Validation of Plasmalyte in Cordwashing - Cord blood processing and quality control
This detailed documentation covers the validation process of using Plasmalyte in cord blood washing procedures, providing insights into the reasons for washing cord blood units, current standards, acceptance criteria, and the use of validated methods like Sepax cord wash. It emphasizes the importance of maintaining viability and potency of cord units for successful transplantation, along with detailed checklists for pre-processing and post-thaw evaluation. The content also highlights the significance of decision-making on whether a cord requires washing based on HLA matching, TNC dose, CD34 dose, post-cryo viability, and CFU-GM results. The document is useful for biomedical scientists, stem cell researchers, and healthcare professionals involved in therapeutic stem cell applications.
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Validation of Plasmalyte in Cordwashing Claire Donohue Lead Biomedical Scientist Therapeutic Stem Cell Laboratory
CONTENTS Why wash? Dextran Shortage July 2021 Validation plan Validation Data PPD approval Patient Data What next?
WHY WASH? JACIE Standards: D8.4.3 Cord blood units that have not been red blood cell reduced prior to cryopreservation shall be washed prior to administration D8.4.4 Cord blood units that have been red blood cell reduced prior to cryopreservation should be diluted or washed prior to administration Maintain viability and potency of the cord units during thawing Clinical teams select cords based primarily on HLA matching, Pre cryo TNC, CD34 dose, post cryo viability and CFU-GM results. Whether it requires washing is not high on the selection checklist as we have always had a validated washing process
WHY WASH? MFT- and in fact Royal Manchester Children s Hospital are one of the biggest users of cords in the UK. Decision to wash or not is a regular discussion point Use of a pre acceptance checklist enables us to decide if a cord will require washing. Existing validated method Sepax cord wash using Dextran
CORD ACCEPTANCE Pre Delivery Cord Acceptance Checklist This list does not include the usual cord blood selection criteria (HLA match, confirmatory type, TNC dose, CD34 dose) The factors listed below should be taken into consideration prior to the consultant/department accepting the date of shipment. Patient name Hospital Number D.O.B Weight Checklist Information Required 1.Pre-processing Pre cryopreservation results Total TNC: Total CD34: Total CFU-GM: 2.Post Thaw Post thaw(contiguous bleed-line segment or vial) Ideal:CD34 viability >80% by 7AAD CFU-GM > 5 x 10*4/kg If above targets are not met inform referring Clinician 3.Unit 1.Red Cell Deplete/Replete/Reduced? delete as appropriate RBC replete units (refer to Risk Assessment and Consultant to assess if Dextran wash required) 2.Product cryopreserved volume v weight of patient (kg) 3.DMSO content to be taken into consideration 4.CBU 1.Received and reviewed against cord report. 2.Verification typing (delete as appropriate) Cord Details/No Cord Blood Bank Accredited? Yes/No FACT/AABB Bld Group: Sex: M/F dob: Comments TNC dose = /kg CD34 Cell Dose= /kg Viability= % Method= CGU-GM cell dose = /kg Post Cryo Viability= State Method= Post Cryo CFU dose= %age CFU-GM recovery = Red cell content HCT= Total red cell content = mls Total volume = mls DMSO Content= Dextran Wash required YES/NO Viability Characteristics Yes / No / Not applicable Stored sample Unit segment @CBB Unit segment @ MFT Qualification Form 5. Virology & Additional Virology requested: Please list, note request dates and confirm when received below: Any additional virology required? Overseas/not tested Request additional samples required for testing- either before shipment or with the cord shipment. Please confirm mandatory results: Microbiology HIV 1 & 2 Ab HTLV 1 & 2 Ab HepBs Ag and HepBc Ab Hep C Ab Syphillis HBV PCR, HCV PCR, HIV PCR, HEV PCR West Nile Virus (EDTA) overseas only Chagas (Clotted sample) America s CMV, VZV, EBV, Toxoplasma (not mandatory) Request temperature logger shipment information Does the cord need quarantine on arrival? YES/NO Confirm tank availability: Micro culture: Date: 6. Received & within limits YES/NO
DEXTRAN SHORTAGE 2021 Previous shortage in 2016 and 2018. RMCH had sufficient stock which didn t cause significant issues- but still had to manage appropriately July 2021, RMCH had a planned cord wash and 5 days before the transplant we noted that the Dextran had expired. Panic! Many emails and phone calls to SCUG community but to no avail as everyone was in the same boat. Discussions with clinical team, decision to infuse over two days as the cord was in two bags and recipient was a large child so red cell content in one bag was acceptable. Non-conforming product, deviation to procedure completed
VALIDATION PLAN Plan was needed asap, Literature research: Pasha et al 2017, used 16 CBUs compared plasmalyte A based diluent and Dextran. Showed there was no significant decrease in viability of CD45+ or CD34+ cells even after 4 hours post thaw. Dextran actually seemed to increase occurrence of necrotic cells Plasmalyte A seemed to show an increase in potency of the cord units as seen by greater number of CFUs at all time points- but the differences were not significant.
VALIDATION PLAN Cloutier et al 2016 compared Plasmalyte A, Plasmalyte 148, Dextran 40 and Hespan. Used 10 dilution variants, compare by assessing viability, CD34, Cd45 and CFU. All results obtained relative to Dextran-40 and HAS, lower recovery was observed for all solutions without HAS Showed that it could actually be Human albumin solution that is the critical reagent in the stabilization of stem cells during thawing. No significant differences between Plasmalyte A or 148 with HSA Pharmacy reported previous issues with supply for Plasmalyte-A Plasmalyte 148 was chosen for our validation- we had previously used in clinical trials, and was readily available from our pharmacy.
