procedure of bone marrow stem cell harvesting for bone marrow transplant by apheresis. mobilization of stem cell from bone marrow to peripheral blood. GCSF mobilization. Apheresis principle and procedure. stem cell from bone marrow collection. effect of stem cell collection form different sources. newer drugs for stem cell harvesting. adequate dose of stem cell to be collected. minimal invasive procedure.

1. Hematopoietic Stem Cell
Mobilization and Harvesting
Speaker- Dr. Sudip Roy
Moderator- Dr. Sumit Mitra

2. Overview
• Introduction
• Brief history
• Stem cell sources
• Mobilization
• Harvesting
• Comparison of stem cells from different sources
• Take home message

3. Introduction
• Hematopoietic stem cell transplantation is often life-saving treatment for
many hematological and solid malignancies
• Helps in reconstitution of blood cells following high dose chemotherapy
• Use of GCSF -mobilized peripheral blood stem cells has largely replaced bone
marrow (BM) as a source of stem cells
• For both autologous and allogeneic cell transplantation
Copelan EA. Hematopoietic stem-cell transplantation. New England Journal of Medicine.
2006 Apr 27;354(17):1813-26.

4. • Stem cell infusion with minimum number of 2 × 106 CD34+ cells/kg ensure
successful multi-lineage engraftment and sustained hematopoietic recovery
• For autologous stem cell transplantation (ASCT), CD34+ cell dose >5 × 106/kg
leads to rapid and sustained recovery
• On the other hand, <2 × 106 CD34+ cells/kg often leads to failure in
multilineage engraftment, especially of platelets
• For allogeneic stem cell transplants (AlloSCT), CD34+ cell dose 4.2 – 4.5×106/kg
is associated with improved overall survival in the matched unrelated donor
setting
• Higher doses (>8-14 × 106 cells/kg) is associated with increased risk of GVHD
Copelan EA. Hematopoietic stem-cell transplantation. New England Journal of Medicine.
2006 Apr 27;354(17):1813-26.

5. History of Stem Cell Transplant
19 years old school girl, case of Aplastic
Anemia, in October, 1937 received 1st
intravenous autologous marrow aspirate
Initially Hb was improved but gradually
declined and died of infections

6. E. Donnall Thomas
• At the Fred Hutchinson Cancer Research Center in b/w 1950s and 1970s
• Bone-marrow cells infused intravenously could repopulate the bone marrow
and produce new blood cells
• Discovered genetic markers to confirm donor matches
• Awarded with with a Nobel Prize in Physiology in 1990

7. John Kersey
• University of Minnesota in 1975
• Performed the first successful bone-marrow
transplant to cure Burkitt’s lymphoma,
• 16-year-old-boy
• The longest-living transplant survivor till now

8. George Judson
• Invented the apheresis technology
• Was an IBM engineer
• Worked for NCI to remove the cancer cells from his AML affected son’s blood
• IBM 2990- could harvest WBCs from PB in continuous flow manner
• Processed 2-3 times of patient's blood volume in 2-3 hours
• Blasts were removed from the blood and neutrophils were separated from
CML patients to reduce infection

9. Curt Civin
• Developed the CD34 monoclonal antibody
• Identify, isolate, and collect hematopoietic
stem cells, the immature cells
Donald Metcalf
• Identified the hormones that regulate the
cells' proliferation and differentiation
• G-CSF in 1960

10. Forecast from US FDA for HSCT across the globe, published in 2022

11. Incidence of Stem Cell Transplants by cell sources from NMDP

12. Choice of HSC Product
• All patients undergoing AutoHSCT have PBSCs as the source of HSC
• Ease of collection,
• Greater quantities of HSCs
• Potentially lower risks for tumor cell contamination of the graft
• Allogeneic transplant- wider range of sources
• Bone marrow, PBSCs, or umbilical cord blood (UCB) products
• HLA-compatible or partially compatible, related or unrelated donors
Horowitz MM, Confer DL. Evaluation of hematopoietic stem cell donors. Hematology Am Soc Hematol Educ
Program. 2005:469–475

13. Comparisons of Different Sources of HSC
• PBSC products v/s BM HSC
• Greatest quantity of HSCs and result in faster hematologic recovery and
survival advantages compared with marrow or UCB
• But higher risk for difficult to-control chronic GVHD
• PBSC may not be appropriate for non-malignant disease
• Cord Blood HSCs
• Slower hematologic recovery
• Greater risk for post-transplant infection because of the relative immature
immune system of the donor
Sacchi N, Costeas P, Hartwell L, et al. Haematopoietic stem cell donor registries: World Marrow Donor Association
recommendations for evaluation of donor health. Bone Marrow Transplant. 2008;42(1):9–14

14. Meisel Ret al. Similar Survival Following HLA-Identical Sibling Transplantation for Standard Indication in Children with Haematologic Malignancies: A Single Centre Comparison of Mobilized
Peripheral Blood Stem Cell with Bone Marrow Transplantation. Klinische Padiatrie. 2015 Jan 1;217(3):135–41.
UCB
BMSC
Takaaki Konuma et al. Single Cord Blood Transplantation Versus Unmanipulated Haploidentical Transplantation for Adults with Acute Myeloid Leukemia in Complete Remission.
Transplantation and Cellular Therapy. 2021 Apr 1;27(4):334.e1–11.

