HISTOCOMPATIBILITY TESTING,

CROSSMATCHING

PRESENTER : DR. KASHYAP DAHAL

DM SR
NAMS, BIR HOSPITAL
 THE MAJOR HISTOCOMPATIBILITY
COMPLEX
• Human MHC Gene cluster:

 0.1% of the genome

 Located on chromosome 6p21.31

 50% is expressed and 40% of the expressed

gene is involved in immunity
 THE MAJOR HISTOCOMPATIBILITY
COMPLEX
• Human MHC Gene cluster:
 The MHC has been divided into three regions:
class I (telomeric), class II (centromeric), and
class III
 INHERITENCE
• There is a 25% chance that siblings will share
the same haplotypes (two-haplotype match)

• A 50% chance they will share one haplotype

(onehaplotype match)

• 25% chance that neither haplotype will be the

same (zero-haplotype match).
 HLA MATCHES AND MISMATCHES
1. A2, -, B27, B13, DR17, DR4

2. A2, A3, B8, B14, DR17, -

 Failure to identify the second antigen

 The inheritance of the same antigen

HLA NOMENCLATURE

Handbook of kidney transplantation . Danovitch . GM. 6th edition

 HLA TYPING TECHNIQUES
 Immunological typing method

• Serology
• Cellular typing

 DNA based typing

• DNA-based typing using sequence-specific oligonucleotide

probes
• DNA-based typing with direct Sanger sequencing
• Next-generation DNA-based typing
• DNA-based typing with genotyping microarray
 HLA TYPING TECHNIQUE
 SEROLOGY:
• Lymphocytes from the patients to be typed
are added to the antiserum

• Complement and dye is also added

• Proportion of dead cells in each well is

examined
 HLA TYPING TECHNIQUE
 CELLULAR TYPING:

• Human T cells can distinguish more division among

some of the HLA recognized by the sera

• Mixed lymphocyte typing allow more exact typing

• A panel of reference cells that are homozygous for

different antigens, called homozygous typing cells
(HTCs)are used
DNA BASED TYPING

Patel R et al. N Eng J Med. 1969;280(14):73

5
DNA-based typing using sequence-specific
oligonucleotide probes
• DNA PCR amplificationis done using a set of carefully
selected primers designed to amplify the desired portion of
DNA

• Different primer sets are used for different loci, such as HLA-
DRB, -DQA, -DQB, etc.

• The amplified section of DNA is then probed with various

SSOPs, which are typically labeled by nonradioactive means

• Designed to bind only to specific sequences that

discriminate among alleles
 SEQUENCE-SPECIFIC PRIMERS (SSP)
TYPING
• SSP depends on DNA amplification using group- or
allele-specific primers

• Detects an amplified product of the correct size by gel

electrophoresis

• The size is determined by running an agarose gel that

separates the PCR products according to their size

• A major advantage of the SSP method is its rapid turn

around time of two to three hours
DNA-BASED TYPING WITH DIRECT SANGER
SEQUENCING
• DNA-based typing with direct Sanger sequencing, also
called sequence-based typing (SBT)

• Consists of the amplification and direct sequencing of

relevant exons

• Based on the incorporation of chain-terminating

dideoxynucleotides by the DNA polymerase during in vitro
DNA replication

• It will give the exact DNA sequence at the nucleotide level

of the DNA fragment under consideration
 REAL-TIME PCR (RT-PCR)-BASED TYPING
• This form of typing is based upon the use of allele-specific
PCR similar to SSP methods

