TM

1. ROLE OF TRANSFUSION MEDICINE IN
HEMATOPOIETIC STEM CELL
TRANSPLANT
Dr Atif Irfan Khan
Moderator: Dr Hem Chandra Pandey

2. OUTLINE
• INTRODUCTION
• HEMATOPOEITIC STEM CELLS
• SOURCES AND TYPES OF HSC
• INDICATIONS OF HSCT
• ROLE OF TRANSFUSION MEDICINE
• Donor evaluation
• Collection
• Preservation
• Immunohematologic compatibility
• Transfusion support
• CONCLUSION

3. INTRODUCTION
• HSCT has been on the rise
over the past decades in
India
• Transfusion Medicine plays
an integral role in a process
with several specialties
involved
Autologus Stem Cell Transplantation for Cancers: India: 2017 Raut, J Blood Disord Transfus 2017, 8:3 DOI:
10.4172/2155-9864.1000379
YEARWISE GROWTH IN AUTOLOGOUS AND ALLOGENIC STEM CELL TRANSPLANT
IN INDIA

4. HEMATOPOIETIC STEM CELL
• Stem cell- undergo both self-renewal and differentiation.
• Hematopoietic stem cells (HSCs)
• Capable of self-renewal
• Can also form all lines of blood cells (Haematopoiesis).
• Formation start during embryonic development.
• Found in bone marrow and umbilical cord blood.
• They express CD34
• Multipotent

5. HEMATOPOIETIC STEM CELL
wikipedia

6. HSC
Allogenic Autologous
Mismatched Haploidentical
Less than fully
matched from a
relative
Donor has one HLA
haplotype of two in
common (half-matched,
e.g. 3/6, 4/8, 5/10)
Matched
Unrelated Donor
Matched
HLA matched
sibling or rarely
from a close
relative or
unrelated
Related
Donor

7. INDICATION FOR AUTOLOGOUS TRANSPLANT
Malignant disorders
• Non-Hodgkin lymphoma
• Hodgkin’s disease
• Multiple myeloma
• Neuroblastoma
• Ovarian cancer
• Germ-cell tumors
Non-Malignant disorders
• Autoimmune disorders
• SLE, Systemic sclerosis
• Amyloidosis

8. INDICATION FOR ALLOGENIC TRANSPLANT
Malignant disorders
• Acute myeloid leukemia (AML)
• Acute lymphoblastic leukemia
(ALL)
• Chronic myeloid leukemia (CML)
• Chronic lymphoblastic leukemia
(CLL)
• Myeloproliferative disorders
MPD
• Myelodysplastic syndromes
Non-Malignant disorders
• Aplastic anemia
• Thalassemia
• Sickle Cell Disease
• SCID
• Fanconi’s anemia
• Blackfan-Diamond
• Wiskott-Aldrich
• Inborn errors of metabolism

9. SOURCES OF HSC
1. Bone Marrow – BM
2. Peripheral blood by Apheresis- PBSC
3. Umbilical cord blood- UCB

10. BONE MARROW HSC
• Was initially the main source of hemopoietic
stem cells,
• Under Local anaesthesia by repeated
aspiration from the posterior illiac crest,
anterior illiac crest or sternum can be used.
• Removal of mature erythrocytes from the
graft necessary
• to avoid hemolytic transfusion reaction in
allogeneic HSCT with major incompatibilities.

11. UMBILICAL CORD HSC
• At the time of delivery by clamping the cord and nicking the umbilical
vein at the newborns first cry.
• Normal blood bags are used for collection.
• Volume 100 ml (60-140 ml).
• Samples sent to determine cell counts and for culture.
• cryopreserved in liquid nitrogen till further use.
• RBCs are removed while retaining mononuclear cells, including
hematopoietic stem and progenitor cells

12. PBSC-Peripheral blood stem cells
• Collected by peripherally apharesis after mobilization

13. HSC-SOURCES UCB BM PBSC
Collection
process
safest Needs
anaesthesia
Mobilisation can
cause discomfort
to donor
Engraftment 3weeks 16-18 days 13-35 days
Infection rare common common
GVHD Less and less
severe
Higher chance Higher chance of
chronic GVHD as
compared to BM
HLA matching Less stringent Much
stringent
Much
stringent
HSC cells low high high
Repeat
harvest
Not possible possible possible