15. Peripheral blood stem cell
transplantation
• Presence of PBSC identified in 1951
• But the number of stem cell in peripheral blood is very low (0.01- 0.05%)
• So large quantity of PB had to be processed than BM as a source
• 1st performed in 1986
• Almost replaced BM a stem cell source
• 100% autologous and 75% allogenic
Horowitz MM, Confer DL. Evaluation of hematopoietic stem cell donors. Hematology Am Soc Hematol Educ
Program. 2005:469–475

16. • Advantages of PBSC
• Ease of collection
• Greater quantity of HSCs- faster recovery
• Lower risk of tumour cell contamination
• Robust graft-vs- leukaemia effect
• Complication
• Graft versus host disease-
• PBSC may not be useful for benign diseases where GVHD is not
beneficial
Dercksen MW, Rodenhuis S, Dirkson MK, et al. Subsets of CD34+ cells and rapid hematopoietic recovery after
peripheral-blood stem-cell transplantation. J Clin Oncol. 1995;13(8):1922–1932

17. Development of clinical apheresis technology
• Main concerns were-
• “victims of clonal senescence, expelled as waste product from BM”
• In 1960s, 1st attempt to clinical application of apheresis as attempted in MD
Anderson Centre
• This was capable to process 2-3 times of whole body blood volume in 2-3 hours
• Allowed to harvest small population of cells by precession large amount of blood

18. First successful autologous PBSCTs
• in 1981, at Hammersmith Hospital in London
• Patient with accelerated phase CML
• With the apheresis and cryopreservation technologies
• SCTs were preserved during chronic phase of disease
• Intended to achieve second chronic phase
• In the same year, at Johns Hopkins
• PBSC collected in CML-CP after cytogenetic remission
• Myeloablative therapy and PBSC infusion
• First evidence of fast and complete hematopoietic reconstitution

19. Strategies to increase PBSC concentration
• Main challenge in 1980s for PBSCT was inadequate stem cell dose collection
• In the late 1980s, non-myelotoxic chemotherapy and short-term myelo-
suppression were popular
• Rebound of PB stem-cell concentration increases
• Stem cell mobilization- the release of HSCs from bone marrow into peripheral
blood following stimulation
• No increase in stem cell number, they come out from their “home”
De Arriba F, Lozano ML, Ortuño F, et al. Prospective randomized study comparing the efficacy of bioequivalent doses of
glycosylated and nonglycosylated rG-CSF for mobilizing peripheral blood progenitor cells. Br J Haematol. 1997;96(2): 418–420

20. Bone Marrow Niche
• The BM niche is a highly organized microenvironment which anchors HSCs
and regulates their self-renewal, proliferation and trafficking
• Subdivided into vascular and endosteal compartments
• Vascular niche contains sinusoidal endothelial cells and nestin+ mesenchymal
stem cells
• Endosteal niche is located closer to the trabecular or cortical bone and is
composed of osteoblasts and osteoclasts
Kandarakov, O.; Belyavsky, A.; Semenova, E. Bone Marrow Niches of Hematopoietic Stem and Progenitor Cells. Int.
J. Mol. Sci. 2022, 23, 4462.

22. Mobilizing agents
• Therapeutic molecules those help to release HSCs from bone marrow into
peripheral blood
• These are classified into 3 categories-
• Cytokines
• works in days
• Chemokines
• Works in hours
• Myelosuppressive agents
• Lunger duration
Schmitz N, Beksac M, Hasenclever D, et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk
leukemia. Blood. 2002;100(3):761–767

23. Cytokines
• Small proteins that are crucial for the growth and activity of immune system
and blood cells
• Currently approved
• G-CSF
• Peg- G CSF
• GM- CSF
• Other experimental targets
• Stem Cell Ligand (SCF) [KIT Ligand]
• FLT3 ligand
• Thrombopoietin
• interleukins
Nervi B, Link DC, DiPersio JF. Cytokines and hematopoietic stem cell
mobilization. J Cell Biochem. 2006 Oct 15;99(3):690-705

24. Hematopoietic growth factors
• G-CSF developed for limiting the side effects of chemotherapy-induced
cytopenia
• It was noticed that G-CSF also expand the circulating hematopoietic stem cells
by mobilizing CD34+38− hematopoietic progenitor cells
• In 1988, it was established that colony stimulating factor increases PBSC 60-
100 times ( Dana-Farber and Royal Melbourne Hospital)
• By the mid 1990s, chemo-mobilization and/or G-CSF combination approved
• PB- SCT collection increased to optimum dose
• Autologous PBSCT became increasingly accepted and replaced AutoBMT

