INTRODUCTION TO HAEMATOPOIETIC STEM CELL TRANSPLANT(HSCT)

1. INTRODUCTION TO HAEMATOPOIETIC STEM CELL TRANSPLANT(HSCT)

2. INTRODUCTION • HSCT is an approved curative option for the treatment of malignant and non-malignant diseases.

3. STEM CELLS • Are primitive and undifferentiated cells with the capacity of self-renewal. • Have regenerative influence on irreversible damaged tissues or severely damaged by genetic diseases. • Identified by the expression of CD34 antigens as adhesion protein to the marrow. • Hematopoietic stem cells(HSCs)express as CD34 cells. • Majority of CD34+ cells have limited proliferative capacity and restricted spectrum of cell type. • NB:- Only a small fraction of CD34 cells are capable of given rise to all peripheral blood cell lineages-pluripotent cells.

4. SUITABILITY/COMPATIBILITY • HLA- human leukocyte antigen is the human version of major histocompatibility complex(MHC) • HLA antigens are located on the short arm of chromosome 6. • Allogeneic donor (related or unrelated) suitability is determined by HLAtyping of both patient and donor. • It is desirable that HLA typing of the patient and donor are identical.

5. REDUCED INTENSITY CONDITIONING(RIC) • Induced bone marrow failure leading to aplasia. • Immune suppression of the patient to allow for the incoming graft components. • Creates space in the patient’s marrow.

6. SOURCES OF STEM CELLS • Bone marrow stem cells(BMSC) • Peripheral blood stem cells(PBSC) • Umbilical cord blood(UCB)

7. BONE MARROW HERVEST/TRANSPLANTATION • Harvest site- Pelvic bones under anaesthesia • Multiple punctures into the pelvic bones. • Mixed with special anticoagulant. • Transplant by intravenous infusion.

8. LAB. INVESTIGATIONS • FBC/Retics/Films. • C-Reative Protein. • E/U/Cr • CMV • Bilirubin • BLOOD TRANSFUSION SUPPORT • Packed red cell • Platelet Concentrate

9. IRRADIATION OF BLOOD/COMPONENTS • Inactivate T-cells. • NB: All blood/components for SCT transfusion must be irradiated with standard dose of 25GY.

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11. HAEMOPOIETIC STEM CELLS(HSC) • HSCs are derived from pluripotent stem cells capable of self renewal and differentiation into all haemopoietic lineages: myelopoiesis, monopoiesis,erythropoiesis, megakaryopoiesis and lymphopoiesis including stromal cells or dendritic cells. • Stem cells(SC) give rise to mature haematopoietic cells • Stem cells(SC) provide haematopoietic cells for the entire life span.

12. NB: Many blood disorders originated from SCs. • leukaemia, aplastic anaemia, myelodysplastic syndrome etc. • SCs are very rare representing less than 0.01% of all the bone marrow nucleated cells • Human SCs are expressed on the surface protein: CD34 and C –Kit and negative for CD38 and lineage –specific markers. • Majority of SCs are dormant under normal body condition. • Bone marrow in the main site of haematopoiesis and haematological disorders.

13. PRIMARY FUNCTION OF SC. • Maintainance of homeostasis • Replacement of dying cells due to injury or diseases • Adult stem cells behave differently depending on their local environment and tissue origin eg. HSC are located in the BM.

14. DEFINITION OF TERMS • TOTIPOTENT STEM CELLS :- Gives rise to all cells and tissues of the developing embryo ; the only type is the fertilized egg. • PLURIPOTENT STEM CELL:- It has the ability to give rise to cell originating from all the three germ layers: ectoderm , mesoderm and endoderm. • MULTIPOTENT STEM CELL:- eghaematopoitic stem cell can give rise blood cell of different lineages. • UNITIPOTENT STEM CELL:- Indicate a cell population usualy present in adulttissues capable of differentiating along only one lineage.

15. TYPES OF STEM CELLS There are two types of stem cells • The embryonic stem cells • The non- embryonic stem cells EMBRYONIC STEM CELL(ESC) • Embryonic stem cells are derived from the fertilized egg in the inner cell mass of blastocyst. • Blastocyst is a stage of an embryo- prior to implantation in the uterine wall. • ESC gives rise to all stem cells both embryonic and adult organs. • ESCs have unrestricted differentiation potential to support the development of a foetus. • ESC self- replication give rise to cells derived from all three germ layers. These cells proliferate extensively in the embryo.

17. They can be asolated and grown invivo where they continue to replicate and show potential to differentiate • ESCs can be propagatyed under invitro condition almost indefinitely • The culture of human pluripotent stem cells have active telemerase indicating ability to replicate for many generations.

