The target of Hc examination is to recognize the immunological risk of a TR with a candidate donor.
HLA sensitization & crossmatch testing
Abbreviations: (To facilitate your reading, read twice please):
o +ve: Positive
o a.a.: amino acid
o Ac Rj: Acute rejection
o Ag (s): antigen (s)
o AHG: anti-human globulin
o Alm: Alemtuzumab
o AMR: Antibody-mediated rejection
o AT1: angiotensin II type 1 receptor
o ATG: antithymocyte globulin
o BTx: blood transfusions
o C/I: contraindication
o C: complement
o CDC: complement-dependent cytotoxic
o Chm: chemiluminescent
o Chr: chromosome
o CMX: crossmatch
o cPRA: calculated panel reactive antibodies
o CREG: cross-reactive epitope group
o CTOT: Clinical Trials in Organ Tx
o DDKT: Deceased donor kidney transplant.
o DFU: Direct fluorescence units.
o DSAs: donor-specific anti-HLA antibodies
o DTE: Dithioerythritol
o DTT: Dithiothreitol
o Dsnz: Desensitization
o EDTA: ethylene diamine tetraacetic acid
o ELISA: enzyme-linked immunosorbent assay
o Fc: flow cytometry
o Flr: fluorescence
o GE: gel electrophoresis
o HA: hyperacute
o Hc: histocompatibility
o HLAs: human leukocyte antigens.
o HSCT: hematopoietic stem cell transplant
o Htp: haplotype
o I-I/H database: IPD-IMGT/HLA database
o im/m: immunosuppression/immunosuppressive
o IVIG: IV immunoglobulins
o KAS: kidney allocation system
o mAB: humanized monoclonal ABs
o MCS: Median channel shifts
o MESF: Molecules of equivalent soluble fluorescence,
o MFI: Median fluorescence intensity
o MHC: major histocompatibility complex
o MICA: MHC class I polypeptide-related sequence A
o NGS: Next-generation sequencing
o ONT: oligonucleotide
o OPTN: Organ Procurement and Transplantation Network
o PE: plasmapheresis
o Pmph: polymorphism
o PRA: The panel of reactive antibodies
o Prn: pronase
o Rj: rejection
o Rsl: resolution
o rSSO: reverse sequence-specific oligonucleotide probes
o RT-PCR: real-time polymerase chain reaction,
o Rtx: rituximab
o SAB: single-antigen bead
o SBT: Sequence-based typing
o Snz: sensitization
o SOT: solid organ transplant
o SPA: solid phase assays
o SSOP: Sequence-specific oligonucleotide probes
o SSP: Sequence-specific primers
o TR: transplant recipient
o Tx: transplant
o Un-Ags: unacceptable antigens
o UNOS: United Network of Organ Sharing
o -ve: Negative
o W/L: waitlist
The target of Hc examination is to recognize the immunological risk of a TR with a candidate donor. If Tx is between genetically variable subjects, the allograft is determined as foreign primarily because of the variability between donor/TR MHC molecules (= HLAs). The resultant immune response can be seen via 2 major mechanisms: T cell-mediated (cellular) response & AMR (humoral) response. The current Hc testing focuses mainly on anticipating AMR. The figure is describing how HLA examination is used in the assessment of a candidate TR. Before Tx, HLA typing is proceeded to evaluate the magnitude of donor/TR mis-matching, and anti-HLA AB screening & CMX are prepared to assess the TR 's likelihood to reject a potential allograft from the donor. With lack of a potential living donor, pre-Tx HLA testing can be also utilized to recognise the TR chance to receive a Tx from a cadaveric donor and to define criteria prohibiting Tx if the TR has experienced immunological memory against a specific donor's HLA Ags (i.e., "Un-Ags"). After Tx, detecting the presence of DSAs is beneficial in diagnosing AMR as well as assessing ptn's risk of Rj and the therapeutic options.
I will discuss applying, methodology, and limitation of assays via Hc tests.
OVERVIEW OF HLA
Recognizing any graft as foreign/non-self, can be obtained via the host's immune system's function to identify mismatched donor related Ags, including variabilities in the MHC molecules (HLAs molecules) expressed on the allograft. In human being, the MHC gene (s) found on the short arm of Chr 6 & including class I genes HLA-A, -B, & -C & class II genes HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRA, -DRB1-5. . Expression of class I molecules are usually seen on all nucleated cells, whilst the expression class II molecules are observed mainly on Ag-presenting cells (e.g., B cells, dendritic cell, & macrophages) but can be also observed with inflammatory states on the endothelial & epithelial cells. MHC molecules are greatly polymorphic with each MHC locus expressing 1000-5000 allelic types, encoding variable molecules. This intensity in Pmph is what is resembling a fundamental obstacle for a successful Tx requiring a robust im/m to prevent Rj.
Molecular structure: Class I MHC molecules are composed of a polymorphic a chain that is consisting of 3 external domains + transmembrane zone + intracellular domain, + non-polymorphic B2 microglobulin chain. Class II MHC components are hetero-dimers composed of 2 polypeptide chains, an a & B chains, each of them has a transmembrane domain, 2 external domains + short intracellular domain. HLA-DR, -DQ, & -DP molecules showed polymorphic B chains. HLA-DR molecules show a conserved a chain, whilst HLA-DQ & -DP showed polymorphic a chains. However, both classes showed a peptide-binding groove that can specifically binds to peptides to be presented to T cells.
Despite the higher level of Pmph observed in the MHC genes, only specific zones of the MHC molecule seem to be immunogenic (i.e., has the ability to elicit an AB response). In regard to class, I molecules, this corresponding mainly to the zone encoded by exons 2 & 3 of a chain (a1 & a2 subunits that are farthest from the cell membrane). MHC molecules that are differing only in zones outside these antigenic regions tend to be appeared as "serologically identical" and can be considered as serologic Ag equivalent (serotype). On the other hand, in class II molecules, the most antigenic zones are usually encoded by exon number 2 of the B chain for the HLA-DR molecules and exon 2 of the a & B chains for HLA-DQ & -DP. Whilst a chain of HLA-DQ &-DP molecules is also severely polymorphic, most recognised ABs seem to be directed against the B chain. However, there’re reports recognizing that anti-HLA ABs can be directed only against class IIa chain or directed to the tertiary structure of both a & B chains.
The partition of the molecule where an AB can bind is = epitope, and each HLA Ag may have > one epitope. Moreover, HLA Ags may be sharing a common ("public") epitope that is belonging to a CREG.
Genetic structure: The HLA genes are usually encoded on Chr6 with a Mendelian fashion inheritance; one linked set of genes (Htp) is inherited intact from each parent. So, a child can be expressing one representative set of Ags from each of class I & II loci of each parent, as HLA molecules are co-dominantly expressed. According to the definition, a child is a one-Htp match with each parent. Statistically wise, a child will get 25 % chance sharing 2 inherited Htp with a sibling, a 50 % chance sharing 1 Htp with a sibling, and a 25 %chance of being a zero-Htp matching with a sibling.
