Chapter 22

The Lymphocyte Crossmatch by Flow Cytometry for Kidney

Transplantation
Jonathan Downing

Abstract
The flow cytometric lymphocyte crossmatch is a standard technique for evaluating the compatibility of
potential kidney transplant recipients and donors. Recipient serum is incubated with donor lymphocytes
and the latter are analysed in a flow cytometer for the presence of bound IgG antibodies. An increase in
the level of IgG binding compared to a negative control indicates the presence of donor-specific antibodies
which may lead to deleterious graft function. Described here is a method to perform T and B lymphocyte
crossmatching in the same tube to detect IgG donor-reactive antibodies.

Key words: Flow cytometry, Lymphocyte crossmatch, Kidney transplantation, Human leukocyte
antigen, Pronase treatment

1. Introduction

The flow cytometric lymphocyte crossmatch (FCXM) was first

described by Garovoy et al. (1), and has since become a standard
test performed prior to kidney transplantation. The FCXM exam-
ines potential donor lymphocytes for recipient antibody binding by
passing the cells one at a time through a laser and measuring the
amount of a fluorescently labelled secondary anti-Ig antibody.
With the use of labelled monoclonal antibodies differentiation can
be made between IgG and IgM antibodies and between reactivity
towards T and B lymphocytes (2).
A positive complement-dependent cytotoxicity (CDC) cross-
match in the presence of CDC detectable donor-specific HLA anti-
bodies is a contraindication to transplant, and its link to hyperacute
rejection is well established (3). However, it was recognised that a
proportion of transplant recipients experienced early graft loss
despite a negative CDC crossmatch test. The FCXM is able to

Frank T. Christiansen and Brian D. Tait (eds.), Immunogenetics: Methods and Applications in Clinical Practice,
Methods in Molecular Biology, vol. 882, DOI 10.1007/978-1-61779-842-9_22, © Springer Science+Business Media New York 2012

379
380 J. Downing

detect HLA antibodies of low titre or of a non-complement binding

nature that may be responsible for this early graft loss. Numerous
studies have since described a link between positive T and B lym-
phocyte FCXM and graft rejection and, or loss in both primary and
re-graft recipients and in highly sensitised patients (4–12). Despite
these studies, controversy has persisted as to the clinical relevance
of a pre-transplant T or B cell positive FCXM test.
During the interpretation of the crossmatch result, it is essen-
tial to determine the nature of the antibody causing the reactivity.
The most clinically relevant antibodies are donor reactive, HLA-
specific, IgG antibodies (10, 12). Irrelevant positive reactions can
be caused by antibodies reacting with self epitopes shared by the
donor cells; Fc receptors that bind antibodies in a non-specific
manner; or antibodies against non-HLA antigens. Thus, extensive
characterization of patient HLA antibody must always occur along-
side the crossmatch test.
The era of fluorescent bead solid phase HLA antibody detec-
tion has led to a rethink of FCXM relevance. It is now reasonable
to consider the strength of positivity of FCXM in relation to quan-
titative, precise detection of donor-specific antibody (DSA) and
assess a degree of risk of rejection for each individual patient and
donor pair (12–14). Thus, a positive FCXM result even in the pres-
ence of DSA may not be considered as a contraindication to trans-
plant. Rather it allows the clinician to stratify risk for each patient.

2. Materials

1. 2 × 10 mL anti-coagulated peripheral blood sample on donor

and patient (see Note 1).
2. 1 × 10 mL clotted peripheral blood sample on patient.
3. Phosphate buffered saline (Invitrogen, Carlsbad, CA, USA)
(see Note 2).
4. Ficoll-Paque™ Plus (GE Healthcare Biosciences, Uppsala,
Sweden).
5. 50-mL plastic centrifuge tubes (Thermo-Fisher, Waltham,
MA, USA).
6. 15-mL plastic centrifuge tubes (Greiner-Bio-one, Monroe,
NC, USA).
7. RPMI 1640 (Invitrogen) with 20% v/v foetal bovine serum
(Invitrogen).
8. Pronase/Protease XIV (Sigma, St. Louis, MO, USA).
9. 3-mL flow cytometry tubes (Becton Dickinson Labware,
Franklin Lakes, NJ, USA).
 22 The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation 381

