BASICS OF HSC COLLECTION AND PROCESSING INCLUDING ALL THE THREE SOURCES, A BRIEF ABOUT STEM CELL MOBILIZATION, STEM CELL SELECTION CRYOPRESERVATION AND DMSO

1. The Collection and Processing of
Hematopoietic
Stem Cells
DR AKSHAYA TOMAR
MD IMMUNOHEMATOLOGY AND BLOOD TRANSFUSION
AFMC,PUNE
6/18/2019

2. INTRODUCTION
• HEMATOPOIETIC STEM CELLS (HSCs) are primitive pluripotent cells
capable of self-renewal and differentiation into any cells of
hematopoietic lineage
• Regardless of tissue source
– Marrow
– Mobilized peripheral blood
– Umbilical cord blood (UCB)
• Utilized to treat a diverse array of hematologic and non hematologic
diseases and conditions.
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3. DISEASES / CONDITIONS TREATED
• Hematological malignancies
– Leukemia
– Lymphoma
– Myeloma
• Marrow failure state/clonal disorders of marrow
– Aplastic anemia
– Myelofibrosis
– Paroxysmal nocturnal hemoglobinuria
– Myelodysplasia
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4. DISEASES / CONDITIONS TREATED
• Inborn errors of metabolism/congenital immunodeficiencies
– Mucopolysaccharidoses
– Severe combined immunodeficiency
– Wiskott aldrich syndrome
– Osteopetrosis
• Pediatric cancers
– Wilms tumor
– Neuroblastoma
– Rhabdomyosarcoma
– Ewings sarcoma
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5. DISEASES / CONDITIONS TREATED
• Hemoglobinopathies
– Thalassemia
– Sickle cell disease
• Autoimmune diseases
– Rheumatoid arthritis
– SLE
• Solid tumors
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6. AUTOLOGOUS vs ALLOGENIC TRANSPLANT
Autologous
• Used for hematopoietic rescue
after antineoplastic therapy
• Antitumor effect –
Chemo/radiotherapy
• Patient must be healthy
enough to undergo
mobilization
• Product collected generally
cryopreserved
Allogenic
• Used in the treatment and it
can offer potential cure
• Antitumor effect – GvT along
with chemo
• It can replace the deficient
cellular machinery
• Histocompatibilty / blood
group matching is mandatory
• Rarely require cryopreservation6/18/2019

7. DONOR SELECTION
• Allogenic stem cell transplant is potentially curative with major
limitation in the form of identifying a suitable donor
• Donors can be
– Related donors / haploidentical donors / Syngenic donors
– Matched unrelated donors (From existing donor registries/Volunteers)
– UCB donors (private/public cord cell banks)
• Things to consider:
– HLA matching ; Age ; Viral markers ; Alloantigen status ; ABO titers etc.
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9. 6/18/2019

