4. Types of stem cells
1. Embryonic stem cell (E S
Cells ) :
Are Pluripotent.
Can generate all tissues of
body.
Derived from
embryos(blastocyst).
Developed from in vitro
fertilization.
5. Types of stem cells
2. Adult stem cell ( somatic
cell ) :
Undifferentiated cell found
among differentiated cells in
a tissue or organ.
Can renew it self in to
specialized cells.
e.g. Bone marrow , Skin ,
lining of G.I.T., pancreas ,
liver adipose tissues & many
others.
6. HSCs (Hematopoietic stem cells)
Adult stem cells that are
found in bone marrow and
blood.
HSCs are capable of
producing all of the cells
that make up the blood
and the immune system.
7. Hematopoietic Stem cells
1/ 25,000 - 1,00, 000 cells in the bone marrow
Characteristic:
• CD34
• CD133
• C-kit (CD117)
• ADH
8. Hematopoietic Stem Cell Transplantation
• Hematopoietic stem cell transplantation ( HSCT) is a therapeutic procedure
involving intravenous infusion of autologous or allogenic stem cells
collected from bone marrow, peripheral blood or umbilical cord blood to re-
establish hematopoietic function in patients whose bone marrow or
immune system is damaged or defective.
• More than 50,000 first HSCT’s – 53% autolgous and 47% allogenic are
performed every year worldwide . ( World Wide Network of Blood and
Marrow Transplantation)
• The list of diseases for which HSCT is being used is rapidly increasing and
currently numbers more than 70.
9. Types of HSCT
I. Allogeneic HSCT
Patients receive stem cells from another individual ( donor)
•syngeneic (from identical twins)
•from HLA identical sibling/relative
•from matched unrelated donor (MUD)
II. Autologous HSCT
Patients receive their own stem cells.
11. BONE MARROW TRANSPLANT
A bone marrow transplant is a process to replace unhealthy
bone marrow with healthy bone marrow.
Bone marrow transplants can be used to treat patients
with:
• life-threatening blood cancers like leukemia
• diseases which result in bone marrow failure like aplastic
anemia
• other immune system or genetic diseases
12. TYPES OF BONE MARROW TRANSPLANTS
Autologous bone marrow transplant- auto means “self”. Stem
cells are removed from recipient’s BM before he receives
chemotherapy or radiation. After these treatments stem cells are
re infused into the body. This is Aka “rescue” transplant.
Allogenic bone marrow transplant- allo means “other”. Stem
cells are removed from another individual of the same species
called the “donor”. He can be either a HLA identical
sibling/relative or a matched unrelated donor (MUD)
Syngeneic bone marrow transplant – Donor is an identical twin.
13. HISTORY OF BMT
Early attempt was in 1891
Oral administration of healthy BM to patient with defective blood
formation.
not successful, but physicians had the right idea: donor marrow
cells----may find their way into the BM cavity.
Some unsuccessful BMTs in 1930s and 1940s.
After World war II
Nuclear bombings inspired the research into the effects of the radiation on the
bone marrow of survivors in Hiroshima and Nagasaki.
BM is very sensitive organ to radiation.
This led to major breakthroughs for bone marrow transplants in the 1950s
14. 1956 – The First successful Transplantation Between Identical
Twins with total body irradiation.
E. Donnall Thomas
Performed total body irradiation followed by infusion of bone
marrow from an identical twin and found that it could result in
complete remission of leukemia.
The Nobel Prize, 1990
15. 1958 – an Important Discovery
Allogeneic BMT was not performed on large scale until
Jean Dausset, a French medical researcher, made a critical
discovery about the human immune system : Human
histocompatibility antigens “HLA”
Dausset described HLA as “proteins on the surface of most cells
in the body. The immune system uses these proteins to identify
which cells belong in the body”
The better the antigen match = less likely T cells of the donor
will react against the patient’s body.
The Nobel Prize, 1980
1968 – First Bone Marrow Transplant Between HLA
matched Siblings.
18. Investigation Rationale
PET / CT scans; Bone marrow
aspirate / trephine
To assess disease status
Echocardiogram or MUGA
scan
To assess cardiac function
Lung function tests To assess pulmonary function
Blood tests: FBC, ABO, U&Es,
clotting screen, syphilis, Viral
To assess suitability for HSCT
Pre-Transplant Evaluation
19.
20. 1. Bone Marrow Harvest-
•Under anaesthesia
•Posterior iliac crest/Anterior iliac crest
•Several bone punctures are required to get a sufficient supply of bone marrow for transplant.
After the procedure, a sterile surgical dressing is applied to the site where the bone marrow was
collected.
