TISSUE TYPING K R.pptx

1. TISSUE TYPING
K R MICRO NOTES
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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…
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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.
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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.
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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).
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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
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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
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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.
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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.
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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.
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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
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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

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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.
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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.
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21. The types are :-
➔ Hybridization with Sequence Specific Oligonucleotide Probes
[SSOP]
➔ Sequence Specific primers [SSP]
➔ Sequence Based Typing [SBT]
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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.
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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.
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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.
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28. CLINICAL SIGNIFICANCE OF HLA TYPING
➔ In organ transplantation
➔ In transfusion therapy
➔ Disease association
➔ Disputed paternity
➔ In cancer prevention
➔ Anthropological studies
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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/
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