10. COLLECTION OF STEM CELLS
• MARROW HSC COLLECTION
– Post donor screening for diseases/infections & HLA matching
– Donor should be physically suitable for donation as it’s a invasive procedure
performed under GA in operating room
– Contraindications for BM HSC collection include – multiple sessions of
chemo/radio; significant tumor burden in marrow etc
– Maximum limit of collection is 20ml/kg with targeted yield of 2 to 3 X 108
nucleated cells/kg (TNC)
– Checking the TNC or CD34 count midway during collection procedure is
required
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11. POINTS TO PONDER
1. 11 to 14G syringe flushed with
anticoagulant
2. Inserted in PSIS
3. Initially aspirate small volume (5ml)
4. Rotate the needle along different vectors
and aspirate repeatedly, multiple sites may
be required
5. Do not aspirate vigorously
6. Collected product to be transferred into
blood bag with ACD (14ml/100ml)
Complications:
- Fatigue/insomnia/dizziness
- Pain at aspiration site
- Anemia (need for transfusion)
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12. COLLECTION OF STEM CELLS
• PERIPHERAL BLOOD HSC COLLECTION
– Most common method now a days, due to advent of newer and better
mobilizing agents
– Done on outpatient basis with minimal side effects
– Peripheral line is preferred/central line is required only during poor peripheral
access as per AABB
– Minimum dose required is 2 X 106/kg however 5 X 106/kg is desirable
– Poor mobilization is one of the limiting factor in collection can be curtailed by
using mixed mobilization regime (G-CSF with chemotherapy or CXCR4
antagonist)
– Complications include : Citrate toxicity, hypotension, allergic reaction, syncope
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13. HEMATOPOIETIC GROWTH FACTORS USED IN STEM
CELL MOBILIZATION
CYTOKINES CHEMOKINES
Granulocyte colony-stimulating factor (G-CSF)
Granulocyte-macrophage colony-stimulating
factor*
Stem cell factor
Interleukin-3
Flt 3 ligand
Erythropoietin*
Interleukin-6
Interleukin-8
Stromal-derived-factor 1
AMD3100 (PLERIXAFOR)
*In combination with G-CSF.
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14. FACTORS AFFECTING MOBILIZATION
• Increasing age
• Increasing cycles and regimens of chemotherapy
• Prior radiation to active marrow sites
• Prior treatment with purine analogues especially fludarabine
• Female gender
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15. PBSC MOBILIZATION
• MOBILIZATION WITH G-CSF
– G-CSF is the standard of cytokine mobilization at a dose of 10-15µg/kg/day s.c.
– CD34+ cells enter circulation on the fourth or fifth day and peak during the 5th/6th day.
– Advantage : scheduling of apheresis is usually easier.
– Side effects: injection site erythema , bone pain, headaches, and fevers. Rarely,
splenic rupture has been reported.
• MOBILIZATION WITH CHEMOTHERAPY
– Depends on underlying disease (should mobilize as well as has activity against it)
– Most common is cyclophosphamide for 10 to 14 days followed by G-CSF
– Can be used only in Autologous harvest
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16. PBSC MOBILIZATION
– Advantage : it can be coordinated as part of the salvage chemotherapy regimen.
– Side effects: risk of myelosuppression including potential need for transfusions,
risks of infections, and bleeding.
In a retrospective analysis the mobilization failure rate (defined as inability to
collect at least 2 ×106 CD34 cells/kg) was equal in the patients mobilized with
G-CSF alone (failure rate, 18%) vs those mobilized with G-CSF plus
chemotherapy (failure rate, 18%).
Highest in NHL (28%) and lowest in MM(8%)
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17. PBSC MOBILIZATION
• MOBILIZATION WITH CHEMOKINES
– Plerixafor (mozobil) is a reversible bicyclam inhibitor of hematopoietic stem
cell binding to SDF-1α on marrow stromal cells via CXCR4.
– Alone or in combination of G-CSF, 240µg/kg single dose s.c. 10 to 12 h prior to
collection
– Advantage : More patients were able to reach the goal of 5×106 CD34 cells/kg
in fewer apheresis days.
– Side effects : injection site erythema, nausea ,vomiting , flatulence, and
diarrhea.
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18. 6/18/2019

19. COLLECTION OF STEM CELLS
• COLLECTION OF CORD BLOOD STEM CELLS
– Collection objective is to maximize the volume of blood harvested from the
placenta while reducing risks of contamination
IN UTERO EX UTERO
•Blood is aspirated from the placental vein
while the placenta is still in utero.
•Avoids the possibility of a failed collection
•Associated with an increase in volume and
reduced incidence of clotted collections.
•Done by trained obstetricians
•Collections after the infant and placenta
are delivered
•Robust cleaning of the umbilical cord and
aspiration of the blood from the placental
vein.
•Handled by trained UCB bank staff in a
dedicated area outside of the delivery room
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20. COLLECTION OF STEM CELLS
• The collection kit includes a large-bore needle attached to a closed
collection bag containing an anticoagulant—usually citrate-
phosphate-dextrose(CPD).
• Blood drains into the bag under gravity and takes only a few minutes
to complete.
• The volume harvested needs to be maximized so collection should
continue until the blood stops flowing
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21. COLLECTION OF STEM CELLS
ADVANTAGES DISADVANTAGES
Availability , no risk to mother or baby Genetic diseases might be present but not
apparent esp preleukemic states
HLA matching minimum 4/6 Storage is costly ; inconclusive storage window
Low incidence of GvHD and strong GvL effect Low yield , only one donation can be obtained
per cord
Less risk of infectious disease transmission Delayed engraftment/increased length of
hospital stay
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22. Neutrophil and Platelet Engraftment Time
STEM CELL SOURCE ANC > 500/µl
(median day)
PLATELET COUNT >20,000/µl
(median day)
MARROW 15 25
PBSC 11 21
UCB 18 27
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23. PROCESSING OF STEM CELLS
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24. METHODS
• Routine methods
– Centrifuge based : plasma reduction, RBC reduction, buffy coat preparation
– Filtration
– Thawing/washing
• Specialized methods
– Elutriation
– Cell selection
– Cell expansion
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25. SPECIALIZED METHODS
• ELUTRIATION
– Counter flow centrifugal force is used
– Separates cell population on the basis of sedimentation coefficient (size &
density)
– Historically , used in T cell depletion
– Presently it is used in enrichment of monocytes during dendritic cell therapy
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26. 6/18/2019