18. NON- EMBRYONIC STEM CELL(NESC)Adult stem cells(ASC)/somatic stem cell/multipotent stem cell. • Adult stem cells originated from the embryo yolk sac • Yolk sac mesenchyme differentiate into endothelial cells and haematopioetic stem cells. • Adult stem cells have long- term self- renewal capacity (through life- time). • Give rise to mature cell types with specialized function. • Are undifferentiated cells capable of developing into other cell types of other tissues refered to as stem cell plasticity.

19. Generate intermidiate cell types (progenitor cells and differentiated precursors) before achieving their full differentiation. • Progenitor and precusor cells are regarded as commited to differentiate along specific cellular pathway. • Adult tissue specific stem cells is an important therapantic tool for regenerative medicine.

21. NB:- Amniocentesis fluid are rich source of multipotent, mesenchymal ,haemotopoietic, neural , epithelial and endothelial stem cells.

22. SOME ADULT TISSUE CONTAINING STEM CELLS Bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelial of skin and digestive system, cornea, retina, liver, pancreas, heart and central nervous system.

23. NB: In vertebrates most adult tissues and organs contain SCs capable of self- renewal, proliferation and differentiation into mature functional progeny.

24. SCs are more abundant in tissues with a high renewal rate e.g blood, epithelia or vasculature; and less abundant in tissue or organs with little renewal capacity e.g. myocardial muscle or central nervous system.

25. NORMAL RANGE OF STEM CELLS • BONE MARROW STEM CELL 1) Nucleated cell count:- 2 – 4.5x10 8/kg body weight 2) CD34+ cell count :- 2.8 – 4.5x 10 6/kg body weight 3)T. cell count :- 2.2- 4.5x10 7/kg body weight

26. PERIPHERAL BLOOD STEM CELL 1)Nucleated cell count: - 9x10 8/kg body weight 2)CD34+ cell count :- 7x 10 6/kg body weight 3)T. cell count :- 2.7 x10 7/kg body weight

27. CORD BLOOD STEM CELL 1)Nucleated cell count :- 0.3x10 8/kg body weight 2)CD34+ cell count : 0.2x10 6/kg body weight 3)T. cell count :- 0.4x10 7/kg body weight

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29. UNDERSTANDING HLA • CONTENT • Distribution of MHC molecules • HLA and Race • Classes of MCH molecules • HLA class I molecules • HLA class II molecules • Class I molecules and viral or bacteria cells • Different between class I and II • Class II molecules • Genetics of MHC molecules

30. B. HISTOCOMPATIBILITY TESTS • Types of histocompatibility test • Serologic assays • Microhymphocytotoxicty test • Principles of micro----- • Extraction of HLA for serologic assay

31. (HLA) • Human leukocyte antigen (HLA) are the human version of the major histocompatibility complex (MHC) • MHC molecules are cell surface glycoproteins that present a protein antigen called antigenic peptide to T-cells. • HLA are located at the short arm of chromosome 6(6p21:31) • HLA region is a multigenic system that encode structurally homologous cell surface glycoproteins

32. HLA genes are located within MHC class 1 and 11 region with different proteins MHC antigens are integral to the normal functioning of the immune response RULE OF HLA • HLA antigens lie in the control of self recognition and defence against microorganisms and survellance. • MHC molecules allow T-cells to recognize protein antigens which they are unable to recognize on their own • T-cell upon the recognition of the protein antigens can direct the destruction of antigens.

33. DISTRIBUTION OF MHC MOLECULES • MHC molecules are found on almost all body cells • I n blood MHC are present on leukocytes and platelet • T hey are not present on mature d red cells.

34. HLA AND RACE • HLA cause humans to be immunologically different from one another • Differences in HLA between donor and recipient provide important antibody information and activation of cytotoxic T-cells against donor cells bearing foreign HLA. • MHCs form the most polymorphic genes in humans which make it difficult to find MHC match donor within the general population.

35. CLASSES OF MHC MOLECULES • There are three classes of MHC molecules; class I, I I and III. • The class I and II are essential for presentation of antigenic peptides to T-cells for the generation of immune response. Class III molecules produce complement proteins and certain cytokines e.g tumor necrosis factors (TNF).

36. HLA CLASS I MOLECULES • Class I molecules is made up of HLA-A,-B and –C, others are :HLA –E,-F,-G and –H • HLA –A B C are most significant in transplantation • Found in almost all nuclaeted cells and have the soluble form in plasma. • All the circulating peripheral blood lymphocytes contain HLA-ABC • While the B- lymphocytes contain HLA class I (HLA-ABC) and class II (HLA- DR, DQ) • HLA-B is most significant followed by-A and C. • They are adsorbed onto platelets (some antigens more readily than others)

37. HLA class I present antigens in form of peptide to cytotoxic CD8+ T-cells in short cytoplasmic tail

38. HLA CLASS II MOLECULES • Found on the immune competent cells ; B-and activated T-lymphocytes, macrophages, endothelial cells • They have five loci; HLA- DQ,DP, DM and DO. HLA-DR,-DQ and DP are most important. • HLA class II molecules present peptide to CD4+ T-helper cells. (i.e general immune reaction with, cellular and humoral defence). HLA class II presentation aids the helper function to set up general immune response. ROLE OF HLA CLASS II They are expressed on immunologically active cells to initiate a general immune response.