14. ROLE OF TRANSFUSION MEDICINE

15. DONOR EVALUATION
• Proper history including details of chemotherapeutic drugs
received in past, hx of infections-TB, fungal
• Detailed general physical examination, and venous access
• CBC.
• Viral infection markers-hepB, HepC, Syphilis, HIV, CMV
• ABO grouping

16. HLA TYPING/MATCHING
• Essential for Allogeneic HSCT.
• HLA identical donor is one that matches the recipient’s HLA antigens:
• HLA-A,HLA-B,HLA-C,HLA-DR,HLA-DQ ( Also called 6/6 match ).
• At least 5/6 or 6/6 matching should be there
• UCB transplant can be done with more HLA disparity
• Not needed in autologous transplant

17. STEM CELL COLLECTION- autologous
Preparative(or conditioning) regimen.
• Chemotherapy or chemotherapy and growth factors together can be used
in mobilisation
• Eradicate any residual cancer.
• High-dose therapy is employed to overcome tumor resistance.
• HPCs- increase more than 50-fold and remain elevated for 3 to 5 days
• Chemotherapy alone mobilizes HPCs sufficiently well in many patients to collect an
adequate dose of HPCs in one or more apheresis procedures

18. STEM CELL COLLECTION - Allogenic
• Stem cells in circulation – 0.1% of PB mononuclear cells
• Need to mobilize them from marrow
• Mobilizing agents used
• HGF – GM-CSF, G-CSF.
• CXCR4 blockers - Mozobil (plerixafor injection)

19. MOBILIZING AGENTS - Hematopoietic growth
factor
• Agents – GM-CSF, G-CSF(Filgrastim)
• Moa
• cause the dissociation of HPC adhesive interactions
• release of metalloproteases (eg, MMP9, elastase, and cathepsin G) that are
hypothesized to cleave one or more receptor-ligand pairs (eg, SDF1) and to release
HPC tethers to their stroma.
• DOSE – 10 micrograms/kg each morning for four days before harvesting
• Side effects- bone pain, headache, muscle ache, fatigue, sweating

20. ,
• AGENTS- Mozobil (plerixafor)
• MOA
• directly block HPCs adhesive
receptors by agents as VLA4
antibodies or oligopeptides that
block CXCR4
• DOSE – 0.24mg/kg 11hrs prior to
apharesis
MOBILIZING AGENTS – CXCR4
Blockers

21. STEM CELL MOBILISATION -REGIMEN
The optimal time for collecting PBSC is when a peripheral blood sample contains at least 20X103 circulating CD34+
cells/ml

22. CRITERIA FOR POOR MOBILISATION
•Failure to achieve a minimum level of
• 5-20 CD34+ cells/μL in peripheral blood after completion of
mobilisation regimen
• 1-2X106 CD34+ cells/μL during a single apheresis procedure
• 5X106 CD34+ cells/μL in all collections

23. EQUIPMENT FOR PBSC COLLECTION
Comtec Amicus Spectra

24. TARGET YIELDS IN APHERESIS
Apheresis is generally continued till the following doses (per Kg body
weight of the patient) are collected.
Parameter Optimal Dose
CD34+ve cells / Kg >2 x106
MNCs / kg 4-8 x 108
CFU-GM/Kg >2x105

25. STEM CELL COLLECTION - PROCESSING
Positive selection
• selection of HPCs expressing CD34.
• used to reduce tumor cells in autologous transplants or to reduce T-cells in allogeneic transplants.
• separation of the mononuclear cells from the RBCs and neutrophils by density-gradient
centrifugation.
• Monoclonal anti-CD34 antibodies bound to a solid-phase matrix- used to selectively adsorb the
CD34 cells.
• bound cells are then eluted from the solid matrix

26. STEM CELL COLLECTION - PROCESSING
Negative selection(purging)
• destruction of the malignant cells
RBC depletion
• Hydroxyethyl Starch Sedimentation
• Cell Separator
• Sedimentation and Dilution

