Published on February 23, 2014
Hatim Alabbas Mostafa ,PhD Royal Care International Hospital Pathology and laboratory Medicine Dep.
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
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.
The Major Histocompatibility Complex
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)
Gene low high resolution typing “subtype”=01 Allele: Haplotype: Genotype: HLA-DRB1*0401 HLA-DRB1*0401 HLA-DQB1*0302 HLA-DRB1*0301 HLA-DQB1*0201 DRB1*02 HLA-DRB1*04 HLA-DQB1*0302 J. Noble
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.
Positive crossmatch ⇒Recipient’s antibodies attack donor’s ⇒ Not suitable for transplant Negative crossmatch ⇒Recipient’s antibodies do not attack donor’s ⇒ Suitable for transplant
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
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 .
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
CDC Results Exclusion dyes Fluorochromes
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 .
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.
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 antidonor .
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
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 antihuman CD3 monoclonal antibodies were used to detect T and B cell populations, respectively.
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 semiquantitative measure of antibody binding, it can be less subjective than visual assessment of cell death in complementdependent assays.
HLA antibody screening and specificity analysis
Pregnancy Transfusion Transplant
Generation of DSA A1,24; B8 A1,2 B 7,8 + What antibodies are likely to develop?
Generation of DSA A1,24; B8 A1,2 B 7,8 + Anti-A2 Anti-B7
Generation of DSA DSA are rarely generated alone and generally antibodies to HLA molecules related to the donor HLA are also often found: A68 A1,24; B8 A1,2 B 7,8 Anti A2 A28 A23 B57 A69 B58 A24 + B42 B13 Anti B7 Due to shared epitopes with donor HLA B27 B60 B61 B55 B48 B54 B47 B41
Cell-based Solid phase immunoassays (SPI): • ELISA • bead based o flow cytometry o Luminex®
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,
(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.
Solid phase assays Purified HLA molecules are immobilized onto the solid surfaces Higher sensitivity than CDC-based assays ELISA Anti-IgG Flow Cytometry (Latex beads) Luminex Array (Polystyrene beads) HLA alloantibody Anti-IgG-FITC Anti-IgG-PE Gebel and Bray. Transplantation Reviews 20: 189-194, 2006
Advantages Increased sensitivity and specificity High throughput, automation, and rapid turn-around time Reactions scored on a continuous scale
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
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)
• 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
Tells the instrument how much antibody is bound to the bead Tells the instrument which bead is being examined PE Laser 2 Laser 1
MFI Single antigens present on each bead
OUTPUT: PURPOSE: HLA antibody screening (initial) LSMix: Class I or Class II HLA antibody +/ - Determine whether HLA class I or class II antibodies are present HLA antibody screening (Subsequent) LumPRA: MFI of each bead containing a specific haplotype Identify which HLA antigens antibodies are directed to and levels at which they are found HLA antibody specificities LSA: MFI of each bead containing a single antigen Identify specific HLA antigens that antibodies are directed to and approx. levels at which they are found
Sensitivity of DSA identification methods DSA negative Low Moderate High Very high DSA levels Luminex SAB Flow cytometry ELISA CDCAHG CDC
Calcification Inhibitors in CKD and Dialysis Patients
Volume-4 | Issue-2 | July-December 2015 Narayana Medical Journal 90 Evolution in Cross Matching & HLA Typing Techniques And their Clinical Significance
Evolution in Cross Matching & HLA Typing Techniques And their Clinical Significance. Mahaboob V Shaik1, Subrahmanyam G2.
Official Full-Text Publication: Evolution in Cross Matching & HLA Typing Techniques And their Clinical Significance on ResearchGate, the professional ...
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