This document discusses antibody-mediated rejection (AMR) in organ transplantation and methods for detecting donor-specific antibodies (DSAs). It first describes how AMR occurs when antibodies react with antigens on vascular endothelium, causing complement activation and vascular damage. Diagnosis requires histologic lesions, detection of C4d factor, and identifying DSAs in serum. It then discusses different antibody detection methods like panel-reactive antibodies (PRA), Luminex single antigen beads, and crossmatch tests. Flow cytometric crossmatching is more sensitive than CDC crossmatch in detecting low-titer or non-complement-fixing antibodies. Preformed DSAs, especially against HLA Class I and II, increase rejection

1. 23/10/2017
1
Panel Reactive Antibodies and
Crossmatch by Flow
Cytometry
Katherina Psarra
Immunology – Histocompatibility Dept
Evangelismos Hospital
Athens, Greece
Renal transplantation
In spite of the enormous progress in the
techniques
• Surgery
• Graft preservation
A main limitation for the successful result of
a transplantation especially renal and to a
lesser extent of other organs
remains
the recipient’s immune response to the donor
cells/tissues
rejection
Graft loss
Variable factors contribute to στην
progressive malfunction
Loss of the allograft
(chronic rejection remains a challenge)
antibody-mediated rejection – AMR
consists the dominant cause of graft loss
Antibody – mediated rejection (AMR)
• Abs react with antigens of the
vascular endothelium 
• complement activation
• Rapid endovascular thrombosis,
• PLT accumulation 
• Vascular endothelium necrosis
worse prognosis asking for a
different therapeutical
approach than T-cells
dependent rejection treatment
AMR diagnosis confirmation (revised Banff criteria)
demands
 histologic lesions in renal biopsy (acute tissue destruction)
 recent interaction of abs with vascular endothelium
C4d factor on peritubular capillaries (PTC)
 detection of DSA (Donor-Specific Antibodies) in the serum
- HLA or non-HLA
- preformed or de novo
Antibody – mediated rejection (AMR) Antibodies against
 Major Histocompatibility Systems (polymorphic)
• RBC antigens (mainly ABO system)
• MHC - Major Histocompatibility Complex antigens
known in humans as
• HLA- Human Leukocyte Antigens
Minor Histocompatibility Systems
(no significance in clinical practice)
 Endothelial cells antigens
(most non polymorphic)
Antiendothelial Cell Antibodies – AECAs

2. 23/10/2017
2
ΑΒ
Β
Α
Ο
ΑΒ
Β
Α
Ο
recipient
donor
τα συγκολλιτινογόνα Α και Β εκφράζονται στο ενδοθήλιο των αγγείων
People without Α or Β antigens have anti-Α and anti-Β antibodies in serum
Transplantation with major ΑΒΟ incompatibility leads to hyperacute rejection
Tranfusion rules are
followed
Significance of ABO compatibility
in renal transplantation
Trials of ΑΒΟ incompatible
renal transplantations
 Α2 Ο (due to A2 low
antigenicity)
 transplantation from live
ΑΒΟ incompatible donor
after:
• plasmapheresis
• B cells depletion (in the
place of splenectomy)
• immunosuppressive
treatment
MHC region
• situated on the short arm of chromosome 6
• it covers 4Χ106 base pairs (4Μb)
• genes are classified into class Ι, ΙΙ και ΙΙΙ
HLA antigens
 Glycoproteins
cell membrane
 classified
• class Ι
HLA-A, -B, -Cw
• class ΙΙ
HLA-DR, -DQ, -DP
sensitization: generation in the recipient of abs against
allogeneic HLA antigens
• blood transfusions (multiple)
• pregnancies (women with many
children)
• HLA incompatible allograft
rejection
• Venus or arterial allograft
• infections
• trauma
• inoculations
Donor HLA :
A1,A2
B7,B8
recipient HLA :
A1,A3
B8, B52
+
anti-A2
anti-B7
One single sensitization event may lead
to multiple specificities abs generation

