Summary of the Review Article
Modern crossmatch techniques and human leukocyte antigen (HLA) typing play a crucial role to ensure better organ allocation and provide the recipient with a more favourable match. 

Foreign major histocompatibility complex (MHC) proteins are recognized by recipient alloreactive T cells via three pathways: direct, indirect and semi-direct .

• In the direct pathway recipient T lymphocytes recognize MHC molecule–peptide complexes on donor antigen-presenting cells. This leads to activation of CD4 helper or CD8 cytotoxic T cells. 
• In the indirect pathway, recipient’s antigen-presenting cells present donor peptides to recipient CD4 helper T cells via MHC class II. This also leads to interaction with B-lymphocytes resulting in alloantibody production.
• The semidirect pathway is a more recently proposed pathway whereby recipient antigen-presenting cells acquire intact donor MHC–peptide complexes via cell- to-cell contact or exomes and present them to recipient T cells.

 Activated allospecific T cells include those with regulatory function and those with effector function. The former attempt to prevent allograft rejection while the latter mediate graft rejection. 

Despite new and more potent immunosuppressive regimens in the modern transplantation era, the crossmatch still remains an essential tool to identify preformed donor- specific antibodies in recipients who have already been primed to foreign antigens. Allosensitization to HLA proteins occurs after pregnancy, blood transfusion or transplantation. 

A positive complement-dependent cytotoxicity crossmatch (CDC-XM) has generally been considered as a contraindication to transplantation in view of the associated high risk of hyperacute rejection. This type of rejection occurs immediately upon reperfusion and is attributed to preformed donor-specific antibodies to HLA. 

One of the important purpose of tissue crossmatch is as a risk assessment tool for antibody-mediated rejection, which is one of the main barriers to improve long-term graft outcomes.

HLA typing 

• HLA typing and quantification of donor-specific antibodies are prerequisite investigations which facilitate the assessment of the recipient’s overall immunological risk. HLA plays a central role in both cellular- and antibody- mediated alloresponses, which determine the outcome of a transplanted organ.

• It is well established that a better HLA match is associated with significantly better patient and graft survival.
• HLA mismatches have been significantly associated with death secondary to cardiovascular disease and infection, especially during the first year after transplantation. Furthermore HLA-DR mismatches have also been associated with a higher risk of acute rejection, overall graft failure and death-censored graft failure. 

TISSUE TYPING: TYPES ,ADVANTAGES AND DISADVANTAGES

1.Lymphocytotoxic Crossmatch Techniques 

(i)Complement-dependent cytotoxicity crossmatch:

• Good quality T- and B-lymphocytes are isolated from the donor’s blood and incubated separately with the serum of potential recipients. 
• Rabbit complement is subsequently added and after an appropriate incubation period (which can vary between different methods of CDC-XM).
• A vital dye (usually eosin) is added together with formalin to fix the cells. 
• Longer incubation periods and additional wash steps, known as the Amos technique, have been introduced to eliminate unbound antibodies before the addition of complement. 
• If donor-specific antibodies are present in recipient serum, these will bind to HLA antigens on donor cells resulting in complement activation via the classical pathway. This ultimately generates the membrane attack complex, which inserts into the lipid bilayer causing cell rupture and uptake of vital dye. These cells are visualized as red (dead) when seen under the microscope. 
• The result can be scored on a spectrum from two to eight, with two being equivalent to approximately 20% cell lysis and generally indicating a positive result. A score of eight indicates the strongest possible reaction.
• Serial doubling dilutions of the recipient serum is another way to determine the strength of the crossmatch. The more dilutions necessary for the test to become negative, the higher the level of donor-specific antibodies present.

• The CDC-XM may be applied to both T cells and B cells. The former reflects the presence of HLA class I antibodies, while the latter reflects both HLA class I and II antibodies. Since B cells express higher amounts of class I antigens, a positive B cell CDC-XM associated with a negative T cell CDC-XM may indicate low levels of class I antibodies. 
• The advantage of CDC-XM is detection of donor-specific cytotoxic antibodies 
• The disadvantages of CDC-XM : (i) Detects immunoglobulin M. (ii)False positive rate of 20%. (iii)There have been reports of a false positive B-cell CDC-XM following treatment with rituximab and basiliximab.

This may arise because of auto-antibodies, which are generally of the IgM and non-HLA IgG type. In order to remove the confounding influence of IgM on the crossmatch result, its activity can be eliminated by heating the serum to 55°C since IgM antibodies are cold agglutinins that react best at 4°C. 

(ii) Antihuman globulin CDC-XM:

• The sensitivity of the CDC-XM is enhanced if anti-human globulin is added before the complement factors. This promotes complement fixation by binding to HLA antibody on donor cells and increases the number of Fc-receptors available for complement binding. 
• Low-titre antibodies detected by this method were associated with a 36% 1-year allograft loss compared with 18% loss in those with a negative test.
• The advantage of Antihuman globulin CDC-XM: Increased sensitivity over the CDC-XM.
• The disadvantages of Antihuman globulin CDC-XM:Detects immunoglobulin M.

