HLA AND HLA TYPING [HLA-Rupendra Shrestha]

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CLINICAL IMMUNOLOGY:

CLINICAL IMMUNOLOGY LEUCKOCYTE ANTIGEN AND HLA TYPING RUPENDRA SHRESTHA DEPARTMENT OF HUMAN GENETICS IMMUNOGENETICS

SYNOPSIS:

SYNOPSIS HUMAN LEUKOCYTE ANTIGEN HLA TYPING SEROLOGICAL METHODS MOLECULAR METHODS CELLULAR METHODS ANTIBODY PROFILE PANEL REACTIVE ANTIBODY TEST CROSS MATCHING TEST

PowerPoint Presentation:

HUMAN LEUKOCYTE ANTIGEN

HLA SYSTEM:

HLA SYSTEM Also named as the Major histocompatibility complex (MHC) in humans . It is a Cluster of genes responsible for immune response, transplantation Ag and proteins of the complement system Produces a set of protein called histocompatible molecules which are located on the cell membranes of all nucleated cells of the body as well as in the blood serum . Responsible for allograft rejection, immune recognition etc . Present on surface of leukocyte & hence called as leukocyte antigen.

HLA SYSTEM:

HLA SYSTEM The group of HLA genes resides on short arm of chromosome -6. HLA Antigens can be classified as products of a number of different genetic loci and can be ordered according to their similarity or different function. MHC Class I (A,C,B loci) MHC Class II (DP,DQ,DR) MHC Class III (In between them)

PowerPoint Presentation:

Location and Organization of the HLA Complex on Chromosome 6

MHC Class I:

MHC Class I Include the serological defined products of the HLA-A,B and C loci. highly polymorphic glycoprotein found on surface of virtually all nucleated cells, abundantly on lymphoid cells and sparsely on liver, lung and kidney. Differences in heavy chain are responsible for graft rejection. Processing of Antigen by cytosolic pathways.

MCH Class- II :

MCH Class- II Represented primarily by Ag of the HLA-DR,DQ and DP loci. Glycoprotein found on the surface certain cells including Macrophages, B-cells, dendritic cells of the spleen, Langerhans cells of the skin and activated T- cell. Antigen presentation, T-B cell co-operation & Macrophage –B cell co-operation and processing of Ag by endocytic pathway.

MHC Class III :

MHC Class III Product of gene locus present in between class I and Class II loci. Contains several immunological important genes, encoding 2 cytokines (TNF & Lympho-toxin) and 2 complement (C2 & C4).

HLA TYPING:

HLA TYPING

Serological Method (Micro-lymphocytoxicity technique or NIH Method) GOLD STANDARD:

Serological Method (Micro-lymphocytoxicity technique or NIH Method) GOLD STANDARD Principle lymphocytes are separated from peripheral blood and incubated with antisera containing cytotoxic antibodies. When rabbit serum complement is added to the system cell death occurs. This can be detected by the dye exclusion technique. (the cytotoxic effect of antibody is detected by the entry of dye such as eosin or trypan blue entering into the cell),phase contrast microscopy is used for the detection of stain cell.

MATERIALS REQUIRED:

MATERIALS REQUIRED Equipment's Deep freezer(-20 to -80°C), Refrigerator, Inverted Phase Contrast microscope, Microsyringe dispenser, Terasaki Trays, Haemocytometer, Cold Swing head centrifuge etc.

PowerPoint Presentation:

Chemicals Lymphoprep/ Ficoll (Sp. Gr 1.075) Sodium Heparin PBS(PH - 7.2) RPMI-1640 Media HLA-antisera (minimum 3 of each specificity obtained from different Companies) 5%Eosin formalin mineral oil pipettes and tubes.

BASIC STEPS OF HLA TYPING:

BASIC STEPS OF HLA TYPING

STEP 1 - Preparation of Typing Trays:

STEP 1 - Preparation of Typing Trays Pre-dropped plates are available commercially but it is expensive hence can be prepared self MANUAL PREPARATION Label the trays(contain small wells) and fill up the wells with 5µl of heavy mineral oil. Prepare the antiserum to be used and arrange them accordingly in a stand on ice Pour 1µl of antiserum into each well, below the oil. The plates are ready and can be stored at-20°C or at -70°c for long time storage.

