For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry Cytotoxic Crossmatches Comparison of Methods

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For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry
Cytotoxic Crossmatches Comparison of Methods
C. Cervelli, F. Pisani, A. Aureli, R. Azzarone, M. Scimitarra, C. Battistoni, B. Di Iulio, D. Fracassi,
M.A. Scarnecchia, A. Famulari, and F. Papola
ABSTRACT
Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of the transplant
(hyperacute rejection) that today happens rarely due to immunologic studies
d
prospective
crossmatch
d
of patients awaiting the kidney graft. The usual approach for pretransplant
donor/recipient evaluation is based on 2 methods: (1) the cytotoxic complement cross-
match (CDC) and (2) the flow cytometric crossmatch (FCX). The CDC crossmatch is
positive when complement-fixing antibodies are present, an absolute contraindication to
kidney transplantation. The more sensitive FCX-positive crossmatch detects low concen-
trations of unable to fix performed antibodies complement. It is an “index”of possible
damage due to accelerated rejection. The target of our study was to develop a cytotoxic
flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies move sensitively
than the traditional CDC method and also was less subjective and more standardized
for interpretation studying sera from 23 patients; the cFCX showed the requested effi-
ciency characteristics even in an emergency. In addition, the new method permited one
to calculate a cutoff for positivity (average value of the negative control þ2 stan-
dard deviations), assuring an “objective”interpretation of the results that agreed with
the CDC but was more sensitive and accurate allowing solution of ambiguous results for
cases of “doubt”-positive CDC crossmatch. Furthermore, our aim was to correlate the
effect of the strength of the anti-HLA antibodies determined by mean fluorescence
intensity value of LabScreen Single Antigen beads with results of CDC, cFCX, and FCX
methods.
THE complement-dependent cytotoxicity (CDC) cross-
match (CDC-XM) and the flow cytometric crossmatch
(FCXM) are routinely used to allocate a kidney graft. The
FCXM, which is widely accepted as the most sensitive
crossmatch test in current use, gates out and thus disregards
dead and dying cells, reducing the need for high initial cell
viability (>85% for CDC assays) and requiring less perfor-
mance time. FCX assay detects the IgG versus IgM class of
anti-HLA antibodies by the secondary antibody. Since IgG
immunoglobulins class are believed to be responsible for
early graft loss, they must be detected, rapidly. FCXM
permits simultaneous detection of anti-HLA alloantibodies
that bind to either T (CD3-positive cells) or B cells (CD19/
CD20-positive cells), eliminating the need for a physical
separation of the cell populations. This information can
facilitate the interpretation of crossmatch results due to the
differential expression of HLA molecules, since T cells
display predominantly HLA class I molecules while B cells
express both class I (at a higher density than T cells) and
HLA class II molecules. The CDC technique, which
requires viable cells, simultaneously detects IgM and IgG
cytotoxic antibodies but requires more time and technical
experience. Even if some authors claim that the FCXM is
“too sensitive,”“inappropriately denying individuals a
transplantation,”we believe that the test should be viewed
From the Regional Center of Immunohematology and Tissue
Typing (C.C., R.A., M.S., C.B., B.D.I., D.F., M.A.S., F.P.) and Organ
Transplant Centre (F.P., A.F.), S. Salvatore Hospital, and C.N.R.
Institute of Translational Pharmacology (A.A.), L’Aquila, Italy.
Address reprint requests to Dott Carla Cervelli, Regional
Center of Immunohematology and Tissue Typing, S. Salvatore
Hospital, Lorenzo Natali street, Coppito, 67100 L’Aquila, Italy.
E-mail: ccervelli@asl1abruzzo.it
ª2013 by Elsevier Inc. All rights reserved.
360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$
e
see front matter
http://dx.doi.org/10.1016/j.transproceed.2013.07.023
Transplantation Proceedings, 45, 2761
e
2764 (2013) 2761

in the context of the immunologic history of the patient. A B
cell- positive FCXM without evidence of anti- class II HLA
antibody may can be due to the recognition of non-HLA
surface molecules; it does not necessarily contraindicate
kidney transplantation.
1
e
4
For this reason we have elabo-
rated herein a new method that sought to combine the
characteristics of specificity and sensitivity of the FCXM
with the possibility to detect both complement-fixing and
non-fixing antibodies.
MATERIALS AND METHODS
Cell Suspension and Sera
Peripheral blood mononuclear cells were obtained from the buffy
coat of four healthy donors, who were completly typed for HLA
class I and II antigens. After separation using Lympholyte medium
(CEDERLANE), the phosphate-buffered saline (PBS) þ2% fetal
calf serum (FCS) washed cells were resuspended at 2 10
6
/mL for
a serologic CDC-XM and at 10 10
6
/mL for FCXM and cytotoxic
FCXM (cFCX) methods Sera, which were undiluted and 1:2, 1:4,
1:8 dilutions in the CDC technique are obtained from 23 patients,
already tested by flow panel reactive antibody screening and Lab-
screen Single Antigen class I and/or II (One Lambda). They were
used undiluted in the FCX and cFCX methods. A negative control
serum (AB human serum, Euroclone) was employed in conjunction
with a positive control serum prepared from a pool of sera from
patients with PRA >90% class I and/or II antibodies.