3. 23/10/2017
3
anti A2
anti B7
A28
A23
A69
A68
B57
A24
B58
B27
B60
B61
B13
B42
B54
B55
B48
B41
B47
+
Sensitization against multiple HLA antigens in one transplantation
Due to shared epitopes in different HLA antigens
The role of transfusions in sensitization
• Transfusions increase sensitization risk
 20% sensitization or panel reactive antibodies (PRA) increase
• If it is possible transfusions should be avoided in all candidate
renal recipients
anti-HLA antibodies detection methods
 Panel-reactive antibodies (PRA)
 Donor-specific antibodies (DSA)
detection and identification methods
1. Using cells
 Complement-dependent Cytotoxicity (CDC)
 Flow cytometry (Flow PRA)
2. Solid phase
 Enzyme-linked Immunosorbent Assay (ELISA)
 Bead Array Assay (Luminex)
- single antigen beads (SAB)
Hypersensitized patients: with PRA > 70%
(class Ι or/and class ΙΙ)
they are checked in renal failure patients in the lists
• every 3 months
• 2-4 weeks after each sensitization event (e.g tranfusion)
PRA using cells and CDC
an example
Robert A. Bray et al 2011
The Concept of FlowPRA™ Test
FITC
Sheep Anti-
Human IgG
Alloantibody
Class II Bead
Mixture of
Multiple
Beads
Class II
Bead
Class I
Bead
Class I
Bead
Class I
Bead
HLA Polymorphic
Antigens
FlowPRA Class I Specific Tests
Pioneer Multiplexed Bead Assay

4. 23/10/2017
4
FlowPRA™ II Specific Tests
MFI
Mean Fluorescence Intensity
single antigens
present on each bead
Bead Array Assay (SAB) results
MFI
it depends on the quantity and the
strength of the ab
Bead Array Assay (Luminex)
Main advantage
Identification of Abs against specific HLA antigens (one antigen per
bead)  Virtual Xmatch
Disadvantages
•Very sensitive method  false positive results 
the patient remains in the list
•It doesn’t discriminate between abs activating or not complement
•It is not clear whether these abs have any clinical significance (on
the beads there are native or denatured HLA antigens)
•The method is not standardized
an important debate:
what is positive and what is negative (cut-off)?
no agreement regarding clinically significant MFI
- peak MFI › 6000 correlates with graft failure
- Most of the centers agree that DSAs with MFI › 2000 have clinical
significance
- DSAs with 1000 › MFI › 2000 shouldn’t be ignored (possible accelerated acute
rejection in some patients)
Specific crossmatch - ΧΜ
methods
•CDC- XM
•T/B Flow Cytometry- XM
Checking specific sensitization against a particular donor

Recipients with donor specific abs are excluded

Hyperacute rejection is avoided
CDC-XM
advantages
• The only method detecting only abs complement
activating (greater clinical significance)
• The only functional method
(it imitates in vivo reality)
disadvantages
• It does not detect low titer abs
false negative results
• False positive results in the presense of
autoabs, non-HLA abs, high background
(especially regarding B cells)
• Less sensitive than FC-XM
• It does not detect clinically significant abs not
activating C

5. 23/10/2017
5
FC-XM
advantages
• less subjective, quantitative
• it detects low titer abs (10-250 times
more sensitive than CDC-XM )
• it detects complement activating and
non-activating abs (IgG1, IgG2, IgG3 IgG4, IgA,
IgM)
disadvantages
•Not a functional method
•Abs detected do not reflect their
capacity to activate C  do they harm
the graft in vivo?
•False positive results especially
regarding B cells
DSA detection methods
Clinical significance of preformed DSA abs
• HLA-A,-B, (-Cw), -DR DSA: their role has been documented years ago
•HLA-DQ,-DP DSA (detected with Luminex & FC):
- correlated with low/intermediate immune risk
- If their presence combined with IgG CDC-XM (+)  high risk
• HLA-DP DSA: they will possibly lead to acute or chronic rejection (although weakly
expressed on renal endothelial cells)
• C activating DSA (subclass determination, C1q assay): It is suggested as the method
determining whether the detected Abs are involved in rejection pathogenesis
DSA (+) double risk of AMR
CDC-XM (-) +
FC-XM (-) 76% greater graft loss (meta-analysis)
It is generally accepted that the presence of preformed HLA class Ι or/and ΙΙ DSA
(IgG) is correlated with  graft rejection risk
Sensitization (anti-HLA abs development) clinical
significance in renal transplantation
Annals of the New York Academy of Sciences 2013, 1283: 30-42
• CDC-XM (+): absolute contraindication for transplantation
• FC-XM (+): relative
• DSA detected only with solid-phase methods (Luminex):
-  risk for graft rejection and graft loss
- generally accepted cutoffs have not been determined and they vary
between centers
- stronger immunosuppression and stricter follow-up
IgM anti-HLA abs
• Clinical significance not clear
• They are not generally harmful for the graft
• positive IgM FC-XM (T cells): could be a risk factor
for graft rejection (not performed in most centers)