STEP 2 - Preparation of cell Suspension:

STEP 2 - Preparation of cell Suspension Density gradient centrifugation using Ficoll/Lymphoprep solution. (for Lymphocyte are separation) Dilute 10ml of heparinized blood with equal volume of PBS an mix gently. Count the lymphocyte cell suspension and adjust the count to 2000cell /µL and viability to 95-100%.

STEP 3. Separation of T &B-Cells (Nylon-wool Method):

STEP 3. Separation of T &B-Cells (Nylon-wool Method) Gently tease the 100µg of nylon wool in RPMI media and pack it onto sterile syringe. Wash and equilibrate the nylon wool with selected media RPMI 1640 with 10% FCS. Mononuclear cells suspended in 2ml of media are pipetted onto sterile nylon wool. Incubate at 37% for 45 – 60 minutes to allow B- cells to adhere to nylon column. Collect the non adherent T-cells by washing with RPMI and resuspend which can be directly used for typing HLA Class I. Collect B-cell enriched cells in another tube by adding 1ml of RPMI to the syringe column and gently sterile plungers of syringes are forced the medium through the nylon wool. Repeat till 5ml of media is used. Centrifuge and resuspend the pellet in 1 ml medium and adjust the count an viability.

STEP 3. Separation of T &B-Cells (Nylon-wool Method):

STEP 3. Separation of T &B-Cells (Nylon-wool Method) Gently tease the 100µg of nylon wool in RPMI media and pack it onto sterile syringe. Wash and equilibrate the nylon wool with selected media RPMI 1640 with 10% FCS. Mononuclear cells suspended in 2ml of media are pipetted onto sterile nylon wool. Incubate at 37% for 45 – 60 minutes to allow B- cells to adhere to nylon column. Collect the non adherent T-cells by washing with RPMI and resuspend which can be directly used for typing HLA Class I. Collect B-cell enriched cells in another tube by adding 1ml of RPMI to the syringe column and gently sterile plungers of syringes are forced the medium through the nylon wool. Repeat till 5ml of media is used. Centrifuge and resuspend the pellet in 1 ml medium and adjust the count an viability.

STEP 4. Preparation of Complement:

STEP 4. Preparation of Complement Collect 8-10ml of de-fibrinated blood on ice by cardiac puncture from 15-20 rabbits. Separate the serum in cold and mix to make a pool . Put in several aliquots and store at-70°C and thaw just before use when needed. Avoid exposure to heat or repeat thawing and freezing.

STEP 4. Preparation of Complement:

STEP 4. Preparation of Complement Collect 8-10ml of de-fibrinated blood on ice by cardiac puncture from 15-20 rabbits. Separate the serum in cold and mix to make a pool . Put in several aliquots and store at-70°C and thaw just before use when needed. Avoid exposure to heat or repeat thawing and freezing.

STEP 5. HLA Typing Procedure:

STEP 5. HLA Typing Procedure Bring the typing trays to 22°C and check for the presence of serum in each well. Dispense 1µl of cell suspension into each well and ensure mixing and avoid carry over. Incubate at 22°C 30 min for HLA- A, B, C. 60 min for HLA- DR, DQ. Add 5µl of complement to each well Mix and incubate at 22°C. 60 min for HLA- A, B, C. 90 min for HLA- DR, DQ. Add 5µl of Eosin to each well. After 5 min add 5µl of formalin to each well. Allow trays to stand at RT for 30-60min to settle the cells.

STEP 6. Microscopic Evaluation:

STEP 6. Microscopic Evaluation The plate is read for the result in inverted phase contrast microscope and scoring is done by estimating the percent of cell lysis. Viable lymphocytes:- small, bright and refractile Damaged cells:- large, dark and non-refractile (stained with vital dye appear larger than live cells)

International Scoring System:

International Scoring System Score Interpretation Dead Cells (%) 1+ Negative 0-15 2+ Doubtful Negative 16-25 4+ Weak Positive 26-50 6+ Positive 51-80 8+ Strong Positive 81-100 0 Not readable -

MOLECULAR METHODS:

MOLECULAR METHODS All commonly used molecular methods require good quality genomic DNA. There are numerous methods for extraction of DNA from whole blood. Salting Out method commercial kits available

MOLECULAR METHODS:

MOLECULAR METHODS The application of molecular techniques to HLA typing began around 1987 when the Southern Blot technique was used to identify restriction fragment length polymorphisms (RFLP’s) associated with known serological DR/DQ and cellular Dw defined specifities. Around 1992 polymerase chain reaction (PCR) methods were developed.