25. • But till long period, healthy donors were excluded from G-CSF mobilization
• Concerns about triggering lymphohematopoietic malignancies in
genetically predisposed healthy sibling donors
• 5 years after the introduction of G-CSF as a stem-cell mobilization agent
• Data on healthy donors were published by researchers at Sapporo Medical
College in Japan in 1992
• Initial guideline formulated- biannually checkup and once a 3 years BM
examination of donor
• In 1999, NMDP and EBMT approved G-CSF for healthy unrelated donor

26. In 2008 conference of NMDP concluded
1.Severe but rare side effects of G-CSF mobilization treatment, including
splenic rupture and sickle cell crisis, have been reported.
2.Overall rate of 0.6% serious adverse events attributed to stem-cell
mobilization treatment and collection.
3.No definite conclusions can be made regarding the long-term effects of
G-CSF treatment in healthy donors.
McCullough J, Kahn J, Adamson J, et al. Hematopoietic growth factors—use in normal blood and stem cell
donors: clinical and ethical issues., Transfusion, 2008, vol. 48 9(pg. 2008-2025)

27. Granulocyte Colony-Stimulating Factor
• G-CSF has 2 effects on mobilization
• Proteolysis
• Increased PMN differentiation
• Increase PMN protease release
• Elastase
• Cathepsin G
• Matrix Metalloprotease 9
• Leads to VCAM 1, SDF1, CXCR4 and C-KIT proteolysis
• Release of HSC in peripheral blood
• Decreased SDF1 mRNA expression and Osteoblastic activity

28. Effect of G-CSF in HSC Niche- Proteolysis and decreased mRNA expression

29. Effect of G-CSF upon different stem cells
• G-CSF enhances expansion of lymphoid (Ly)-biased, short term HSC
• Maintain their repopulating potential.
• Does not have a direct effect on myeloid (My)-biased, long-term HSC
• Increase myelopoiesis and impair lymphopoiesis
Chen Y. Granulocyte colony-stimulating factor acts on lymphoid-biased, short-term hematopoietic stem cells. Haematologica. 2020 Jun 5;106(6):1516–8.

30. Dose of G-CSF
• Dose of G-CSF is 5- 32 µg/kg/day
• Standard dose is 10 µg/kg/day
• Mobilization starts after 3 days
• Peak at day 4-6
• Then PBSC declines despite repeated doses
• G-CSF is discontinued when WBC > 1,00,000/ dl

31. Side effect of G-CSF
• Bone pain (76%)
• Headache and flu-like symptoms like malaise, nausea, subfebrile body
temperature and night sweats (67%)
• Muscle pain (67%)
• Insomnia (24%)
• Anorexia and vomiting (43%)
Hölig K. G-CSF in Healthy Allogeneic Stem Cell Donors. Transfus Med Hemother. 2013 Aug;40(4):225-35

32. Post G-CSF biochemical changes
• Increased Alkaline Phosphatase
• Osteocalcin level decreases
• Urinary free deoxypyridinoline
level increased
Decreased osteoblastic activity and
increased osteoclastic activity
Bone pain
• Increases LDH
• Increase Na+/ Decrease K+

33. Rare Complications
• Anaphylaxis
• Vasculitis
• Arterial thrombosis
• stroke
• Immune Cytopenia
• Splenic rupture
• Leukaemia and Myelodysplasia (Debatable)
https://www.cancerresearchuk.org/

34. Incidence of AML among Healthy PBSC Donors Mobilized with G-CSF
Transplant physicians must explain the potential risk of MDS/AML to all donors
Can G-CSF Cause Leukemia in Hematopoietic Stem Cell Donors? Biology of Blood and Marrow Transplantation. 2011 Dec 1;17(12):1739–46.

35. Splenic Enlargement
• Seen in 95% donor
• Mean length increase by 13%
• Size returns to normal within 10 days of last GCSF dose
• Contact sports should be avoided during this period
• Life threatening rupture may occur

36. Peg- G CSF
• Poly-ethylene glycol conjugated with recombinant G-CSF
• PEG reduces sensitivity to proteases- increase self life
• Longer t ½ (80 hrs v/s 3.5 hr)
• Only one subcutaneous injection is needed
• Action and side effect similar to conventional G-CSF
• More expensive but daily injection not needed

37. GM-CSF
• Sargramostim
• Very limited role in mobilization
• Mobilize significantly fewer CD34+ cells than G-CSF
• But higher number of CD14+ monocytes, dendritic cells and CD4+ CD25+
regulatory T cells
• lower rates of acute GVHD

