Hatim Alabbas Mostafa ,PhDRoyal 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)

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

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 transplantNegative 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 anti-donor .

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 anti-human 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 semi-quantitative measure of antibody binding, it can be less subjective than visual assessment of cell death in complement-dependent assays.

HLA antibody screening and

specificity analysis

Pregnancy

Transfusion

Transplant

A1,2 B 7,8

A1,24; B8

+

What antibodies are likely

to develop?

Generation of DSA

A1,2 B 7,8

A1,24; B8

+

Anti-A2

Anti-B7

Generation of DSA

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

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.

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

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

PEPE

Laser 1

Laser 2

Tells the instrument which bead is being

examined

Tells the instrument how much antibody is

bound to the bead

MFI

Single antigens present on each bead

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:

Sensitivity of DSA identification methods

DSA levels

Very high

HighModerate

LowDSA negativ

e

Luminex SAB

Flow cytometry

CDC-AHG

CDCELISA