Any subject sharing all HLA Ags with another is said to be phenotypically identical. If these subjects also sharing similar allelic variants encoding these Ags, they are genotypically identical. However, if they’re NOT known as common descent, such subjects will have shared genotyping "by state" but not exactly "by descent." This can be seen with cadaveric donors who are HLA matched at ALL loci with a Tx candidate.
HLA mismatches: In SOT, a "mismatch" means that an HLA Ag that is present on the donor allograft’s cells but not in the TR. The more disparity between the donor & TR, the more "foreign" the allograft will appear and the higher the liability of an alloimmune response evolution. For KTx allocation, ONLY the HLA-A, -B & -DR loci are usually compared between the donor & TR. So, a zero-Ag mismatching refers to concordance involved at these loci but does NOT excluding disparity at other loci (HLA-C, -DP, or -DQ). As HLA genes are usually inherited as a set, related donor/TR pairs are likely sharing the same Ags at the other loci, e.g., a 2-Htps matching would indicating that this pair is not only a zero-Ag mismatching at HLA-A, -B, & -DR loci but also matching at the HLA-C, -DP, & -DQ loci. However, if the donor & TR are unrelated, such setting cannot be expected.
HLA nomenclature: HLA typing nomenclature are usually expressed at the Ag or allele level. Matching process for a KTx, the HLA typing is mostly expressed at the Ag level. Serological typing has been greatly replaced with molecular typing with the ability to report at allele level. HLA allele names are usually expressed with designed gene (locus), followed by an asterisk to indicate that it was typed via molecular methodology; colons are usually separating the remained fields. A high-Rsl, "full" HLA typing can involve up to 4 fields:
o Numerical digits after the asterisk (1st field) are corresponding to the HLA allele g. Historically, this 1st field was identifying the equivalent serological Ag of an expressed molecule. Whilst this’s still widely true, the nomenclature convention for the 1st field has performed alterations with the wide numbers of the new HLA alleles for which referring cells may have no serological identification. Many of the new alleles have been provided 1st field identifications entirely based on the sequence homology to a definite serological variant.
o The 2nd set of digits (2nd field) corresponding to a distinct HLA protein; allele encoding a unique a.a. sequence having variable digits in this field.
o The 3rd field differentiate among alleles with synonymous nucleotide substitution translating into the same a.a. cascade.
o The 4th field declares the variabilities in the non-coding zone.
o Finally, a suffix may be utilized to recognize alterations in cell surface expression (e.g., N = null allele & L = low expression).
In SOT, we may typically focusing only on the disparities identifying serologically specific proteins. Sometimes, variabilities in the 2nd field (representing the variabilities in a.a. sequence and, so, a unique protein) are considered as these disparities could potentially indicate variable immunologic responses. HLA-A*02:01 & HLA-A*02:05 - for example-belong to the same serological g., and the equivalent Ag name would be HLA-A2. However, they do vary in the a.a. cascade, and there are reported cases where an AB observed to be reacting against one not reacting against the other. As definite reactivity is discovered, the "parent" Ag can be split into a narrower specificity. HLA-A* 02:03 - For example - used to be dealt as the same serologic Ag equivalent (A2) but has been recognized as distinct and can be re-categorized as its own serological Ag (A203).
The precise HLAs typing is crucial to determine the magnitude of mismatching between the donor and TR and in to avoid Tx of organs from donor expressing HLA Ags against which the TR has expressing pre-formed AB. Historically wise, HLA typing of a donor for KTx was focusing on HLA-A, -B, -DR & -DQ loci. This was, partially attributed to the shortage in the available typing methods but also due to preformed ABs directed against the mentioned loci were thought to identify the robust alloimmune response and may induce HA/accelerated Rj. HLA typing was previously performed via serological-based assay, but this has been greatly replaced by the DNA-based molecular technique that allow a higher Rsl and more precise typing of ALL loci (HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA, -DQB, & -DPB).
By Oct 2020, 27,599 classical HLA class I & II alleles, encoding > 16,000 distinct HLA proteins have been reported in the I-I/H database. This distinct allelic variability makes high-Rsl HLA typing seems like incredible challenge. High-Rsl typing include a more precise recognition of an allelic variants via methodology sequencing the whole a.a. length of protein. Low-Rsl DNA typing, however, denotes typing results resolving the difference (s) at the Ag level. For SOT, only low-Rsl typing is required.
In the US, HLA typing of ALL loci by molecular methodology is now mandatory by the UNOS. Although organ allocation relies primarily on HLA-A, -B & -DR matching, allocated algorithm for a cadaveric-donor KTx is considering now ALL loci when a ptn's suitability for Tx from a specific donor to be determined based on the presence/ lack of preformed DSAs. However, the current algorithms still only considering the HLA-A, -B, & -DR loci to provide points of preferability for zero-Ag mismatches.
A particular methodology applied for HLA typing in SOT differs between HLA labs. Variables in choosing specific method may include the Rsl level, ability of resolving an ambiguity, turnaround timing, expertising and costs. HLA typing of a cadaveric donor is typically proceeded via RT-PCR or pre-made, rSSO tray. The required rapid turnaround timing impedes the high-Rsl typing methods application, e.g., sequence-based typing, NGS that may be used in TR typing or in living-donor assessment. However, serologically based assays are still applied by certain labs outside North America & Europe.
Serologic methods: For > 30 ys, serologically based typing was the 1st standard manoeuvre utilized to identify donor & TR HLA typing. This technique utilizes a reference sera panel (often from multiparous lady) containing ABs to various HLA Ags. Donor or TR’s lymphocytes can be added to multiple wells of plates containing different sera. After initial incubation allowing the binding between AB & Ag, C can be added to the wells, and a viability dye used to detect cell lysis. The finding of dead cells = +ve test. Comparing the serological specificities of variable reacting sera may allow the lab to identify the HLA typing. Major limitations for serological assay may include:
o Wide panels of sera with sufficient AB power & specificity were needed for reliable identification of the wide number of HLA specificities.
o Anti-sera were rarely monospecific containing ABs directed against > one specified HLA molecule leading to inconclusive pattern of reaction.
o Difficult typing of Ags with low cell surface expression (e.g., HLA-C & -DP Ags).
So, most labs have moving to use molecular methodology for HLA typing.