10. Negative control serum—male blood group AB blood donor

serum tested for the absence of HLA antibodies (see Note 3).
11. Positive control serum—human serum known to contain broad
specificity IgG HLA antibodies (see Note 4).
12. PBS-Azide: 1 L PBS containing 10% sodium azide (Scharlau
Chemicals, Sentmenant, Spain).
13. Anti-human IgG conjugated to fluorescein isothiocyanate
(FITC) (Jackson Immunoresearch, West Grove, PA, USA)
(see Note 5).
14. Anti-CD3 conjugated to peridinin chlorophyll protein
(PERCP) (Becton Dickinson, San Jose, CA, USA).
15. Anti-CD19 conjugated to phycoerythrin (PE) (Becton
Dickinson).
16. Three colour flow cytometer (Becton Dickinson FACSCalibur).

3. Methods

3.1. Isolation Peripheral blood lymphocytes (PBL) are the main target of the
of Peripheral Blood crossmatch test. Anti-coagulated blood samples should be stored
Mononuclear Cells at room temperature and should be no older than 48 h before
isolating the lymphocytes, where possible.
1. Transfer 10 mL of anti-coagulated peripheral blood into two
labelled 50-mL centrifuge tubes.
2. Dilute with PBS to a total volume of 30 mL.
3. Underlay diluted blood with Ficoll-Paque™ Plus taking care
not mix the blood-Ficoll interface.
4. Centrifuge for 10 min at 1,600 × g.
5. Carefully aspirate the mononuclear cell layer at the Ficoll—
plasma interface from each 50-mL centrifuge tube into clean
labelled 15-mL centrifuge tubes and top up to 15 mL with
PBS.
6. Centrifuge for 3 min at 650 × g.
7. Decant the supernatant and re-suspend the cell button in
15 mL of PBS.
8. Centrifuge for 3 min at 650 × g.
9. Decant the supernatant.
10. Pool and re-suspend the cell buttons from the two 15-mL cen-
trifuge tubes into 1 mL of RPMI cell medium (see Note 6).
11. Adjust the mononuclear cell count to 8 × 106 cells/mL (see
Note 7).
382 J. Downing

3.2. Cleavage of Fc T and B lymphocyte cell surface Fc receptors can bind IgG molecules
Receptors Using non-specifically and lead to a high anti-IgG FITC measurement on
Pronase the cell. This can lead to difficulty in discriminating between nega-
tive and positive crossmatch results. Cleavage of Fc receptors using
Pronase at can lead to a reduction in the incidence of false negative
crossmatch due to the presence of excess irrelevant IgG bound to
Fc receptors (15) (see Note 8).
1. Add 0.5 mL of 1 mg/mL Pronase to 1 mL of donor or recipient
cells.
2. Incubate for 30 min at 37°C.
3. After incubation top up to 15 mL with RPMI.
4. Centrifuge for 3 min at 650 × g.
5. Decant the supernatant and re-suspend the cell button in
0.5 mL RPMI.
6. Re-adjust the mononuclear cell count to 8 × 106 cells/mL.