•A sufficient harvest ( one that provides atleast 1.2 ×10 CD8
34+ cells/kg b.w)
•Possible complications- bleeding, infections or anesthetic side effects.2.
BM Harvest Filters:
180-263µ filters
Removal of fat/ bony spicules/clots
Types- Ethylene tetrafluoroethylene/Mesh monofilament filter/ plastic mesh filter.
Stem Cell Collection and Processing
22. Peripheral Blood Stem Cell collection is a non-surgical procedure.
• The donor receives daily injections of G- CSF for five days prior to the harvest. This
injection increases the number of stem cells in the bloodstream. ( Mobilisation)
•The stem cell are then collected by a process called apheresis which takes 4 – 5 hours.
During apheresis donor's blood is removed through a needle in one arm and passed
through a machine that separates out the mononuclear cells. The remaining blood is
returned to the donor through the other arm.
23.
24.
25. • 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.
• Then cryopreserved in liquid nitrogen till further use.
• Due to immaturity of immunogenic T cells in the cord blood,
risk of GVHD is low.
However , since only a small quantity of blood is obtained from an umbilical
cord , the stem cell yield is usually poor. it is therefore more suitable for
transplantation in children.
Umbilical Cord
26. Why Cord Blood Banking ?
•1 in 200 individualsmay need a haematopoietic stem cell (HSCs)
transplant by the age of 70 according to a scientific paper
published in 2008.
• It is once-in-a-life time chanceto collect cord blood – a readily
available source when needed in the future.
•60% higher chance of locating a match versus bone marrow in a
family.
27. • Approx. 3 × 108
nucleated cells / kg of recipient's body wt.—for
bone marrow.
• 5 × 108
mono nuclear cells / kg or
2 × 108
/ kg CD 34+ cells (markers of stem cells) – for Peripheral
blood.
• > 2-5 × 107
/ kg nucleated cells or 2-5 × 105
/ kg CD 34+ cells –
For UCB.
Number Of Stem Cells
28. Processing
3. In vitro purging:
autologous stem cell grafts are contaminated with tumor cells which may lead
to relapse .
Detected by Flow cytometry, PCR assay, Tumor clonogenic assay.
Removal by-
a) chemical agents- Cyclophosphamide/ Mafosfamide.
b) Monoclonal antibodies- positive selection of CD 34 + cells and negative
selection of tumor cells.
29. 4. Quantitation of HSCs:
• Mononuclear Cell Count- indirect estimate ( includes Lympho, mono,
immature myeloid cells )
• CD 34 counting- Flow Cytometer
• Colony Forming Units
It is the Body weight of the recepient that determines the adequate dose of
stem cell for engraftment. (1.2 ×108
CD34+ cells/ kg body weight).
5. Enrichment of HSCs:
CEPRATE stem cell concentrator.
30. 6. Quality control and VIABILITY check of Stem cells :
• To ensure purity ( cells free from microbial contamination)
• Identity ( cells are what they are claimed to be)
• Stability ( genotype and phenotype are stable during growth and passage in vitro )
a. Dye Exclusion test : (Trypan blue, Naphthalene black) dyes that penetrate cells
are excluded by the action of cell membrane in viable cells.
b. Flow - Cytometry : viability, cell counts, potency
c. Karyology : Karyoanalysis facilitates monitoring of genetic stability in cell cultures
and also recognition of transforming cells.
d. Sterility check : Microbiological control in stem cell banks.
e. PCR and ELISA
f. HLA directory : in house HLA testing process
g. Clean room environment
31. Storage of stem cells
In case of autologous transplants stem cells must
be harvested months in advance of the
infusion, therefore Stem cells need to be kept
frozen for prolonged periods.
In allogenic transplants fresh stem cells are
preferred to avoid stem cell loss during
freezing/thawing.
CRYOPRESERVATION:
Preservative- DMSO (Dimethyl Sulphoxide) and
cooled very slowly in a controlled rate freezer
to prevent osmotic cell injury during ice crystal
formation.
HSCs can be presrved in cryofreezers for years
( liquid Nitogen at -196 C)
33. Conditioning/ Preparative regimens
Prior to transplant to-
a) eradicate the disease process from the marrow
b) create a physical space / niche to allow growth of donor cells
c) Immunosuppression ( in case of allogenic tx )
Classified as-
1. Myeloablative
2. Non Myeloablative Conditioning Regimens
34. • Myeolablative conditioning
Irreversibly destroys the haemopoietic function of the bone marrow
with high doses of chemotherapy +/- TBI.