DNA-based molecular methods: 2 major progresses encouraging DNA-based molecular methodology to identify an individual’s HLA typing more precisely:
I. Introduction of the PCR technique.
II. The wide sequence of the polymorphic zones of the HLA genes.
DNA-based molecular methodology includes the following:
SSP typing - SSOP typing - RT-PCR-based typing - SBT- NGS
 SSP typing include using of primer (s) designed to identify certain HLA sequence (allele) or groups of similar alleles, such that the Pmph to be recognized is localized at the 3' ending of the primer. DNA can be extracted from a blood sample and magnified by PCR via these primers. If both primers can bind to the DNA, then magnification occurs that can be identified via GE. The amplicons’ pattern may allow the assigning the HLA genotyping. Commercially present typing class I kits show primer set that can identify Pmph in exons No. 2 & 3, whilst class II kits often identify Pmph in exon No. 2. These zones have been selected as they are covering mostly the known Pmph in many alleles. SSP methodology may be used for either low-Rsl, by recognizing allele groups of certain Ag, or for high-Rsl typing that identify certain allele.
The main advantage of the SSP technique is its fast turnaround timing of 2-3 hs, to be suitable for application if a rapid result is required, e.g., typing of a cadaveric donor. However, it is NOT suitable for typing large quantities of samples. Moreover, the ongoing rise in the quantity of HLA alleles showed a difficulty in resolving even with a single-step process. Even with utilizing additional primer set, ambiguity persisting that made a high-Rsl typing with increasing difficulty. Moreover, Pmph outside a sequenced exon cannot be easily resolved.
 SSOP typing: Here, DNA is magnified via a set of primers that identify a specific HLA locus. As in SSP technique, primers designation allow amplification most of the polymorphic zones of HLA genes (exons 2 & 3 for class I & 2 for class II genes). However, primers utilized in SSOP for DNA magnification are less specified than those utilized in SSP that cannot differentiate specific alleles or g.s of the same alleles. For alleles discrimination within a locus, the magnified DNA can be blotted into a membrane, then labelled SSOPs recognizing certain sequence can be added. A Chm or colorimetric reaction is then utilized to declare the finding of bound ONT, and the pattern of +ve reaction will declare the HLA typing. Further probes may be added to another membrane with an amplifying DNA to declare any ambiguity.
SSOP typing is well prepared to type wide number of samples, with each assay able to test 80-180 samples but taking about 2 days to get results. For a smaller number of samples, a modified assay, reverse-SSO (rSSO) can be used. In the rSSO technique, ONT probe can be bound to the membrane and each membrane have all the SSOs bound that are needed to type a specific HLA locus. Magnified, and then biotinylated, DNA can be added to the commercially available, premade membrane. Hybridize amplicon is then identified via a Chm or colour-depending detecting system. ONT probe can also be linked to a bead (solid phase hybridizing) to permit multiplex analysis of HLA typing via the Luminex platform. The rSSO typing technique still has the same disadvantage of ambiguities Rsl as the SSO technique but may allow much rapid turnaround timing.
 RT-PCR-based typing: This kind of typing is primarily relied on using allele-specific PCR like SSP methodology. However, instead of GE, amplicon can be identified in real time via the fluorescent dye or probing. Every well may contain a sequence-specific primer such that if one allele is found, the DNA became amplified. Adding cyanine dye can bind to any magnified, double-stranded DNA & fluoresce. Variable fluorescent readings of each well can be obtained at various degrees of temperature. Once the temperature rises, the DNA can be dissociated ("melted") and the Flr declines. The resulted melt-curve analysis permits an easy visualization of the presence or lack of certain allele (s), and the behaviour of the reactive wells can identify the desired HLA typing.
An alternate, the labelled SSOP can be utilized instead of the lowered specific cyanine dyes. This probe is designed for binding the location between primers and then labelled with a fluorescent reporting dye. If the primer able to bind to the present DNA, DNA polymerase will start copying the DNA. Once it can reach the location of the probe that is bound to the same DNA strand, the dye became freed and can be identified. With repeated rounds of magnifications, more dye can be freed. This Flr can be monitored via real timing and used to detect if the reaction can be "+ve." The advantage of using a probe-dependent method is the permission of variable fluorescent reporting molecules to be utilized in the same reacting wells that allow a higher degree of multiplexity. Moreover, as the specificity of this reaction is not limited to the primer pairing but also on the binding of the sequence-specified probing, this augment the typing Rsl of the assay. Utilizing RT-PCR-based typing may shorten the turnaround time to about one h. requiring much lesser hands of the technicians. Moreover, the interpreted automatised data may be also induce significant simplification of the analysis.
 SBT: depends on direct magnification and sequencing of the related exons via fluorescence-labelled di-deoxynucleotides. As this technique can unmask the specified nucleotide sequence of the magnified zone, it permits a higher Rsl typing. Then this sequence can be readily compared with a known sequence of HLA allele (s) in the IPD-MGT/HLA database to accomplish the HLA typing. However, in sample (s) with heterozygous allele (s), it is still hard assigning the basal calls of one allele or the other that may induce potential ambiguity.
 NGS: The introduction of NGS manoeuvres has allowed high-Rsl typing with significant reduction in ambiguities as it permitting basal calls to be accomplished to the same (cis) or variable (trans) alleles (= phasing). Despite the advent of this technology to be used in HSCT, the limited throughput, scalability, costs, and its speed restricted its application in certain SOT plans.
ANTI-HLA ABs SCREENING
Almost 30 %of ptns on the W/L are proved to have ABs against one or more HLAs, owing to Snz related to previous exposure to HLA Ags, e.g.,
o Pregnancy &
o Prior organ Tx.
The reason of screening ptns for anti-HLA ABs before commencing a Tx is to:
o Recognize pre-existing anti-HLA ABs,
o Determining their specificity, &
o Assessing their relative strength.
These data will assist the physician to evaluate the ptn's likelihood to receive an HLA-compatible Tx & classifying TR with high immunologic risk with mandated requirement of a more robust im/m regimens and/or more intense post-Tx monitoring.
The DSAs identified via cell-based cytotoxic assay can be considered an absolute C/I to Tx owing to their avidity to induce HA Rj. DSAs identified by more sensitive techniques (e.g., ELISA, Fc, or bead-based technique) usually representing variable levels of risk. Despite the pre-existing DSA is accompanied by a higher risk of Rj and allograft loss, it still debatable if these ABs identified via the solid phase tools impact the long-term allograft survival. However, avoidance of these Ags against which the ptn has a lowered-level of AB may induce in a better "matching" kidney graft for the TR improving the chance of long-term allograft outcome considering that lowered-level AB is indicating of previous exposure & immunologic memory against Ags. However, this attitude may also limit the ptn's access to SOT that may result in longer waiting time and higher risk of morbidity & MR whilst on the W/L.
Previous consideration was that only preformed ABs against HLA-A, -B, & -DR Ags were considered as risks for AMR and decreased graft survival. However, all HLA proteins including HLA-C, -DQ, & -DP are now dealt as antigenic agents with the potential to induce an AB response, despite it still uncertain whether the ABs against these loci can exert a similar effect on allograft survival in comparison to those against the HLA-A, -B, or -DR Ags.