VALIDATION PLAN Plan was to follow same SOP as used with Dextran but using Plasmalyte-148 instead. 8 cords- Roger Horton at Anthony Nolan provided FOC in return for sharing results Sepax 2 Cordwash programme Plasmalyte 148 and 4.5% HAS (50:50 mix)
VALIDATION RESULTS The Critical Quality Attributes assessed for the procedure were the same as the previously used Dextran wash procedure: Pre and Post total nucleated cell count will be obtained and the recovery calculated. The target recovery will be greater than 75%. Pre and post CD34 count will be obtained. The recovery will be calculated and the target will be greater than 75% recovery. Pre and Post CD34 Viability recovery will be >75%. Pre and post CFU-GM samples will be cultured and following 14 days incubation the colonies will be counted. A target of greater than 60% recovery will be set. Post processing bacteriology samples will be taken, a negative result will be required.
VALIDATION RESULTS TNC Mean recovery was 94.3% Init Vol ml Final vol ml TNC % recovery CFU-GM recovery CD34 viab rec %Red Cell reduction Sterility : growth? Ref Date Notes CD34 recovery 82.2% % recovery CFU-GM 89.8% none PW01 21.0 100 98.5 100.0 75.0 24.3 Neg 14/10/2021 none Negative sterility following 10 days incubation PW02 21.0 100 85.9 100.0 77.2 33.1 Neg 14/10/2021 none PW03 21.3 100 98.5 85.5 69.0 59.4 Neg 18/10/2021 none PW04 20.8 100 88.8 66.5 77.6 34.8 Neg 20/10/2021 none All environmental monitoring within limits. PW05 20.9 100 95.4 89.3 87.1 49.1 Neg 20/10/2021 none PW06 21.6 100 100.8 97.8 97.8 33.5 Neg 21/10/2021 none PW07 20.8 100 99.4 100.0 85.7 42.6 Neg 25/10/2021 none PW08 Mean 20.7 100 86.8 76.6 88.2 28.7 Neg 29/10/2021 94.3 89.8 82.2 39.0
2021 DEXTRAN PATIENT DATA Comparison of real life data Init Plasmalyte TNC recovery 94.3%, compared with Dextran 80%. Final vol ml TNC % recovery CFU-GM recovery %Red Cell reduction Sterility: growth? Ref Date CD34 viab rec Notes Vol ml CT2946-1 24.7 100 84 100 100 57.9 Neg 22/01/2021 none CT2935-1 26.4 100 84 97.9 100 76.0 Neg 25/01/2021 none AN cords will have been red cell depleted- may not have needed washing CT2955-1 50 100 77 89.3 100 65.9 Neg 01/02/2021 none CT2964-1 25.8 100 80 89.4 91 65.3 Neg 19/02/2021 none CT3002-1 53.2 100 81 92.1 73 58.9 Neg 26/04/2021 none Age of cords? Validation cords all <2years. CT3004-1 26.4 100 73 100.0 100 63.1 Neg 04/05/2021 none CT3043-1 25.27 100 84 100.0 82 17.6 Neg 29/06/2021 none Mean 80 96 92 57.8
PPD APPROVAL All testing performed in 2 week period in October. Submitted findings to HTA as PPD on 15th November 2021, following some further discussions and clarification the PPD approval letter was received on 23rd December 2021. A welcome Christmas present!
PATIENT DATA Final red cell volume (ml) Red Cell Reduction % TNC % recovery CD34 % recovery Loss of viability % Sterility testing Days to Engraftment Laboratory no. Instrument CFU-GM % recovery TNC recovery mean 75% 16 19 22 14 19 20 19 16 * * 14 7 CT3146-1 CT3151-1 CT3152-1 CT3159-1 CT3143-1 CT3178-1 CT3177-1 CT3197-1 CT3103-3 CT3199-2 CT3230-1 CT3234-1 6619 6619 6619 6619 6619 6619 6619 6619 6619 6619 6620 6619 74 82 83 72 75 83 73 88 62 78 85 82 93 98 92 75 97 159 86 88 95.2 95.8 120.0 86.0 7.40 5.30 4.60 7.30 8.20 4.10 10.98 6.60 4.50 6.40 5.70 10.10 33 55 39 52 32 54 30 53 43 41 38 35 -13% 0% 4% -6% -16% -28% 2% 1% -32% -2% -10% 1% Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 100.6 56.6 134.9 171.5 211.1 419.0 UTR 175.1 430.3 303.8 64.1 114.7 CD34 % recovery 91.5% All negative sterility CU-GM recovery >100% All environmental monitoring within limits 2 failed to engraftment- Both relapsed ALL, 2nd transplant following referral for Granulocyte trial. 13 121.6 332.7 N/A CT3282-1 CT3286-1 CT3315-1 6619 6619 6620 91 86.6 86.2 88.3 91.3 99.4 17.20 11.40 9.90 0 0% 3% -1% Neg Neg Neg 30 22 55.54 32.36 Mean 75.0 91.5 7.5 39.5 -0.1 182.3 18.4 123.4 SD 4.99 29.14 1.70 10.65 0.11
WHAT NEXT? Plasmalyte dilution for CD34 selection validation working with Miltenyi and Mike Watts Currently under conditional authorisation for plasmalyte wash on Cobe 2991 for cryopreserved Bone marrow Then ? Cobe removal, Sepax removal Dextran now available but haven t been able to obtain any yet ..
THANKS To all my amazing staff in Stem Cell Lab at MFT Wendy Ogden Roger Horton and the team at Anthony Nolan Mike Watts Rest of the SCUG group for replying to my panicked emails and phone calls
