HSCT

INTRODUCTION TO HAEMATOPOIETIC STEM CELL TRANSPLANT(HSCT)1HSCT is an approved curative option for the treatment of malignant and non-malignant diseases.INTRODUCTION2Are 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.

STEM CELLS

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.

SUITABILITY/COMPATIBILITY

Induced bone marrow failure leading to aplasia. Immune suppression of the patient to allow for the incoming graft components.Creates space in the patients marrow.

REDUCED INTENSITY CONDITIONING(RIC)Bone marrow stem cells(BMSC)Peripheral blood stem cells(PBSC)Umbilical cord blood(UCB)SOURCES OF STEM CELLSHarvest site- Pelvic bones under anaesthesiaMultiple punctures into the pelvic bones.Mixed with special anticoagulant.Transplant by intravenous infusion. BONE MARROW HERVEST/TRANSPLANTATIONFBC/Retics/Films.C-Reative Protein.E/U/CrCMVBilirubin BLOOD TRANSFUSION SUPPORTPacked red cellPlatelet Concentrate

LAB. INVESTIGATIONS

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

THANK YOUHSCs 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 cellsStem cells(SC) provide haematopoietic cells for the entire life span.HAEMOPOIETIC STEM CELLS(HSC)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 cellsHuman 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.Maintainance of homeostasisReplacement of dying cells due to injury or diseasesAdult stem cells behave differently depending on their local environment and tissue origin eg. HSC are located in the BM.PRIMARY FUNCTION OF SC.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:- eg haematopoitic 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.DEFINITION OF TERMS There are two types of stem cellsThe embryonic stem cellsThe 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.

TYPES OF STEM CELLSThey can be asolated and grown invivo where they continue to replicate and show potential to differentiateESCs can be propagatyed under invitro condition almost indefinitelyThe culture of human pluripotent stem cells have active telemerase indicating ability to replicate for many generations.Adult stem cells originated from the embryo yolk sacYolk 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.NON- EMBRYONIC STEM CELL(NESC)Adult stem cells(ASC)/somatic stem cell/multipotent stem cell.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. TYPES SOURCES 1 EMBRYONIC STEM CELLFertilized egg2 NON EMBRYONIC STEM CELL bone marrow stem cell(BMSC)Peripheral blood stem cell (PBSC)Umblical Cord blood (UCB)Amniotic stem cell(ASC)Skill stem cell(SSC)Menstrual stem cell (MSC)Advipose stem cell (AdSC)

Borne marrow eg Pelvic and Stemum bonesBlood VesselsUmblical cordAmniocentic fluidSkillmenstrual fluidAdvipose tissue NB:- Amniocentesis fluid are rich source of multipotent, mesenchymal ,haemotopoietic, neural , epithelial and endothelial 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.

SOME ADULT TISSUE CONTAINING STEM CELLS NB: In vertebrates most adult tissues and organs contain SCs capable of self- renewal, proliferation and differentiation into mature functional progeny.

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. myo cardial muscle or central nervous system.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 NORMAL RANGE OF STEM CELLSPERIPHERAL 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 weight26CORD 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 weight27GOD BLESS YOU ALL CONTENTDistribution of MHC moleculesHLA and RaceClasses of MCH moleculesHLA class I moleculesHLA class II moleculesClass I molecules and viral or bacteria cellsDifferent between class I and IIClass II moleculesGenetics of MHC molecules

UNDERSTANDING HLATypes of histocompatibility testSerologic assaysMicrohymphocytotoxicty testPrinciples of micro-----Extraction of HLA for serologic assay

B. HISTOCOMPATIBILITY TESTSHuman 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

(HLA)HLA genes are located within MHC class 1 and 11 region with different proteinsMHC antigens are integral to the normal functioning of the immune response RULE OF HLAHLA 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 ownT-cell upon the recognition of the protein antigens can direct the destruction of antigens. MHC molecules are found on almost all body cellsI n blood MHC are present on leukocytes and plateletT hey are not present on mature d red cells.DISTRIBUTION OF MHC MOLECULES33HLA cause humans to be immunologically different from one anotherDifferences 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. HLA AND RACEThere 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).

