Cord Blood: Thawing & Infusion Cord Blood: Thawing & Infusion ––Practical MethodsPractical Methods

Donna M Regan, MT(ASCP)SBBDonna M Regan, MT(ASCP)SBBSt. Louis Cord Blood BankSt. Louis Cord Blood Bank

@ SSM Cardinal Glennon Children@ SSM Cardinal Glennon Children’’s Medical Centers Medical Center

ObjectivesObjectives

Present variety of product configurationsPresent variety of product configurationsSummarize current thawing practicesSummarize current thawing practicesSuggest a menu of post thaw testing, indicative Suggest a menu of post thaw testing, indicative of quality and potencyof quality and potencyDetermine the method appropriate for thawDetermine the method appropriate for thawRecognize adverse events associated with HPC, Recognize adverse events associated with HPC, Cord Blood thawingCord Blood thawing

Disclaimer Disclaimer –– not endorsing particular products or not endorsing particular products or vendorsvendors

CB Product TypesCB Product Types

Physical configurationPhysical configurationSingle bagSingle bag

Baxter CryocyteBaxter CryocyteOrigen CryostoreOrigen Cryostore

Compartment bag Compartment bag Pall (Med Sep)Pall (Med Sep)

VialsVials

Product CompositionProduct Composition

UnmanipulatedUnmanipulatedPlasma reducedPlasma reducedRed cell reduced / plasma depletedRed cell reduced / plasma depletedBuffy coat enrichedBuffy coat enriched

Cryopreserved ProductsCryopreserved Products

Thawing PracticesThawing Practices

WashWash

Dilution or ReconstitutionDilution or Reconstitution

BedsideBedside

Rubinstein MethodRubinstein Method

Rubinstein, Dobrila, Rosenfield, Rubinstein, Dobrila, Rosenfield, et al.et al. Processing & Processing & cryopreservation of placental/umbilical cord blood for cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Nat Acad Sci unrelated bone marrow reconstitution. Proc Nat Acad Sci USA 1995; 92: 10119USA 1995; 92: 10119--10122.10122.

StatisticsStatisticsTNC recovery improved to 61% from 35%TNC recovery improved to 61% from 35%Mononuclear recovery much greater than PMN (grans)Mononuclear recovery much greater than PMN (grans)CFU losses were undetectableCFU losses were undetectable

Wash MethodWash MethodTraditionally thawed using a 5% Traditionally thawed using a 5% albumin/dextran wash protocol albumin/dextran wash protocol Rationale that reconstituting and washing frozen Rationale that reconstituting and washing frozen products would:products would:

Restore osmolarity & extend cell viabilityRestore osmolarity & extend cell viabilityReduce hemoglobin loadReduce hemoglobin loadDiminish DMSO toxicityDiminish DMSO toxicityDecrease infusion volumesDecrease infusion volumesRemove potentially ABORemove potentially ABO--incompatible plasmaincompatible plasma

Pediatric SettingPediatric Setting

When this procedure was developed, products:When this procedure was developed, products:

were not red cell or plasma reducedwere not red cell or plasma reduced

were cryopreserved in larger volumeswere cryopreserved in larger volumes

contained greater amounts of DMSOcontained greater amounts of DMSO

EquipmentEquipment

Laminar Flow HoodLaminar Flow HoodWaterbathWaterbathScaleScaleRefrigerated CentrifugeRefrigerated CentrifugePlasma ExtractorPlasma Extractor

Reagents & SuppliesReagents & Supplies

Sealable bagSealable bagSyringes & NeedlesSyringes & NeedlesUSP Albumin, 5%USP Albumin, 5%10% Dextran10% DextranAlcohol wipesAlcohol wipesSampling Site couplers (transfer sets)Sampling Site couplers (transfer sets)Transfer packsTransfer packsHemostatHemostat

PreparationPreparation

Verify physician orderVerify physician orderPull product and recipient filesPull product and recipient filesPrepare thawing solutionsPrepare thawing solutions