CDC
The CDC-XM was performed according to the “long incubation”
method. Briefly, serua from patients was dispensed into wells of
Terasaky microplates (1 sera
m
L) at 1:1, 1:2, 1:4 and 1:8 dilutions
followed by 1
m
L of cell suspension (w210
6
cell/mL) for a 1-hour
incubation at room temperature before addition of rabbit comple-
ment (One Lambda). After a further 2 hours incubation at room
temperature, the cells were stained with FluoroQuench (One
Lambda) that allowed detection of both intact and lysed cells by
fluorescence microscopy.
cFCX
To 25
m
L of cell suspension (10 10
6
mL) were added to 50
m
Lof
undiluted serum in a test tube, for a 30-minute incubation at room
temperature. Then 200
m
L of rabbit complement (One Lambda)
were dispensed and 2 washed with PBS þ2% FCS at 800 g for 5
minutes were performed after a further 30-minute incubation at
room temperature. Following addition of 50
m
L of IgG FITC
(DAKO; at 1:40 dilution), 5
m
L CD3 phycoerythrin (Becton Dick-
inson, BD) were added before incubation the tubes for 30 minutes
at 4C in the dark. After washing with cold PBS þ2% FCS, 5
m
L
of 7 amino-actinomycin-D (BD) were added for a 10-minutes
Fig 1. a) Serum with cytotoxic and non cytotoxic antibodies; Fig.1 b) serum mainly with non cytotoxic Ac.; Fig.1 c) serum with
completely cytotoxic Ac; Fig.1 d) serum with cytotoxic “factors”.
2762 CERVELLI, PISANI, AURELI ET AL

incubation 4C in the dark. Finally 225
m
L of PBS were added to the
pellet before on the flow cytometer reading (FACSCalibur, BD).
FCX
To 25
m
L of cell suspension (10 10 cells
6
/mL) were added 50
m
L
of undiluted serum in a test tube. After a 30-minute incubation at
room temperature, we performed 2 washes with PBS þ2% FCS at
800g for 5 minutes. The final incubation included 50
m
L IgG FITC
(DAKO; 1:40 dilution), 5
m
L CD3PerCP (BD) and 5
m
L CD19PE
(BD) for 30 minutes at 4C in the dark. After 2 washes with cold
PBS þ2% FCS, 225
m
L of PBS were added to the pellet for the flow
cytometric analysis (FACSCalibur, BD).
Statistical Analysis
The data acquisition and subsequent analysis were performed using
CellQuest Software (Becton Dickinson).
RESULTS
Among 11 of 23 sera studied by the 3 crossmatch methods,
the new cFCX technique demonstrated high levels of cyto-
toxic and noncytotoxic IgG antibodies (Ab; Table 1,Fig
1a,b,c). There were cases of noncytotoxic Abs associated
with cytotoxic Abs (Fig 1a); one, of only cytotoxic Abs
(Fig 1c) and a last one, mainly non complement fixing
antibodies in high percentage (Fig 1b), while 5/23 sera
revealed negative PRA screening without anti-HLA anti-
bodies or very low PRA, the CDC and cFCX were positive,
suggesting that the new technique showed some non-IgG,
non-IgM cytotoxic factors (data not shown). Their features
and clinical meanings are not clear (Fig 1d). The 23 sera
were all studied for the presence/absence of anti-HLA Abs
with the cytofluorimetric Flow PRA Screening method
(One Lambda). Positive sera were analyzed by the Lab-
screen Single Antigen class I method (One Lambda) to
identify individual specificities. Anti-HLA-specific donor
antibodies (DSA) antibody specificities which were scored
in mean fluorescence intensity (MFI), were correlated with
crossmatch results obtained by CDC, FCXM, and cFCX
techniques. Among our patients we observed some partic-
ular cases; the first was a patient with anti-HLA-A2 DSA
with a MFI medium value of 8229 (cutoff 5000MFI), a PRA
screening class I and II >80%, a highly positive CDC-XM
(1:8 dilution) and positive FCXM and cFCX. In contrast,
a second patient showed only high titre non-complement
fixing antibodies directed toward HLA B57 allele with an
MFI value of 14500, PRA class I >80% as well as negative
CDC and cFCX but a positive FCXM.