27. CRYOPRESERVATION
• Cells are suspended in
• autologous plasma or
• another source of protein
• Use Of dimethylsulfoxide (DMSO) to make 10% solution
• DMSO+albumin
• DMSO+plasma
• controlled-rate freezing.
• Controlled rate freezing
• 1-2°C/min to -30 °C ,then 2-10 °C/min to -90 °C
• Minimise hemolysis because incompatible donor red cells are typically
lysed as a result of the freezing process
• Stem cell are stored in aliquots

28. FREEZING
• Controlled rate freezing
– 1-2°C/min to -30 °C ,then 2-10 °C/min to -90 °C
• Uncontrolled rate freezing
– -70 to -80 °C
• Mechanical freezer
(<-80 °C) or in liquid nitrogen freezer(-196 °C)

29. THAWING
• Product is thawed rapidly by immersing in a 37°C water bath just
before infusion.
• Rapid thawing may lead to bag breakage.
•Infusion done as soon as it thaws
• Done bed side

30. INFUSION
• May be infused rapidly if patients condition permits at 5-20 ml/min with a
BT set. (without line filter : 170-210 μ)
• Look out for adverse effect: nausea, vomitting, hypertension, hypotension,
flushing, chest tightness, cramps, bradycardia.
• If DMSO >0.8to1 g/kg, cardiac dysfunction and fatal dysrhythmias may
occur, do multiple bag infusions over 2 days.

31. ENGRAFTMENT
• The number of days after infusion of the graft until a defined
threshold of circulating neutrophils or platelets is reached
• first of 3 days with PMN>500/μL and platelet>20,000/μL, untransfused.
• Shorter engraftment times are associated with fewer complications.

32. IMMUNOHEMATOLOGIC COMPATIBILITY
Relationship between donor and recipient
The relationship between the ABO types of the donor and recipient can
fall into :
• Major incompatibility: occurs when the recipient has antibodies
against blood group antigen(s) present on the surface of the donor’s
red cells
• Minor incompatibility occurs when the donor has antibodies against
blood group antigen(s) present on the recipient’s red cells .
• A bidirectional incompatibility is the existence of both major and
minor incompatibilities

33. ABO COMPATIBILITY IN HEMATOPOIETIC STEM CELL TRANSPLANTATION
AABB 17th Edition

34. MAJOR INCOMPATIBILITY
Challenges:
• The potential for acute intravascular hemolysis when ABO-incompatible
donor red cells are infused with the graft to the recipient
• The continued production of circulating ABO antibodies directed against
donor red cells and erythroid progenitors produced by the engrafted HPCs.
• Can also lead to pure red cell aplasia
Prophylactic measures :
• Red cell depletion:
currently acceptable volumes range from 10 to 20 mL.

35. MINOR INCOMPATIBILITY
• Donor red cell antibodies can be readily removed by Depletion of
plasma from the graft product.
• if Isoagglutinins are not completely removed, the ensuing Hemolysis
is mild and self-limited.
• Passenger lymphocyte syndrome(PLS)- 5 to 15 days post transfusion,
donor, transplanted B lymphocytes produce incompatible blood
group antibodies after transplantation
• If hemolysis is severe and potentially life threatening, rapid institution
of erythrocytapheresis to exchange incompatible recipient red cells
with donor compatible red cells can be therapeutic and lifesaving.
Non-ABO-Incompatible Transplants- require similar attention as the
ABO grouping

36. LAB INVESTIGATIONS
• DCT
• ICT
• Phenotyping
• Blood grouping
• Ab titration

37. STANDARD PROCEDURES FOR ABO
MISMATCHED TRANSPLANTS
Hematopoietic stem cell transplantation between red cell incompatible donor–recipient pairs, Bone Marrow
Transplantation (2001) 28, 315–321