CLASSES OF MHC MOLECULESClass I molecules is made up of HLA-A,-B and C, others are :HLA E,-F,-G and HHLA A B C are most significant in transplantationFound in almost all nuclaeted cells and have the soluble form in plasma.All the circulating peripheral blood lymphocytes contain HLA-ABCWhile 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)

HLA CLASS I MOLECULESHLA class I present antigens in form of peptide to cytotoxic CD8+ T-cells in short cytoplasmic tailFound on the immune competent cells ; B-and activated T-lymphocytes, macrophages, endothelial cellsThey 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 IIThey are expressed on immunologically active cells to initiate a general immune response.

HLA CLASS II MOLECULESClass I MHC are CD8+ cytotoxic cells, MHC are required for recognition and destruction of cells infected by viruses and intracellular bacteria eg Tuberculi baciliThe 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-cellCD8+T-cell recognize only antigenic peptides bound to MHC class I molecules called MHC class I restrictedCLASS I MOLECULES/VIRAL OR BACTERIAL CELLSWhen 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.CLASS I MOLECULES /DONOR CELLS40They differ in their moleculal structuresThey are found in different body cellsThey present different types of antigens to either CD4+ or CD8+T-cells. DIFFERENCES BETWEEN CLASS I AND IIClass II MHC are CD4+T-cell which are CD4+helper T- cellThese molecules are essential for CD4+T-cell recognition of an antigenic peptideIn the absence of class II MHC , CD4+ helper T-cells can not produce an immune response to protein antigenCD4+ helper cells must have protein antigens presented to them by class II molecules on surface of antigen presenting cells (APC)

CLASS II MOLECULESAPCs are B-cells, monocytes, macrophages, activated T- cells , dendritic cells and endothelial cells etc.APCs phagotize exogenous antigens (extracellular microorganisms) and their toxinsThe 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.

ANTIGEN PRESENTING CELLS (APC)CD4+T-cells are called MHC class I restrictedThe most powerful immune response is directed against MHC class II molecule coded for by genes at DR locus on chromosome 6Major 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 parentsAn offspring receives either one of the parental haplotypes

GENETICS OF MHC MOLECULESAn 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 HLAIdentical twins always share the same HLA genes and are called syngeneicMHC are codorminant genes and share equal expression in the MHC molecules producedMHC 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.Donor-recipient matching is determine by histocompatibility testing to find out a better match for the recipientThe test identifies class I and II HLA on the cells of the recipient and the prospective donor TYPES OF HISTOCOMPATIBILITY TESTS.SerologicalCellularMolecular (DNA) assays.HISTOCOMPATIBILITY TESTINGBlood/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 recipientThe 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:

HLA/ BLOOD/ COMPONENTS TRANSPFUSIONFebrile nonhaemolytic transfusion reaction,Platelet refractoriness,transfusion- related acute lung injury (TRALI)Transfusion- related graft versus host disease (TA-GvHD). ROLE OF LEUKODEPLETION OF BLOOD/COMPONENTSLeuko-reduction filters help to prevent alloimmunization to hla by leukocyttes removAL from blood/components.