27. SPECIALIZED METHODS
• CELL SELECTION SYSTEM
– Based on immunomagnetic and monoclonal antibodies based technologies
– CLini- MACS (Miltenyi Biotec Bergisch,Gladbach, Germany)
– Positive/negative selection (enrichment/depletion)
– CD34+/CD38- contains higher number of primitive HSC
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28. CELL SELECTION
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29. PRINCIPLE OF Clini MACS (ENRICHMENT)
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STEM CELLS WITH SURFACE
CD34 EXPRESSION
MONOCLONAL Ab (Anti CD34)
linked with MAGNETIC BEAD
MAGNETIC ASSISTED CELL
SORTING OF CD34 + CELLS
POSITIVE SELECTION

30. SPECIALIZED METHODS
• CELL EXPANSION
– CD34 dosage has positive correlation with patient outcome therefore focus has
been shifted to ex-vivo cell expansion
– UCB is the main target for cell expansion trials worldwide
– Particularly important for adults (requires increased dosage of TNCs)
– Since most primitive HSCs are thought to be in resting state, the goals to
achieve rapid expansion of HSCs and retain primitive HSCs stand at odds to
each other
– Stem cell factor/FLT3 ligand/thrombopoietin analogue generally constitute the
cytokines cocktail of cell expansion
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31. EXPANSION VS In-VIVO RECONSTITUTION
• Expanded cells are of no use if they cant engraft and reconstitute
normal hematopoiesis
• Rapid ex vivo expansion can:
– Exhaust the primitive HSC pool
– Increase the chances of genetic mutations
– Delays homing and engraftment
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32. STEM CELLS WITHOUT CRYOPRESERVATION
• The actual freezing/crystallization point of human plasma has been
shown to be at -0.8° Celsius
• Storage around the freezing point is more appropriate in short term
storage for periods of less than 72 hours than the usual deep
freezing temperatures
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33. CRYOPRESERVATION
• Cryopreservation is required as HSCs need to be stored for weeks to
years before transplantation
• Cryoprotectant – DMSO (dimethyl sulphoxide)
• DMSO : Colligative cryoprotectant
– Reduces the osmotic stress on cell membrane
– It slows down extracellular ice formation
• Overall most frequently reported side effects are of gastrointestinal
and cardiovascular nature.
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34. A WORD ABOUT DMSO
• Lovelock and Bishop in 1959, first described DMSO as Cryoprotectant
• It also found medical application in a wide spectrum of musculoskeletal,
autoimmune and metabolic diseases, including gonarthrosis, interstitial
cystitis and amyloidosis
• As a small amphiphatic molecule, DMSO penetrates into stem cells and acts
as a strong hydrogen bond disrupter and hence exerts colligative effects
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35. SIDE EFFECTS OF DMSO
• Pulmonary excretion : Garlic like smell during infusion
• Vagolytic effects : DMSO induced histamine release
• Substantial amount of frequently observed bradycardia and
hypotension is calorically induced
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CVS Respiratory MISC
Hypotension Bronchospasm Anaphylaxis
Bradycardia pulmonary capacity Renal failure
Arrythmias Seizures
Cardiac arrest Acute hepatoxicity
Nausea
Abdominal cramps

36. EFFORTS TO REDUCE DMSO TOXICITY
• Reduction of DMSO concentration in stem cell conc
• Stem cell product with high CD34 cell count
• Prolonged duration of infusion/ divided dose over days
• Depletion /Washing of DMSO during post thaw processing
• Using alternatives to DMSO
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37. CRYOPRESRVATION
• DMSO is tried in various concentration from 5% to 20% , but preferred
concentration is 8% to 10%
• Some laboratories add hydroxyethyl starch (HES), which allows the use of
a decreased concentration of DMSO (e.g., 5% DMSO and 6% HES).
• HES is a non-penetrating (extracellular), macromolecular cryoprotectant.
• HES forms a glassy shell, or membrane, around the cell, retarding the
movement of water out of the cell and into the extracellular ice crystals
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38. CRYOPRESRVATION
• DMSO alternatives
– Trehalose
– Sucrose
• Sucrose/trehalose/DMSO combinations with DMSO concentrations
as low as 2.5% have been shown to be comparable with standard
10% DMSO as cryo-additive.
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39. CRYOPRESERVATION
• The Freezing Process
– Controlled rate freezing(CRF) procedure still remains the defined standard in many
countries
– Principal rationale is the limited cell damage during the freezing process
– Costly, sophisticated technique which require expertise (not available in many
centers)
– Non-controlled freezing on the other hand is cheap,less labour intensive and time
saving (No need of programmable freezing protocol, liquid N2 refrigeration) ; also
known as Dump freezing (at -80 ° C)
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40. CONTROLLED RATE FREEZING ALOGRITHM
Finally the product is placed in liquid N2
Stem cells continued to be cooled @ -1° C/min till -40° C
Increased rate of cooling @ -5° C/min till -140° C
Cooling @ -1° to -2° C till -5° C
Rapid cooling at eutectic point to avoid damage from heat of fusion
Precooling of stem- cells and placement in cryocassettes
At 6° C
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41. CRYOPRESERVATION
• Liquid Nitrogen storage
– Products cryopreserved using a programmable freezer are generally stored in a
liquid nitrogen
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LIQUID PHASE VAPOUR PHASE
•Upto -196° C
•Products are completely
immersed in liquid N2
•Temperature fluctuations are
minimal
•Risk of cross contamination, as
pathogen can survive the liquid
phase
•Upto -156° C
•Temperature fluctuations are
more
•Major advantage is decreased
risk of cross contamination