1. PCR - SSP (Sequence Specific Priming):

1. PCR - SSP (Sequence Specific Priming) Can be used for HLA Class I and II typing using a panel of primer pairs either for low to medium resolution whereby primers amplify groups of alleles or high resolution whereby primer pairs amplify specific alleles.

2. PCR - SSOP (Sequence specific Oligonucleotide probe):

2. PCR - SSOP (Sequence specific Oligonucleotide probe) It involves following steps:- Amplification of specific region of the locus to be typed. Dot blotting of the amplified DNA. Labeling of oligonucleotide probes. Hybridization of blotted product with labeled probe. Detection of hybridized probes. Interpretation of signals

3. PCR - SBT (Sequence Based Typing) :

3. PCR - SBT (Sequence Based Typing) DNA sequencing is the determination of the sequence of a gene and thus is the highest resolution possible. Sequence based typing involves PCR amplification of the gene of interest eg HLA DRB1 followed by determination of the base sequence. The sequence is then compared with a database of DRB1 gene sequences to find comparable sequences and assign alleles. This method also allows for detection on new alleles.

4. Reference Strand Conformational Analysis (RSCA):

4. Reference Strand Conformational Analysis (RSCA) It Offers sequence level typing without the need to sequence. Assigns HLA type on the basis of accurate measurement of conformation i.e shape dependent on DNA mobility in polyacrylamide gel electrophoresis (PAGE). Complex and difficult technique

5. Luminex technology:

5. Luminex technology It is SSOP based. Just beginning to be introduced into laboratories for routine use on non urgent samples.

CELLULAR TYPING:

CELLULAR TYPING Not / Rarely used by laboratories these days. Requires panels of homozygous typing cells. Cell culture method therefore takes a long time. Labour intensive involves use of radioisotopes.

HLA PROFILES:

HLA PROFILES

HLA ANTIBODIES:

HLA ANTIBODIES HLA antibodies are not naturally occurring and arise following pregnancy, transfusion, or previous transplantation. HLA antibodies may be IgG or IgM type. Reason for testing HLA Antibodies. To look for Abs which may affect the outcome of future transfusion or treatment, especially in patients who have transfusion reactions. To look for antibodies suitable for use as typing reagents.

HLA ANTIBODY DETECTION:

HLA ANTIBODY DETECTION Panel Reactive Antibody (PRA), including HLA Specificity Analysis Luminex ELISA Complement Dependent Cytotoxicity, including AHG (anti-human globulin) Titration/Quantitation studies for pre-transplant immunotherapy protocols Cross matching for transplant compatibility Flow cytometry Complement Dependent Cytotoxicity, including AHG (anti-human globulin)

PANEL REACTIVE ANTIBODIES:

PANEL REACTIVE ANTIBODIES Anti HLA antibodies in the serum of a person can be assessed as PRAs Testing the serum of the patient against a panel of cells or antigens prepared from many different donors using cytotoxicity or flow cytometry Results are expressed as percentage of positive donors

CROSS MATCHING TEST:

CROSS MATCHING TEST Serum of potential recipient is incubated with cells from possible donor If recipient has anti-donor antibodies there is a strong likelihood that recipient would destroy transplant by antibody mediated rejection

Comparison of HLA Typing Techniques :

Comparison of HLA Typing Techniques

CLINICAL APPLICATION:

CLINICAL APPLICATION Kidney transplantation. Bone marrow transplantation in leukaemia. Blood component therapy. Paternity testing. Diseases association (alkylosing spondylitis – HLA B27)

Reference:

Reference DS Smith, 1987, A guide to laboratory transfusion practice; Blackwell Scientific Publications Ltd. http://www.kisker-biotech.com/shop/categorie-101-nylon-wool-pap-pen.htm http://www.dolcera.com/wiki/index.php?title=Transplant_Diagnostics_(HLA)_Market_Landscape

THANKS ALL FOR KIND ATTENTION:

THANKS ALL FOR KIND ATTENTION