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.(I.) SEROLOGIC ASSAYSCellular assay identifies the degree of similarity and not specific HLA identification between donor-recipient HLA class II DP,DQ and DR.These HLA are identified on lymphocytes hence called cellular defined antigens.Mixed lymphocyte culture ( or mixed lymphocyte reaction are various cellular assays used to identify the similarity between donor recipient.(ii)CELLULAR ASSAYSThis method uses DNA fragments to identify HLA gene.Molecular assay are very specific and highly sensitive but complex system. (III.) MOLECULAR TESTING HISTORY /POINTS TO NOTE:Lymphocyte HLA is use for detection of human antigens in a complement-dependent microlymphocytotoxicity test.Lymphocyte HLA consist of micro plates containing pre-loaded anti-HLA reagents e.g. monoclonal HLA antibodies or human polyclonal HLA antisera.HLA antigens are glycoprotein present on the cell membrane and are divided into two classes-class I and class II.Class I antigens (HLA-A,-B and C) are found on almost all nucleated cells.MICROLYMPHOCYTOTOXICITY TEST Class II antigens (HLA-DR and DQ) are found on the surface of only a few cell populations.All circulating lymphocytes in peripheral blood contain HLA-A, -B and C antigens.B-lymphocytes possess HLA DR and DR antigens in addition.In HLA A,-B and C typing, either a whole lymphocyte or a T-lymphocyte populations can be used.For HLA DR and DQ typing , a sample rich in B-lymphocytes must be used as these account for only 10-15% of the whole lymphocyte population.Standard HLA specificity antigens are incubated with a sample of lymphocyte suspension in the present of complement.The lymphocytes are lysed in the present of corresponding antibody and complement.A stain (e.g. eosin) is added to makes it visibleThe lysed and non-lysed lymphocytes are assessed using an inverse phase contact microscope.PRINCIPLE OF MICROLYMPHOCYTOTOXICITY TSETPatient HLA antigens + standard HLA antibodies complement incubation and stain lysed and non-lysed colored cells.Centrifuge patient heparinized whole blood and remove the plasma. Dispense packed cells in a 10ml tube and add 100ul Dynabeads of various HLA class I into each tube.Mix by inversion and incubate at room temperature for 90 sec.Place the tube in a Dynal magnetic particles concentrator to attract the cells to beadRemove the non-cell bead (i.e. non-magnetized cells.EXTRACTION OF HLA ANTIGENS FOR SEROLOGIC TESTWash the magnetized cell-bead in phosphate buffer saline (PBS).Wash finally with non citrated PBS solution The solution now become colorless.

Organ transplantationStem cell transplantation Disputed paternity testImmune regulation Disease associationsDisease susceptibilityGranulocyte and platelets transplantation.

IMPORTANT ASSOCIATION OF HLA CONTENTSChromosomesStructure of DNA Restriction endonucleasesMolecular HLA typingExtraction of DNA (see h)DenaturationHybridizationStringency conditions of DNAProbesGel electrophoresis.

BASIC CONCEPTS OF MOLECULAR BIOLOGYNormal human somatic cells contain 46 chromosomes arranged in pairs.One of each pair is inherited from the father and other from the mother .The paired chromosomes are 22 autosomes and I pair of sex chromosomes :(X and Y) 22 X+X for female 22 X+Y for maleCHROMOSOMESChromosomes are threadlike bodies seen within nuclei during cell divisionsEach chromosome has a bifid structure formed by two chromatids lying side by side and linked at one point, the centromere.Individual chromosome consists of supercoiled DNA strand.DNA strand is a double helix STRUCTURE OF DNA

Deoxyribonucleic acid (DNA) is composed of a phosphate and sugar (deoxyribose) back bone attached to bases (adenine, thymine cytosine , guanine)The combination of base, a sugar and a phosphate group is called a nucleotide e.g. deoxyadenosine triphosphate ( dATP) and deoxyguanosine triphosphate (dGTP)Nucleotides are basic building blocks of DNANucleotides are linked together to form a linear chain of nucleotides with one and called 5 and the other 3. E.g. 5TAAGGCT 3

The two end of nucleotides (DNA) have different structureTwo strands of DNA form a double helix (or ladder) by base pairing with hydrogen bonds (G=C and A=T).The hydrogen bonds linking G=C and A=T are 3 and 2 respectively making the G-C bonds harder than A-T bondsThe two strand of DNA that form a double helix run in opposite directions and are complementary to one another.Restriction endonucleases are enzymes that cut double strand DNA at a specific sequence of base pairs called Palindromes.Palindromes sequence read the same forwards (coding strand) as backwards (non-coding strand) ---GAATTC--- (coding strand) ---CTTAAG--- (noncoding strand)Restriction enzyme (RE) cleaved ends are blunt ( or flat) or have a 5 or 3 single strand protrusion (cohesive or sticky end