38. Chemokines
• Family of small signaling proteins- induce directional movement of leukocytes
• Receptors are named
CXCR1 through CXCR6
• CXCR4 (fusin) is the receptor for CXCL12 (or SDF-1)
• Utilized by HIV-1 to entry into target cells
• Expressed on most immature and mature hematopoietic cell

39. Plerixafor
• Reversibly inhibits SDF-1 binding to CXCR4
• Water soluble , small molecules (52 Dalton)
• Initially used to block the entry of HIV virus in T Cell
• Failed to block mutated HIV
• Observed very high level of PBSC and rapid transient leucocytosis

40. • Dose is 20 mg fixed or 0.24 mg/kg of body weight for patients weighing
≤83 kg
• Peak plasma concentrate within 30- 60 min
• 50% remains plasma protein bound
• Not metabolized in liver, major route of excretion is kidney
• Combination of G-CSF 10 μg/kg daily and plerixafor 0.24 mg/kg subcutaneously,
10-11 hours prior to apheresis
• Up to 4 consecutive days before apheresis

41. DiPersio JF et all, Phase III prospective randomized double-blind placebo-controlled trial of plerixafor plus
granulocyte colony-stimulating factor compared with placebo plus granulocyte colony-stimulating factor for
autologous stem-cell mobilization and transplantation for patients with non-Hodgkin's lymphoma. J Clin Oncol.
59% v/s 27%

42. Stem cells anchored in the bone
marrow through the CXCR4/SDF-1α
interaction
Mozobil disrupts the CXCR4/SDF-1α interaction
Stem cells moving into the bloodstream

43. Side effect of Plerixafor
• Injection site erythema (67%)
• Nausea (53%)
• Headache (32%)
• Peri-oral paraesthesia (12%)
• Abdominal distention and flatulence (58%)
https://www.mayoclinic.org/drugs-supplements/plerixafor-subcutaneous-route/side-effects

44. Stem Cell Factor
• Hematopoietic growth factor also known as c-kit ligand
• Produced by endothelial cells and perivascular stromal cells
• Differential splicing of SCF leads to the soluble and membrane-bound forms of
the protein
• Membrane-bound form binds with to c-kit and important for HSC maintenance
• Activates multiple downstream signals, including VLA-4-mediated BM HSC
adhesion Lapierre V et al. Ancestim (r-metHuSCF) plus filgrastim and/or chemotherapy for mobilization of blood progenitors in 513
poorly mobilizing cancer patients: the French compassionate experience. Bone Marrow Transplant. 2011; 46:936–42

45. Schematic representation of SCF-induced c-Kit activation
Lennartsson J, Rönnstrand L. Stem Cell Factor Receptor/c-Kit: From Basic Science to Clinical
Implications. Physiological Reviews. 2012 Oct;92(4):1619–49.

46. • C-Kit receptor can be proteolytically cleaved from the surface of hematopoietic
cells and circulate as s-Kit in human plasma
• This circulating s-Kit binds to SCF and blocks c-Kit/SCF interaction
• Can be used for stem cell mobilization
• Recent study
• 550 patients of Myeloma, NHL and HL with failed prior mobilization with G-
CSF or a combination of G-CSF and chemotherapy
• Recombinant SCF and G-CSF leads reach target of >2 × 106 CD34+ cells/kg in
31% of patients
Lapierre V et al. Ancestim (r-metHuSCF) plus filgrastim and/or chemotherapy for mobilization of blood progenitors in 513 poorly
mobilizing cancer patients: the French compassionate experience. Bone Marrow Transplant. 2011; 46:936–42

47. Alternative target for SDF-1/CXCR4 axis
• POL6326 (Polyphor, Switzerland)
• Synthetic cyclic peptide which reversibly inhibits CXCR4
• Phase I study: well-tolerated and effective
• Adverse events limited to minor infusion site reactions
• Phase II study in myeloma patients indicate sufficient stem cell
mobilization in 66% of patients in 1 or 2 apheresis sessions
• BKT 140 (Biokine Therapeutics, Rehovit, Israel)
• Highly selective CXCR4 antagonist,
• Originally designed to inhibit binding of HIV to CXCR4
• Synergized with G-CSF, leading to a 78-fold increase in PB progenitor cells
over controls

48. Sphingosine-1-phosphate (S1P) agonists
• Sphingosine-1-phosphate is a phospholipid stored and released into peripheral
blood mainly by erythrocytes
• Plays key role in immune surveillance and differentiation
• While plasma S1P levels remain high, most tissue S1P is quickly degraded and
dephosphorylated
• This results in a gradient of S1P which is important in lymphocyte egress from
lymphoid organs.
• This gradient also helps to weaken BM- HSc interaction and SCt comes out to PB
Juarez JG et all. Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice.
Blood. 2012 Jan 19;119(3):707-16

49. VCAM/ VLA4 inhibitors
• Natalizumab
• Recombinant humanized monoclonal antibody against α4 subunit of VLA-4
• Approved for treatment of multiple sclerosis (MS) and Crohn’s disease
• Has been found to increase peripheral blood CD34+ cells
Ramirez P, Rettig MP, Uy GL, Deych E, Holt MS, Ritchey JK, DiPersio JF. BIO5192, a small molecule inhibitor of VLA-4,
mobilizes hematopoietic stem and progenitor cells. Blood. 2009 Aug 13;114(7):1340-3.

