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).
HLA TYPING
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
[1] 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.
[2] 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.
[3] 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.
[4] 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.
[5] 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 BTx,
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):
[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.
[2] 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.
[3] 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.
[4] 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.
[5] 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
|
SAB |
Flow CMX |
CDC CMX |
Impression |
|
+ve |
+ve |
+ve |
o Higher DSA burden. o Higher risk of HAR. |
|
+ve |
+ve |
- ve |
o Moderate DSA burden. o Non-C-fixing DSA |
|
- ve |
- ve |
+ve |
o IgM AB (anti-HLA or
non-HLA). |
|
+ve |
- ve |
- ve |
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) |
|
- ve |
+ve |
+ve |
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) |
|
- ve |
+ve |
- ve |
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).
Other varaities:
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.
COMMENTS