Assays screening AB:
Cell-based cytotoxic assay: Historically, anti-HLA ABs were recognized by testing TR sera against a panel of donor cells representing the HLA Ag frequency within the donor cohort. TR serum was mixed to the donor lymphocytes, with added exogenous C and a specific dye. If the serum was containing AB that can bind to the donor cells fixing the C, cell death ensues. The observed pattern of reactivity can be utilized to assess the TR 's degree of Snz and liability for Tx. For example, if cell death is seen in 45 out of the 60 different cell donor(s) in the panel, the TR has a PRA = 75 % with ineligibility for receiving a graft from 75 % of the donor’s cohort based on the finding of preformed DSA that may induce +ve cytotoxic CMX against this cohort of donors.
The major disadvantage of this technique is that recognizing the anti-HLA AB specificity is difficult, especially for a very sensitized subject, as the possibility to define specificity relied mainly on the represented target Ag exclusive from other antigens.
Another hard limitation of the cytotoxic panel for AB survey is that the broad list reacting ABs against non-HLA Ags can entirely impede the analysis process. False-+ve readings could result from the finding of non-HLA ABs or IgM HLA + non-HLA ABs, whilst false -ve results can result from lowered titre AB. Each donor cell may express up to 12 distinctive HLA molecules (2 from each HLA-A, -B, -C, -DP, -DQ, -DR loci) against each, the serum can react, and TR often have ABs against several HLA Ags. Even with precise testing of the pattern of reactivity and wider panels of cells, it is usually difficult to recognize their exact specificity. For distinct recognition of PRA, panel should include cells from volunteer (s) representing donors pool but including only the most observed phenotypes.
So, HLA labs worldwide have mostly shifted to utilize the new technology for AB screening. Since 2009, the UNOS has mandatory recommendation to use SPA to recognize HLA AB in candidate TR in the US; however, as explained below, SPA is a completely variable technique that has no concordance with cytotoxicity findings.
Solid phase assay (SPA): The introduction of solid phase multiplex techniques has allowed the recognition of anti-HLA AB specificity with higher sensitivity & specificity. TR serum can be added to a mixture of polystyrene beads where purified HLA Ags being attached. A fluorochrome-conjugated anti-IgG detecting AB can be then added, with the finding of anti-HLA IgG isotype AB can be recognized via the flow cytometric methods (Luminex).
Currently, to save more costs, lab will 1st screen sera through pooled Ag or phenotype bead (s) that’re coated with several HLA Ags. If +ve results have been obtained, then the SAB assay can be utilized to identify the exact specificity of the HLA Ag against which the AB is currently directed. A single survey may allow the recognition of ABs against up to 100 distinctive HLA molecules, each of them has been expressing on a specific bead that has been impregnated on 2 fluorescent dye (s). Bead specificity can be identified via combining these signals and the finding of allo-AB determined by a 3rd "reporting" channel. The magnitude of Flr exerted by the allo-AB in this reporting channel may result in terms of its MFI and may provide certain clues regarding the magnitude and strength of found allo-AB. Despite these results are usually given as a numerical value, the MFI values cannot be utilized quantitatively. Moreover, MFI threshold above which an AB is dealt as "+ve" are not precisely standardized. Physicians should argue how best the interpreted findings within their current HLA labs.
Specificity of SPA: SPA testing is obviously more sensitive as compared to the cytotoxic assay. However, traditional SPA utilizes secondary AB recognizing ONLY IgG do NOT detect Ig M ABs against HLA even though IgM anti-HLA ABs can induce +ve CMX. SPA may also show false +ve findings as the reactivity against latex bead, can denaturize HLA Ag, or non-HLA protein coating these beads. Accurate identification of the anti-HLA ABs specificity can help determining which HLA Ags to avoid with considering a candidate donor. The current commercially present kits may allow detecting ABs against >200 variable HLA Ags, including the most found phenotypes in the US cohorts.
The class I HLA molecules are mostly representing via a single bead with some of them are sharing common epitope (s) (= public epitopes) against which a single AB may react against ALL beads expressing that specific epitope. Public epitope (s) is that being common to ALL members of a CREG, whilst private epitope (s) declaring the individual, serologically identified Ags. B7 & B8 can be identified via distinctive AB (private epitope) but also via a commonly found AB directed to the BW6 public epitope.
Class II HLA molecules are usually shown as heterodimers consisting of 2 polymorphic chains (a & B chains) and are commonly named considering their B chain only. The DR-a chain is widely monomorphic, and the anti-DR ABs are mainly targeting epitopes on the B chain. In contrary, DQ & DP Ags are composed of a chains that are greatly polymorphic, and whilst most recognised ABs seem targeting epitopes on B chain, there’re evidence that some can be directing to a chain or requiring a specific a/B combination. Almost 25 % of ABs reacting to DQ Ags may need recognizing both subunits. So, in each subject, up to 4 distinctive a/B combination must be identified as possible immune target (s). Of note, not all a/B combinations of the class II Ags expressing within the US cohorts are present in the commercially current kits. ABs reacting against all loci with DPA as an exception can be considered as an " Un-Ags" for DDKT in the US. Physicians should go in a deep discussion with their HLA labs regarding their current criteria for recognizing these settings.
+ve result: The MFI threshold cut-off is not standardized along HLA lab (s) nor are they utilized in the same mode by the Tx program. Each HLA lab stabilizes its threshold cut-off via validated assay using well-known -ve & +ve sera to recognize a cut-off value optimizing the true -ve & +ve rates of the assay. Certain labs may also choose setting their cut-off level for better correlation with CMX findings. Tx protocols may change their threshold according to the clinical risks with variable cut-off (s) according to the type of the organ, TR 's Snz history, whether their donor is living or cadaveric, and whether the test has been performed before or after Tx.
Study: by CTOT to determine standardized SAB testing has reported that MFI +ve cut-off (s) ranging from 1000-1500 may yield a higher level of agreements (>90 %) among HLA labs in identifying the presence/absence of an HLA AB. MFI values may be influenced by many technical effectors that may include:
o Ag density expressed upon the beads,
o The applied fluorochrome detecting AB, &
o The flow cytometer/Luminex instrumentation setup.
However, recognizing a threshold cut-off is often balanced between the assay’s sensitivity & its false +ve rates. Certain labs may test sera from 20 normal, healthy males without previous HLA-sensitivity and revealing a 7 % false +ve rate with an MFI threshold = 1000 that was declined to 0.5 % if the cut-off was elevated to 3000. However, the "true" anti-HLA AB that results from previous exposure can be identified at MFI values between 1000 & 3000. Testing a ptn's screened findings longitudinally may allow one to discriminate these true +ve that remain stable, from false +ve results that are usually sporadic.