50. Alternate Mobilizing Agents
• Myelosuppressive drugs
• hr- PTH
• Proteosome inhibitors
• HIF stabilization

51. Myelosuppressive agents
• Chemotherapy
• Number of HSCs is moderately increased during early hematologic recovery
• Chemotherapy plus cytokine generally mobilizes greater numbers of PBSCs
than either agent alone
• No differences in tumor cell contamination, speed of hematological
recovery, or survival identified
• Cyclophosphamide or Ifosfamide based regimens most commonly used
• Cyclophosphamide, etoposide, cisplatin, paclitaxel
• Cyclophosphamide- High dose (7 gm/m2) has greater toxicity but superior to
intermediate (3-4 gm/m2) or low dose (1-2 gm/m2)

52. Parathyroid hormone (PTH)
• Positive correlation between PTH levels in patients with pituitary adenomas
and a number of circulating HSCs was established by Brunner et al
• Phase I study
• Treated with escalating doses of PTH over 14 days, followed by filgrastim
10μg/kg on days 10-14
• Adequate mobilization in 47% of patients who had failed 1 prior
mobilization and 40% of patients who had failed 2 prior mobilization
attempts
Okechukwu Nwogbo et all; Stem Cell Mobilization Using Parathyroid Hormone Analog:
A Single Institutional Review. Blood 2020; 136

53. Impact of PTH on mobilization and homing of stem cells
• Left axis: PTH administration results in endogenous release of G-CSF.
• Right axis: PTH results in down-regulation of DPPIV, which activates of SDF-1
and promotes homing of CXCR4+ BMCs.
Huber BC, Grabmaier U, Brunner S. Impact of parathyroid hormone on bone marrow-
derived stem cell mobilization and migration. World J Stem Cells. 2014 Nov 26;6(5):637-43

54. Proteosome inhibitors
• Bortezomib increases peripheral blood CFU-Cs 6.8-fold in animal studies
• Combined bortezomib with G-CSF or Plerixafor in animal studies
• Resulted in the mobilization of significantly higher number of CFU-Cs than
produced by G-CSF or Plerixafor alone
• Reduces CXCL12 expression and vascular permeability
Matsumoto T et al. Bortezomib enhances G-CSF-induced hematopoietic stem cell mobilization
by decreasing CXCL12 levels and increasing vascular permeability. Experimental Hematology.
2021 May;97:21–31.

55. Gro β
• Member of CXC chemokine family
• Stimulates chemotaxis and activation of neutrophils by binding to the CXCR2
receptor
• N-terminal 4–amino acid truncated form of Groβ can mobilized HSC within
15 minutes following administration
Bonig H, Papayannopoulou T. Mobilization of hematopoietic stem/progenitor cells: general
principles and molecular mechanisms. Methods Mol Biol. 2012;904:1-14

56. Stabilization of Hypoxia-inducible Factor (HIF)
• Hypoxia is an important factor for BM HSC quiescence and self renewal
• O2 concentration rises above 2%, the HIF-1α subunit is quickly degraded
• HIF has a key role in neutrophil function
• Induce expression of SDF-1 and VEGF-A
• In animal study, G-CSF and cyclophosphamide promote hypoxia within the BM
microenvironment
• Expansion of BM sinusoids, vasodilation and enhancement mobilization
Forristal CE et ell. HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α.
Leukemia. 2015 Jun;29(6):1366-78

57. PB Stem Cell Harvesting
• Some amount of PBSC taken out from body for future transfusion
• Therapeutic apheresis improved in recent years-
• 1st gen-
• 1950s
• Dr Edwin Cohn
• To separate albumin from plasma
• Further developed by Alan Latham to introduce “Latham bowl”
• Single-use version from plastic centrifugal element
• For washing or deglycerolizing RBCs
• “intermittent-flow” platelet (PLT) donation

58. • 2nd gen-
• By George Judson
• Continuous-flow centrifugal blood separator
• “reusable” metal bowl
• Capable in exchanging plasma or RBCs
• 3rd gen-
• Herbert Cullis in 1970s
• Sealless centrifuge mechanism
• Subsequently adopted by all manufacturers of continuous-flow centrifugal
separators

59. Migration of Stem Cell
• MMP-9 mediated proteolysis
of ECM
• Decreased SDF1 expression
• Increased gap junction
diameter via sympathetic
stimulation
Hoover-Plow J, Gong Y. Challenges for heart
disease stem cell therapy. Vascular Health and
Risk Management. 2012 Feb;99.

