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TISSUE TYPING
K R MICRO NOTES
1
INTRODUCTION
➔ Organ and Tissue donation is often referred to as the gift of
life, for good reason.
➔ A single donor has the potential to save up to nine lives
through organ donation and heal more than 75 lives
through tissue donation.
➔ While the steps to sign up to be an organ donor are simple,
the scientific process of matching donor and recipients for
transplant is a complex one.
➔ It begins with TISSUE TYPING…
2
3
WHAT IS TISSUE TYPING ?
Medical definition of tissue typing :
➔ The determination of the degree of
compatibility of tissues or organ from
different individuals based on the
similarity of Histocompatibility
antigens especially on lymphocytes
and used especially as a measure of
potential rejection in an organ
transplant procedure.
4
TISSUE TYPING
➔ Tissue typing ensures that an organ from a donor will be compatible
with its recipient.The process starts with identifying the unique Human
Leukocyte Antigens[HLA] for the organ donor and recipient, either
from blood or tissue.
➔ This test is also called Human Leukocyte Antigen [HLA] typing.
➔ The genetic loci involved in the rejection of foreign organs are known
as the Major Histocompatibility Complex-MHC. The Human MHC is
called the HLA system because these antigens were first identified and
characterized using alloantibodies against leukocytes.
➔ The Human MHC maps to short arm of chromosome and spans
approximately 3,600 Kilobases of DNA.
5
TISSUE TYPING
➔ HLA acts as barcode to distinguish ‘Self’ from ‘Nonself’.
➔ To date, more than 35,000 variations of HLA protein have been
identified. This makes it extremely difficult to find perfect HLA-
matched donor for transplants.
➔ During HLA typing, the recipient’s blood is checked for presence of
preformed anti-HLA antibodies, created by the immune system
when exposed to foreign tissue (Usually by a previous transplantation,
pregnancy or blood transfusion).
6
SNEAK PEEK OF TISSUE TYPING HISTORY…
➔ Tissue matching was first suggested by Alexis Carrel and was
studied in animals by George Snell [Snell 1948].
➔ It began to emerge as a reality for human transplants in 1958,
when Jean Dausset discovered the first Human Leukocyte
Antigen[HLA][Dausset 1958].
➔ Antibodies against this antigen were identified in transfused
patients and multiparous women by Rose Payne [1957] and Jon
Van Rood [Van Rood et al. 1958].
➔ In 1960s,UCLA professor Paul Terasaki invented a tissue typing
test that became an international standard for matching
7
➔ He developed a microcytotoxicity assay in
1964 [Terasaki 1964].
➔ His test, which mixed recipient serum and
donor lymphocytes in tiny wells quickly
became standard.
➔ For several years, Terasaki did the typing for
most U.S. transplant centres.
➔ Histocompatibility matching remains
crucial in bone marrow transplantation and
important in selection of family
donors.Unless their is perfect match it is less
Paul Terasaki
8
HLA TYPING
➔ In this test, donor’s antigens expressed on the surface of
leukocytes or genes are matched with that of the recipient.
➔ The closer the HLA antigens on the transplanted organ match the
recipient, the more likely that recipient’s body will not reject the
transplant.
➔ Value of HLA matching between donor & recipient varies in
different solid organ transplantation. In kidney transplants, there
is substantial benefit if all the polymorphic HLA alleles are
matched.
9
➔ Every person inherits each of the following
antigens from each parent:
➔ HLA-A antigen
➔ HLA-B antigen
➔ HLA-C antigen
➔ HLA-DR antigen
➔ HLA-DQ antigen and
➔ HLA-DP antigen
➔ 6 HLA antigen are looked at for each person
➔ When performing an HLA typing test for a
kidney transplant, HLA-A, HLA-B and HLA-DR
antigens are looked majorly.
10
➔ By analyzing which of these 6 HLA
antigens both the donor and
recipient have, scientists are able
to determine the closeness of
tissue typing.
➔ 6 antigen match is best when took
from blood cord and usually
doctors recommend 8 to 10
antigens for best compatibility.
➔ This match occurs 25% for siblings
and 50% for parents.
11
12
TISSUE TYPING METHODS
➔ PHENOTYPIC METHODS
◆ Serological method
◆ Mixed lymphocyte culture (MLC)
➔ MOLECULAR METHODS
◆ Sequence specific oligonucleotide probe hybridization
◆ Sequence specific primers
◆ Sequence based typing
13
PHENOTYPIC METHODS:
1. SEROLOGICAL METHODS:-
➔ Potential donor’s and recipient’s lymphocytes are isolated from blood
by immunomagnetic beads coated by monoclonal antibodies
against T cells or B cells.
➔ Once the cells adheres to beads a strong magnet is used to pull them
on to the surface of test tube.
