Overview of Specialised BMT Cell Processing€¦ · Overview of Specialised BMT Cell Processing....
Transcript of Overview of Specialised BMT Cell Processing€¦ · Overview of Specialised BMT Cell Processing....
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Prepared by Vicki Antonenas
Senior Scientist, Blood and Marrow Transplant Laboratory,
Westmead Hospital
Overview of Specialised BMT Cell Processing
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Topics to be presented today:
Introduce and explain indications for:
Bone Marrow Processing
CD34 flow testing and reporting
CD34 positive selection of stem cell products for the purpose of T cell depletion
Cord Blood thaw and wash prior to infusion
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Apheresis 200 ml
Bone Marrow1 L
Cord Blood25ml
Types of HPC Products used for BMT
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Steps in BMT Cell Processing:
1) Collection 2)Processing for Freezing & Storage 3)QC testing 4)Thaw & Infusion
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Bone Marrow Harvesting
Performed in theatre under general anaesthesia, collected by trained doctors
HPC(M) is obtained by multiple aspirations from the iliac crest or sternum
Maximum marrow can be removed 20ml/Kg body weight
This process takes approx. 2 hours
Target TNC dose >2 x10^8/Kg
Common anti-coagulant is ACD-A
Ratio 1:5, example
100mls ACDA + 500mls BM, total 600 mls in 1bag
BM is filtered in BMT Lab to remove fat, clots and bone spicules, in a closed bag system
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Bone Marrow Processing in Lab
Common machine used for BM processing is the Cobe 2991 machine
Operates on centrifugal principals
Spin BM for 10mins at 3000rpms
Multiple spins for 1-2 L BM
2-3 hours
Centrifugation into 3 layers; RBC/Buffy coat cells/plasma
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Processing BM on the Cobe 2991
COBE centrifuge bowl
plasma
buffy coat
red cells
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Why Bone Marrow Process?
Debulk BM in preparation for cryopreservation, want to freeze down BM buffy coat cells
Plasma Depletion (or volume reduction) Adult donation for a pediatric patient, large volume of collected cells,
remove some of the plasma to obtain a safe infusion volume of <20 ml/Kg.
RBC DepletionMajor ABO incompatibility of donor graft to patient Starting BM volume for adults is 1-2 LTypically have a starting RBC volume of 300 to 500 mls After RBC depletion on Cobe 2991, have approx 90% removal of RBC,
>75% TNC recovery and >80% CD34 recoveryTarget is to infuse a safe incompatible RBC volume of < 0.5 ml/Kg in graft
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Challenges with the Cobe 2991:
• Machine is reaching its retirement (>25 years old)
• Manual, hands-on
• No walk-away
• Typical 1.5 L marrow will require >3 hrs of processing on Cobe 2991
• Intimidating for new lab staff in training
2018/6/4
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New Marrow Processing:
Spectra Optia: Commonly used for Apheresis collections
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Cobe 2991 Optia
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Processing time on the Optia:
• Optia = 40-75 mins
• Cobe 2991= > 3 hrs for 1.5L marrow
• Bone Marrow MNC and not BM buffy
• Operator adjustments- Optia 1/10 compared to Cobe 2991 10/10
2018/6/4
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Results:Median Cell Parameters
Published ResultsN=30 (28/20 Allogdonors)
Westmead’s ResultsN=82015-current
Volume reduction 93% 91%
MNC recovery 79% 75%
RBC reduction 98.8% 98%
CD34 recovery 88% 89%
CD3 recovery 79% 78%
Neut > 0.5 (days) 20 19.5
PLT > 20 (days) 19.6 25
2018/6/4
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Summary on BM Processing:
•The Spectra Optia can effectively volume reduce and RBC deplete marrow before transplantation
•Fast cell processing
•Small volume for infusion (<100 ml), great volume reduction > 90% reduction
•Produces consistent results with CD34 recovery
•Extreme RBC reduction >96%, great for major ABO incompatible donor marrow grafts
•Safe for adults and pediatrics for BMT
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Apheresis Collection
MNC collected by apheresis machines
Time approx 4 hours
10-12 L blood volume processed for an adult
Average volume of PBSC collected
200 to 250 ml (adult)
40-100 ml (paediatrics)
Target: To collect >2x10^6 CD34/Kg
Request for 100 mls plasma
ACD-A added as anti-coagulant (1:12)
Requires good venous access
Can collect on multiple days to achieve CD34 target if donor is fit
Cobe Spectra Machine
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Cytotherapy 2006
Optimum Transport of Fresh HPC Products is 2 - 8 oC,not at RM temp
Apheresis
Marrow
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How we perform volume reduction for next cell processing steps:
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Testing of stem cell products
forCD34
Apheresis
Marrow
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Why a CD34+ Assay?