Higher level of disease control
Younger patients with a good performance status
Quicker engraftment of donor cells
Higher toxicities associated with higher transplant related
mortality
35. Reduced intensity/ Non - Myeloablative conditioning
• Regimens that have been developed to reduce the morbidity and
mortality of particularly allogeneic transplants.
• It aims to use enough immunosuppression to allow donor cells to
engraft without completely eradicating the recipients bone marrow.
• Can be given to older patients.
• Less regimen related toxicities . Reduction in morbidity and transplant
related mortality.
36. Stem Cell Infusion
• 24-48 hour rest period
• Stem cells removed from freezer, thawed at room
temp in a water bath.
• Infusion through a central venous line over 2-4 hours
Stem cells travel through the blood and settle in the
bone marrow.
DMSO ( preservative) related side effects during infusion-
a) Nausea/ Vomitting/ Diarrhoea
b) Anaphylactoid reactions ( flushes/rashes to
bronchospasm and resp compromise)
( premedications can be used)
c) Hypertension (smooth muscle)
37. POST TRANSPLANT SEQUELAE
A. NEUTROPENIC PHASE
✦Few days to 2-4 week period
✦No effective immune system
✦Poor healing and high susceptibility to infections.
✦Supportive care and empiric antibiotic therapy
✦HSV infection
✦Nosocomial infections
✦Mucositis ( parentral nutrition is recommended)
✦Fever ( empirical anti fungals )
38. B. ENGRAFTMENT PHASE
Recovery of cells is called engraftment.
•PB Neutrophil count > 500/µl ( G-CSF can accelerate this),
•Platelet > 20,000/ µl
•Red cells are the last to return.
Commonly patients require blood and platelet transfusions after transplant.
Graft Failure-
Failure to attain N count > 500/ µl and platelet > 20, 000/ µl by 28 days post
transplant.
Secondary / Late Graft failure- redevelopment of pancytopenia at any time of
the primary engraftment.
40. i. Infections
EARLY
< 30 days
MIDDLE
30-100 days
LATE
>100 days
Staphylococcus Aspergillus Pneumococcus
Streptococcus Pneumocystis Carnii Hemophilus influenzae
Gram negative
Enterobacter
Toxoplasma Gondii Varicella Zoster virus
Candida Cytomegalovirus
Herpes Simplex Adenovirus
41. •In autologous transplants risk of bacterial infections decreases with
hematopoietic recovery.
•However, recovery of cell mediated immunity and humoral immunity may
take upto 3 -12 months
•Therefore there is increased risk of viral infections particularly reactivation
of Varicella Zoster.
42. ii. Mucositis-
(2nd week after ASCT)
Injury of the mucosal lining of the mouth and the throat.
Common regimen related toxicity following myeloablative ASCT.
Painful- hampers eating/drinking.
parental alimentation to prevent dehydration and malnutrition and opioids for
pain relief.
Human Keratinocyte Growth Factor (pelifermin)
iii. Pulmonary Complications-
High dose chemo prior to ASCT predisposes- injury to the pulmonary artery.
Diffuse pul haemorrhage ( dyspnoea, fever, cough, hemoptysis, hypoxia).
Pulmonary infiltrates on Xray and CT chest .
43. iv. Engraftment syndrome-
second /third week following engraftment
weight gain, ascites, edema
associated with kidney and liver abnormalities
Pathogenesis : poorly understood, role of circulating leucocytes
Prophylactic steroids.
v. Hemorrhagic cystitis-
Preparatory regimens containing ifosfamide and cyclophospahamide ( chemical
inflammation of bladder).
Infections- polyoma, BK virus or adenovirus.
a. Mild Cystitis (Grade 1)- dysuria and micro hematuria
b. Overt Cystitis (Grade>2)- gross hematuria, clot retention, UT obstruction, RF.
44. vi. Hepatic Veno- Occlusive disease/ Sinusoidal Obstruction syndrome-
Jaundice ( s.bilirubin > 2mg/dl)
Hepatomegaly, ascites, and unexplained weight gain (> 2% of baseline b.w) within 20
days of ASCT.
Incidence- 15-25 % ( lower in autologous)
Pathogenesis-
a. injury to sinusoidal endothelial cells and hepatocytes
b. deposition of fibrinogen, factor VIII and and fibrin within the venular walls and
sinusoids- micro thrombosis
c. Centri lobular hepatic injury- portal hypertension, hepatorenal syndrome, multi
organ failure and death.
Rx with Difibrotide- fibrinolytic and anti thrombotic.