➔ All the antibodies to unwanted cells are washed and only T
lymphocytes are obtained.
➔ Cells are then stained with carboxyfluorescein in fluorescence test.
➔ These cells are added to pre dotted tissue typing tray (microtitre
plates).
➔ These trays contain highly selected antisera either from pregnant 14
➔ Each antibody reacts to an epitope which is unique to a certain HLA
specificity.
➔ Typically, the tray will have 72 or 96 different antisera with 0.001ml
volume.
➔ Cells are incubation for 30 minutes, then 0.005ml rabbit
complement is added and incubated for an hour.
➔ Ethidium bromide or propidium iodide is added and read by
fluorescence test.
➔ Living cells stains with carboxyfluorescein while dead cells stains red
with ethidium bromide.
➔ In eosin dye test dead cells are stained red with eosin and living cells
appear bright white under phase-contrast microscope.
➔ In both tests, dead cells denote a positive reaction in which Ab and
complement reacted. 15
16
2. MIXED LYMPHOCYTE CULTURE (MLC)-
➔ It has been observed that lymphocytes from one donor,when
cultured with lymphocytes from unrelated donor stimulates
proliferation.
➔ This is due to a disparity in class II MHC [DR] antigens and T cells of
one individual interacts with allogenic class II MHC antigen bearing
cells [B cells, dendritic cells, langerhans cells, etc] of another.
➔ This reactivity is also known as Mixed Leukocyte Reaction[ MLR].
➔ In this test, lymphocytes [responder cells] of donor are mixed with
irradiated or mitomycin C which is treated leukocytes from the
recipient, containing B-lymphocytes and monocytes [stimulator cells].17
➔ Cells are cultured for 4-6 days.
➔ Responder cell will recognise the foreign class II antigens found
on the donor and undergoes transformation and proliferation
[mitogenesis].
➔ The T cells that respond to foreign class II antigens are typically
CD4+ TH-1 type cells.
➔ These changes are recorded by addition of radioactive (tritiated,
3H) thymidine into the culture and monitoring its incorporation
into DNA.
18
19
MOLECULAR METHODS
➔ DNA based procedures give accurate HLA typing and lead to the
identification of serologically undetected alleles and of many
subtypes of serological specificity.
➔ Focused on analysis of nucleotide variation occurring in both exon 2
and 3 of class I genes and exon 2 of class II genes.
➔ Techniques are based on PCR polymorphism.
➔ All the techniques uses same method for extraction of genomic DNA
and Amplification of gene of interest and only deferes in detection of
sequence polymorphism that defines the alleles.
20
The types are :-
➔ Hybridization with Sequence Specific Oligonucleotide Probes
[SSOP]
➔ Sequence Specific primers [SSP]
➔ Sequence Based Typing [SBT]
21
1. SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBES [SSOP]:-
➔ Method involves selective amplification of target followed by
hybridization to a panel of oligonucleotide probes.
➔ Specificity for a particular HLA locus is achieved by selecting PCR
primers specific for a sequence in conserved region of the second
exon.
22
23
2. SEQUENCE SPECIFIC PRIMING [SSP]:-
➔ It's a rapid method of HLA typing that uses sets of primer pairs to
amplify region of genomic DNA.
➔ The efficiency of the amplification reaction is controlled by the
primers that amplify conserved sequences of a selected gene.
24
25
3. SEQUENCE BASED HLA TYPING:-
➔ Involves determining the nucleotide sequence of an amplified
segment of an HLA gene.
➔ It is advantageous over other procedures because of relatively fast [
in 24-48 hrs] with high level resolution.
➔ It is more reliable and specific method.
26
27
CLINICAL SIGNIFICANCE OF HLA TYPING
➔ In organ transplantation
➔ In transfusion therapy
➔ Disease association
➔ Disputed paternity
➔ In cancer prevention
➔ Anthropological studies
28
REFERENCES
➔ Encyclopedia of Immunology, Second Edition by Peter J Delves and
Ivan M Roitt
➔ Immunology, Fifth Edition by Richard A Goldsby, Thomas J Kindt,
Barbara A. Osborne and Janis Kuby
➔ https://www.slideshare.net/drnisha22/hla-92546736
➔ https://www.slideshare.net/debbarma1989/hla-typing-and-its-role-
in-tissue-transplantation
➔ https://wexnermedical.osu.edu/blog/tissue-typing
➔ https://www.invent.org/inductees/paul-
terasaki#:~:text=In%20the%201960s%2C%20UCLA%20professor,pote
ntial%20organ%20donors%20with%20recipients.