• Instead of using gel electrophoresis, amplicons are detected

in real time with the use of fluorescent dyes or probes

• Each well contains sequence-specific primers such that if

the allele is present, the DNA becomes amplified

• The added cyanine dye binds to any amplified, double-

stranded DNA and fluoresces
 REAL-TIME PCR (RT-PCR)-BASED TYPING

• Fluorescent readings of each well are obtained at

different temperatures

• As the temperature increases, the DNA dissociates

("melts") and the fluorescence decreases

• The resulting melt-curve analysis allows for easy

visualization of the presence or absence of specific
alleles, and the pattern of reactive wells determines
the HLA typing
 SCREENING FOR HLA ANTIBODIES
• Donor-specific anti-HLA antibodies (DSAs)
detected by cell-based cytotoxicity assays are
considered an absolute contraindication to
transplantation because of their propensity to
cause hyperacute rejection

• DSA detected by other assays represent

varying degrees of risk
Patel R et al. N Eng J Med. 1969;280(14):735
 CELL-BASED CYTOTOXICITY ASSAYS

• Anti-HLA antibodies identified by testing recipient sera against

a panel of cell donors with known HLA specificities that are
representative of the HLA antigen frequency within the donor
population

• Recipient serum is mixed with donor lymphocytes, along with

exogenous complement and a viability dye

• If the serum contains antibody capable of binding to the

donor cells and fixing complement, cell death occurs

• The pattern of reactivity is used to calculate the recipient's

degree of sensitization and likelihood of transplantation
CELL-BASED CYTOTOXICITY ASSAYS
• False-positive results arise from the presence
of non-HLA antibodies or immunoglobulin M
(IgM) HLA and non-HLA antibodies

• False-negative results can occur with low titer

antibody
 SOLID PHASE ASSAYS
• Recipient serum is added to a cocktail of
polystyrene beads, to which purified HLA antigens
are attached

• A fluorochrome-conjugated anti immunoglobulin

G (IgG) detection antibody is then added

• The presence of anti-HLA IgG isotype antibody is

identified by flow cytometric methods (Luminex)
 SOLID PHASE ASSAYS
• Laboratories first screen sera using pooled antigen or
phenotype beads that are coated with multiple HLA
antigens

• If this screening test is positive, then the single-antigen

bead (SAB) assay is used to determine the precise
specificity of the HLA antigen against which the antibody is
directed

• A single assay allows for detection of antibodies directed

against up to 100 distinct HLA molecules
 SOLID PHASE ASSAYS
• The degree of fluorescence exhibited by the
presence of alloantibody is given in terms of
its median fluorescence intensity (MFI)

• Provide some clue as to the amount and

strength of alloantibody present

• MFI thresholds above which to call an antibody

"positive" are not standardized
 SPECIFICITY OF SOLID PHASE ASSAYS

• Solid phase assays are more sensitive than

cytotoxic assays

• More specific for anti-HLA antibodies

• Solid phase assays do not detect IgM

antibodies against HLA
• Most of the class I HLA molecules are
represented by a single bead

• Among these, some share common epitopes

against which a single antibody can react
against all beads expressing that particular
epitope

• Private epitopes delineate the individual,

serologically defined antigens
 MFI
• MFI-positive cutoffs ranging from values 1000
to 1500 yielded a high level of agreement (>90
percent) among HLA laboratories in
determining the presence or absence of an
HLA antibody

Reed EF et al. Am J Transplant 2013

 MFI
 MFI levels can be affected by:

• The density of antigen expressed on the beads

• The fluorochrome detection antibody used

• The setup of the flow cytometer

 False-positive results:

• Improper conformation of protein and /

or denaturation  unveiling epitopes that are not
naturally found

• Binding of the patient's IgG antibody to the latex

beads themselves, or to a non-HLA protein used
in bead manufacture a high background signal
and obscure the true results of the assay
 False positive results:

• This is often detected by the high MFI values of

the negative control bead, which does not
contain any bound antigen

• To eliminate this, sera can be pretreated with

adsorption beads to remove interfering factors
 False-negative results:

• Inhibitors such as various complement components (including

C1q and C3/C4 activation products) can bind to the anti-HLA
antibody and stereotypically hinder the ability of the
detection antibody to bind