Several Labs may be also utilizing variable MFI cut-offs for ABs against variable HLA loci to examine variabilities in their ability eliciting a +ve CMX. The observed density of HLA Ag present on an individual bead may not be corresponding to its density expressed on the cell surface. The HLA-C & -DP may have 13-18-fold lesser cell surface expressions compared with Ags of other loci. So, a larger burden of allo-AB-directed HLA-C/DP is currently required to detect a +ve CMX. Consequently, many labs apply the implementation of elevated threshold levels above which to call an HLA-C or -DP AB "+ve”. It still uncertain whether ABs against these loci express a similar impact on allograft longevity as compared to those who target other HLA Ags. However, many case reports have recognized their ability to induce AMR.
Nephrologist should be aware that inter-assay variations can also impact whether an AB is diagnosed present or absent if it is identified at an MFI value that’s adherent to the threshold cut-offs. Variations has been observed among kits from several manufacturers, variable lots of the same kits, and variable runs utilizing the same lots. Studies: have observed inter-assay variabilities in MFI levels of up to 25-50 %, even when implemented by a solitary lab with the recommended strict standardized operating manoeuvres. Furthermore, MFI levels may not be comparable between various labs examining the same sample; even with standardized reagents and strategies, one study found 25 % variable findings observed by the participating labs.
Recognizing the magnitude/strength of AB: MFI value on the bead, via undiluted ptn serum is representing a relative magnitude of AB bounding to the Ag on the bead and may be variable between individual beads. Of note, MFI readings are not synonymous with the AB titre, and several studies have observed their inaccuracy in the assessment of the AB strength & concentration. MFI values have been applied successfully to anticipate the possibility of the finding of -ve CMX below specific threshold (-ve predictive value), however, lacked ability to anticipate a +ve CMX is very problematic. Moreover, MFI levels of pre-Tx DSA are NOT persistently predicting allograft outcome.
However, the given information about the allo-AB burden can be currently useful, e.g., in evaluating the efficacy of AB removal via PE in Dsnz programs or in AMR therapy. Ideal assay should provide physicians with a trustable quantification. However, standardizing MFI values still do not consider assay variability. The current guidelines suggested that quantifying the AB burden is best evaluated via titration (serial dilution) reporting.
Correlating MFI values and the magnitude of AB present can be also problematizing in detecting their upper limit. Beads are usually saturated at MFI levels of 10,000, any more AB present in the serum will not be capable of binding. This defect may result in:
1) Underestimation of the AB burden, &
2) 2 variable ABs with similar MFI levels near the upper threshold may show greatly different serum levels.
For better evaluation of the AB loads, titration studies should be implemented. An AB detecting out past a 1:128 dilution is found at much greater concentration than AB detected only at 1:8 dilution, even if they both show the same MFI levels in the neat (undiluted) serum. The monitored AB load via titration seems to be more precise and predictable than depending on the MFI levels in estimating the efficacy of Dsnz regimens and can also be efficacious in anticipating a ptn's responding possibility.
Moreover, the number of bound Ag is variable from bead to another. MFI variabilities between different beads in single assay may be attributed to the different magnitudes of present target Ags, rather than variable magnitude of ABs, especially at near-saturation levels. Moreover, the relation between Ag concentration on the beads in comparison with cell surface expression is not well recognized; 2 variable ABs with the same MFI level may show variable CMX findings. MFI levels may be also underestimating the magnitude of AB found in the serum sample. This occurs in conditions where the allo-AB is reacting against a public/shared epitope. The bound AB is currently distributed across all beads that contain Ags expressing the common epitope, efficaciously "diluting out" the AB. This result in much declined MFI levels than if a single bead with the specific epitope is present with underestimated true AB burden.
False +ve results: Sometimes, generation & coupling of HLA Ags to the beads results in improperly conformed and/or denaturated protein, so, unmasking epitopes that are normally not found. If AB have bound to these neo-epitopes, a false +ve detected AB can be identified with no associated clinical value. The bound ptn's IgG AB to latex beadings, or to another non-HLA protein utilized in bead manufacturing, may induce a high background signal, and may be masking the true results of the performed assays. This is usually identified via the higher MFI levels of the -ve control beads that does not involve any bound Ag. To remove this effect, sera of the ptns can be pre-treated via adsorption beadings to eliminate the interfering agents.
False -ve results: The finding of interfering agents in the ptns' sera can also induce under-estimation of the DSA magnitude. Various inhibitors e.g., C constituents (e.g., C 1q & C 3/C4 activating products) may bind to the anti-HLA AB and stereotypically impede the ability to detect AB of binding (prozone effect) leading to a diminished MFI values and inaccurate conclusions that allo-AB is absent or having a lowered level. The finding of IVIG in a ptns' sera and/or IgM AB of the same HLA speciality can also impede the recognizing of IgG allo-AB.
Documented methodology preventing this phenomenon may include pre-treatment of the ptns' sera with EDTA to impede C 1q bound and/or titration studies diluting out the impact of the inhibitor revealing the finding of the allo-AB at greater dilutions. Other techniques e.g., DTT treatments or heat inactivation have also been documented.
False -ve results have also been observed if a lower-level AB has been directed against a public/shared epitope(s). As binding of the AB is distributed across > one bead, the MFI level of a single bead is underestimating the true magnitude of the ABs. This cascade may induce a -ve SAB testing but a +ve result if the phenotype bead is utilized or if CMXs have been performed. Proper testing of SAB histogram reports for allo-AB binding to CREG (s) may also help identifying this phenomenon.
C1q binding assay: All IgG ABs can be detected via the standardized SAB assays, regardless their sub-classes and their ability for C binding. Certain sub-classes (IgG1 & G3) have been observed to be more effective in C activation and consequently more amenable for inducing graft injury that has led to the evolution of the modified SAB manoeuvre (=C1q binding assay). After adding ptns' sera to the SAB mixture, exogenous C can be added. The finding of any bound C can be identified via a fluorescent conjugated anti-C1q AB.
Several studies have suggested that C-fixing IgG DSAs can be complicated by greater rates of graft loss compared with non-C-fixing DSAs, although the latter still portending a worse survival if compared with TR with no DSA. So, the order of survival benefit can be interpretted as follows:
1) TR with no DSA.
2) Non-C-fixing DSAs
3) C-fixing IgG DSAs
However, it’s not clear if this’s related to an inherent ability to discriminate between the AB's ability to activate C or if the positive results are reflecting the magnitude of ABs and/or better sensitivity of the assay detecting allo-AB that could be obscured by an interfering protein affecting the standardised SAB assays.
Any AB identified at MFI level of >10,000 on the standard assay is strongly correlated with C1q positivity, and, if adjusted to MFI readings, the C1q assay utilization did not seem to discriminate functionally distinctive and clinically significant DSA. The C1q binding assay use in KTx still uncertain, and not routinely applied in clinical transplantation.
