INTRODUCTION TO HAEMATOPOIETIC STEM CELL

TRANSPLANT(HSCT)

HSCT is an approved curative option for the

treatment of malignant and non-malignant diseases.

INTRODUCTION

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.

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 patient’s marrow.

REDUCED INTENSITY CONDITIONING(RIC)

Bone marrow stem cells(BMSC) Peripheral blood stem cells(PBSC) Umbilical cord blood(UCB)

SOURCES OF STEM CELLS

Harvest site- Pelvic bones under anaesthesia Multiple punctures into the pelvic bones. Mixed with special anticoagulant. Transplant by intravenous infusion.

BONE MARROW HERVEST/TRANSPLANTATION

FBC/Retics/Films. C-Reative Protein. E/U/Cr CMV Bilirubin BLOOD TRANSFUSION

SUPPORT Packed red cell Platelet 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 YOU

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.

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 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.

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.

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 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.

TYPES OF STEM CELLS

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.

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.

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 CELL Fertilized egg

2 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 CELLS

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

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

GOD BLESS YOU

ALL

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

UNDERSTANDING HLA

Types of histocompatibility test Serologic assays Microhymphocytotoxicty test Principles of micro----- Extraction of HLA for serologic assay

B. HISTOCOMPATIBILITY TESTS

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

(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 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.

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.

DISTRIBUTION OF MHC MOLECULES

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.

HLA AND RACE

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).

CLASSES OF MHC 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)

HLA CLASS I MOLECULES

HLA class I present antigens in form of peptide

to cytotoxic CD8+ T-cells in short cytoplasmic tail

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 IIThey are expressed on immunologically active cells to initiate a general immune response.

HLA CLASS II MOLECULES

Class I MHC are CD8+ cytotoxic cells, MHC are required

for recognition and destruction of cells infected by viruses and intracellular bacteria eg Tuberculi bacili

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

CLASS I MOLECULES/VIRAL OR BACTERIAL 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.

CLASS I MOLECULES /DONOR CELLS

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.

DIFFERENCES BETWEEN CLASS I AND II

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)

CLASS II MOLECULES

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.

ANTIGEN PRESENTING CELLS (APC)

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

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

GENETICS OF MHC MOLECULES

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.

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.

HISTOCOMPATIBILITY TESTING

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:

HLA/ BLOOD/ COMPONENTS TRANSPFUSION

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 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 ASSAYS

Cellular 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 ASSAYS

This 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 visible The lysed and non-lysed lymphocytes are

assessed using an inverse phase contact microscope.

PRINCIPLE OF MICROLYMPHOCYTOTOXICITY

TSET

Patient 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 bead

Remove the non-cell bead (i.e. non-magnetized cells.

EXTRACTION OF HLA –ANTIGENS FOR SEROLOGIC TEST

Wash the magnetized cell-bead in phosphate

buffer saline (PBS). Wash finally with non citrated PBS solution The solution now become colorless.

Organ transplantation Stem cell transplantation Disputed paternity test Immune regulation Disease associations Disease susceptibility Granulocyte and platelets transplantation.

IMPORTANT ASSOCIATION OF HLA

CONTENTS Chromosomes Structure of DNA Restriction endonucleases Molecular HLA typing Extraction of DNA (see h) Denaturation Hybridization Stringency conditions of DNA Probes Gel electrophoresis.

BASIC CONCEPTS OF MOLECULAR BIOLOGY

Normal 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 male

CHROMOSOMES

Chromosomes are threadlike bodies seen

within nuclei during cell divisions Each 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 DNA Nucleotides are linked together to form a linear chain of

nucleotides with one and called 5’ and the other 3’. E.g. 5’TAAGGCT 3’

The two end of nucleotides (DNA) have different

structure Two 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 bonds

The 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

together RE 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/ elagation

MOLECULAR HLA TYPING

Denaturation 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.

DENATURATION

Heating 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 DNA The 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 DNA

NB: Tetramethylammonium chloride (TMAC)

chemical which causes DNA to reanneal at a rate related only to it’s length that the time of annealing does not depend on the numbers of GC pairs

Is 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 DNA

Salt concentration Temperature 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 Conditions

Low 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 TEMPERATURE

Denaturation + 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.

PROBES

Detection 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 ELECTROPHORESIS

NB: 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 pairs

DNA 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 ug

DNA EXTRACTION

NB: Hemoglobin released by the blood cells interfere with the gene amplification reaction and must be washed out completely.