38. PHASES OF HSCT
ROLE OF TM
Phase I Pre Transplantion Begins when the patient is identified as a
transplant candidate
Selection and screening of donor, donor
preparation, transfusion support-
leukodepletion, irradiation
Phase II Peri
Transplantation
Begins when the patient receives
chemotherapy and/or irradiation and
includes haematopoietic cell infusion and
engraftment phase of the transplant, which
may last for 2–4 weeks or longer
Harvesting, stem cell QC, transfusion
support-irradiation, leukoreduced products,
RBC, Granulocytes, Platelets, ICT/DCT, Ab
titrarion
Phase III Post
Transplantation
Begins when the RBC direct antibody test
(DAT) is negative and patient
isohaemagglutinins/RBC are no longer
detectable; forward and reverse types are
consistent with the donor ABO type
ABO Mismatch monitoring- and
interventions; plasma exchange
Transfusion support as above with irradiated
products at least 1yr post transplant
38

39. TRANSFUSION SUPPORT
• Required before, after transplantation and before engraftment, may
take up to 6 weeks.
• The decision to initiate red cell transfusion is based on symptomatic
anemia.
• Most patients with a haemoglobin of less than 7 g/dl should be
transfused.
• recipients of allogeneic HSCT- there is a prolonged period of
inappropriately low endogenous EPO levels. red cell transfusions
may be required for up to a year following engraftment.

40. AABB 17th Edition

41. PLATELET SUPPORT
• Low platelet counts is a predictor of transplant-related mortality.
• Following transplantation, patients will lack megakaryocytopoiesis
until platelet Engraftment occurs.
• Platelet engraftment, defined as a sustained platelet count of
20000/µl.
• transfusion of ABO-incompatible plasma and platelets is a common
practice for routine transfusion, it cannot be readily applied to the
recipients of allogeneic HSCT.
• Transfusion of platelet concentrates containing ABO-incompatible
plasma increases the risk of hepatic veno-occlusive disease in young
children Treated with a busulfan- containing regimen.

42. PLATELET SUPPORT
• Prophylactic or Therapeutic???
• platelet threshold
• 10,000 /µL for prophylaxis
• 20,000/µL in febrile & bleeding patients.
• Low dose/ Standard dose/ High dose???
• REFRACTORINESS-
• Alloimmunization to (HLA) class I or platelet- specific antigens
• ABO incompatibility
• Drug-induced thrombocytopenia
• Immune thrombocytopaenic purpura (ITP)

43. FFP AND CRYOPRECIPITATE
• There are no specific recommendations regarding the usage of any of
these products for HSCT patients.
• Indications remain the same as in any non transplanted patient

44. GRANULOCYTE SUPPORT
• Haematopoietic cell transplantation patients are profoundly neutropenic
following their conditioning Regimen until neutrophil engraftment, defined as a
sustained absolute neutrophil count (ANC) of 500/µl occurs.
• Treatment resistant fungal infection
• Granulocytes are collected from donors who have received a combination of
granulocyte colony-stimulating factor (G-CSF) and dexamethasone which permits
collection of 1x1010 granulocytes that need to be irradiated and infused as soon
as possible, and no later than 24hours, after collection owing to their short half-
life.

45. SPECIAL PROCESSING OF BLOOD
COMPONENTS
• Leucocyte-reduced blood components
• CMV-reduced risk blood components
• Gamma irradiation of blood components
• Pathogen inactivation technologies

46. LEUCOREDUCED BLOOD COMPONENTS
Benefits:
• Alloimmunization to human leucocyte antigens (HLA)
• Platelet refractoriness
• Febrile non-haemolytic transfusion reactions
• Transfusion-associated graft-versus-host disease
• Cytomegalovirus (CMV)
• Epstein–Barr virus (EBV)
• Human T-cell lymphotropic viruses types I and II (HTLV
I/II)

47. GAMMA IRRADIATION
• Gamma irradiation induces chemical crosslinks in the DNA of irradiated donor
lymphocytes, preventing their Proliferation.
• Gamma irradiation of all transfused erythrocyte and platelet components is
the only reliable method for preventing TA-GVHD.

48. CMV NEGATIVE PRODUCTS
• The CMV status of the recipient and the donor should also be considered
before a decision is made regarding transfusion of leukocyte-reduced vs
CMV-seronegative units.

49. CONCLUSION
Role of transfusion medicine encompasses through large part of the
hematopoietic stem cell transplant process from planning, selecting the
donor, harvesting and storage of stem cells.
Transfusion support during different stages with specific requirements
and therapeutic apharesis amongst many others