42. CRYOPRESERVATION
• DURABILITY
– The maximal possible cryopreservation time span is still unknown
– In clonogenic assays , it has been shown that BFU-E and CFU-GM are
compromised early whereas recovery of nucleated cells and CD34 cells
remains relatively well preserved
– As per recent systematic reviews, life of cryopreserved unit is estimated to be
around 7 to 11 years
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43. CRYOPRESERVATION
• STORAGE CONTAINERS
– Properties which needs to be fulfilled by storage containers:
• Closure integrity; sample stability ; easy accessibilty
• Low cost ; environmental strain ; potential for reuse ; economical
– Container should be resistant to significant gravitational forces.
– Ethylene vinyl acetate(EVA) based products are the most commonly used
storage containers
• Cryocyte/Baxter®
• CellFlex/Maco Pharma®
• Pall Medical® Freezing Bag 791-05
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44. THAWING AND POST THAW PROCESSING
• Thawing should be done at the bedside just prior to infusion
• Product thawed to a slushy state and handed over (Caloric induced
hypothermia and bradycardia is common)
• Washing : to remove cryoprotectant (DMSO)
– Has negative impact on engraftment kinetics
– Washing solution consist of acd-saline mixture.
– Thawed product mixed with washing solution & centrifuged at 850g for 6
minutes at 22°c
– Supernatant discarded ; with resultant volume, maximum 150ml
– Holds a higher relevance for UCB products
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45. THAWING AND WASHING FOR UCB
• Thaw-and-wash method
• The thaw-and-dilution technique
• The bedside thaw method
Despite the potential advantage of the bedside product preparation method in
minimizing potential cell loss from post thaw manipulation,this approach is not
recommended (as it lacks the capacity of process control and product assessment)
Dilution solution : 10% Dextran + Albumin (final conc – 4.2%)
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46. VOLUME REDUCTION
• The usefulness of volume reduction using an automated method has
advantages compared with processing performed through centrifugation
and manual plasma reduction.(done for minor ABO incompatibility)
• The “PBSC—volume reduction” Sepax S100 protocol is designed for
volume reduction by plasma depletion of PBSC units obtained by HPC-A.
• Allows for the processing of samples from 80 to 600 mL using multiple
cycles
• The fractions must contain a CD34+ cells quantity able to guarantee the
graft dose (>2 X 106/kg patient body weight).
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47. SEPAX S -100
• Manufacturer – Biosafe
• It uses a rotating syringe technology that allows separation of blood
components through rotation of the syringe chamber
• Blood components are detected by an optical sensor
• Volume of syringe sample – 220ml (multiple cycles are required if
BM processing is done), better for PBSCs , time taken is 15min/220ml
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48. RBC DEPLETION
• Done in cases of major ABO incompatible Allogenic stem cell transplants
• Strategies adopted:
– Gravity sedimentation using HES/Dextran
– Buffy coat separation using apheresis/cell separators
– Separation on a density gradient – Counterflow centrifugal elutriation
– Automated Sepax system can also be used to remove RBC contamination
6/18/2019
These two can be combined

49. INFUSION OF STEM CELL PRODUCT
• @ 10-15 ml/min
• If total volume to be infused is>10ml/kg , divide the product
concentrate into aliquots of equal volume
• To be transfused in 2 sessions or over 2 days
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50. 6/18/2019
Mobilization / gradient
separation/CD34 selection
Reduction of cell bulk, with centrifugation and red cell depletion
Addition of diluent and cryopreservative soution
Freezing of stem cells
Thawing at bed side at 37 C
Post thaw processing (red cell depletion; DMSO removal)
Infusion into recipient
Bone
marrow
PBSC UCB

51. REFERENCES
• AABB Technical Manual of Transfusion Medicine 18th edition
• Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians
2009
• Rossi’s principles of transfusion medicine 5th edition
• Umbilical cord blood transplantation – how, when and for whom?
Blood 2011
• Optimization of immunomagnetic separation for HSC ; BMC Cell
Biology 2008
• Ex vivo expansion of HSCs: Recent Advances ; WJH 2015
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52. THANK YOU
6/18/2019