RESTRICTION OF ENDONUCLEASES (RE)Any blunt ends DNA fragments can be ligated togetherRE will not cut DNA which restriction site appears at the end of a DNA fragment .The molecular HLA typing process requires a post amplification to discriminates between the different alleles using a group specific primer.The process of amplification involves ; i. denaturation ii.annealing/ hybridization iii.extension/ elagationMOLECULAR HLA TYPINGDenaturation disrupts the hydrogen bonds that hold the bases of the double stranded DNA together .Factors Affecting Denaturation Are; i. heating the DNA ii. Treating DNA with alkali e.g. sodium hydroxide (NaOH). iii. Using polar solvents e.g. dimethyl sulfoxide (DMSo) or formamide.DENATURATIONHeating DNA at 94% or higher will denature all double stranded DNA regardless of the length or base composition.Melting temperature (TM) ; Temperature at which 50% of DNA is hybridized (DNA in double form) and 50% is denatured (i.e. separated into two strands)Melting temperature is influenced by the base composition and the length of the double stranded DNA . Hybridization reannealing of DNA regenerates the base pairs and yields double stranded DNAThe efficacy of hybridization depend on : i. concentration of DNA ii. Time taken by hybridization iii. Base composition of DNA ;stretches of GC pairs tend to anneal faster than stretches of AT pairs to form double stranded DNA.HIBRIDIZATION OR REANNEALING OF DNANB: Tetramethylammonium chloride (TMAC) chemical which causes DNA to reanneal at a rate related only to its length that the time of annealing does not depend on the numbers of GC pairsIs refers to as the binding of a single strands DNA to another single strands DNA under: i. low stringency ii. High stringency Low stringency condition is when single strands of DNA bind imperfectly matched single strands DNA.High stringency condition is when the single strands DNA bind perfectly match DNA single strands.STRINGENCY CONDITIONS OF DNASalt concentrationTemperature of the reactionSALT CONCENTRATION: High salt concentrations allow imperfectly matched hybrids to be form in low salt concentration , only perfectly matched hybrids are formed.TEMPERATURE OF THE REACTION: High temperature favor's DNA strands rapid movement .The imperfectly matched DNA hybrids will dissociate more rapidly at a high temperature. High temperature favor's the generation of perfectly matched hybrids. NB: If the temperature is too high , the two strands will not reanneal.

Factors Affecting Stringency ConditionsLow salt and high temperature create high stringency conditions The stringency of the match is control during the incubation of single stranded ( hybridization ) or during the wash following hybridization reaction.The final high stringency wash is often carried out 3-5 C below melting temperature to keep the perfectly matched strands hybrids but eliminates imperfectly matched hybrids.LOW SALT AND HIGH TEMPERATUREDenaturation + single strands synthetic DNA (oligo)Hybridization control stringency wash to remove imperfectly matched hybrids . Probes are oligonucleotides single stranded DNA used to detect specific DNA sequence by hybridization .They have 12- 26 nucleotides in length and are called sequence specific digonucleotideprobes (SSOP)One strand of the DNA is usually attached to a solid support ( e.g. membrane or bead) to increase the speed of hybridization and aid detection of a successful hybridization reaction.PROBESDetection of hybridization is performed by using the radioactive phosphate or unusual nucleotide to a probe.Adding label to a probe during or after synthesis of a probe.Gel electrophoresis is the standard method to separate , identify and purify DNA fragments.DNA is negatively charged (due to the phosphate backbone ) and moves away from the cathode towards the anode in an electric field.DNA are identified by their sizes and small pieces of DNA move faster than large pieces of DNA.DNA visualized on the gel by Uv light after staining with ethidium bromide or X- ray film when the DNA IS labeled with radioactive isotope.GEL ELECTROPHORESISNB: i. Polyacrylamide gel are used to separate small DNA fragment (5-500 base pairs) and are used for DNA sequencing or digonucleotide purification. ii. Agarose gel are used for DNA from 200- 50,000 base pairsDNA can be extracted from peripheral blood , bone marrow and cultured cells (B-and T-lymphocytes)Sample containers: EDTA, citrate and heparin etc can be used.DNA extracted from cells is by the use of organic solvents or salt precipitation and aqueous DNA is purified by ethanol precipitation.After extraction DNA concentration is measured in ugDNA EXTRACTIONNB: Hemoglobin released by the blood cells interfere with the gene amplification reaction and must be washed out completely.