(iii) Flow cytometry crossmatch: 

• It served as a more sensitive tool in order to detect donor- specific antibodies that were being missed by the CDC-XM. 
• The technique involves adding recipient serum to donor lymphocytes and then incubating them with fluorescently conjugated anti-human globulin . Additional antibodies with different fluorochromes specific for T-cell and B-cell surface proteins respectively are later added to identify both cell groups. 
• The flow cytometer is calibrated using serum from blood group AB unsensitized male donors as a negative control and a pool of sera from highly sensitized patients as a positive control. 
• Electrical impulses generated from the specific forward and side scatter of the laser beam are converted to a numerical value using special software algorithms.
• The relative median fluorescence of a particular sample is calculated by dividing the median fluorescence of that sample by the median fluorescence of the negative control. 
• The relative median fluorescence is then compared to a predetermined cut-off value. Results from a flow cytometry crossmatch may also be expressed as a median channel shift.
• The advantage of Flow cytometry crossmatch (i)One advantage when compared to the CDC-XM is that it gives a semi-quantitative result, which is therefore less subjective. (ii)Sensitive for low titre antibody
• (iii)Detects non-complement binding antibody.
• The disadvantages Flow cytometry crossmatch (i)May exclude patients unnecessarily
• (ii)Specificity for human leucocyte antigen antibodies is low (iii)May be falsely positive in patients having previously received monoclonal antibodies like rituximab.

2.Solid-Phase Assays:

• The use of solid-phase assays has largely superseded serological methods because of higher sensitivity, specificity and reproducibility. 
• They come in the form of enzyme-linked immunosorbent assays (ELISA) or microbeads. 
• Luminex technology is an example of solid phase assay , which has revolutionized the detection of donor-specific antibodies. Indeed, Luminex single antigen beads (Luminex-SAB) are 10% more sensitive than ELISA, which in turn are 10% more sensitive than serological methods. For this particular reason most transplant centres use Luminex-SAB even though this technique is more expensive. 
• Analysis of donor-specific antibodies using Luminex-SAB entails (i)the addition of recipient serum potentially containing HLA antibodies to a mixture of synthetic beads each with a unique dye signature (usually red). (ii)Each bead is also coated with a specific type of antigen so that anti-HLA antibodies in the potential recipient serum can bind to the corresponding bead. (iii)Phycoerythrin-labelled anti-human globulin is subsequently added to the mixture. (iv)The Luminex machine itself is a special type of flow cytometer, which is able to report on 100 different types of simultaneous interrogations (flow is usually able to differentiate about 3–15 different beads or cells at best). (v) Beads are channelled in a single file through the flow chamber where the two laser beams intersect.(vi) Each unique bead can then be interrogated for the presence of the reporter dye on its surface and phycoerythrin-labelled anti-human globulin. (vii)The light detectors measure the intensities of forward and side scatter, which are converted into electrical impulses. (viii) Software will then convert these electrical signals into a meaningful result. (ix) Donor-specific antibodies can be defined according to which kind of cell they interact with (B cell vs T cell).

• Luminex-SAB can also be used to identify antibodies directed towards minor histocompatibility antigens. The downside of this technique is the detection of both complement and non-complement binding antibodies and the detection of low level donor-specific antibodies which may refute a potential transplant unnecessarily as it would ultimately result in a negative crossmatch. Another factor to keep in mind when interpreting these assays is the panel of HLA antigens used by the local laboratory. Luminex-SAB may also give false positive results because of denatured antigens on the microbeads.

• The advantage of Luminex-SAB ; higher sensitivity, specificity and reproducibility. 
• The disadvantages Luminex-SAB is the (i)detection of both complement and non-complement binding antibodies and the (ii) detection of low level donor-specific antibodies which may refute a potential transplant unnecessarily as it would ultimately result in a negative crossmatch.

3.The Virtual Crossmatch 
Since the introduction of Luminex-SAB it is now possible to

• It ‘virtually’ compare specific anti-HLA antibodies in the recipient with the HLA profile of the donor. This is known as the virtual crossmatch.
• The correlation of the virtual crossmatch with flow cytometry crossmatch is greater than 85%. 
• It requires HLA typing of the donor and a recent anti-HLA profile of the potential recipient. To ensure a correct anti-HLA profile at the time of the transplant call, regular collection of sera every 3 months is required for antibody screening via solid-phase assays, because antibody titres and specificities can change over time. Any potential sensitizing events like pregnancy, blood transfusions and previous transplantation must be documented accurately. 

• A false negative virtual crossmatch can arise for a number of reasons;(i) Incomplete typing of the donor,(ii) as well as donor- specific antibodies in the recipient serum against a unique HLA epitope which is not available on the SAB panel, can give rise to a false negative result.
• Of note, not all donor-specific antibodies detected by Luminex- SAB are detrimental to graft outcomes.
• Studies have shown that donor-specific antibodies detected by SAB but with a negative CDC-XM are a major risk factor for early allograft rejection and long-term graft loss. Nonetheless, these are not an absolute contraindication for transplantation if one is prepared to perform desensitization when required, or to use more potent immunosuppressant protocols. False positives may also occur, as mentioned previously, as a result of antibodies directed at HLA epitopes that come about secondary to denatured HLA antigens on the SAB. Yet another cause for a false positive virtual crossmatch is the presence of null alleles, which are not expressed as antigens on the cell surface in vivo. 
• One of the principal advantages of performing a virtual crossmatch is the detection of acceptable and unacceptable antigens . This avoids unnecessary shipping of organs resulting in less surgery delays, reduces cold ischaemia time, encourages cost savings and improved odds of transplanting highly sensitized patients. 
• A number of studies have reported low risk for early antibody- mediated rejection and good allograft survival even in sensitized patients when using the virtual crossmatch.