3.3. Crossmatch PBLs should be prepared from the donor sample for the allo-cross-
Procedure match and from the patient sample where an auto-crossmatch test
is required (see Note 9).
1. Label 7× 3-mL flow cytometry tubes in the following sequence:
3 negative control serum vs. donor cells; 3 patient serum vs.
donor cells; 1 positive control serum vs. donor cells. Additionally,
if required, label a series of flow tubes for an auto control cross-
match to include a negative control serum vs. patient cells and
at least two replicates of patient serum vs. patient cells (see
Note 10).
2. In each tube, add 50 μL of appropriate serum to 50 μL of
appropriate cells, mix by gentle vortexing and incubate at room
temperature for 30 min.
3. After incubation, wash each tube by adding approximately
2 mL of cold PBS-azide (see Note 11) and centrifuging for 2 min
at 1,800 × g. Decant the supernatant and gently re-suspend the
cell button. Alternatively an automated cell washer can be
employed.
4. Repeat the wash process twice. After the third wash remove as
much supernatant as possible and gently re-suspend the cell
button (see Note 12).
5. Add 50 μL of diluted anti-human-IgG FITC to each tube, mix
by gentle vortexing, and incubate for 15 min in the dark at 5°C
(see Note 13).
6. Wash once as described in step 3 above.
7. To each tube add 2 μL each of anti-CD3 and anti-CD19
reagents (see Note 14). Mix by gentle vortexing and incubate
for 15 min in the dark at 5°C.
 22 The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation 383

8. Wash once as described in step 3 above. Re-suspend the cells in

200 μL of PBS-azide. The tubes are ready for flow analysis (see
Note 15).

3.4. Flow Cytometric The flow cytometer instrument must be set up correctly before
Analysis use. The manufacturer’s daily start-up and shut-down procedures
must be followed and a daily calibration using commercial stan-
dard reagents performed. In addition, optimisation (see Note 16)
and colour compensation must be performed (see Note 17).
1. Collect data on 1,200 B lymphocyte events as follows, see
Fig. 1 (see Note 18).
2. Use the cytometer acquisition and analysis software to create a
1,024 channel 4-decade log plot of forward scatter (FSC) vs.
side scatter (SSC) (Fig. 1a).
3. Draw a polygon gate around the lymphocyte population and
use this gate to create a dot plot to show FL2 (PE) vs. FL3
(PerCP) to discriminate the B and T lymphocytes, respectively
(Fig. 1b) (see Note 19).
4. Draw a rectangular gate around each of the lymphocyte popu-
lations, and use these to plot an FL1 (FITC) vs. cell count
histogram for each population (Fig. 1c, d). Draw a histogram

Fig. 1. Flow cytometer analysis histograms. Flow analysis histograms created using Becton Dickinson Cell Quest Pro analysis
software. Lymphocytes are displayed by plotting forward scatter (FSC) against side scatter light on a 1,024 channel log-
scale plot (a). A gate is drawn around the lymphocytes and gated data used to populate a CD19-PE against CD3-PERCP
plot to distinguish the B and T population, respectively (b). Rectangular gates are drawn around each lymphocyte popula-
tion and gated data used to plot anti-IgG FITC on a log scale against count on a linear scale for T lymphocytes (c) and B
lymphocytes (d).
384 J. Downing

marker around the main peak of the B lymphocyte FITC

histogram (see Note 20).
5. Obtain the median FITC channel value from each histogram,
in the case of the B lymphocytes this will be the M1 median
value (see Note 21).

3.5. Interpretation To determine whether a crossmatch result is negative or positive,

the median FITC channel in the patient serum—donor lympho-
cyte reactions is compared to that of the negative control serum—
donor lymphocyte reactions. The following method is recommended
as it uses median FITC values simply obtained from the analysis
software of the cytometer and applies a statistical method to deter-
mine whether the result is in the normal range expected if no
donor-reactive antibody is present. It must be made clear, however,
that there are a number of alternative methods (see Note 22).
1. For each lymphocyte population, subtract the mean of the
median FITC channel values of the negative serum vs. donor
lymphocyte replicates from the mean of the median FITC
channel values of the patient serum vs. donor lymphocyte rep-
licates to give the median channel shift (MCS).
2. An absolute MCS value can be established as a positive thresh-
old by testing the negative control serum used in the assay
against a panel of lymphocytes from 30 healthy individuals
and deriving the standard deviation (SD) of the median FITC
value for each of the T and B lymphocytes. A threshold of the
mean median FITC value plus three SD of the MCS can then
calculated. This will give the threshold above which a patient
result can be described as having a positive crossmatch (see
Notes 23 and 24).