• The presence of IVIG in a patient's serum and/or IgM

antibody of the same HLA specificity

• Low-level antibody is directed against a public/shared epitope

 C1q binding assay

• After addition of the patient's sera to the SAB

mixture  exogenous complement is added

• The presence of any bound complement is

then detected using a fluorescent-conjugated
anti-C1q antibody
 CROSSMATCH TESTING FOR DONOR-
SPECIFIC ANTIBODIES
Complement-dependent cytotoxic (CDC) cross
match:

• T cells express class I HLA molecules, and a

positive T cell crossmatch identifies the
presence of a class I DSA

• B cells express both class I and class II HLA

molecules, and a positive B cell crossmatch
identifies class I and class II DSAs
 CROSSMATCH TESTING FOR DONOR-
SPECIFIC ANTIBODIES
Complement-dependent cytotoxic (CDC) cross match:

Donor lymphocytes are separated (usually by magnetic bead

isolation) into a CD3+ T cell fraction and a CD19+ B cell
fraction

Serum from the recipient is then added to the cells

Addition of complement and a viability dye

If DSA is present, cell lysis is observed, and the crossmatch

is deemed positive
 Complement-dependent cytotoxic (CDC) cross match:

• The CDC crossmatch depends upon the titer of DSA present in sera,
immunoglobulin isotype, and cell surface density of the target HLA antigen

• Low titer, but potentially relevant, antibody may not cluster in sufficient density
to be able to crosslink complement and activate the membrane attack complex
to induce cell lysis

• To enhance the assay's sensitivity, anti-human globulin (AHG), a complement-

fixing antibody capable of binding human immunoglobulin, is added following
the addition of the recipient's sera

• By binding any DSA already bound to the donor's lymphocytes, it increases the
density of antibody present, thereby increasing the likelihood of activating
 Complement-dependent cytotoxic (CDC) cross match:

• AHG also binds non-complement-binding DSA 

detection of non-complement-binding DSA that would
not result in reactivity in the unenhanced assays

• This can result in false-positive results due to the

presence of non-HLA IgG antibody (directed against
antigens present on lymphocytes) or IgM HLA or non-
HLA antibody.
 Flow cytometry crossmatch

• The flow cytometry crossmatch assay is more sensitive than the

CDC crossmatch and has greater ability to detect IgG DSA
regardless of its capability to activate complement

• Recipient serum is added to the donor lymphocytes, followed by

the addition of a secondary fluorochrome-conjugated antibody
that detects human IgG

• Donor lymphocytes do not need to be separated into their T/B cell

fractions
 Flow cytometry crossmatch

• Additional detection antibodies (conjugated to different

fluorochromes) are added to distinguish between the two subsets

• Samples are analyzed on a flow cytometer and results read out in

a quantitative fashion as a unit of median fluorescence intensity
(MFI)

• Samples with the patient's sera incubated with the donor cells
(allocrossmatch) are compared with a negative control containing
pooled sera from normal, healthy, unsensitized individuals

• A shift in fluorescence intensity above a predetermined cutoff

signifies the presence of an alloantibody. The strength and amount
 Flow cytometry crossmatch
• Common false-positive results encountered in flow cytometry
crossmatch testing include high background signal
(particularly with B cell flow cytometry crossmatch)

• The presence of non-HLA antibody and a strict threshold

cutoff

• Individuals who have only anti-HLA IgM antibody may have a

negative flow cytometric crossmatch since the fluorochrome-
labeled detection antibody is selective for IgG
 Virtual crossmatch :

• The results of two actual laboratory tests (the anti-HLA

screening results and the HLA type of the donor) is used
to deduce what the result of an actual crossmatch might
be if performed

• If a candidate is found to have antibody against an HLA

antigen for which the donor is mismatched with the
candidate (DSA), and if the "strength" of the antibody is
thought to be sufficient, there is some predictive value
with a positive or negative actual crossmatch
Handbook of kidney transplantation . Danovitch . GM. 6th edition
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