6. 23/10/2017
6
endothelial XM (XM-ONE)
detecting abs on endothelial cells
PB precursor endothelial cells (1-2%)
HLA I
HLA II
non-HLA
Tie-2-r
it is not clear
- Whether it should become a routine
test
- If a positive result
should affect
graft distribution
or treatment
Do Abs detected by XM–ONE link on the graft differentiated
endothelial cells??
It is very important to follow up the development of de novo anti-
HLA abs.
Chronic rejection may be predicted by the development of HLA-
specific antibody following transplant (Terasaki and Ozawa,
2004)
Flow cytometry crossmatch
FCXM
history
Introduced by Garavoy et al (1983)
Cook et al., 1987; Lazda et al., 1988;
Mahoney et al., 1990; Ogura et al., 1993
2 color FC (separate tubes for T and B cells
3 color FC (one tube for T and B cells) Bray
et al., 1989, 2004a, 2004b, Bray and Gebel,
2007.
The procedure
 Sample preparation
 Donor PBMCs
Donor PBMCs
Overlay WB (preferably in
heparin) on Ficoll
Serus liquid
Cells band
RBC and grans
Collect PBMCs
Wash and prepare a
sample of 3*106/ml
concentration

7. 23/10/2017
7
Xmatch incubation
Τ + negative control
50 μl cells + 50 μl
negative serum
T + positive control
50 μl cells + 50 μl
positive serum
T + recipient serum
50 μl cells + 50 μl
recipient serum
Β + negative control
50 μl cells + 50 μl
negative serum
Β + positive control
50 μl cells + 50 μl
positive serum
Β + recipient serum
50 μl cells + 50 μl
recipient serum
Negative: AB serum pool or commercial preparation e.g. NIBSC
Positive: multisensitized patient serum or commercial preparation
Anti-human IgG incubation
Anti-human (goat? Sheep?) IgG –FITC (Fab)2
Fc specific
CD3 for T cells
CD19 or CD20 for B cells
2 or 3 colour
Washing steps
Robert A. Bray et al 2011
THREE-COLOR Anti-T and Anti-B Cell CROSS-MATCH
Control
Control
CD3 PE
CD19 APC
CD3 PE
CD19 APC
Positive
T-Cell
XM
Positive
B-Cell
(and T Cell)
XM
Test
Test
sIg+ B Cells
T negative T positive

8. 23/10/2017
8
T donor - recipient
B negative
B positive B donor - recipient
Ratio MFI
T cells
0,38/0,25 = 1,52
B cells
0,77/0,55 = 1,4
UKNEQAS FCXM

9. 23/10/2017
9
Our results The report
FREQUENT OCCURRENCE OF UNCORRECT
BEHAVIOUR OF POLYCLONAL ANTISERA
BEHAVIOUR OF POLYCLONAL
Whole Sheep-Anti-Hu IgG FITC
FLOW CYTOMETRIC CROSSMATCH
CORRECT BEHAVIOUR
“ANGLE OF DANGLE”
BEHAVIOUR:
Non Specific Binding
Methods for the determination of the
cut-off value for the positive

Many more methods for the determination of
the cut-off value for the positive
 median difference
 Ratio of median fluorescence of test to negative
 Linear channel shift
 Test of negative control MESF ratio
 Fluorescence index
 8000-mean negative control
Clinical decisions
If negative ok to proceed?
Most yes
1. We also use the term weakly positive if the
test/neg MESF value is between 1.2 and 1.5.
2. As no other clinical data is available to review
with the flow crossmatch results then pos/neg
along with equivocal is adequate.
3. The option "Equivocal result" is interesting

