The contents of the wells are then run by electrophoresis through an agarose gel with the amplification product 24 K. Tinckam appearing as a band on the gel; the HLA typing is assigned by matching the primers of resulting amplification products to the DNA sequences of the various candidate alleles. Unique fluorescent tags distinguish those probes that are complementary to the DNA, such that the unique HLA alleles may be identified. Sequencing determines the exact order of nucleotides in the gene of interest and the HLA type is assigned by comparison to published HLA allele sequences .
Regardless of the specific method, molecular typing more precisely identifies the differences in HLA antigen between donor and recipient, frequently with resolution to the amino acid level which may provide better quantification of the risk associated with mismatched donor—recipient antigens, amino acids, and epitopes [12, 13]. Historically, as HLA antigens were serologically discovered, they were named in order of that discovery by gene locus, e.
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Refinement of serologic methods identified even more antigens, previously thought to represent single allotypes, which in fact were serologically and genetically unique. For example, B60 and B61, which were identified as unique antigens with therefore unique private epitopes , were earlier thought to be just one antigen, B40, based on sera binding to a shared public epitope between the two.
Public epitopes are those common to all the members of a CREG whereas private epitopes delineate the individual serologically defined antigens. Serological antigen nomenclature does not represent the true heterogeneity of the HLA system. Indeed, early studies with mixed lymphocyte cultures detected this heterogeneity in HLA antigen recognition that could not be discerned by serology alone; for example, HLA A2 was found to consist of several subtypes stimulating different lymphocyte reactivity.
DNA sequencing subsequently confirmed that indeed multiple alleles of each HLA antigen are known to exist, despite the fact that they may react to a single common typing serum at the antigen level. A new molecular typing nomenclature was introduced in where the locus is followed by an asterisk, then the first two digits describe the type, and then the next two digits represent a unique allele differing by at least one amino acid difference.
Beginning April 1, , this system was modified further, adding a colon between each two digit designation, thus allowing for greater than 99 unique alleles within each allele family. For the purposes of solid organ transplantation, it is important for the clinician to recognize which system is used by their laboratory in the assignment of HLA type as well as in the assignment of antibody specificities.
Failure to appreciate that different nomenclature is used may result in missed recognition of a donor specific antibody DSA. Such concerns may be easily addressed through communication with the HLA laboratory, and in addition several references are readily available [9, 10, 14]. A table is provided here as a reference for the clinician listing the most common HLA serologic antigen names where the molecular typing may not be congruent or obviously related Table 2.
In addition, for Class II molecules, that are at the protein level, composed of unique alpha and beta chains, the serologic equivalent name reflects the beta chain polymorphism only. Communication with the laboratory is required to discuss if the alpha chain typng is clinically relevant for example, if a DSA is present to the alpha chain as the serologic nomenclature will not reflect differences in the alpha chain, and the molecular alpha chain typing may need to be specified.
However, if the donor and recipient typings were reversed then the recipient immune system would see 4 HLA antigens 1-A, 2-B, 1-DR as nonself. The number of affirmative matches should not routinely be used. These represent up to 18 unique protein products in donors and recipients where mismatch may 26 K.
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Programs differ widely as to what extent these typing are used in their clinical decision making, however it is important for the clinician to be aware of all of these typing possibilities. HLA Antibody Screening Up to one third of waitlisted patients may have some HLA antibodies detected when the most sensitive screening methods are used.
Sensitization to HLA antigens occurs with previous exposure to nonself HLA during pregnancy or after blood transfusion or prior transplant. A major consequence of preformed antibodies is decreased access to transplantation; antibodies to a greater number of HLA antigens will result in higher rates of positive crossmatches and exclusion of these donors. Indeed, even if the crossmatch is negative, permitting transplant to proceed with short-term safety, low titers of antibody directed to donor HLA are associated with higher rates of early  and late  antibody mediated outcomes including rejection and graft loss.
Therefore, both sensitive and specific identification of HLA antibodies is necessary to identify the risks faced by sensitized recipients and also to permit novel strategies for successfully transplanting these patients, such as desensitization , acceptable mismatching, and paired exchange [13, 18]. Repeated pretransplant antibody screening for waitlisted patients comprises a majority of solid organ transplant work in most HLA laboratories. If the serum contains antibodies that bind to the cell surface with sufficient density, complement will be activated, and the vital dye uptake allows the dead cells to be easily identified Fig.
Serum is added to each well in the panel. On the top, the antibody in the serum does not bind to the cells, on the bottom, donor specific antibody DSA does bind. Bound DSA remain after wash steps, so that when complement is added, it forms the membrane attack complex, killing the cell and allowing the vital dye is taken up by and visualized. No donor specific antibody leaves live cells i , and when DSA are present, the vital dye identifies the dead cells ii.