Determining ptn Snz: PRA is a technique that clinician can utilize for assessing ptns' level of Snz to HLA Ags and their amenability for Tx. Later, PRA was recognised by the reactivity pattern of the ptns' sera against a panel of cells derived from volunteer (s) with HLA phenotypes representing the Tx donor pool.
In Dec 2007, UNOS recommends the cPRA to be mandatory replacement to this practice providing a more precise and accountable tool to assess ptns’ Snz. The cPRA is calculating the liability for Tx via utilizing the findings of the SAB assay to recognize the anti-HLA ABs specificity, combined with the common frequencies of HLA Ags within the donor cohort, e.g. if ptn showed an AB against the HLA-A2 Ag that’s found in 48 % of the US donor cohort (A2 Ag phenotypic frequency), their cPRA level will be 48 %, and they will be disqualified to receive 48 % of renal grafts considering they present DSA against the A2 Ag. If the ptn had an AB against B44 that’s found in 27 % of the population, their cPRA will be 27 %. If the ptn showed ABs against both A2 & B44, their cPRA level will be 59 % that is < the sum of the single Ag frequency as certain donors may be expressing both A2 & B44 Ags (i.e., cPRA = the % of donors expressing A2 alone + the % of donors expressing B44 alone + the % of donors expressing both A2 & B44).
The cPRA calculator that is based on HLA frequency derived from the US donor cohort can be available on the OPTN.
Ptns with high sensitization (cPRA ≥80 %) have ABs against several common HLA Ags that would make >80 % of doated organs ineligible for Tx. Ptns with broad Snz have ABs against several and variable Ags, but, if these are rarely found, they may not have a high cPRA. According to the current KAS in the US, a candidate's cPRA is applied in an algorithm for organ allocation to permit greater parity between individual TR on the W/L, regardless their magnitude of Snz.
Ptns with higher cPRA values can be given additional W/L "points" in trial to limit the difference in W/L timing between the greatly sensitized ptns as compared to ptns free of any anti-HLA ABs. The matching algorithm applied by UNOS is calculating the candidate cPRA via the Un-Ags list for the ptns.
Unacceptable Ags (Un-Ags): The term " Un-Ags " may refer to a donor HLA Ag against which a candidate TR showed pre-formed AB and should be discouraged due to a higher risk of AMR. Donated organs expressing these Un-Ags cannot be offered to the TR.
The finding of preformed DSA is currently identified via the SAB assay, but individual Tx protocols utilize different criteria and threshold levels through calling an Ag unacceptable, considering the balance between ptn's access to Tx & the magnitude of immunological risk that is considered acceptable. As low values of preformed DSA (with +ve flow CMX) may be complicated by early/late AMR, considering Un-Ags via a strict threshold will provide a better "matched" allograft for the TR, improving the long-term graft outcome. However, this attitude may also limit a TRs' access to the donated organs and result in prolonged waiting time with increasing risk of morbidity/MR while still on the W/L that is currently crucial especially in highly Snz TR. Despite the "priority points" provided by the recently designed KAS has increasing the number of Tx, it is anticipated that 25 % of TR with cPRA of 100 % are not possibly offered a single graft according to their Un-Ags list. Moreover, some reports have observed that exclusive detection of ABs in sensitive, SPA are not currently influencing the long-term allograft survival.
Un-Ags can be introduced for the following HLA loci: HLA-A, -B, -C, -DR B1, -DRB3/4/5, -DQA, -DQB, & -DPB. ABs against the HLA-DPA are usually not considered in the allocated schema. Deciding to consider certain loci and their threshold cut-off utilized to decide whether considered "unacceptable" may vary through various clinical Tx protocols.
CMX TESTING FOR DSA
The current reason of CMX testing is to recognize any pre-formed DSA found in a ptns' sera and directed against a specific donor. In 1969, Patel & Terasaki reported that ptns with pre-formed DSA rendering a CDC +ve posing higher threat of HA Rj with primary allograft non-function and made the implemented CDC CMX against donor T cells a standard prerequisite prior to Tx, with the +ve CMX became an absolute C/I for Tx. Since that time, other varieties and more sensitive CMX tests (e.g., flow CMX & "virtual" CMX) have been implicated in clinical practice. While +ve results from these tests may not preclude moving forward with Tx, they do highlight the presence of increased immunologic risk for graft injury.
Assays for CMX testing
CDC CMX: CMX assay is used to recognize the finding of preformed AB in a TR 's serum that is particularly directed against a potential donor(s) (= DSA). This ideally referring to the finding of anti-HLA AB, as HLA molecule exhibit a higher degree of Pmph, representing the primitive antigenic matter against which the immune system is reacting. Typically, CMX assay using the target donor allograft tissue may permit one to identify any ABs with potential reaction against the allograft. This may include HLA AB as well as ABs against non-HLA Ags found on the target cell surface, e.g., endothelial Ags, AT1 receptor, & MICA that have been sometimes associating AMR.
CMX assay utilizing allograft-derived cells is not practical nowadays. So, the currently performed CMX assay utilizing donor lymphocytes as surrogative: T cells expressing class I HLA molecules, with +ve T cell CMX recognizing the finding of a class I DSA; B cells expressing both class I & II HLA molecules, with +ve B cell CMX recognizing class I & II DSAs. Separation of donor lymphocytes are 1st performed (via magnetic bead isolation) into (1) CD3+ T cell & (2) CD19+ B cell fractions.
The TR’s sera are then added to these cells, followed by adding the C with a viability dye. If DSA is found, cell lysis can be seen, and the CMX is considered +ve.
The CDC CMX is usually depending on the magnitude of DSA found in ptns’ sera, Ig isotype, and cell surface density of the targeted HLA Ag. The lowered titter, AB may not gather in enough density to crosslinking C and activating the MAC inducing cell lysis. To augment the sensitivity of the assay, AHG, a C-fixing AB that can bind human Ig, can be added after addition of the TR 's sera. Via binding any DSA already bounding donor's lymphocyte, it can increase the density of the found AB, consequently increasing the possibility of C activation. Moreover, as AHG usually binding not only to C-binding DSA but also to non-C-binding DSA, its utilization permits the identification of non-C-binding DSA that cannot induce reactivity in an un-enhanced testing. Both tests may provide false +ve result(s) owing to the finding of:
1) IgM HLA AB or
2) Non-HLA AB or
3) Non-HLA IgG AB (against Ags on lymphocyte)
Fc CMX: It varies from CDC CMX in its higher level of sensitivity and its ability to identify IgG DSA whatever its ability to activate C. TR sera can be added to the donor’s lymphocytes, followed by adding secondary fluorochrome-conjugated AB capable of detecting the human IgG. With test, donor lymphocytes have no need to be isolated into their T/B cell fractions. Instead, additional detecting ABs (conjugating to various fluorochromes) can be added to discriminate the 2 subsets. Sample (s) analysis via a flow cytometer with results could be read quantitatively appear as MFI units. Ptn's sera can be incubated with donor’s cells (allo-CMX) and compared to -ve controls containing pooled sera from normal, healthy, non-sensitized subjects. Shifting in Flr severity above a pre-identified cut-off may signify the finding of an allo-AB.