60. Procedure of PBSC Apheresis
ebmt.org

61. Schematic diagram of apheresis- centrifugation dependent blood component separation

62. Anticoagulation During Apheresis
• To prevent clotting in the extracorporeal circuit and clumping of the product
• Citrate anticoagulants are most commonly used
• ACD-A contains 10.67 g of citrate per 500-mL volume in the form of trisodium
citrate and citric acid (2.13% free citrate ion)
• Major concern is “citrate toxicity”
• Chelate calcium and magnesium ions
• Symptoms depend upon muscle mass, citrate metabolism rate
• Initial signs of citrate toxicity
• Circumoral or acral paresthesia

63. • May progress to
• Nausea, vomiting, loss of consciousness, tetany, and seizures
• In paed, suspected when
• Any change of behavior, such as crying
• Slowing the speed of blood flow, changing the blood-to-citrate ratio
• Management
• Oral calcium – may cause abdominal discomfort
• Heparin may be used with additional citrate in collection bag
• Intermittent or continuous infusions of calcium gluconate
• Excessive calcium replacement can induce cardiac dysfunction
Min YS, Kwon SW, Choe WH, Kim BJ, Cho KJ, Kim SS. Analysis of the adverse effects associated with
therapeutic plasmapheresis. The Korean Journal of Blood Transfusion. 2011 Aug 30;22(2):161-70.

64. Venous Access
• Continuous-flow apheresis devices require two-lumen access with blood flow
capacity greater than 20 mL/min
• Single lumen access may be used with discontinuous-flow apheresis devices
• Donors with small veins and undergoing multiple procedures, require
temporary venous catheter
• Hickman catheters are usually unsuitable due to subcutaneously placed ports
• For adults- catheters of 10 F or larger
• Pediatric patients- catheter sizes of 5 to 7 F

65. Apheresis procedure at MCK

66. Adverse Reactions
• Relatively safe for the patient and healthy donors
• Procedure-related mortality rates are low, at an estimated 3 deaths per 10,000
procedures
• But apheresis-associated morbidity is more frequently reported
• Citrate toxicity
• Hypovolemia
• Thrombocytopenia
Babic A, Trigoso E. Cell Source and Apheresis. 2017 Nov 22. In: Kenyon M, Babic A, editors. The European Blood and Marrow
Transplantation Textbook : Under the Auspices of EBMT. Cham (CH): Springer; 2018. Chapter 5

67. Hypovolemia related symptoms
• Dizziness, light-headedness, tachycardia, hypotension, and diaphoresis
• Life threatening cardiac dysrhythmia
• Due to large fluctuations in blood volume
• More common in children
• Pulse and blood pressure monitoring hourly
• Hemoglobin and hematocrit monitored after procedure
• Prevented by by priming the apheresis machine with red blood cells and fresh frozen
plasma

68. Thrombocytopenia
• Platelets can stick to the bowl used during the centrifuge process
Michael A. Pulsipher, Pintip Chitphakdithai, John P. Miller, Brent R. Logan, Roberta J. King, J. Douglas Rizzo, Susan F. Leitman, Paolo
Anderlini, Michael D. Haagenson, Seira Kurian, John P. Klein, Mary M. Horowitz, Dennis L. Confer; Adverse events among 2408 unrelated donors of
peripheral blood stem cells: results of a prospective trial from the National Marrow Donor Program. Blood 2009; 113 (15): 3604–3611

69. GVHD and GVL
• PB stem-cell allograft contain 10-fold greater number of CD3+ T cells
• G-CSF mobilization immunomodulatory effect on GVHD
• 50 X more number of CD14+ monocytes and monocyte progenitor
• Th2-inducing dendritic cells,
• Diminished GVHD-inducing ability
• Graft v/s Leukemia
• Significantly higher number of T cells
• 3 -fold greater numbers of natural killer cells
• more pronounced GVL effect
Zi-Min, S., Hui-Lan, L., Liang-Quan, G. et al. HLA-matched sibling transplantation with G-CSF mobilized PBSCs and BM
decreases GVHD in adult patients with severe aplastic anemia. J Hematol Oncol 3, 51 (2010).

70. • Clinically significant acute GvHD > grade I developed in 12/16 (75 %) patients allografted with PBSC
compared to 7/18 (39 %) BM transplant recipients
• Incidence of chronic GvHD almost similar between PBSC and BMT allograft recipients
• No difference in survival noted
Meisel Ret al. Similar Survival Following HLA-Identical Sibling Transplantation for Standard Indication in Children with Haematologic Malignancies: A Single Centre Comparison of Mobilized
Peripheral Blood Stem Cell with Bone Marrow Transplantation. Klinische Padiatrie. 2015 Jan 1;217(3):135–41.