In this case, the antibody in the serum is only specific to the bead on the bottom. Increased fluorescence defines positive beads with DSA bound to them Limitations of Cytotoxic Antibody Screening An obvious limitation of this method is that the PRA percent may numerically change without a change in amount or type of antibody depending on the cell panel that was used in the screening. The interpreting clinician must not overinterpret small changes in PRA as a significant change in alloimmune potential.
Frequently, commercially made cell panels are used, however they may not accurately represent the HLA distribution of a particular donor region depending on the racial differences in that region, which can alter HLA antigen frequencies. Furthermore, substantial false positive results may occur due to non-HLA antibodies and autoantibodies or nonspecific IgM antibodies, as well as false negative results from low sensitivity dependence on complement activation which requires higher titer antibodies.
Complement activation requires that antibody must be of sufficient density to link complement between Fc receptors; with lower titer antibody, 28 K. Tinckam Table 2. Finally, accurate and complete lists of antibody specificities and unacceptable antigens are almost impossible to obtain with this methodology as there are multiple antigens per reaction well Table 2.
Cellular or cytotoxic PRA testing may therefore be best thought of as estimating the risk of a given recipient of having a positive cytotoxic crossmatch to a potential organ donor drawn from a comparable population as the cell panel donors. Solid Phase Antibody Screening Antibody-mediated damage has been reported in the absence of detectable antibody by cytotoxic screening methods as described earlier; the development of more sensitive assays was needed. The desire to discriminate HLA antibody from non-HLA antibody, as well as to clearly differentiate Class I and Class II antibodies stimulated the development of the currently available solid phase methodologies.
Purified HLA molecules are applied to solid phase media enzyme-linked immunosorbent assay [ELISA] [20, 21] platforms or microbeads  , and therefore will bind only HLA antibody when recipient serum is added. By virtue of controlling the antigens placed on the beads, these assays are specific for HLA antibody only, Class I and Class II HLA antibody may be easily distinguished by utilizing class-specific beads, and isotype detection can be limited to IgG. Finally, precise specificities may be determined by utilizing beads that each binds only one unique HLA antigen Table 2.
Limitations of Solid Phase Antibody Testing Although the use of these platforms has addressed many of the problems associated with cellular assays, they too have their limitations including detection of both noncomplement and complement binding antibody simultaneously which may have different clinical implications , and detection of antibody well below the level associated with a positive crossmatch.
The detectable antibody may not always be associated with a meaningful clinical outcome, yet if this information is used to exclude potential donors, it could limit transplants with negligible net benefit. The role of non-HLA antibodies in certain clinical outcomes is increasingly recognized, so it is important that we do not view solid phase HLA test results in isolation. As the number of HLA alleles identified continues to grow into the thousands, it is clear the full spectrum of unique HLA antigens cannot be practically represented on solid phase assays.
Clear examination of donor and recipient typing must also be considered in the interpretation of any solid phase PRA result. The outputs of solid phase assays are fluorescence or optical density readouts; these are continuous variables and considerable controversy exists as to what thresholds should be considered positive. As a result, there can be substantial interlaboratory variability; it is recommended that the clinician review how antibodies are called and how they are correlated to crossmatch results in their own HLA laboratory .
Crossmatching In a landmark paper, Patel and Terasaki  demonstrated for the first time that recipients with DSA in their serum at transplant had substantially higher rates of hyperacute rejection and primary nonfunction. The test described in the paper was the cytotoxic assay described in the previous sections of serologic typing and cytotoxic PRA testing.
Thus, the T cell cytotoxic crossmatch was implemented almost universally as the requisite immune assay before transplant  and resulted in a significant reduction in hyperacute rejection. Detection of donor-specific cytotoxic antibodies a positive crossmatch was a contraindication to transplant.
In contrast to a PRA, which identifies all antibodies to a potential pool of donors, the crossmatch identifies whether a recipient has antibodies to a particular single donor of interest. Tinckam Cytotoxicity with flow cytometry Yes Yes No Yes Low to very low Yes demonstrating it was insufficient to define all relevant antibodies and may be unnecessarily excluding patients from transplant.
Over time, assays have been developed to address these limitations [26—29], and the improved sensitivity has lead to a critical examination of which antibodies identified by more sophisticated techniques are predictive of significant clinical outcomes Table 2. The solid phase antibody screening data should always be used in conjunction with crossmatch results to help classify them as immunologically irrelevant or relevant high risk of rejection or graft loss, or transplant contraindicated .