The intensity/magnitude of AB found can be reflected on the shift magnitude.
It is crucial for physicians to be understand that Fc CMX regimens are not standard in their assay. An individual HLA lab established its own threshold cut-off via validated testing of known -ve/+ve ptns’ sera and correlated them with the SAB assay. The reported Fc CMX findings also show wide variabilities that can be expressed as channel shifting of median Flr MCS above the baseline or normalized against MESF, ratio (s), or as DFU. Moreover, Flr severity measuring can vary according to cytometer criteria, variabilities in detecting AB used (e.g., manufacturers policy, fluorochromes, and concentrations), modifying protocol, & variabilities in cell concentration. This inconsistency induces some difficulties in threshold cut-off standardization among HLA labs and represents some challenges in study interpretation or clinical plans utilizing these findings quantitatively to identify AB strength/magnitude. Physicians must be communicated with their related HLA labs for better interpretation of CMX reports at their Tx centre. Commonly unanticipated +ve findings observed in Fc CMX assay may include:
1) Donor’s cell viabilities,
2) A distinctive threshold cut offs.
3) The finding of AB reacting against lymphocyte specified Ags,
4) Higher background signalling (especially with B cell Fc CMX),
Subjects having only anti-HLA IgM AB may show -ve flow cytometry CMX as the fluorochrome-labelled determining AB is only IgG selecting.
Virtual CMX: The title "virtual" CMX may refer to a tool that a clinician or lab be utilizing the 2 actual lab assay findings (the anti-HLA assay findings & the HLA donor typing) deducing which results of an actual CMX might be, should one be performed. If a TR having AB against an HLA Ag for which the donor is mismatching with the candidate (DSA), and if the "power" of the AB is considered fair enough, there’s some anticipated values with +ve/-ve actual CMX. The correlated value of this DSA or "virtual CMX " naturally depending on which kind of CMX could be predicted.
In a report provided by the UNOS/OPTN, the virtual CMX may show a >85 % +ve predictive value on a subsequent flow CMX. On the other hand, its -ve predictive value was only about 50 %, supposing to be lower owing to an incomplete profile of the ptns' HLA ABs. During this study, ABs against HLA-C, -DQ, &-DP Ags were not ideally dealt as un-Ags. Moreover, the anticipating value of the virtual CMX is greatly related to the threshold cut offs each HLA lab utilizing to identify the finding of an AB.
Unexpected +ve CMX findings: A +ve CDC or flow CMX with lack of DSA (-ve virtual CMX) may be induced by a variety of factors (Tab. 1):
 Presence of "true" DSA: Sometimes, +ve CMX finding can be attributed to "true" but un-recognized DSA. The utilized serum for screening HLA ABs may be variable than the serum utilized for the CMX assay. Here, if a sensitizing effector has been introduced, a +ve CMX but a -ve SAB screening may be reflecting the newly developed DSA. Moreover, AB levels may be fluctuating by time, either normally or responding to therapy. Lack of a comprehensive profiles of ptns' HLA ABs and/or donor HLA typing insufficiency can also provide an incorrectly assessed virtual CMX with discrepancy between these findings & the physical CMX testing.
For example, if HLA-DP ABs were found in a ptns' sera but not currently identified by an HLA lab, or if the donor has not been typed at the DP locus to identify the donor specification to the AB, then the virtual CMX could be wrongly considered as -ve (false -ve). Failed consideration of the sharing epitope(s) may also result in under-estimation of the DSA recognized by SAB assay. If several beads containing a shared epitope, all these beads may register MFI levels under the considered threshold cut-off. Recognizing a shared epitope is currently crucial to interpret the SAB testing properly.
 The finding of non-HLA AB: The finding of AB against self-/non-HLA Ag present on T/B cells may induce a +ve CDC or flow CMX results. As AB reactivity is directed to a non-HLA Ag expressing on a lymphocyte but NOT on graft tissue, these ABs are not likely to induce graft injury. If the ptns' sera tested against their own lymphocytes (auto-CMX) or against surrogated donor(s) may be revealing a repeated +ve CMX findings with lack of DSA.
 Finding of IgM ABs: IgM ABs may induce false +ve CDC CMX, if directed against non-HLA Ags, or true +ve CDC if directed against HLA.
IgM could be auto-AB, mostly detected in ptns with autoimmune diseases and usually not pathological. IgM auto-AB could be detected via an auto-CMX assessing the reactivity of a ptn's serum to his own lymphocytes. To confirm this test, we can add DTT or DTE to the assay, breaking the disulfide bond of the IgM pentamer, rendering the CDC CMX to be -ve. If DTT/DTE added to the allo-CMX; a +ve result that is rendered -ve after DTT/DTE treatment can be attributed to the IgM AB, whilst remain +ve with finding of IgG AB. However, not all IgM ABs considered benign; those owing an anti-HLA specificity can be observed with HA/accelerated Rj. With exposing to a foreign HLA via recently sensitizing events, ptn will 1st mount an IgM anti-HLA ABs response before to be class-switched to an IgG isotype (along 2-4 wks).
Using DTT/DTE does not differentiate between benign IgM auto-AB and potentially pathological IgM allo-AB. IgM ABs cannot be currently identified by the traditional Fc CMX or SAB assay. The latter tests may be employed in detecting AB that specifically recognizing only IgG. Theoretically, these tests may be modified using a secondary AB that is able to detect the IgM, however, this is not routinely implemented. It is believed that the anti-HLA IgM may be interfering with a flow CMX via steric hindrance of IgG inducing false -ve results.
 Therapeutic mAB: A growing number of ptns on W/L are usually treated by mAB that can interfere with the CMX assay. The most commonly observed scenario is a TR treated with Rtx, an anti-CD20 mAB. If found in the TR serum, Rtx can bind in vitro to the donor B cells (expressing CD20), resulting in false +ve B cell CMX (both CDC & Fc CMX). T cell CMX testing have not been implicated, as T cells is not expressing CD20. Other therapeutic ABs known to be interfering with these tests may include Alm (binding to CD52 on T/B cells), (ATG; polyclonal AB binding to Ags on T/B cells), and daratumumab (binding to CD38 on B cells and, to less extent, T cells). Their impact on the tests depending on the pharmacokinetic/pharmacodynamic of the agent and most robust with greater concentration of the seral mAB. The interfering Rtx may persist for a complete year after infusion. Adding Prn to the test can help eliminating the contribution of the mAB.