➔ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684003/
29
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TISSUE TYPING .pptx

  • 1. TISSUE TYPING K R MICRO NOTES 1
  • 2. INTRODUCTION ➔ Organ and Tissue donation is often referred to as the gift of life, for good reason. ➔ A single donor has the potential to save up to nine lives through organ donation and heal more than 75 lives through tissue donation. ➔ While the steps to sign up to be an organ donor are simple, the scientific process of matching donor and recipients for transplant is a complex one. ➔ It begins with TISSUE TYPING… 2
  • 3. 3
  • 4. WHAT IS TISSUE TYPING ? Medical definition of tissue typing : ➔ The determination of the degree of compatibility of tissues or organ from different individuals based on the similarity of Histocompatibility antigens especially on lymphocytes and used especially as a measure of potential rejection in an organ transplant procedure. 4
  • 5. TISSUE TYPING ➔ Tissue typing ensures that an organ from a donor will be compatible with its recipient.The process starts with identifying the unique Human Leukocyte Antigens[HLA] for the organ donor and recipient, either from blood or tissue. ➔ This test is also called Human Leukocyte Antigen [HLA] typing. ➔ The genetic loci involved in the rejection of foreign organs are known as the Major Histocompatibility Complex-MHC. The Human MHC is called the HLA system because these antigens were first identified and characterized using alloantibodies against leukocytes. ➔ The Human MHC maps to short arm of chromosome and spans approximately 3,600 Kilobases of DNA. 5
  • 6. TISSUE TYPING ➔ HLA acts as barcode to distinguish ‘Self’ from ‘Nonself’. ➔ To date, more than 35,000 variations of HLA protein have been identified. This makes it extremely difficult to find perfect HLA- matched donor for transplants. ➔ During HLA typing, the recipient’s blood is checked for presence of preformed anti-HLA antibodies, created by the immune system when exposed to foreign tissue (Usually by a previous transplantation, pregnancy or blood transfusion). 6
  • 7. SNEAK PEEK OF TISSUE TYPING HISTORY… ➔ Tissue matching was first suggested by Alexis Carrel and was studied in animals by George Snell [Snell 1948]. ➔ It began to emerge as a reality for human transplants in 1958, when Jean Dausset discovered the first Human Leukocyte Antigen[HLA][Dausset 1958]. ➔ Antibodies against this antigen were identified in transfused patients and multiparous women by Rose Payne [1957] and Jon Van Rood [Van Rood et al. 1958]. ➔ In 1960s,UCLA professor Paul Terasaki invented a tissue typing test that became an international standard for matching 7
  • 8. ➔ He developed a microcytotoxicity assay in 1964 [Terasaki 1964]. ➔ His test, which mixed recipient serum and donor lymphocytes in tiny wells quickly became standard. ➔ For several years, Terasaki did the typing for most U.S. transplant centres. ➔ Histocompatibility matching remains crucial in bone marrow transplantation and important in selection of family donors.Unless their is perfect match it is less Paul Terasaki 8
  • 9. HLA TYPING ➔ In this test, donor’s antigens expressed on the surface of leukocytes or genes are matched with that of the recipient. ➔ The closer the HLA antigens on the transplanted organ match the recipient, the more likely that recipient’s body will not reject the transplant. ➔ Value of HLA matching between donor & recipient varies in different solid organ transplantation. In kidney transplants, there is substantial benefit if all the polymorphic HLA alleles are matched. 9
  • 10. ➔ Every person inherits each of the following antigens from each parent: ➔ HLA-A antigen ➔ HLA-B antigen ➔ HLA-C antigen ➔ HLA-DR antigen ➔ HLA-DQ antigen and ➔ HLA-DP antigen ➔ 6 HLA antigen are looked at for each person ➔ When performing an HLA typing test for a kidney transplant, HLA-A, HLA-B and HLA-DR antigens are looked majorly. 10
  • 11. ➔ By analyzing which of these 6 HLA antigens both the donor and recipient have, scientists are able to determine the closeness of tissue typing. ➔ 6 antigen match is best when took from blood cord and usually doctors recommend 8 to 10 antigens for best compatibility. ➔ This match occurs 25% for siblings and 50% for parents. 11
  • 12. 12
  • 13. TISSUE TYPING METHODS ➔ PHENOTYPIC METHODS ◆ Serological method ◆ Mixed lymphocyte culture (MLC) ➔ MOLECULAR METHODS ◆ Sequence specific oligonucleotide probe hybridization ◆ Sequence specific primers ◆ Sequence based typing 13
