Human Stem Cells- Introduction Types of HSC transplants Indications Sources of stem cells Collection and mobilization Types of Mobilizing agents Processing Cryopreservation and storage PBSC Transplant Quality control Complications

1. A seminar on-
Peripheral Blood Stem
Cell Transplantation
Presented By- Dr. Shiny
Moderator- Dr. Yadwinder Kaur Virk

2. TOPICS
 Human Stem Cells- Introduction
 Types of HSC transplants
 Indications
 Sources of stem cells
 Collection and mobilization
 Types of Mobilizing agents
 Processing
 Cryopreservation and storage
 PBSC Transplant
 Quality control
 Complications

3. INTRODUCTION
Hematopoietic stem cells (HSC)
 Primitive pluripotent cells, multipotent in adult stem cells (restricted
differentiation capacity depending upon location)
 Capable of self-renewal
 Differentiation into any cells of haematopoietic lineage
(lymphocytes, monocytes, granulocytes, erythrocytes, and platelets).

4.  Clinically, HSCs can fully reconstitute the functions of
marrow when transplanted into susceptible recipients.
 Stem cells are forming the core of a new field called
“Regenerative Medicine”.
 HSC transplantation has been increasingly utilized to treat
different hematologic and non-hematologic conditions.

5. PROPERTIES OF STEM CELLS
 SELF MAINTENANCE- Ability to maintain their own population at constant level
 ASYMMETRIC REPLICATION- With each stem cell replication, some daughter
cells differentiate into other mature cells while remaining cells form stem cells
 STEM CELL IDENTITY- At present, CD34 is widely used to identify HSCs

6. HAEMOTOPOIETIC GROWTH FACTORS
 SCFs (Stem Cell Factors)- responsible for proliferation and self renewal of these cells.
 Glycoprotein Growth Factors- for proliferation and maturation of cells that enter blood from
marrow.
 Colony Stimulating Factors (CSFs)
 Others- Erythropoietin, Thrombopoietin
Granulocyte-macrophage CSF (GM-CSF)
Macrophage- CSF (M-CSF)
Granulocyte- CSF (G-CSF)

7. Haematopoietic Stem Cell (HSC) Transplantation
 Defined as ability to achieve long term reconstitution of both myeloid and lymphoid
lineages.
TYPES
ALLOGENIC
• HSCs from donor having
different genetic constitution
• For defective marrow function or
when tumour cells are extensively
infiltrating bone marrow.
• Also requires graft vs host disease
prophylaxis.
SYNGENEIC
HSCs from
identical twin
AUTOLOGOUS
HSCs from patient’s own BM or
peripheral blood, later infused
back after intensive chemo or
radiotherapy.
• Performed when bone marrow
function is normal.
• Eg.- Multiple Myeloma

8. INDICATIONS FOR STEM CELL TRANSPLANTATION

9. SOURCES OF STEM CELLS
MOST COMMON OTHER UNCOMMON
Bone Marrow Wharton’s Jelly
Peripheral Blood Liposuction Waste
Umbilical Cord Blood Mesenchymal Cell Structures
etc.

11. STANDARD PROTOCOL FOR HSC
TRANSPLANTATION
DONOR EVALUATION BLOOD GROUPAND
INFECTION TESTING
STEM CELL
HARVESTING
1. Marrow assay- to see
sufficient mobilization
of stem cells
2. Detailed history,
thorough physical
examination
3. HLA compatibility
between donor and
recipient
ABO-Rh, Hep B, Hep C,
Syphilis, TORCH etc. within
30 days of scheduled
collection of stem cell
product.
Collection
Processing
Storage

12. STEP 1-
COLLECTION

13. Collection of peripheral blood stem cells
(PBSC)
 PBSCs- used in most autologous and allogeneic stem cell transplantation.
 Mobilization of stem cells into the peripheral blood- defined as the increased
release of immature and mature haematopoietic cells from the marrow into the blood
circulation.
 Initially, stem cell mobilization was achieved by- chemotherapy alone.
 Presently-
Recombinant growth factors such as granulocyte-colony stimulating factors (G-
CSF)- considered the standard mobilizing agent.

14.  First, Hematopoietic progenitor cells are measured using the CD34+ cell
surface marker
 A dose of G-CSF, 5-15 µg/kg for a period of 5 days, is usually sufficient
to increase HSCs in peripheral circulation.
 A Dose of G-CSF alone- From a baseline concentration of <5x106/L, blood
CD34+ cell counts increase 10 to 30 folds at 96-144 hours after first daily
dose of G-CSF or single injection of pegfilgrastim; peak comes at 120
hours.
COLLECTION- Mobilization

15. COLLECTION- Mobilization
 PREFERRED MOBILIZATION STRATEGY for autologous
transplant in oncology patients-
 Most reliable Chemomobilization drug- Cyclophosphamide
 Advantage- enhances more progenitors to be collected with fewer
apheresis procedures
Growth Factor Administration + Chemotherapy
(Chemomobilization)