4. Notes

1. Common anti-coagulants, such as ethylenediaminetetracetic

acid (EDTA), acid citrate dextrose or heparin, are all suitable
for the collection of blood used to obtain lymphocytes for flow
cytometry analysis.
2. Phosphate buffered saline can be purchased as a solution or as
tablets to be dissolved in a specified volume of water.
3. The negative control serum is a human serum that has been
shown to contain no reactivity to T or B lymphocytes. This
serum can be sourced locally from male blood group AB blood
donors or can be obtained commercially from suppliers, such
as Invitrogen or One Lambda (Canoga Park, CA, USA). The
negative control serum should be evaluated for lack of reactivity
22 The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation 385

before used routinely. This should consist of testing to confirm

lack of HLA antibodies using a solid phase method, and by
testing by flow cytometry using the above FCXM method with
lymphocytes from a number of individuals to show lack of
reactivity.
4. The positive control serum is required to establish that correct
binding of the anti-IgG FITC secondary antibody has occurred
during the assay. A pool of locally available highly sensitised
transplant patients is a common source for this positive con-
trol. Pools of up to ten individuals known to possess strong
IgG HLA antibodies can be made and aliquots stored at −30°C
for continued used. Each pool should be evaluated for reactiv-
ity before use, by testing by flow cytometry using the above
FCXM assay. Alternatively, a commercial source of a monoclo-
nal antibody, such as w6/32 (Abcam, Cambridge, MA, USA)
could be used.
5. The fluorophore-labelled antibodies, anti-human-IgG-FITC,
anti-CD19-PE, and anti-CD3-PERCP fluoresce at particular
light wavelengths so that they can be detected by the flow
cytometer fluorescence (FL) detectors, FL1, FL2, and FL3,
respectively. These detectors collect signals at 530, 585, and
>670 nm, respectively, in a BD FACSCalibur instrument. The
fluorophores emit at 519, 578, and 675 nm for FITC, PE and
PERCP, respectively. Thus, if a particular labelled antibody is
not available, any replacement needs to emit in the correct
range for it to be collected by a specific FL detector. The anti-
CD19 and anti-CD3 antibodies are required to discriminate
between B and T lymphocytes and this can give valuable infor-
mation as to the nature of the specificity and strength of patient
antibody binding. It is possible to perform the crossmatch
without these, but no B and T lymphocyte discrimination will
be possible.
6. If the cell buttons are quite large, increase the re-suspension
volume. This will ease the process for adjusting the cell count
to the optimum.
7. It is important to adjust the cells to the correct optimum con-
centration. Too few cells will make interpretation of the results
difficult. With too many cells, there is a risk of diluting any
allo-antibody to levels below detection. In addition, an exceed-
ingly high cell event rate can lead to inaccurate data capture.
8. The pronase cleavage of FC receptors could be considered as
an optional step and is not performed by all laboratories.
Pronase can potentially reduce the incidence of false negative
results by reducing background fluorescence caused by irrele-
vant binding of IgG to Fc receptors (15). The requirement for
pronase treatment is difficult to predict as the incidence of
386 J. Downing