 Impact of pronase (Prn): B cell flow CMX can be sometimes hardly interpreted owing to the higher Flr background related to binding of the fluorochrome-conjugated that detects AB to the cell surface IgG (i.e., B cell receptor) and Fc receptor (s) found on B cell.
The higher background Flr decreases the sensitivity of the test to identify additional Flr that is due particularly to DSA. To decrease this background Flr with better test specificity, some labs may utilize Prn to manage these cells. Prn = cocktail composed of non-specified proteases that isolated from Streptomyces griseus, and comprising of neutral protease(s), trypsin, chymotrypsin, carboxy peptidase, aminopeptidase, and phosphatase (s). Commercially present Prn formulas may vary in their composition and magnitude of activity with variabilities between various companies & lots.
Each lot can be examined for its rate of Folin +ve a.a. & peptides from casein that is reported as units of reactivity allowing standardized testing. However, utilizing Prn may impact HLA expression reducing its sensitivity to identify the finding of DSA. It may also induce false +ve CMX findings by unmasking cryptogenic (hidden) epitopes.
Incubating time, concentrations, lot-to-lot differences, and products from variable manufacturers are effectors that should be evaluated if an HLA lab considered this modified test. The threshold for +ve CMX identification should be modulated if Prn is utilized. As mentioned before, Prn has been frequently utilized to prevent false +ve CMX result related to the finding of therapeutically provided monoclonal AB in ptn's serum. With these circumstances, incubation period is too long and/or Prn levels is increasing as compared to that utilized in regimens decreasing the background alone.
However, a similar consideration may be applied regarding the unintended consequences of decreasing sensitivity related to an effect on HLA expression level. Consequently, some physicians prefer to identify the immunologic risk of class II DSA via SAB assay findings alone, excluding B cell CMX result.
HLA TESTING FOR IMMUNOLOGICAL RISKS
The findings of all HLA assays should be interpreted providing the immunological risk evaluation between a donor & TR pair. Furthermore, deciding to declining/proceed a Tx should also take in consideration the clinical criteria e.g., organ typing, urgent events, liability to receive compatible grafts, availability, appropriate im/m regimens for a TR, and various donor factors.
Common scenarios: Common clinical scenarios of HLA assay findings and their interpreted results can be summarized in table 1:
Interpretation of CMX results in KTx
o Higher DSA burden.
o Higher risk of HAR.
o Moderate DSA burden.
o Non-C-fixing DSA
o IgM AB (anti-HLA or non-HLA).
o Lowered DSA burden
o Variable sera for SAB vs CMX testing (historical DSA)
o Recognizing an AB specific to an allele that donor does not have
o False +ve SAB testing (non "true" DSA) owing to:
1) Binding to denatured Ag
2) Lowered threshold to call an AB present (overcalling)
3) Higher background (serum factors bound to latex beads)
o Non-HLA IgG bound to cell surface Ags present on lymphocytes
o Medication effect (e.g., Rtx, ATG, Alm, IVIG) where therapeutic AB bound to lymphocytes can be identified via the assay
o ABs against specific loci may not be routinely recognized by HLA lab
o Variable sera used for SAB vs CMX (DSA present, new DSA occur within interval sensitizing event, or greater burden in serum used for CMX)
o False -ve SAB:
1. Donor Ag/allele is not representing in the bead panel.
2. Class II: Combined donor a/B chain not represented by bead panel
3. The finding of inhibitors in serum ("prozone" impact)
4. IgM/IVIG bound to beads masking IgG allo-AB identification.
5. Lowered anti-HLA AB level against a shared epitope "diluted out" along multiple beads (under-represented true AB burden)
o Low-level IgG non-HLA AB
o False - ve SAB testing.
Potential variabilities in lab strategies must be considered when correlating the assay findings and the allograft outcome and may also declaring the disparities between different studies. Unexpected discrepancies between the assays may invite a deep discussion with the HLA lab personnel.
o +ve CDC CMX: A +ve CDC CMX owing to DSA is an indication of higher AB burden and considered C/I to Tx as it can be associated with HA/accelerated Rj.
o -ve CDC & +ve Fc CMX: A +ve flow CMX with -ve CDC is representing an intermediate risk for AMR but is NOT a C/I to Tx. However, many reports showed: a +ve flow CMX due to DSA can be complicated by a higher rate of Ac Rj with both early & late allograft loss.
o -ve CDC & Fc CMX + DSA by SAB: clinical effect of ABs identified only by SPA, with many reports: lowered-level ABs have NO clinical impacts reflecting oversensitivity of threshold utilized to identify the ABs. However, the finding of DSA is indicative previous exposure to donor-specific HLA Ag with a higher risk of a latent memory responses.
o +ve CMX without DSA: With DSA lack by SAB assay, a +ve CMX can NOT be correlated to allograft outcome. Here, CMX is +ve due to clinically irrelevant, non-HLA AB. This can be seen where a "true" anti-HLA AB is found in the TR serum but currently not detected by SAB assay (false -ve results).
Historical DSA: The lack of pre-Tx DSAs does not reflect an absence of previous Snz, as the test is reflecting ONLY the AB level found in the currently tested serum samples. Thorough testing of longitudinal SAB assay and previous CMX findings may declare the finding of a historical DSA with anti-donor Ag immunological memory. Whilst the finding of immunological memory reflects a greater risk degree, it still difficult to anticipate its impact on allograft survival.
There is not currently found, clinically valid testing challenging a latent memory response or identifying if a low-titter DSA will persistently low or rapidly increasing after Tx after repeated exposure to an Ag.
However, considering the potential memory recall responses may impact the physician's policy to augment his im/m strategy and/or post-Tx monitoring plans.
Clinically significant MFI value of pre-Tx DSA: MFI value of pre-Tx DSA do not seem to anticipate allograft outcome. One study: increased rates of AMR & allograft failure were observed if the pre-Tx DSA showed MFI levels >10,000 but were comparable between all other g.s (moderate MFI [5000-10,000] vs lowered MFI [1000-5000]).
Study: The difference in allograft longevity was primarily related to determining the finding of DSA (MFI >1500) but Not to the actual value of MFI. Failing to discriminate these groups of ptns may be reflecting the limited technology of the test (with MFI levels is a poor indicator of the magnitude/strength of the ABs) in addition to the failure of prediction of whether a lowered level of DSA will be persisting lowered or be rapidly increasing after Tx with repeated exposure to the Ag.
Lack of pre-Tx DSA impact on outcome: Even with lack of DSA, certain reports have observed that sensitized TR (with non-DSA allo-AB) are at greater risk for allograft loss. However, it is still controversial, as other reports have showed No effect on TR having non-DSA on allograft survival. The discrepancy of these findings may be also attributed to whether more/less aggressive im/m have been administrated or may be reflecting incomplete DSA determination depending upon whether ABs against all loci (including class IIa chains) have been considered.