16.  Mobilization is followed by collection using the apheresis method
 Poor mobilization of PBSCs might occur due to old age, female
gender, prior radiation to active marrow sites, prior treatment with
purine analogues (especially fludarabine), or due to increasing cycles
and regimens of chemotherapy.
 Mobilizing agents can be divided into- chemotherapy, cytokines,
or chemokines

17. A. CHEMOTHERAPY
 used as a mobilization agent only in autologous transplant settings
 Typical chemotherapy agents used for mobilization include:
• Single-agent cyclophosphamide, especially in patients with Multiple myeloma
• Combination chemotherapy regimens like ifosfamide,carboplatin,etoposide (ICE)
 Advantage: It can be coordinated as a part of a salvage chemotherapy regimen
 Disadvantage: The timing of apheresis is not as predictable as it is with G-CSF alone
 Side effects:
• Side effects of individual chemotherapies
• Risk of myelosuppression, risk of infections

18. B. CYTOKINES
The standard agent for cytokine mobilization -> G-CSF
 Has been shown to mobilize more CD34+cells with less toxicity than other GFs.
 A standard dose of G-CSF is 10-15μg/kg/day given subcutaneously
 Advantage: Predictability of apheresis scheduling is easier as CD34+ cells peak by
day 4 or 5 of G-CSF mobilization
 Disadvantage: Sometimes, G-CSF mobilization fails to provide an adequate
collection and is termed as poor mobilization
 Side effects: Injection site erythema, Bone pain, Headache, Fever, Splenic rupture

19. C. CHEMOKINES
 Chemokines: Plerixafor- reversible bicyclam inhibitor of haematopoietic stem cell
binding to stromal cell-derived factor-1 alfa (SDF-1) on marrow stromal cells
 Used alone or in combination with G-CSF
 On the evening of day 4 of G-CSF dose, plerixafor (240 μg/kg given subcutaneously) is
administered approximately 10-11 hrs before apheresis, followed by apheresis on day 5.
 Non-responders to G-CSF often respond to a dose of plerixafor administered on the
previous night of collection.
 Side effects: Injection site erythema, Vomiting, Flatulence, Diarrhoea

20. Collection of PBSCs by apheresis
 Commonly used machines- Optia Spectra, Cobe Spectra, CS 3000 Plus, etc.
 PBSC collection can be either started when peripheral leukocyte counts rise to ≥ 1.0 x
109/μl or when peripheral blood CD34 level is above the centre’s cut-off (typically 5-
20 CD34+ cells/ μL).
 Because PBSCs segregate in the mononuclear cell fraction of blood, apheresis
devices capable of mononuclear cell concentration and harvest may be used to collect
these cells.
 Processing 2-3 blood volume (10-15 L) per procedure requires approximately 3-5
hours.

23. Adverse effects of PBSC Collection
 Procedures for mobilization and collection of PBSCs from patients and
normal donors are well-tolerated by most of them.
 Common adverse effects of mobilization with growth factors include bone
pain, myalgia, headache, and fatigue.
 Subside on their own or on mild medication and subside completely when
growth factors are stopped.
 Citrate toxicity in apheresis procedure
 Sometimes complications can result from peripheral/central line placement
(including hematoma, thrombosis, infection, etc.).

24. Appropriate Time and Target of collection
1. When Leukocyte count exceeds 5x109/L. (However Leukocyte
concentration does not always correlate with no. of HSCs in peripheral
blood)
2. Analysis of CD34+ cells by Flow cytometry (Standard- when CD34+
cells > 10 cells/µL)
3. Collection Targets- Minimum threshold of CD34+ cells necessary
for engraftment is 2-5x106 cells/kg body weight of patient.
4. In terms of MNC count- 4-6 x 108 MNCs/kg

25. STEP 2-
Processing

26. VARIOUS METHODS FOR PROCESSING
ROUTINE METHODS SPECIALIZED METHODS
1. Volume Reduction- to prevent fluid overload in
patients
2. Red Cell Reduction- to prevent haemolytic
transfusion reactions
3. Buffy Coat preparation
4. Thawing- in a 37°C water bath
5. Washing- removes lysed red cells, haemoglobin,
and cryoprotectant (DMSO)
6. Filtration- to remove bone spicules, aggregates,
and debris
1. Elutriation- separates cell populations based on
two physical characteristics— size and density
(sedimentation coefficient)
2. Cell Selection Systems- the isolation of the cell
type of interest by either positive
selection(target cells retained) or negative
selection (target cells depleted) using target
antibodies as magnet
3. Cell expansion- includes stem cell factor, FMS-
like tyrosine kinase 3 (FLT-3) ligand, and
thrombopoietin,

27. CELL PROCESSING METHODS for PBSCs
Fluorescent- Activated
Cell Sorting
Immunoadsorption Systems Physical Parameter
separation
-Combines flow
cytometry with physical
separation to segregate
individual cells based on
the expression of
molecules.
-Not used on large scale
1. Positive Selection- CD34+ cells
magnetically retained, unwanted
cells removed. (passive
depletion)
2. Negative selection- Tumor cells
magnetically retained, desired
cells released and collected.
(active depletion)
Magnetic bead- Anti CD34 antibody
Machines- Isolex 300i system
(magnetic cell separator),
CliniMACS
1. By centrifugation on the
basis of density of
progenitor cells.
2. By increasing density of
unwanted cells and
removing them.