Dextran to equal oneDextran to equal one--half total volume of producthalf total volume of productAlbumin to equal oneAlbumin to equal one--half total volume of producthalf total volume of product

E.g.: 20 ml product + 5 ml cryoprotectant = 25 mlE.g.: 20 ml product + 5 ml cryoprotectant = 25 ml12.5 ml Dextran, 12.5 ml Albumin12.5 ml Dextran, 12.5 ml Albumin

Remove from StorageRemove from Storage

Verify identity of Verify identity of product & recipient product & recipient with paperworkwith paperworkLeave in vapor for 5 Leave in vapor for 5 minutes before minutes before thawingthawingRemove segments (if Remove segments (if present)present)

Thawing the ProductThawing the Product

Place into clean bagPlace into clean bagSubmerge into 37Submerge into 37ººC C waterbathwaterbathPorts up out of waterPorts up out of waterWarm just until slushyWarm just until slushy

Add Wash SolutionsAdd Wash Solutions

Insert sampling site Insert sampling site coupler or transfer setcoupler or transfer setInject one half of wash Inject one half of wash solutionssolutions

Drain into Transfer PackDrain into Transfer Pack

Add remaining wash solutionsAdd remaining wash solutionsto rinse the bagto rinse the bag

Drain into transfer bagDrain into transfer bag

AlternativelyAlternatively……..

Prepare wash solution Prepare wash solution from 250 ml Gentran from 250 ml Gentran + 50 ml 25% Albumin+ 50 ml 25% AlbuminPrepare syringes in Prepare syringes in appropriate amountsappropriate amountsInsert spike of wash Insert spike of wash bag into one of the bag into one of the ports of the cryobagports of the cryobag

Reconstitute to 150 mlReconstitute to 150 ml

Insert port of transfer Insert port of transfer pack into second port pack into second port of cryobagof cryobagDrain successive Drain successive additions of wash additions of wash solutions into transfer solutions into transfer packpackRinse bag with wash Rinse bag with wash solutionsolution

CentrifugationCentrifugation

Temperature = 10Temperature = 10ººCCSpeed = 420 x g (1200 rpm)Speed = 420 x g (1200 rpm)Duration = 10 minutesDuration = 10 minutesNo brakeNo brake

Express supernatentExpress supernatent

Expression TipsExpression Tips

Calculate amount of supernatent to Calculate amount of supernatent to remove from the centrifuged productremove from the centrifuged product

12.5 ml Dextran12.5 ml Dextran12.5 ml Albumin12.5 ml Albumin5 ml cryoprotectant5 ml cryoprotectant

Safely remove 30 ml supernatent Safely remove 30 ml supernatent Perform cell count on supernatentPerform cell count on supernatent

Aspirate concentrated product into Aspirate concentrated product into syringe for administrationsyringe for administration

Rinse with fresh wash solutionsRinse with fresh wash solutions

Include with product Include with product for infusionfor infusion

Use for sterility Use for sterility testingtesting

Mix well & remove sample for post Mix well & remove sample for post thaw assaysthaw assays

Nucleated cell Nucleated cell countcountHematocritHematocritViabilityViabilityCD34+ cell CD34+ cell countcountCFUCFU

Limited cells for testing??Limited cells for testing??

Reserve the Reserve the attached segment attached segment prior to thawprior to thaw

Pellet the Pellet the supernatentsupernatent

Rinse of CBU bagRinse of CBU bag

Salvage cellular Salvage cellular material into material into cryovialscryovials

Perform ABO/Rh Perform ABO/Rh testing &testing &crossmatch in crossmatch in allogeneic settingsallogeneic settings

Spot onto Spot onto Schleicher & Schuell Schleicher & Schuell paperpaper

Dilution MethodDilution Method

Regan, D.M., Grunzinger Nelms, L.M., Wofford, Regan, D.M., Grunzinger Nelms, L.M., Wofford, J.D., Alonso, J.F. III, Creer, M.H. J.D., Alonso, J.F. III, Creer, M.H. Comparison of Comparison of Cord Blood Product Thawing Methods on Cell Cord Blood Product Thawing Methods on Cell Recovery and Progenitor Integrity.Recovery and Progenitor Integrity.