background can vary depending on patient serum and donor

lymphocytes and it is recommended that this step be carried
out with all crossmatch tests. In addition, for patients under-
going treatment with Rituximab, the anti-CD20 monoclonal
antibody, binding of Rituximab will lead to high levels of bind-
ing of FITC. Using pronase will cleave the anti-CD20 and
hence remove Rituximab binding.
9. An auto crossmatch result will aid interpretation where the allo
crossmatch result is positive for either T or B lymphocyte, but
no donor-specific HLA antibody can be detected in the patient.
If the auto crossmatch is also positive and of a similar magni-
tude, this can indicate non-HLA auto-reactive antibodies that
are also able to bind to donor lymphocytes. It must be stressed
though, that this interpretation can only be made in the pres-
ence of precise determination of patient HLA antibody using
sensitive solid phase methodology.
10. Each laboratory will have its own requirements for the number
of replicate reactions performed during the crossmatch.
Flexibility may be required in the event of low serum or cell
preparation volumes.
11. Sodium azide is typically included in the wash buffer to pre-
vent the modulation and internalisation of surface antigens
which can produce a loss of fluorescence intensity.
12. Careful removal of most of the wash supernatant is important
at this stage to ensure that the volume of FITC that the cells are
incubated in the following stage is optimum and consistent.
13. The optimum dilution of anti-IgG FITC should be determined
in each laboratory by testing a range of dilutions against known
positive and negative serum/cell combinations. An optimum
dilution will give the best differentiation between the negative
and positive reactions.
14. The anti-CD3 and anti-CD19 can be combined in equal vol-
umes before addition to the cells. This reduces the number of
low volume reagent dispensing steps required. Thus, 4 μL of the
combined reagent can be added to each crossmatch tube.
15. At this stage, the cells should be kept in the dark until ready for
analysis. This should be performed within an hour of the end
of the assay. Alternatively, a cell fixing reagent such as para-
formaldehyde can be used to preserve the cells for a number
of days.
16. The flow cytometer must be set up before analysis of the cross-
match assay in order to optimally record the amount of patient
IgG antibody binding to cells. Using lymphocytes prepared in
the same manner as the crossmatch assay, adjust the FSC amp
gain and SSC voltage to display the lymphocytes appropriately
on the scale of a log-scale FSC vs. SSC plot. When prepared in
22 The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation 387

the manner described above, lymphocytes should represent the

densest population recorded and thus be easily identifiable.
Adjust the FSC threshold in order to exclude the collection of
data on unwanted small particles and debris.
17. At some stage prior to analysis, colour compensation should be
performed in order to optimise the instrument settings for use
with the lymphocyte crossmatch assay. Compensation is
required where the emission spectra of fluorophores used in an
assay overlap, as is the case for example with FITC and PE and
with PE and PERCP. Compensation ensures that fluorescence
emitted by FITC is not detected by the FL2 photomultiplier as
a PE signal. Compensation is performed by individually pre-
paring lymphocytes with the fluorophores to be used in the
assay and then using the flow cytometer acquisition software
compensation controls to ensure there is minimal overlap
between the FL detectors. It is recommended that compensa-
tion be performed using lymphocytes in addition to any manu-
facturer’s calibration reagents. This is because lymphocytes
have different optical properties to these reagents. Once com-
pensation is performed, the saved instrument settings should
be used when subsequently analysing lymphocytes from the
crossmatch test. Compensation does not need to be performed
before each analysis, but should be carried approximately 6
monthly to monitor wear of the cytometer optics or in the
event that changes are made to the optics.
18. As B lymphocytes are usually less numerous than T lympho-
cytes, setting a minimum number of B lymphocyte events is
important to ensure enough data is collected to be able to
make an accurate interpretation. Generally, enough T cell
events will be collected under these conditions. In the case of
samples with low B lymphocyte counts, where 1,200 events
cannot be obtained, a time limit should be placed on each
acquisition such that all 200 μL of the sample is acquired at a
high flow rate. It is useful to have a minimum T and B lympho-
cyte count, for example, 2,000 T and 350 B, so that accurate
interpretation can be made. If the counts obtained fall below
this the crossmatch should be repeated. With very low cell
counts, natural variation in FITC level can introduce inaccu-
racy that may lead to false interpretation of the test result.
19. The T and B lymphocyte population should show as clearly
defined clusters of events segregated to high on the PERCP
axis for T lymphocytes and high on the PE axis for B lympho-
cytes. Quadrant markers are placed on the plot to indicate the
areas of negative and positive cell populations (see Fig. 1b).
If either of these populations or the negative population (which
should appear low on both axis) appear outside of the expected
areas, this may indicate poor compensation set up of the cytometer
388 J. Downing