71. Umbilical Cord Blood Stem Cells
• Source depend upon availability of public cord blood banks
• Relative immaturity of the donor immune system allow HST without
overwhelming GVHD.
• The primary obstacle is the limited quantity of HSCs per cord
• Speed and success of engraftment depend upon
• Total nucleated cell dose
• Quantity of CD34+ cells or colony-forming units (CFUs)
Rogers I, Casper RF. Umbilical cord blood stem cells. Best practice & research Clinical obstetrics
& gynaecology. 2004 Dec 1;18(6):893-908.

72. Procedure of CB collection
• Placental vein after delivery of the infant and transection of the cord
• Cannulation of the umbilical cord veins with aspiration into a collection bag
• Plasma and RBC depleted prior to storage
• Number of collected cells depend upon
• Timing of cord clamping
• Storage techniques
• Birth weight
H. Barreto Henriksson, Hellström A, Hesse C, Nilsson A, M. Dagerås, Falk M, et al. Morphology and biological data in cord blood
eryhtrocyte units resembles adult units after processing and storage - meets current quality recommendations. 2022 Jan 1;103356–6.

73. Complications
• Collection into open containers results in bacterial contamination
• Lower amount of blood collection by only aspiration
• Volume can be increased by perfusion of the placenta with salt solutions-
technical difficulty
• Disadvantages
• Cell dose
• One time supply
• Potential delayed engraftment time
Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation.
Experimental hematology. 2000 Nov 1;28(11):1197-205.

74. Advantages of UC SCT
• Simple harvest with no risk for doner
• Long term storage ability
• Immediately available in stem cell bank
• Immunologically immature- No GVHD
• Higher concentration of stem cell (0.1-0.8% CD34+ cells/ 100 nucleated cells)
• Higher proliferation rate and younger cells with longer telomers
• Low incidence of viral contamination
Willert et all. (2023). Umbilical Cord Blood—Biology, Banking, and Therapeutic Use.

75. • Survival benefit was restricted longest tertile of TL
• 23% reduction in mortality risk for every 1-kb increase in donor lymphocyte TL

76. Bone Marrow Stem Cell Transplant
Gorin NC. Bone Marrow Harvesting for HSCT. In: Carreras E, Dufour C, Mohty M, et al., editors. The EBMT Handbook:
Hematopoietic Stem Cell Transplantation and Cellular Therapies. 7th edition. Cham (CH): Springer; 2019. Chapter 14
Preferences for BM as source of stem cells

77. Technique of BM Collection
• BM collected after 2–4 days of GCSF priming
• G-CSF-primed marrow harvesting
• More mononuclear cells collected and
• Higher CD34(+) stem and progenitor cell doses
• Mobilized marrow speeds up engraftment for both auto- and allo-HSCT
• Possible (unproven) reduction of GVHD rate and severity
Gorin NC. Bone Marrow Harvesting for HSCT. In: Carreras E, Dufour C, Mohty M, et al., editors. The EBMT Handbook:
Hematopoietic Stem Cell Transplantation and Cellular Therapies. 7th edition. Cham (CH): Springer; 2019. Chapter 14

78. • Marrow is collected from the posterior superior iliac crests, usually under
general anesthesia
• Marrow is aspirated with bone needles with multiple holes all around
• Each aspiration is limited to a volume of less than 5 mL to avoid large dilution
with blood
• Transferred the aspirate through a three-way tap to the collection bag
• The collection bag contains ACD anticoagulant solution and the syringes are
rinsed with heparin (5000 U/mL)

79. Bone Marrow Aspiration Procedure and Aspiration Needle

80. Bone marrow aspiration and filtration
(A)With the patient positioned lateral, a 15-
gauge Illinois bone marrow needle with the
lower sleeve removed is inserted into the
central posterior superior iliac spine.
(B)The syringe is capped with an 18-gauge
needle, and the bone marrow heparin
mixture is injected through a coupler into a
150-mL polyvinyl chloride transfer bag.
(C)The contents are run through a 210-mm
filter to remove any bony particulates.
(D)The filtered bone marrow is divided
between two 10-mL red-top serum tubes.