Cytotherapy, Volume 7 Cytotherapy, Volume 7 18th Annual National Marrow Donor Program Council 18th Annual National Marrow Donor Program Council Meeting, November 4Meeting, November 4––6, 20056, 2005Biology of Blood and Marrow TransplantationBiology of Blood and Marrow Transplantation, , Volume Volume 12, Issue 11, Page 1224 (November 2006)12, Issue 11, Page 1224 (November 2006)

Dilution MethodDilution Method

Stabilize the product coming out of thawStabilize the product coming out of thawEliminate safety risks and potential bag breakage during Eliminate safety risks and potential bag breakage during centrifugationcentrifugationReduce tech timeReduce tech timeIncrease confidence that cells are not lost in expression Increase confidence that cells are not lost in expression stepstepProvide material for characterization studies without Provide material for characterization studies without adversely affecting cell doseadversely affecting cell doseLab is a controlled environmentLab is a controlled environmentImprove clinical outcomes from TCs with limited Improve clinical outcomes from TCs with limited experience in manipulating frozen cord blood productsexperience in manipulating frozen cord blood products

Direct InfuseDirect Infuse

Hahn, Bunworasate, George, Hahn, Bunworasate, George, et alet al. Use of non . Use of non volumevolume--reduced (unmanipulated after thawing) reduced (unmanipulated after thawing) umbilical cord blood stem cells for allogeneic umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment. Bone transplantation results in safe engraftment. Bone Marrow Transplantation 2003; 32: 145Marrow Transplantation 2003; 32: 145--150.150.

Product is diluted and stabilized in patient Product is diluted and stabilized in patient circulationcirculationBedside eventBedside eventProduct testingProduct testing

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. ....... ....... ......

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Conventional thaw + wash

(CW)

thaw only (direct thaw or

DT)thaw + albumin reconstitution

(AR)

CFU, TNC, CD34, 7AAD

remo LN2ve from

. ......

. ....... ....... ......

. .....

. ....... ....... ......

. ......

. ....... ...... ......

CFU, TNC, CD34, 7AADCFU, TNC, CD34, 7AAD

EndpointsEndpoints

The results of TNC recovery, CD34 expression, The results of TNC recovery, CD34 expression, CFU growth & viability were compared:CFU growth & viability were compared:

immediatelyimmediatelyat two hour intervals for the first 8 hoursat two hour intervals for the first 8 hoursat 24, 32, and 48 hours post thawat 24, 32, and 48 hours post thaw

0

10

20

30

40

50

60

70

80

90

100

Per

cent

Rec

over

y

Thaw Only 90.9 90.2 88.1 88.7 88.9 88.9 88.4 90.3

Reconstitute 86.3 84.0 84.0 84.4 84.3 87.4 87.7 86.2

Reconstitute and Wash 79.8 78.3 77.7 78.8 78.7 79.4 75.8 74

0 2 4 6 8 24 32 48

TNC RecoveryTNC Recovery

CD34 RecoveryCD34 Recovery

0

10

20

30

40

50

60

70

Per

cent

Rec

over

y

Thaw Only 62.6 64.9 59.2 59.4 53.3 33.9 28.8 19.9

Reconstitute 67.8 68.8 60.0 62.7 60.1 67.9 61.6 64.2

Reconstitute and Wash 59.9 60.3 55.3 58.4 51.1 59.7 50.1 51.5

0 2 4 6 8 24 32 48

CFU RecoveryCFU Recovery

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

Per

cent

Rec

over

y

Thaw Only 83.2 87.0 69.9 62.9 54.5 14.5 1.8

Reconstitute 81.4 87.0 80.6 75.0 78.3 69.6 61.3 46.4

Reconstitute and Wash 81.8 76.6 76.4 78.3 77.6 67.3 58.6 49.3

0 2 4 6 8 24 32 48

Post Thaw Testing & QCPost Thaw Testing & QC

Essential to determining product integrity & Essential to determining product integrity & quality of the manufacturing processquality of the manufacturing processFDA IND FDA IND -- Safety & efficacySafety & efficacyOutcome analysisOutcome analysis

