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Hatim Alabbas Mostafa ,PhDRoyal Care International Hospital
Pathology and laboratory Medicine Dep.
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Transplantation of organs is becoming increasingly successful What was once an experimental and life-saving emergency procedure has now been transformed into a life-enhancing and technologically advanced form of therapy. the key test in the HLA lab are :
HLA matching
T and B cell crossmatching
HLA antibody screening and specificity analysis
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HLA is the name of the major Histocompatibility complex (MHC) in humans. The super locus contains a large number of genes related to immune system function in humans.
This group of genes resides on chromosome 6, and encode cell-surface antigen-presenting proteins and many other genes.
The proteins encoded by certain genes are also known as antigens, as a result of their historic discovery as factors in organ transplantations. The major HLA antigens are essential elements in immune function.
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The Major Histocompatibility Complex
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Low-medium resolution for solid organ transplant
Serology, molecular (SSP – Sequence-Specific Primer, SSOP –
Sequence-Specific Oligonucleotide Probe)
Typing HLA class I (A, B, Cw) and class II (DR, DQ, ?DP)
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HLA-DQB1*0302HLA-DRB1*0401
HLA-DRB1*0401Allele:
Haplotype:
Genotype:
J. Noble
Gene low high resolution typing
“subtype”=01
HLA-DQB1*0302
HLA-DRB1*0301 DRB1*02
HLA-DRB1*04
HLA-DQB1*0201
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To test a recipient for these antibodies, a sample of their blood is mixed with a sample of the potential donor’s blood. This test is called a “crossmatch,” and shows how a recipient’s antibodies react with the potential donor’s.
Test results can be either positive or negative. It may seem confusing at first, but a positive crossmatch means that a donor and recipient are not compatible.
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Positive crossmatch Recipient’s antibodies attack donor’s ⇒ ⇒ Not suitable for transplantNegative crossmatch Recipient’s antibodies do not attack ⇒
donor’s ⇒ Suitable for transplant
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Employs lymphocyte targets to detect complement-fixing
antibodies “transplants in a test-tube”
Binding of antibody to target antigens on cell membrane
induce conformational changes in antibody molecules
exposing the Fc segment that binds to complement
Complement cascade is activated leading to membrane
damage and cell lysis
Is an essential pre-transplant test as it identifies preformed
antibodies responsible for hyperacute rejection
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Donor XM: This test employs serum from the recipient versus cells from
the chosen prospective donor .the test is intended to detect the presence of preformed Abs in the recipient to donor HLA Ags.
Autologous XM: This test employs serum from the recipient tested with the
recipients own cells. Abs detected in this test are termed Auto Abs .
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Standard (Basic, direct or NIH) CDC
Isolated T and B cells CDC XM
Extended incubation CDC XM
Amos-modified (1 or 3 washes) T and B CDC XM: eliminate anti-complementary activity of the serum
Anti-human globulin (AHG): Enhances complement binding
IgM inactivation by DTT/Heat treatment
Flow cytometry
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CDC Results
Exclusion dyes
Fluorochromes
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T cell: Most transplant centers agree that a strongly positive T cell
Cytotoxic crossmatch at warm temperatures is sufficient reason to deny the transplant of that specified donor organ to the recipient , if the CDC XM result is only weakly positive , the transplant is also usually denied. The fact that the antibodies appears weak in a laboratory assay is no assurance it is any less clinically significant in vivo than a strongly Cytotoxic Abs .
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B cell: The interpretation of positive B cell XM remains controversial. Some
centers routinely perform this test while other centers do not perform B cell crossmatching at all for clinical purposes, or may include it only for research purposes. There is data suggesting that transplantation should be denied in the instance of either a high tittered (1:8) B cell reactive antibodies , or one that can be demonstrated to be specifically anti-HLA.
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In the autologous XM It is highly recommended that an
autologous XM be performed when the patient is first evaluated for transplantation . if the XM is positive , there is a high probability that any donor XM performed with that serum will be positive. A determination must be made whether or not there is additional reactivity in the serum that is specifically anti-donor .