Substitue - fludarabine for cyclophosphamide
Px- Ursodiol a day before preparatory regimen
45. vii. Adverse effects a/w cryopreservation and thawing:
a. DMSO ( cryopreservant)
b.Cryoprotectant solution - hyperosmolar 2000 mOsm
that protects the marrow progenitors but causes lyses of other cells in the marrow eg- gran/rbcs / platelets
hemolysis- free hb -kidney damage
cell debris and aggregates- pulmonary emboli.
viii. Prolonged Immunosuppression
ix. Recurrence of disease :
most imp adverse effect of Auto transplant
presence of residual tutor in the autograft ( improper purging)
No auto immune phenomena reported till date.
Acute GVHD like skin rashes have been reported in a few cases of ASCT ,however they have not been
confirmed to be due to rejection.
46. 1.Advantages-
a. No risk of GVHD
b. High Dose therapies prior to harvest helps in curing aggressive component of disease.
c. Produces a durable remission
2. Disadvantages-
a. Engrafted cells may be contaminated with tumor ( relapse)
b. High Dose Chemo regimens : toxicity
c. Infections ( Neutropenic phase : between conditioning and engraftment)
PROs and CONs Of ASCT
47. Tandem Transplant
Type of autologous transplant
Under clinical trial - Hodgkins and NHD/ Multiple Myeloma and Germ cell
cancers
Two sequential courses of therapy:
a. High Dose Chemotherapy with Stem cell transplant
b. Several weeks to several months apart
c. Stem cells are collected for both the transplants prior to the initial course.
d. the second transplant is performed after the recovery from the first one.
Reduced relapse rates
Responce rates & survival rates - higher.
52. • High Dose melphalan 200 mg/ m2
followed by ASCT is associated with CR
rates of 40-60% with improved overall & event free survival.
• Auto-HSCT is clearly indicated for patients less than 70 years of age who
respond to first-line treatment. Age should be considered in conjunction
with the patient’s general health and fitness.
• New agents, such as the proteasome inhibitors (bortezomib) or the
immunomodulatory drugs such as lenalidomide (IMiDs) may change the
place of auto-HSCT.
• Double auto-HSCT has been shown to be superior to one auto-HSCT
although the benefit of the second transplant appears to be restricted to
patients not achieving CR (complete remission) or VGPR (very good
partial response) with the first transplant.
54. •In general, NHL can be divided into two prognostic groups, indolent and
aggressive.
•Follicular lymphoma (FL) is the most common indolent NHL (70%–80% of
cases), and often the terms indolent lymphoma and FL are used
synonymously. Also included in the indolent NHL are SLL/CLL,
lymphoplasmacytic lymphoma, marginal zone lymphomas, and cutaneous T-
cell lymphoma.
•Aggressive lymphomas include DLBCL, MCL, PTCL, anaplastic large cell
lymphoma, and Burkitt’s lymphoma.
55. When ASCT for Non-Hodgkin Lymphoma is covered
1. For patients with non-Hodgkin lymphoma (NHL) B-cell subtypes considered
aggressive (except mantle cell lymphoma), autologous HSCT may be considered
medically necessary:
a. as salvage therapy for patients who do not achieve a complete remission (CR) after
first- line treatment (induction) with a full course of standard-dose chemotherapy;
b. to achieve or consolidate a CR for those in a chemosensitive first or subsequent
relapse; or
c. to consolidate a first CR in patients with diffuse large B-cell lymphoma, with an age-
adjusted International Prognostic Index score that predicts a high- or high-intermediate risk
of relapse.
2. For patients with mantle cell lymphoma:
a. autologous HSCT may be considered medically necessary to consolidate a first
remission. This is often preceded by rituximab (anti CD 20 monoclonal antibody) based
induction and preparatory regimens.
3. For patients with NHL B-cell subtypes considered indolent, autologous HSCT may be
considered medically necessary:
a. as salvage therapy for patients who do not achieve CR after first-line treatment
(induction) with a full course of standard-dose chemotherapy; or
b. to achieve or consolidate CR for those in a first or subsequent chemosensitive
relapse,
whether or not their lymphoma has undergone transformation to a higher grade.
56. AML
1.Therapy in AML :
a.Remission Induction-
Chemotherapy ( daunorubicin + cytosine arabinoside)
b.Post Remission therapy-
i. Consolidation chemotherapy ( High dose cytosine arabinoside)
ii. Intensive chemo or chemo-radio followed by HSCT ( autologous/allogenic)
Criteria for complete remission (CR) :
i.<5% blasts in a normocellular bone marrow
ii. Return of PB counts to normal-
-Neutrophils > 1500/cmm
-Platelets > 1,00,000/cmm
-Haemoglobin > 10.0 g/dl
iii. Disappearance of signs and symptoms
AML is a heterogenous disease with widely different risk of relapse following standard therapy.