Evaluating variables affecting engraftmentEvaluating variables affecting engraftment

QC Testing QC Testing

Regulatory Requirements Regulatory Requirements -- post thaw post thaw not not clearly defined by AABB, FACT or FDAclearly defined by AABB, FACT or FDAEstablishing Reference RangesEstablishing Reference RangesSample size issuesSample size issuesStandardization is crucialStandardization is crucial

QC Testing Recommendations QC Testing Recommendations

Nucleated Cell CountNucleated Cell CountDose infusedDose infusedRecovery Recovery

ViabilityViabilityCFU CFU CD34 CD34 –– total & viabletotal & viableABO/Rh ABO/Rh

Labeling / IdentityLabeling / IdentityPatient post infusion supportPatient post infusion support

HematocritHematocritRed Cell Volume / DoseRed Cell Volume / Dose

Sterility CulturesSterility Cultures

Optional: HLA cases where segment typing not doneOptional: HLA cases where segment typing not done

CB Thaw Averages CB Thaw Averages –– SLCBBSLCBB

848 products thawed at 218 TCs848 products thawed at 218 TCsNC recovery = 81%NC recovery = 81%TB viability = 79TB viability = 79--90%90%

WhatWhat’’s right for you?s right for you?

Laboratory procedures for different HPCLaboratory procedures for different HPC--C C products products

Do laboratories follow CBB recommendations Do laboratories follow CBB recommendations Practice validated inPractice validated in--house procedureshouse procedures

How are infrequent in house procedures How are infrequent in house procedures validatedvalidatedNMDP practice unitsNMDP practice units

To wash or not washTo wash or not wash……

…… what are criteria for this decisionwhat are criteria for this decision

Physician preferencePhysician preferencePatient size Patient size –– 10 kg pediatric or 100 kg adult10 kg pediatric or 100 kg adultDMSO doseDMSO doseRBC doseRBC doseExperience and confidenceExperience and confidence

Adverse EventsAdverse Events

Advance prep will circumvent issuesAdvance prep will circumvent issuesProper thaw suppliesProper thaw suppliesTransportTransportPatient delayPatient delay

Product IssuesProduct Issues

Brittle bag at low tempsBrittle bag at low tempsRapid expansion when removed from LN2Rapid expansion when removed from LN2PortsPortsSegment exposure & Undetected cracksSegment exposure & Undetected cracks

Contain productContain productAvoid introductionAvoid introduction

Bag break in centrifugeBag break in centrifuge

Clinical ConsiderationsClinical Considerations

Filtering Filtering –– not standard practicenot standard practiceRinse product containerRinse product containerPush vs hangPush vs hang

Engraftment/Outcome DataEngraftment/Outcome Data

Essential for evaluating quality of unitEssential for evaluating quality of unitEach institution establishes own outcome criteriaEach institution establishes own outcome criteria

J Wagner et al Blood. Sept 2002. vol 100 pp 1611J Wagner et al Blood. Sept 2002. vol 100 pp 1611

Providing data is crucial Providing data is crucial Stem Cell Transplant Outcomes Database Stem Cell Transplant Outcomes Database (SCTOD)(SCTOD)

Key PointsKey PointsCommunication and advance preparation are essential Communication and advance preparation are essential to contributing to a quality process.to contributing to a quality process.TC need to develop and validate their own thaw TC need to develop and validate their own thaw protocol(s) and utilize those systems.protocol(s) and utilize those systems.QC testing is critical and can be done without QC testing is critical and can be done without compromising the infusion.compromising the infusion.