10. 23/10/2017
10
Clinical decisions
If negative ok to proceed?
 If no
1. "Ok to proceed" - If positive discussion with clinician considering patient history,
sensitization and clinical need
2. Immunological risk. Would need to know HLA mismatch and antibody levels
3. An equivocal range is a good idea.
4. The positive result should be explained in relation to the presence of antibodies.
5. Low positive would also be useful.
6. The results seem to be equivocal because they are close to the positive threshold. In
general our clinical rules consist of investigating the whole historical medical patient
file.
7. A high proportion of samples sent are marginally positive. In practice such results are
evaluated after consideration of other laboratory and clinical data.
8. It would be useful to include a "Weakly reactive/Borderline" result option. In clinical
terms, there is a big difference between "definitely no reaction" and "probably
something“.
9. Sometimes results are indeterminate or borderline. Insufficient samples are sent so
they cannot be tested in duplicates as the patient samples are.
Potential reactivity patterns
 T cell B cell Interpretation
 Negative Negative No HLA antibody or very low titer
 Negative Positive Predominantly Class II antibody
 Negative Wk positive Weak Class I or II antibody
 Positive Positive Strong Class I antibody or a combination of
Class I and Class II
 Positive Negative Likely non-HLA reactivity
 Assumes that HLA specificity has been confirmed via alternative solid-phase
testing methods
Robert A. Bray
Clinical significance of
FCXM
Upon multivariate adjustment
with Cox regression, T
FCXM+/CDCXM- deceased
donor transplantation was
associated with 51% higher
adjusted relative risk of 1-
year graft loss than FCXM-
/CDCXM-.
Relative risks were more
marked at 1 year for the T
groups but stronger in the 1-
to 5-year interval for the B
groups.
R.J. Graff et al (2010)
Effect of blood group
incompatibility on T FCXM
M. Lindemann et al
Cytometry 2016
CDC XM (-) FCXM (+)
 Perhaps due to low levels of abs
 Very significant for 2nd transplantation and for sensitized
patients
Due possibly to memory clones, which give rise to new abs.
Scornik et al, 1992
Clinical significance of B(only)-FCXM+
Specificity of anti-donor antibodies causing the B+FCXM
Detection of noncomplement-fixing antibodies
Level of expression of HLA class II antigens on kidney endothelial cells
Diagnosis and immunological status of patient prior to kidney
transplantation
Immunosuppression protocol
Level of antibody found in the B+FCXM assay
Sensitivity of the flow cytometric instrument
Julio C. Delgado, David D. Eckels (2008)

11. 23/10/2017
11
Specificity of DSA giving rise to
B (only)+ FCXM
Risk category
 Antibody specificity High Low
 Autoantibody X
 Anti-HLA class I antibody X
 Anti-HLA-DR antibody X
 Anti-HLA-DQ, -DP antibody X
 Antibodies against minor antigens (e.g. MICA) X
 IgG1, IgG3 subclass antibody X
 IgG2, IgG4 subclass antibody X
Julio C. Delgado, David D. Eckels (2008)
T (only)+ - FCXM due to non – anti-HLA abs
More advantages of FCXM
 T and B cells XM
 Different classes of antibodies (IgM, IgA)
 Binding assay reduces the chances of technical failure and inconsistencies
 instrumental in helping promote a new approach to living donor transplantation,
namely ‘‘kidney paired donor’’ exchanges (Rees M., et 2009)
Drawbacks of FCXM
 Nonspecific immunoglobulin binding to Fc receptors can be mistaken as a
positive result.
 Non-HLA antibodies (e.g., autoantibody) may also bind to the cells producing a
positive result.
 Monoclonal anti-lymphocyte antibodies such as Thymoglobulin1, Rituxan1, and
Campath1 (used for immunosuppressive therapy) can also interfere with the
FCXM and produce a ‘‘false’’ positive result.
 High dose IVIG, used in some desensitization protocols, can also interfere
with the FCXM.
Guidelines for the detection and
characterization of clinically relevant
antibodies in allotransplantation
Many thanks to my colleagues from the
Histocompatibility Lab
 Katherina Tarassi
 Vasiliki Kitsiou
 Diamanto Kouniaki
Thank you very
much for your
attention