CD34+ cells: typically represent 1% of TNC in HPC (range 0.2 – 5%), thus need an accurate and reproducible assay
The number of CD34+ cells infused for transplantation is a clinically important variable for engraftment
Collection Target for infusion:
Min >2 million CD34/patient weight
Aim >5 million CD34/patient weight
Good internal QC for the monitoring of the laboratory practices for collecting and processing of HPC products
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Add 50µl buffer to
tube
Add 10 µl HPC sample (1-3x106 cells) by reverse pipetting
10 mins incubation RT
Vortex to disperse
bead
Lyophilized Bead pellet with known # of beads
Viable CD34 Assay
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Add 450µl
buffer or warm lysing
solution (NH4Cl)
Flow Cytometer
CD34 count-30 mins
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#CD34+ events in R6 X beads per tube*
#beads in R7 HPC vol µL
3101 X 49,9448535 50
369 CD34+ cells / µL or
CD34+cells/Kg
* Number given by manufacturer
Enumerating Viable CD34+ cells
International ISHAGE Gating Strategy for CD34 counts:
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THAWED Apheresis
HARVESTED Apheresis
No Dead cells
Dead cells
No Dead cells
Dead CD34+cells
Accurate Assay to Enumerate viable CD34+ cellsin HPC Products
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CD34 Positive Cell Selection:
CD34+ Cell Selection: The purification and isolation of CD34+ human stem cells from Apheresis
products
Clinical Applications:
TCD of allogeneic grafts, to reduce GVHD
Predominantly used in the pediatric MUD setting
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• Allogeneic transplantation
• Removal of donor T cells to
prevent GvHD
• Goal: Prevention or reduction of
GVHD
• Laboratory positive selection of
CD34+ selection cells
Why CD34 Positive Cell Selection?
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Based on immunomagnetic bead technology
CliniMACS
CD34+
Anti-CD34 Ab + bead
Magnet
(50nm bead)
How does the CD34 Selection device work?
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CD34 Flow Cytometry Results
Pre-Selection Post-Selection
CD34 Pos Bag CD34 Neg Bag
CD34: 2.8x10^6/kg
CD3 : 6300x10^5/kg
CD34: 2.1x10^6/kg
CD3 : 0.2x10^5/kg
Mean CD34 Recovery>75% Mean Purity >90%
HPC, Apheresis
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CD34 + Selection Process: Costs
• $8.5K to $15K
• Requires speciallab staff training
• Not all BMT labsperform thisprocedure
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The CliniMACS Plus system has been used in our laboratory for over 15 years to remove T-cells from stem cell grafts of the unrelated donors for paediatric CHW patients undergoing BMT
Multiple equipment- 2 F/T staff and approx. 5hrs
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New Technology: The CliniMACS Prodigy
CliniMACS®plus CliniMACS®Prodigy
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Fully automated closed system cell processing:
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Results:
CD34 Recovery %
CliniMACS Prodigy0
20
40
60
80
100
6967
n = 66 n = 9
P value 0.4953CD
34 R
eco
very
%
CD34 Purity %
CliniMACS Prodigy0
20
40
60
80
100
8276
n = 66 n = 9
P value 0.1152
CD
34 P
uri
ty %
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Results:
CD3 or T Cell Log Depletion
CliniMACS Prodigy0
2
4
6
8
4.84.4
n = 63 n = 9
P value 0.0117CD
3 L
og
D
ep
leti
on
CD19 or B Cell Log Depletion
CliniMACS Prodigy0
1
2
3
4
5
3.7
3.3
n = 57 n = 9
P value 0.0099CD
19 L
og
Dep
leti
on
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CONCLUSIONS:• The recovery and purity of CD34+ cells was comparable to the
CliniMACS Plus
• The Prodigy offers a single device for CD34 cell selection, unlike the
CliniMACS Plus which requires other equipment
• The Prodigy process is fully automated and this translates into a
reduction in the time required of experienced laboratory staff
• Our results suggest that the Prodigy can be used for the routine
clinical application of CD34 selection to HSCT products
Why we like the Prodigy:
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The process of receiving, testing and preparing
a CB unit for transplantation:
4) CB Infusion
3) Transplant Center
Laboratory 2) Transport 1) Cord Blood Banks
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Communication:• CBB and TC Registry
• Clinical BMT Team
• Transplant Center Lab Staff
• Courier company
Australia >24-28 hrs
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Cryopreserved CB units come in different bags and vary
in the type of processing performed prior to freezing.