or contamination from other sources of fluorescence that emit

at the same wavelength, e.g. ethidium bromide. Failure to
recognise this problem could lead to a false positive result.
Note also that the count scale for B lymphocytes is lower than
that of T lymphocytes in Fig. 1c, d, so that the peak is appro-
priately sized.
20. B lymphocyte preparations may display low numbers of events
with high levels of bound FITC that manifest as very small
peaks towards the right of the x axis of an FITC vs. cell count
histogram derived from the B lymphocyte gate. This is due to
non-specific IgG binding that can be disregarded from the
analysis by placing a histogram marker (M1 in Fig. 1d) sym-
metrically around the main peak and deriving the median FITC
value from this marker. FITC values outside of the marker will
not be included in the median value, and this has the effect of
lowering the overall median value but potentially lowering the
signal-to-noise ratio.
21. The FITC histograms should show roughly symmetrical peaks
with a clearly defined population based around a single maxi-
mum FITC level. Poorly defined peaks will lead to less accurate
definition of the median FITC level and may be caused by low
cell counts, inadequate mixing of cells, serum or fluorescent
labels during the assay, or by the labelling of abnormal donor
or patient lymphocyte subpopulations.
22. Each laboratory must establish its own criteria for a threshold
for a positive crossmatch and there are a variety of methods
available to do this. The method described above utilises an
absolute cut-off of MCS calculated by testing the negative
control serum against a panel of healthy cells to determine the
normal range of background FITC binding. An alternative is
to calculate the SD of the FITC channel values of the negative
control replicates derived from the crossmatch. If the patient
FITC channel value exceeds the negative value plus three SD
this can be described as a positive crossmatch. Another way of
comparing the patient and negative channel values is to calcu-
late the relative fluorescence by dividing the mean of patient
median FITC channel value by that of the negative control,
and a cut-off value of 1.5 or 2 used to indicate a positive result.
A further method for comparing the patient and negative val-
ues is to employ calibration beads to convert the arbitrary
FITC channel values into molecules of equivalent soluble
fluorochrome (MESF). In this process, fluorescence from com-
mercially available combinations of precisely quantitated
solutions and microbead suspensions is measured in the cytom-
eter. The results are used to create a standard curve which can
be used to convert any cytometer reading into an MESF value.
 22 The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation 389

Thus, median FITC values can be converted into MESF values

and the shift of patient MESF from negative MESF applied to
a threshold level in a similar way as MCS described above.
MESF methods are particularly useful when comparing data
from crossmatch tests performed at different times or in different
centres.
23. The use of a fixed threshold, however it is determined, is prob-
lematic when trying to interpret a result that is close to this
threshold. Whether or not a particular result relates to the
presence of donor-specific HLA antibody is not always well
reflected by a fixed threshold. It might be useful to have a second
threshold to indicate weak positive reactions, for example, the
MCS of the negative plus two SD. A threshold of the negative
median plus two SD relates to the fact that 95% of “normal”
negative reactions, i.e. those reactions that do not show
increased specific FITC binding will fall within this threshold.
Only 5% of reactions that exceed the threshold will be due to
“normal” negative reactions, all others being defined as true
positive due to increased FITC binding. Using three SD
assumes that only 0.3% of negative reactions will exceed this
level and that 99.7% of reactions above this level are of a true
positive nature and hence is a more stringent definition of a
positive result.
24. By testing the negative control serum against a range of differ-
ent lymphocyte samples it is possible to establish ranges of
reactivity that can be used as acceptance criteria for the assay.
For example, it is desirable to have a minimum and maximum
acceptable median FITC value for the negative serum against
any particular donor lymphocyte. If the negative serum median
FITC is too high, this can lead to a false negative interpretation
as a large amount of “background” is subtracted from the
patient FITC value during calculation of the MCS. When
the negative serum is tested against 30 healthy individuals, the
lowest and highest FITC value obtained for each lymphocyte
population can be used as the lower and upper range. If, dur-
ing a crossmatch test, these ranges are exceeded, then the
crossmatch may be repeated or the interpretation questioned.

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