CBBs with cryogenic bags preferred over freezing vials DMSO should be added slowly (up to a final concentration of 10%; samples are kept at 4°C at all times) to prevent an osmotic shock to the cells After performing volume reduction, the amount of the final concentration of DMSO and dextran in the bag was 10% and 15%, respectively

35. CBUs were thawed using a 37°C water bath and diluted 1:3 with the NYCBB washing solution containing 50% dextran 40, 12.5% human serum albumin 20%, and 37.5% PBS. N ot observed any significant difference in nonviable cells between bag, aliquot, and segment. the percentage of early apoptotic and necrotic/late apoptotic cells differed 

36. Controlled-rate comparable uncontrolled-rate freezing and DMSO at 5 or 10% final concentration were used. CD34+ recovery, CFU-GM, and BFU-E recovery in the CBUs were the highest when controlled-rate freezing procedure and 5% DMSO were applied   Slow cooling in a programmable controlled-rate freezing device at rate of 1 C/min will result in adequate recovery of HPCs

37. Both AABB and FACT- Netcord have defined storage temperature limits for cryopreserved UCB to be <−150°C . Once frozen, UCB units are typically transferred to long-term storage into a monitored liquid nitrogen container, either immersed in liquid (at −196°C) or in vapor phase to minimize the potential for cross-contamination

38. The optimal freezing rate for umbilical cord blood units

39. Storage of UCB Cryopreserved using a controlled rate freezing, or equivalent validated procedures. The frozen storage shall be at minus 196ºC and shall not be warmer than minus 150ºC. There will be no shelf life for this class of product( ICMR 2023) UCB can be cryopreserved and stored for >15 years with efficient recovery of stem cells on thawing

40. Reference samples- . At least two reference samples shall be collected from cord blood unit product before cryopreservation and stored at minus 196ºC and shall not be warmer than minus 150ºC. At least one additional reference sample shall be stored at minus 76ºC or colder for purposes other than viability analysis

41. Test Method Cell count Haematology analyser, manual differential CD34+ cell enumeration Flow cytometry( single/ dual platform) Viability assay Dye exclusion(light/ fluorescence microscopy), flow cytometry Clonogenic assay CFU(most commonly used in clinical lab)LTC-ICs Sterility testing Aerobic, Anaerobic, fungal culture HLA typing Molecular ABO/ Rh typing Serology Haematocrit Standard/ Routine QC testing for Umbilical cord blood

42. Thawing and washing of UCB Traditional thaw-and-wash method, the thaw-and-dilution technique, and the bedside thaw method Clean or sterile transparent bag ( ie , a plastic zipper bag) and submerged in a 37 C water bath of clean or sterile water or saline Thaw solution-10% dextran and a protein source, usually human serum albumin in a final concentration of approximately 2.5% to 4.2% Centrifuge and get the cell pellet and resuspended in thaw solution

43. UCB release Designated area with adequate space for procedures and records related to CBU selection and release The CBB shall obtain written or electronic request from the transplant physician or designee for shipment of the CBU

44. Documentation at the time of issue from the cord blood bank shall include indications, contra-indications, caution, instruction for handling and use of the cord blood unit including short-term storage and preparation for transplantation Procedure for transportation of cryopreserved CBU within the facility shall be designed to protect the integrity of the unit and the health and safety of the personnel

45. For a dry shipper to be fully effective, it must be charged or filled with LN2 24 hours before the estimated time of release from the processing laboratory Cryopreserved CBU stored at -150ºC or colder shall be transported in a liquid nitrogen cooled dry shipper that contains adequate absorbed liquid nitrogen and has been validated to maintain temperature below -150ºC for at least 48 hours beyond the expected time of arrival at the receiving facility

46. references National umbilical cord blood banking guidelines-ICMR AABB Technical manual- 20 th edition DGHS Technical manual- 3 rd edition Blood banking and Transfusion medicine Umbilical cord blood quantity and quality: Collection up to transplantation-Asian journal of transfusion sciences

47. Thank you

48. Levels of manipulation Stem cells- autologous or allogeneic, require in vitro or ex vivo processing before their use. This carries the risk of contamination and may lead to alteration in their properties which vary according to the degree and type of manipulation. All laboratory procedures should be carried out under aseptic conditions in a CDSCO certified GMP (schedule M) and GLP (schedule L 1) facility for human applications

49. Minimal manipulation- The processing neither alters the number nor the biological characteristics and function of the cells (or tissue) relating to their utility for reconstruction, repair or replacement Substantial or more than minimal manipulation- This is defined as ex vivo alteration in the cell population (enhancement or depletion of specific subsets), expansion, cryopreservation or cytokine based activation, but does not result in alteration of cell characteristics and function

50. Air-handling system Processing area shall have HVAC (heating, ventilation, and air conditioning) system and be fitted with HEPA (High-Efficiency Particulate Air) filters having Grade C (Class 10,000) environment. The entire processing shall be done conforming to the Grade A (Class 100) Standard of air quality

51. Labelling

52. L2.Label at completion of processing and before issue - Cryogenic Storage Label [Statutory label] shall indicate the following: a.Label at completion of processing and before issue -

53. Issue label at the time of release of Cord Blood Unit shall indicate the following namely

54. Pre-implantation Genetic Diagnosis (PGD) and Tissue Typing (TT) Embryo created by IVF has the exact biological makeup to act as a future UCB donor for an identified recipient, typically a sibling Procedure is performed by the removal of a single cell three to five days after fertilization when the embryo is at the six-to-ten cell stage PGD prevents an identified disease from materializing whilst TT provides a perfect blood match for transplantation purposes

55. UCB (pluripotent) have the potential to improve cardiac function, treat diseases of the central nervous system, and brain injury, including stroke Multipotent (specialized) mesenchymal stem cells (MSCs) found in the bone marrow – cells that possess the lineage of connective and skeletal tissue and can differentiate into such as connective tissues, bone, cartilage and fat, presenting enormous potential for regenerative medicine MSCs from bone marrow, obtaining them is both painful for the donor and yields a low number of cells

56. Cord Blood Banking- Private /public Both public, as well as private cord blood banks, have been established worldwide Private bank-for-profit company for personal or self-directed use Public bank- state-owned by consenting parents to be used for any patient in need of a hematopoietic stem cell transplant (allogeneic umbilical cord blood transfusion). The first publicly funded cord blood bank was established in New York in 1993 India does not have any public UCB banks at present( paucity of public funded UCB banks in India)- ICMR-2023

57. Public V/S Private Cord Blood Banking Several international bodies do not recommend routine private banking for future self-use The likelihood of the stored blood being used for HSCT is very small, probably as low as 0.005 to 0.04% in the first 20 yrs of life- icmr SCT using an individual’s own cord blood (autologous transplant) cannot be recommended for genetic disorders private banking is suggested in cases where there is a relative/sibling with a condition or there is a family history of malignant or genetic conditions that can be treated with HSC transplantation ( Thalassemia ) Banking for allogeneic transplant is recommended when there are shared HLA antigens between parent

58. The likelihood that they will ever be used is remote (range of available estimates is from 1:1000 to 1:200 000 ) The number of cord blood units stored in private banks far exceeds that stored in international stem cell registries for public use [4], and is three times more than that in public bank In one study, quality parameters of privately banked cord blood units are found to be inferior to those stored in public bank

59. The chance of a cord blood being utilized is at least 100 times greater in a public bank as compared to private bank In haematological malignancies, allogenic stem cells are preferred over autologous stem cells due to the proven therapeutic effect of graft-versus leukemia reaction , which occurs only in allogenic transplantation