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The flow cytometric lymphocyte crossmatch is a standard technique for evaluating the compatibility of potential kidney transplant recipients and donors. Recipient serum is incubated with donor lymphocytes and the latter are analysed in a flow cytometer for the presence of bound IgG antibodies. An increase in the level of IgG binding compared to a negative control indicates the presence of donor-specific antibodies which may lead to deleterious graft function
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A fluorescein isothiocyanate conjugated rabbit anti-human IgG antibody was used to detect bound IgG following incubation of donor lymphocytes with patient serum. R-phycoerythrin conjugated anti-human CD19, and Quantum Red conjugated anti-human CD3 monoclonal antibodies were used to detect T and B cell populations, respectively.
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A major advantage of the FCXM is that antibody reactivity can be independently and simultaneously evaluated on donor T and B lymphocytes. In addition, because the FCXM is a semi-quantitative measure of antibody binding, it can be less subjective than visual assessment of cell death in complement-dependent assays.
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HLA antibody screening and
specificity analysis
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Pregnancy
Transfusion
Transplant
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A1,2 B 7,8
A1,24; B8
+
What antibodies are likely
to develop?
Generation of DSA
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A1,2 B 7,8
A1,24; B8
+
Anti-A2
Anti-B7
Generation of DSA
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Anti A2
Anti B7
A28
A23
A69
A68
B57
A24 B58
B27 B60B61
B13
B42
B54
B55B48
B41
B47
A1,2 B 7,8
A1,24; B8
+
DSA are rarely generated alone and generally antibodies to HLA molecules related to the donor HLA are also often found:
Generation of DSA
Due to shared epitopes with donor HLA
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Cell-based
Solid phase immunoassays (SPI):
• ELISA
• bead based
o flow cytometry
o Luminex®
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Complement dependent cytotoxicity (CDC)
CDC was the first technique to detect HLA specific antibodies. It employs the use of live lymphocytes to detect lymphocyte specific antibodies by activation of complement system and killing of lymphocytes. This is a widely used test, but has many drawbacks:
(i) it is limited by the cell panel used, (ii) it depends on the quality of lymphocytes and rabbit
complement,
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(iii) it detects non-HLA antigens and it only detects complement fixing antibodies.
Therefore, patients cannot be tagged as sensitized on the basis of this test.
Results are expressed as percentage of cells reacted with the tested serum.
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Flow Cytometry(Latex beads)ELISA
Anti-IgG-PEAnti-IgG-FITC
HLA alloantibody
Luminex Array(Polystyrene beads)
Anti-IgG
Gebel and Bray. Transplantation Reviews 20: 189-194, Gebel and Bray. Transplantation Reviews 20: 189-194, 20062006
Solid phase assays
Purified HLA molecules are immobilized onto the solid surfacesHigher sensitivity than CDC-based assays
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Advantages
Increased sensitivity and specificity
High throughput, automation, and rapid turn-around
time
Reactions scored on a continuous scale
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Limitations Antigen variability
• amount
• condition
Interference by external factors
• IVIg
• Thymoglobulin™
Interference by intrinsic factors (autoantibody, immune
complexes, and high levels of IgM)
Relevance of low antibody levels
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Problems with flow SA beads
• Only 8 specificities can be
tested in each tube• Initial kit requires 4
tubes/sample • 6 hrs to perform assay (labour
intensive!)• If additional specificities are
required, supplementary kits are
available (↑ cost)
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• controls + 96 specificities (class I) or
76 specificities (class II)/ well
• performed in a 96-well format (8 well
strips)
• assay time = 2 hours
• higher sensitivity than flow SA beads
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PEPE
Laser 1
Laser 2
Tells the instrument which bead is being
examined
Tells the instrument how much antibody is
bound to the bead
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MFI
Single antigens present on each bead
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HLA antibody screening
(initial)
HLA antibody screening
(Subsequent)
HLA antibody specificities
LSMix:Class I or Class II HLA antibody +/ -
LumPRA: MFI of each bead
containing a specific haplotype
LSA: MFI of each bead
containing a single antigen
OUTPUT:
Determine whether HLA class I or class II
antibodies are present
Identify which HLA antigens antibodies are directed to and levels
at which they are found
Identify specific HLA antigens that
antibodies are directed to and approx. levels
at which they are found
PURPOSE:
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Sensitivity of DSA identification methods
DSA levels
Very high
HighModerate
LowDSA negativ
e
Luminex SAB
Flow cytometry
CDC-AHG
CDCELISA
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