Cytogenetic abnormality and time to achieve CR are the two most important prognostic factors.
AML patients are therefore risk stratified based upon cytogenetic and molecular abnormalities.
Therapy is thus individualised and based upon which prognostic category the patient belongs to.
57.
58. A. Allogenic Transplant is the treatment of choice in all patients < 55years
with high risk cytogenetics in CR1.
B. For patients with normal cytogenetics , allogenic HSCT should be
considered if they require 2 cycles of chemotherapy to achieve
complete remission.
C. Autologous SCT should be considered in poor risk category patients in
CR1 if HLA matched sibling donor is not available or if Allo transplant
cannot be performed due to practical reasons.
59.
60. HODGKIN’S LYMPHOMA
•High Dose Chemotherapy (HDCT) supported with autologous
SCT is currently standard of care for relapsed HL & HL refractory
to Primary therapy.
61. ALL
CLL
MDS
CML
•MDS- median age of diagnosis is 70 years, therefore utility of transplants is limited.
Autologous transplants can be considered in selected patients who achieve CR following
induction and do not have an HLA - identical donor.
•CLL- median age at presentation is 65 years. Allo- transplants are associated with
significant morbidity and mortality. Therefore at present the utility of HSCT is limited.
Allogenic SCT
62. Autologous transplants in Non-
Haematological Malignancies
Breast Cancer :
Autologous BMT can produce complete responses in a higher proportion of patients with
breast cancer than seen with traditional doses of chemotherapy.Encouraging results of
incorporation of autologous BMT into primary therapy of patients who present with poor
prognostic factors have led to this treatment being tested in an adjuvant setting in
controlled trials.
Neuroblastoma :
Autologous and allogeneic BMT have been used with some success in patients with
advanced neuroblastoma.
Germ Cell Tumors :
Auto-HSCT is a standard of care for patients who are ( primary) refractory to platinum-
based chemotherapy or for those with a second or further relapse.
Solid tumors :
• Advanced ovarian cancer and limited or extensive small cell lung cancer - under
evaluation.
63. AL amyloidosis
•Patients with systemic immunoglobulin-light-chain (AL) amyloidosis
have been treated by auto-HSCT since 1994.
•A study with matched controls showed that amyloidosis patients
without severe heart failure benefited from high dose therapy and
auto- HSCT.
64. a) Severe systemic sclerosis
b) Multiple sclerosis
c) Systemic lupus erythematosus early in the disease course, with sustained or relapsed activity .
d) Crohn’s disease refractory to immunosuppressive agents and anti-TNF monoclonal antibodies.
e) Autoimmune cytopenias immune thrombocytopenia, autoimmune haemolytic anaemia and Evans’
syndrome refractory to at least two lines of treatment .
f) Rheumatoid arthritis, systemic vasculitis, dermatomyositis and polymyositis can be considered as
exceptional indications.
g) Chronic demyelinating inflammatory polyneuropathy and neuromyelitis optica, type 1 diabetes
mellitus, refractory type II coeliac disease, autism spectrum disorders potentially respond, but
experience of auto- HSCT has been relatively recent and patients should only be treated on approved
prospective clinical trials.
h) For paediatric patients, auto-HSCT is a Clinical Option for carefully selected subpopulations of
patients with juvenile inflammatory arthritis.
Autoimmune Diseases
66. Autologous Vs Allogenic HSCT
PARAMETER ALLOGENIC SCT AUTOLOGOUS SCT
Source of stem cells Family member/
Unrelated donor
Patient himself/herself
Age of recepient Young
<55
Wider range
can be done in older age g
roups ( has been tried upto 70
Main Indications AML, ALL, MDS NHL, HD, MM, solid tumors.
Risk of contamination of graft
with tumor cells
Absent Present
Risk of GVHD Present Absent
GVT effect Present Absent
Main complication GVHD Relapse
Overall procedure related
mortality
20-30% ( sibling), upto 45%
( unrelated)
5-10%
Treatment of inherited
diseases
Possible Not possible
Restoration of Immune
Function
Slower Rapid
67. Bone Marrow Transplant Unit
A. INFRASTRUCTURE
HSCU- The hematology/Oncology special care units : specialised units for
transplant recipients or other patients receiving aggressive therapy.
Each room within the HSCU is HEPA filtered with positive pressure : HEPA
air filter removes 99.97% of all particles greater than 0.3 microns from the air
that passes through.
B. STAFF
Immunologist, Hematologist, Intensivist , Pathologist and Medical
technologist- Transplant team
C. ISOLATION