  • 14. PHENOTYPIC METHODS: 1. SEROLOGICAL METHODS:- ➔ Potential donor’s and recipient’s lymphocytes are isolated from blood by immunomagnetic beads coated by monoclonal antibodies against T cells or B cells. ➔ Once the cells adheres to beads a strong magnet is used to pull them on to the surface of test tube. ➔ All the antibodies to unwanted cells are washed and only T lymphocytes are obtained. ➔ Cells are then stained with carboxyfluorescein in fluorescence test. ➔ These cells are added to pre dotted tissue typing tray (microtitre plates). ➔ These trays contain highly selected antisera either from pregnant 14
  • 15. ➔ Each antibody reacts to an epitope which is unique to a certain HLA specificity. ➔ Typically, the tray will have 72 or 96 different antisera with 0.001ml volume. ➔ Cells are incubation for 30 minutes, then 0.005ml rabbit complement is added and incubated for an hour. ➔ Ethidium bromide or propidium iodide is added and read by fluorescence test. ➔ Living cells stains with carboxyfluorescein while dead cells stains red with ethidium bromide. ➔ In eosin dye test dead cells are stained red with eosin and living cells appear bright white under phase-contrast microscope. ➔ In both tests, dead cells denote a positive reaction in which Ab and complement reacted. 15
  • 16. 16
  • 17. 2. MIXED LYMPHOCYTE CULTURE (MLC)- ➔ It has been observed that lymphocytes from one donor,when cultured with lymphocytes from unrelated donor stimulates proliferation. ➔ This is due to a disparity in class II MHC [DR] antigens and T cells of one individual interacts with allogenic class II MHC antigen bearing cells [B cells, dendritic cells, langerhans cells, etc] of another. ➔ This reactivity is also known as Mixed Leukocyte Reaction[ MLR]. ➔ In this test, lymphocytes [responder cells] of donor are mixed with irradiated or mitomycin C which is treated leukocytes from the recipient, containing B-lymphocytes and monocytes [stimulator cells].17
  • 18. ➔ Cells are cultured for 4-6 days. ➔ Responder cell will recognise the foreign class II antigens found on the donor and undergoes transformation and proliferation [mitogenesis]. ➔ The T cells that respond to foreign class II antigens are typically CD4+ TH-1 type cells. ➔ These changes are recorded by addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA. 18
  • 19. 19
  • 20. MOLECULAR METHODS ➔ DNA based procedures give accurate HLA typing and lead to the identification of serologically undetected alleles and of many subtypes of serological specificity. ➔ Focused on analysis of nucleotide variation occurring in both exon 2 and 3 of class I genes and exon 2 of class II genes. ➔ Techniques are based on PCR polymorphism. ➔ All the techniques uses same method for extraction of genomic DNA and Amplification of gene of interest and only deferes in detection of sequence polymorphism that defines the alleles. 20
  • 21. The types are :- ➔ Hybridization with Sequence Specific Oligonucleotide Probes [SSOP] ➔ Sequence Specific primers [SSP] ➔ Sequence Based Typing [SBT] 21
  • 22. 1. SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBES [SSOP]:- ➔ Method involves selective amplification of target followed by hybridization to a panel of oligonucleotide probes. ➔ Specificity for a particular HLA locus is achieved by selecting PCR primers specific for a sequence in conserved region of the second exon. 22
  • 23. 23
  • 24. 2. SEQUENCE SPECIFIC PRIMING [SSP]:- ➔ It's a rapid method of HLA typing that uses sets of primer pairs to amplify region of genomic DNA. ➔ The efficiency of the amplification reaction is controlled by the primers that amplify conserved sequences of a selected gene. 24
  • 25. 25
  • 26. 3. SEQUENCE BASED HLA TYPING:- ➔ Involves determining the nucleotide sequence of an amplified segment of an HLA gene. ➔ It is advantageous over other procedures because of relatively fast [ in 24-48 hrs] with high level resolution. ➔ It is more reliable and specific method. 26
  • 27. 27
  • 28. CLINICAL SIGNIFICANCE OF HLA TYPING ➔ In organ transplantation ➔ In transfusion therapy ➔ Disease association ➔ Disputed paternity ➔ In cancer prevention ➔ Anthropological studies 28
  • 29. REFERENCES ➔ Encyclopedia of Immunology, Second Edition by Peter J Delves and Ivan M Roitt ➔ Immunology, Fifth Edition by Richard A Goldsby, Thomas J Kindt, Barbara A. Osborne and Janis Kuby ➔ https://www.slideshare.net/drnisha22/hla-92546736 ➔ https://www.slideshare.net/debbarma1989/hla-typing-and-its-role- in-tissue-transplantation ➔ https://wexnermedical.osu.edu/blog/tissue-typing ➔ https://www.invent.org/inductees/paul- terasaki#:~:text=In%20the%201960s%2C%20UCLA%20professor,pote ntial%20organ%20donors%20with%20recipients. ➔ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684003/ 29
  • 30. 30

Editor's Notes

  1. Half of a person's antigens are inherited from their mother and half from their father, so more similar the antigen pattern are from two people, more likely it is that those people are related. Identical twins have the same pattern and brother & sisters have 1 in 4 chance of having an identical match