DISCUSSION
The analysis of these results showed cFCX to highlight 2
antibody types: (1) complement-fixing cytotoxic, and (2)
non-complement fixing, noncytotoxic antibodies, that bind
to the lymphocyte surface. At the moment, the morphologic
and functional difference between the 2 antibody types is
unknown. A possible explanation could be that the test
distinguishes different classes of IgG eg. IgG1 and IgG3 that
are complement-fixing versus IgG2 and especially IgG4,
non-complement-fixing. Five of 23 patients displayed
a negative PRA screening for anti-HLA antibodies
(Table 1), but revealed positive cFCX and CDC. The cFCX
showed the presence of cytotoxic non-IgG and non-IgM
“factors”(data not shown), whose characteristics and clin-
ical significance are still unknown. On the basis of the
guidelines already used in our laboratory for the FCXM, we
established a positive cutoff based on a medium value of þ2
standard deviations above the negative control for the new
assay. This cutoff permitted an “objective”interpretation of
results that corresponded to those obtained with the classic
method. Comparing the CDC, cFCX, and FCXM tech-
niques, we notes (1) perfect correspondence between the
classic CDC and the new cFCX, and (2) a the cFCX to be
more sensitive and less subjective than the CDC method,
particularly in the presence of a doubtful positive cross-
match.
5
Another goal was to use Luminex Labscreen Single
antigen class I to identify a cutoff of positivity of anti-HLA
antibodies, thereby distinguishing “not permissive antigens”
Table 1. CDC, FCX, CFCX, and Flow PRA Comparison
Patient No. CDC FCX CFCX -CD3
Flow pra
screening
Class I Class II
1þNegative þPositive þþ
dd
2þNegative þPositive þ5%
d
3þþ Negative þPositive þþ
dd
4þþ Negative þPositive þþ 18%
d
5þBþTþ Weekly
positive þ
dd
6Negative Negative
dd
7Positive þþ Positive
(CUTOFF)
56% 56%
8þþþ Positive þþ Positive þþþ 96% 30%
9þþþ Positive þþþ Positive þþþ 83% 97%
10 þWeekly
positive þ
Weekly
positive þ
64% 87%
11 þþþ Positive þþ Positive þþþ 97% 96%
12 þþþ Positive þþ Positive þþþ 99% 60%
13 þPositive þþ Weekly
positive þ
60% 74%
14 þWeekly
positive þ
Weekly
positive þ
93% 87%
15 Positive þþ Weekly
positive þ
73%
d
16 þþþ Positive þþþ Positive þþþ 98% 93%
17 þþþ Positive þþþ Positive þþþ 97%
d
18 Negative Negative
dd
19 Weekly
positive 
Negative 87%
d
20 Positive þþþ Negative 95% 91%
21 Positive þþ Negative 94% 71%
22 þþ Positive þþþ Positive þþ 96% 21%
23 Positive þþþ Negative 89%
d
cFCX values 15 is considered to be positive (mean C-7.57 þ2DS(2
3.7) ¼15). CDC, cytotoxic complement crossmatch; FCX, flow cytometric
crossmatch; cFCX, cytotoxic flow cytometric crossmatch; PRA, panel-reactive
antibodies. þ, from 15 to 50; þþ, from 50 to 79; þþþ, from 80 to 100.
COMPARISON OF DSA DETECTION METHODS 2763

to be correlated with “supposed positivity”of the “virtual”
crossmatch. For this reason we related the MFI value of the
anti-HLA antibodies in DSA sera with the crossmatch
results obtained by the 3 methods. Considering a mean
fluorescence intensity (MFI) value of 5000 as the positive
cutoff for “absolutely not permitted”antibodies, we
observed that was not possible to establish an exact corre-
spondence between the MFI and the results of the 3
crossmatches, consistent with the report of Thammanicha-
nond et al.
6
As evidenced in the 2 particular cases cited
above, it was evident that the predictive value of the MFI
for DSA was extremely limited and not always in accor-
dance with the results of different tests especially the CDC.
For this reason a virtual crossmatch is sometimes not
advisable for clinical application. In fact, the virtual cross-
match uses data that (even if theoretically corrected) does
not always reflect the attended prevision. This claim could
be explained because the data are calculated using beads
coated with purified antibodies that can present diversities
from the native antigen expressed on the lymphocyte
surface. Even as our work presents some limitations, such as
the small number of the patients analyzed, and the appli-
cation of the new experimental method only on T cells, we
believe that the cFCX offers advantages: (1) potential
applications in an emergency, (2) availability of an objective
interpretation and evaluation for the simultaneous presence
of cytotoxic and noncytotoxic antibodies, (3) greater sensi-
tivity than the classical CDC, (4) little influence by cellular
viability, or operator experience and (5) a new assay for the
post-transplantation period to highlight cytotoxic versus
noncytotoxic antibodies.
In conclusion, in this study we combined 3 methods with
the MFI value. The cFCX provides clinicians a useful tool to
investigate the type of antibodies and their effects during
the post-transplant period.
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