Similar to the method used in cytotoxic antibody screening, the cytotoxic crossmatch result is considered positive if a significant proportion of the T lymphocytes are killed after the addition of complement, inferring that substantial DSA had been bound to the cell surface Fig. However, as with cytotoxic PRA screening, similar concerns of low titer but nonetheless relevant antibody potentially not detected has lead to improvements to this technique of increasing sensitivity, including longer incubation times, additional wash steps  and most commonly, the AHG-enhanced method . AHG, a complement fixing antibody to human immunoglobulin, is added as a second step, and binds any DSA already on the lymphocyte both complement binding as well as noncomplement binding DSA thereby increasing the antibody density, the likelihood of activating complement, and thereby increasing sensitivity Fig.
Adding AHG increases the overall density of complement activating antibodies on a cell that already has some DSA bound, thereby allowing complement activation with subsequent cell death as with CDC alone. Recipient serum is incubated with donor lymphocytes, and then secondarily stained with a fluorochrome conjugated anti-IgG antibody that remains bound only if DSA from the recipient serum is initially bound to the cell surface.
Additional antibodies with different fluorochromes that are specific to unique B and T 32 K.
Tinckam lymphocyte surface proteins can be added such that when run through a flow cytometer, the B and T cells may be easily distinguished and individually interrogated for the unique DSAs corresponding to those cell types Fig. The output of the flow crossmatch is at least semiquantitative e. Once again, as for flow cytometric-based antibody screening, there is considerable interlaboratory variability in methods routinely used for flow cytometric crossmatching and in the concordance of results between laboratories .
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Again the clinician is encouraged to communicate with their own laboratory to better understand the methods of crossmatch performance and reporting at their center. Simultaneous measurement of complement binding cytotoxic antibodies by various cell death markers over a denominator of total antibody both complement and noncomplement binding can be determined by appropriate staining techniques in flow cytometry .
One cardiac transplant study of complement fixation by antibody on solid phase beads showed an incremental increase in allograft loss over noncomplement fixing antibody . Non-HLA Antibodies With the appreciation that HLA antibodies have a substantial impact on both shortand long-term allograft outcomes, it has also become clear that in some cases, antibody-mediated outcomes are clinically or pathologically suspected, but no circulating HLA antibodies are detected. There is increasing awareness that in some of these cases, immunologically relevant non-HLA antibodies may be contributing.
Whereas this was first postulated over 3 decades ago , recent data from the Collaborative Transplant Study highlighted that even amongst HLA identical sibling transplants, high PRA recipients had worse graft outcomes, suggesting that non-HLA antibodies may be at least partly responsible for this finding . In some cases, it may be seen with newer antibody technologies that HLA antibodies to Cw, DQ, and DP antigens which only recently were able to be reliably detected on a large scale may be responsible for some of these discrepancies, in siblings identical at HLA-A, B and DR.
But in other cases it appears that exploration of non-HLA antibodies is relevant.
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The etiology of these antibodies may be quite different than that of HLA antibodies. In addition to exposure to polymorphic alloantigen — which is thought to be 2 Basic Histocompatibility Testing Methods 33 causative in MICA major histocompatibility complex [MHC] class I-related chain A antibody development — other pathways may include the exposure of otherwise hidden antigens during injury which stimulate autoimmunity, molecular mimicry with antibodies to viruses crossreacting with antigenic epitopes, and nonadherence to immunosuppressive protocols.
It is very important to remember that the target antigens for these antibodies are not expressed on lymphocytes, and therefore not detected on traditional lymphocyte cytotoxic or flow crossmatching. As such, the assertion that non-HLA antibodies detected in lymphocyte crossmatches are not immunologically relevant remains valid. The relevance of non-HLA antibodies detected an assays specific for their detection, remains a large area of investigation. With current immunosuppressive regimens we now have a majority of first allografts with HLA mismatches and still acceptable graft survival.
However, large registries still show a statistically significant though less clinically dramatic impact of HLA mismatches on deceased donor transplants . Results from the Collaborative Transplant Study indicate that shortening of cold ischemia time does not eliminate the effect of HLA matching and argue for consideration of HLA type in deceased donor allocation. Furthermore, even matching of HLA antigens within a given CREG group may be associated with better long-term allograft survival [40, 41].
The development of late antibody-mediated outcomes may require a diagnosis of DSA, which necessitates knowledge of donor typing. Additionally, for the third of waitlisted patients who have preformed antibody to HLA antigens see below , accurate donor typing is paramount in the identification of lower risk donors to whom the recipients do not have alloantibody, as in acceptable mismatch  or paired exchange programs [18, 42]. Molecular typing may be used to ensure only those donors with the allele of interest are potentially excluded, rather than all B44 donors [30, 43].