Old bag
Not
RBC depleted
before freezing
New bag
Plasma and RBC
depleted before
freezing
1 bag
3 bags
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2000- washed all UCB units
As described by Rubenstein et.al. Processing and Cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc. Natl.Acad.Sci. USA 1995: 92: 10119-10122
Antonenas V et al, BMT 2004 -mean TNC cell loss of 27%, (range 11-41% n=21)
2005- CB units were routinely thawed with no washing and infused immediately
No lab processing, infuse at bedside- as per marrow and apheresis infusions
CB infusions for Adults (single and double)
But noticed serious adverse reactions for some paediatric patients and noticed
‘thickness’ of thawed CB for some infusions
2010- present- CB units are routinely thawed and diluted prior to infusion
As described by Barker et al, BBMT 2009;15(12):1596-602 and
As described by Regan, D et al, BBMT 2006
Washing was ONLY performed for:
CB units that have NOT been volume / red blood cell reduced prior to freezing or
For paediatric patient weights <20 Kg
History of our Lab Practice on CB units prior to Infusion:As of 2013, n=112 CB units for 90 patients
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The Thaw and Wash Method:
Traditional wash method- developed by Rubenstein 1995
CB products were not red cell or plasma reduced,
cryopreserved in large volumes, thus greater amounts
of DMSO for paediatric infusions
Same solution mixture as used for the dilution method
Dextran-40 and Albumex-4 solution (1:1) volumes
Centrifugation is required
Reduces DMSO toxicity and the free haemoglobin
Restores osmolarity & extends cell viability
Decreases CB infusion volume
Infuse within 2 hrs of thawing
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Why wash all the non RBC-depleted units?
NMDP and WMDA NOTICE:
Adult CB infusion resulting in Death (March 2013)
Patient death following a double CB infusion
M/39, CML transplant date March 2013
History of cardiac risk factors
CB unit #1- red cell replete B+ and patient O+ in 105 mls in 2 bags-
infused directly
CB unit #2- red cell replete O+ 75 ml in 1 bag-thawed and infused
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Thaw and Wash Method:
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After Centrifugation of a diluted
RBC-depleted CB unit
After Centrifugation of a diluted
Non RBC-depleted CB unit
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The Challenge of Washing CB units:
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Most labs are unfamiliar with the wash method
Difficult to see the separated layers after centrifugation
Some cell loss is associated
Intimidating wash-tubing sets sometimes provided by
the CBB’s
Unfamiliar with the SOP provided by CBB’s as to how
to wash unit
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How should transplant centres treat CB
units for infusion?
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Best and easy method is to perform the thaw and wash
method on all CB units
We have seen a decrease in reported adverse reactions
MUST Wash all non RBC-depleted CB units
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Acknowledgements
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Scientific and Medical Staff of Sydney Cellular Therapies Laboratory,Westmead HospitalSydney, Australia