39. CLASS I MOLECULES/VIRAL OR BACTERIAL CELLS • Class I MHC are CD8+ cytotoxic cells, MHC are required for recognition and destruction of cells infected by viruses and intracellular bacteria egTuberculibacili • The intracellular microorganisms produce antigens peptide s that bind to class I MHC complex to form complexes (bacteria antigen –MHC complex). • These bacteria antigen-MHC complex migrate to cell surface for presentation to and recognition by a CD8+cytotoxic T-cell • CD8+T-cell recognize only antigenic peptides bound to MHC class I molecules called MHC class I restricted

40. CLASS I MOLECULES /DONOR CELLS • When donor cell carrying class I molecules are transplanted ,they stimulate antibody production and cytotoxic T-cell activation by the recipient. • The antibody production and cytotoxic T-cell activation cause destruction of donor cells by lysis. • The most powerful immune response is directed against MHC I molecules coded for by genes of the A and B loci on chromosome 6.

41. DIFFERENCES BETWEEN CLASS I AND II • They differ in their moleculal structures • They are found in different body cells • They present different types of antigens to either CD4+ or CD8+T-cells.

42. CLASS II MOLECULES • Class II MHC are CD4+T-cell which are CD4+helper T- cell • These molecules are essential for CD4+T-cell recognition of an antigenic peptide • In the absence of class II MHC , CD4+ helper T-cells can not produce an immune response to protein antigen CD4+ helper cells must have protein antigens presented to them by class II molecules on surface of antigen presenting cells (APC)

43. ANTIGEN PRESENTING CELLS (APC) • APCs are B-cells, monocytes, macrophages, activated T- cells , dendritic cells and endothelial cells etc. • APCs phagotize exogenous antigens (extracellular microorganisms) and their toxins • The antigenic peptide is bound to a class II MHC molecules to form complex • The complex migrate to the cell surface to present antigen CD4+helper T-cells. • CD4+helperT- cells recognize only antigenic peptide attached to MHC class II molecules.

44. CD4+T-cells are called MHC class I restricted • The most powerful immune response is directed against MHC class II molecule coded for by genes at DR locus on chromosome 6

45. GENETICS OF MHC MOLECULES • Major histocompatibility molecules are produced by group of genes very closely together on chromosome 6 called linked genes. • These molecules are inherited together as a block of genes called haplotype NB: Linked genes are genes located close together on a chromosome. • Individual inherits two genes for MHC molecules each from the parents • An offspring receives either one of the parental haplotypes

46. An offspring(sibling) have a 25% chance of sharing the same MHC genes , hence the first search when requiring organ transplant. • Sibling may or may not share the same HLA • Identical twins always share the same HLA genes and are called syngeneic • MHC are codorminant genes and share equal expression in the MHC molecules produced • MHC form the most polymorphic genes known in humans(greater than 100 million MHC alleles) • The MHC polymorphisms make it difficult to find an MHC match donor within the population.

47. HISTOCOMPATIBILITY TESTING • Donor-recipient matching is determine by histocompatibility testing to find out a better match for the recipient The test identifies class I and II HLA on the cells of the recipient and the prospective donor TYPES OF HISTOCOMPATIBILITY TESTS. • Serological • Cellular • Molecular (DNA) assays.

48. HLA/ BLOOD/ COMPONENTS TRANSPFUSION • Blood/components transfusion can cause alloimmunization to donor HLA. • The donor HLA expressed on platelets and leukocytes in the blood/ components transfused can develop alloantibody on the recipient • The alloantibodies develop from the first transfusion will attack any alloantigen on the component leading to it destruction e.g. platelet refractoriness. • Transfusion reaction resulting from HLA alloantigen's and alloantibodies are:

49. Febrile nonhaemolytic transfusion reaction, • Platelet refractoriness, • transfusion- related acute lung injury (TRALI) Transfusion- related graft versus host disease (TA-GvHD). ROLE OF LEUKODEPLETION OF BLOOD/COMPONENTS • Leuko-reduction filters help to prevent alloimmunization to hla by leukocyttesremovAL from blood/components.

50. (I.) SEROLOGIC ASSAYS • This identifies class I and certain class II HLA and antibodies to HLA. • Lymphocytotoxicity test is a serologic assay that identifies HLA –A,-B and –C class I and class IIHLA-DR and DQ.