28. PERIPHERAL BLOOD PROCESSING
 2 protocols to process PBSC Products-
PBSC Volume Reduction Peripheral Blood Stem Cell
washing
Reducing volume of PBSCs after
leucopheresis to optimize cell
concentration and reduce DMSO
toxicity
Automated safe removal of DMSO
and haemolyzed plasma from thawed
leucopheresis products.

29. STEP 3
Cryopreservation and storage

30. CRYOPRESERVATION AND STORAGE
 The product is collected in Liquid state.
 Can be stored as liquid for 3 days at 4 degrees C without any significant loss of
viability; then cryopreserved until infusion
 No expiry date defined till now.
 Cryoprotectant used- DMSO (Allows controlled freezing and thawing of
mononuclear cells without development of membrane lysis).
 Preparation of cryoprotectant solution-
 hydroxyethyl starch (HES)- 60 ml
 DMSO(Dimethyl sulfoxide)- 15 ml
 Albumin- 25 ml
For every 100 mL of
cryoprotectant solution

32. METHODS OF FREEZING
CONTROLLED-RATE
FREEZING
NON- CONTROLLED RATE
FREEZING
A controlled rate freezer used,
whose temperature is slowly
decreased by 1 degree/minute
till product reached a specific
temperature.
Products are stored at -80 degrees
C mechanical freezer after
mixing with cryoprotectant.
These products have been
engrafted for as long as 2 years of
storage
LIQUID NITROGEN
STORAGE
In liquid nitrogen freezer.
• Liquid state maintains
at -180 degrees C or
lower
• Vapor state maintains
at -140 degrees or lower

33. STEP 4
PBSC TRANSPLANT

34. Thawing and Infusion
 Flow through Central Venous catheter; cells are infused by gravity drip,
calibrated pump or manual push with/without in-line filter.
 Thaw one bag at a time to minimize thawed cells exposure to DMSO ex vivo
 Or, immerse entire bag except access ports, into sterile water or saline at 37-
40 degrees C.
 Infusion- at the rate of 10-15 ml/minute.
 Infusion volume- not more than 10 mL/kg body weight of patient per
infusion

35. Transfusion support in PBSC transplant
 ABO incompatible transplants require more transfusions because of delayed
cellular engraftment and red cell aplasia.
 Plasma reduction may be performed in cases of minor ABO incompatibility
 White red cell depletion may be performed in cases of Major ABO
incompatibility
 For 2-way incompatibility- Group O RBC units and Group AB plasma
should be used
 Leucoreduction and irradiation- widely done to avoid HLA
alloimmunization or transfusion associated GVHD.

37. QUALITY CONTROL AND EVALUATION
1. CELL COUNTS- Total MNC concentration
2. BACTERIAL AND FUNGAL CULTURES- usually just before
freezing in autologous transplants and before infusion in allogenic
transplants.
3. CD34 ANALYSIS- Yield analysis using flow cytometry
4. CELL VIABILITY ASSAY- Acceptable standard of >70%
viability of cells
5. COLONY FORMING CELLASSAY- to demonstrate in-vitro
proliferative capacity of haematopoietic cell sample.

38. MAJOR COMPLICATIONS
 Hemolytic transfusion reactions
 Graft versus Host disease- acute/chronic
 Transfusion associated Dyspnea (TAD)
 Septicemia
 Veno-occlusive disease (VOD)

39. ACUTE GVHD CHRONIC GVHD
• Usually diagnosed before day 100
• Typically occurs near time of engraftment
• Almost always involves skin
• Also can involve intestine, liver, or lung
• Substantial cause of morbidity and
mortality
• SIGNS/SYMPTOMS- Palmar Erythema,
Puritic Maculopapular Rash, etc.
Diagnosed after day 100
Includes-
• Immunodeficiency
• Skin- lichen planus, poikiloderma
• Vitiligo, hyperpigmentation
• Scleroderma
• Limited joint mobility
• Sicca syndrome
• Mouth ulceration/food sensitivity
• Hepatic- vanishing bile ducts
• Pulmonary- bronchiolitis obliterans
• GI- esophageal strictures, fat
malabsorption

41. Quick Summary
 Transplantation- Allogenic, Autologous
 Sources- Bone marrow, Peripheral Blood, Umbilical cord blood
 Mobilization- Chemotherapy, Cytokines(G-CSF-5-10ug/kg), Chemokines
 Processing- Routine(volume reduction,red cell
reduction,thawing,washing,filtration)
- Special methods(Positive & Negative depletion, Elutriation)
 Cryostorage- controlled, uncontrolled, liquid nitrogen
 Infusion- 10-15 mL/min, not more than 10 mL/kg body weight per infusion

42. REFERENCES
 Principles and Practice of Transfusion Medicine, Dr. R. N. Makroo, 2nd edition.
 DGHS, Transfusion medicine, technical manual 3rd edition 2022.
 Denise. Harmening, Modern Blood Banking and transfusion practices, 7th
edition.