81. • Target of collection is at least 3 × 108 nucleated cells/kg of recipient
• Cell count done at the mid time of the collection to ensure proper richness
• If the targeted goal cannot be achieved, additional collection can be made
from the anterior iliac crests
• CD34+ evaluation is not routinely performed for BM
Gorin NC. Bone Marrow Harvesting for HSCT. In: Carreras E, Dufour C, Mohty M, et al., editors. The EBMT Handbook:
Hematopoietic Stem Cell Transplantation and Cellular Therapies. 7th edition. Cham (CH): Springer; 2019. Chapter 14

82. • More pertinent testing consists in the evaluation of CFU-GM which represents
in this setting the most reliable functional viability indicator
• CFUGM/kg are about 1–1.5 log below the expected results in CD34+ cells
/kg
• Therefore expressed in 105/kg
• Maximum accepted volume collected should be < 20 mL/kg donor body weight
• Crystalloid and colloid infusion are used regularly
• Autotransfusion may be required
• Should be prepared 3 weeks before the collection

83. Complications of Bone Marrow Collections
• Very rare are death secondary to general anesthesia
• Major organ damage by mechanical mis-manipulation of the bone needles
• Complications-
• Tiredness (75%)
• Long standing pain at aspiration site (68%)
• Low back pain (52%)
• Mean recovery time is 16 days
Stroncek DF, Holland PV, Bartch G, et al. Experiences of the first 493 unrelated
marrow donors in the National Marrow Donor Program. Blood. 1993;81:1940–6

84. Quality of HSC Product
HSC composition
• Depend upon- source, mobilization used or not, mobilization agent
• Most important is T cell component
• 30% in PBSC, 10% in BMSC, 20% in CBU
• Major role in GVHD, graft versus leukemia, HSC homing and engraftment
• G-CSF increases CD14+ monocytes and CD3+CD4−CD8− suppressor T cells
• Plerixafor increases total T and NK cell in graft
Aljurf M, Snowden JA, Hayden P, Orchard KH, McGrath E. Quality Management and Accreditation in Hematopoietic
Stem Cell Transplantation and Cellular Therapy : The JACIE Guide. Cham: Springer International Publishing AG; 2021.

85. CD34+ Cell Enumeration and Dose
• Quantification of CD34 antigen-positive cells by flow cytometry
• Most cytometers measure at least five characteristics of each cell,
• Size,
• Granularity,
• Three different fluorochromes
• Apheresis can be initiated when PBSC concentration is about 10/μL
• Another method is CFU-GM enumeration from cell culture systems
• Helpful to determinate functional viability of progenitor cell
• Cell viability tested by propidium iodide or 7-aminoactinomycin D in FCM

86. Tumour Contamination
• Tumor cell contamination of HSC product can cause relapse
• Detected by
• Sensitive immunocytostaining techniques,
• Clonality assays
• Flow cytometric analysis, and
• PCR amplification of malignant
• Patients with marrow involvement
• Benefit from several cycles of debulking chemotherapy before
collection BMSC

87. Microbial Contamination
• Bacterial culture is an essential quality-control component
• Most common causes of bacterial sepsis is Gm- ve rods
• Skin flora are the bacteria are usually isolated
• Culture-positive HSC products doesn’t have any clinical sequelae
• Other pathogenic culture-positive products need not be destroyed
• BMSC product shows more risk of bacterial contamination than PBSC

88. Hematopoietic Stem Cell- Quality Control
• Genomic integrity
• Frequently acquire abnormalities in a non-random and sporadic manner
• Concern for future malignancies
• Whole genome sequencing
• Multipotency
• Hanging drop suspension culture method with cocktail of differentiation
inducing cytokines and morphogens
• Gene and marker expression
• In cellular culture media, different cell-surface and intracellular markers
for different stage of maturation for all 3 lineages

89. • Epigenetic Landscape
• Epigenetic reprogramming for committed cell lines
• Using pyrosequencing of different CpG sets analysis
• Cell Morphology
• Under phase contrast microscopy
• Contamination
• Bacterial, tumour cell and somatic differentiated cells
Phase-contrast
microscopy imaging and
HE staining of leukemic
stem cells (A-B), non-
cancer stem cells (C-D)
© ebmt.org

90. Summary
summary of stem cells mobilization strategies and target cells dose for allogeneic stem cells transplantation
Mohammadi S, Mohammadi A, Mohsen Nikbakht, Amir Hossein Norooznezhad, Kamran Alimoghaddam, Ardeshir Ghavamzadeh. Optimizing Stem Cells
Mobilization Strategies to Ameliorate Patient Outcomes: A Review of Guide- lines and Recommendations. PubMed. 2017 Jan 1;11(1):78–88.

91. summary of stem cells mobilization strategy and target cells dose for
autologous stem cells transplantation in adults
summary of stem cells mobilization strategy and target cells dose
for autologous stem cells transplantation in paediatrics

92. summary of apheresis and mobilization strategies
based on CD34 count prior to apheresis
summary of strategies for prediction of poor mobilize patients in
autologous stem cells transplantation

93. Take home message
• Hematopoietic stem cell transplantation allows high dose chemotherapy and
replacement of malfunctioning stem cells
• Peripheral blood stem cells mostly replaced bone marrow as a source
• Umbilical cord blood also another choice but limited by its longer engraftment
time
• For mobilization G-CSF and Plerixafor are most commonly used
• Adequate dose of stem cell infusion helps in early recovery but may cause
significant GVDH

94. THANK YOU