Science
Optimized Flow Cytometry to Measure Anti-ABO
Immunoglobulin G
Dong Il Won, MD, PhD1* and Byung Chang Kim, MD, PhD2
ABSTRACT
Methods: Fixed red blood cells (RBCs) were sensitized with
dithiothreitol-treated sera at 4°C. Residual hemagglutination and
hemolysis were monitored on a time-dependent plot. Only singlet
RBCs were analyzed, excluding RBC agglutinates, to obtain mean
fluorescence intensity (MFI) ratios.
Results: The sensitivity for anti-ABO IgG was compared between
Flow ABO Ab and the column agglutination technique (CAT) in 34 sera
During the past decade, organ transplantations have
overcome humoral barriers of ABO-group mismatch
between donors and recipients. In particular, ABOincompatible kidney transplantation (ABOi KT) has
become common practice. However, recipients
should undergo pretransplant conditioning regimens,
including antibody removal through plasmapheresis or
DOI: 10.1309/LM1O8F7MLLYIPIMZ
Abbreviations
ABOi KT, ABO-incompatible kidney transplantation; CAT, column agglutination technique; FC, flow cytometry; RBCs, red blood cells; IAT, indirect antigloblulin test; IgG, immunoglobulin G; PBS, phosphate-buffered
saline; IgM, immunoglobulin M; DTT, dithiothreitol; FITC, fluorescein
isothiocyanate; SSC, side scatter; FSC, forward scatter; MFI, mean fluorescence intensity; LISS, low ionic strength saline; PE, phycoerythrin;
S/CO, sample/cutoff; HLA, human leukocyte antigen; MESF, molecules
of equivalent soluble fluorochrome; Fab fragments, fragment antigen
bindings
1
Department of Clinical Pathology, Kyungpook National University
School of Medicine, Daegu, and 2Department of Laboratory Medicine,
Maryknoll Medical Center, Busan, South Korea
*To whom correspondence should be addressed.
E-mail: wondi@knu.ac.kr
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Conclusions: Our protocol markedly improved on the previously
reported protocol and provided its analytical performance to be
comparable to that of CAT, suggesting its potential as an additional
effective tool for the measurement of anti-ABO IgG. Further studies
are needed to clarify the target MFI ratio for transplantation and
whether Flow ABO Ab is preferable to CAT for a correlation with a
clinical outcome.
Keywords: ABO antibody, titration, flow cytometry
immunoadsorption and intravenous gamma globulin.
These regimens should be repeated until ABO antibodies
decrease to the target level. Therefore, an accurate
measurement of ABO antibody levels before and after
transplantation is essential.1
Tube hemagglutination techniques have been traditionally
used to measure ABO antibody levels. The column
agglutination technique (CAT) has been preferred recently
because it is simple and decreases variations between
laboratories.2,3 Also, other methods such as enzyme-linked
immunosorbent assay4 and surface plasmon resonance5
have been reported for measuring ABO antibodies.
Flow cytometry (FC) can be used to detect alloantibodies
or ABO antibodies against RBCs. Several methods
have been reported, namely, quantification of anti-D6,
screening of other alloantibodies,7 ABO plasma testing,7
semiquantitative measurements of ABO antibodies in the
protocol proposed by Seebach et al8,9 or Tanabe,3 and
quantification of ABO antibodies using latex beads, rather
than actual red blood cells (RBCs).10
Among several methods, CAT is currently accepted as
the standard method to measure ABO antibody levels.
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Background: An accurate measurement of ABO antibody levels before
and after ABO-incompatible organ transplantations is critical. We
verified several assay steps of a previously reported protocol using
flow cytometry, improved its shortcomings, and developed a more
optimized protocol, which we named Flow ABO Ab, for the anti-ABO
immunoglobulin G (IgG).
diluted to have borderline reactivity of anti-ABO IgG. Flow ABO Ab
yielded a positivity rate of 68% (23/34), whereas CAT yielded 50%
(17/34) (P = .18). The CAT titer was highly correlated with the mean
fluorescence intensity (MFI) ratio of Flow ABO Ab (r = 0.843, P<.001)
in 17 undiluted sera. Using Flow ABO Ab, all group A (30 [100%]) and
group B (30 [100%]) healthy individuals tested positive for anti-B and
anti-A, respectively.
Science
However, CAT is relatively cumbersome and costly
for titration testing because interpretation is not fully
objective and more than 10 gel microcolumns for each
immunoglobulin isotype are required per sample. An
accurate, objective, and simple alternative method is
needed to support CAT, particularly when the levels of
anti-ABO immunoglobulin G (IgG) determined by CAT are
so low that pretransplant conditioning regimens appear to
be theoretically unnecessary.
Technically, the measurement of ABO antibodies by FC
could be hampered by hemagglutination and hemolysis,
ie, agglutination/lysis. To prevent agglutination/lysis,
the Seebach protocol requires fixation of RBCs before
sensitization. Fixation of RBCs cross-links the proteins
and stabilizes their membranes, thereby impeding lateral
movements of ABO antigens, which are required for
efficient hemagglutination.8 However, the Seebach protocol
does not appear to be fully optimized and fails to take into
consideration the potential for ineffective data acquisition
due to residual agglutination/lysis.
This study aimed to obtain a more optimal protocol for
measuring the levels of anti-ABO immunoglobulin G (IgG).
To this end, we verified several assay steps of the Seebach
protocol, improved their aforementioned shortcomings,
and established a more efficient protocol that we have
named Flow ABO Ab.
Materials and Methods
This study was comprised of 2 phases, protocol
optimization and assay validation. In the optimization
phase, all measurements were performed in 2 or more
independent experiments and are displayed as mean (SD)
values. Except for the indicated variations of a single assay
282
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Individual
ABO
Group
No.
Sex, Age
Male:Female (Range), y
Recipient of ABOi KTa
Recipients
O(A)b
6
4:2
O
(B)
6
5:1
O (AB)1 0:1
A (AB)1 0:1
A(B) 10:1
B
(AB) 2
1:1
Healthy Individualb
Negative control
AB
30 30:0
Group A serum
A
30 19:11
Group B serum
B
30 19:11
41 (32-59)
50 (35-65)
56
52
46
36 (33-38)
52 (20-73)
47 (29-70)
47 (26-66)
ABOi KT, ABO-incompatible kidney transplantation.
a
n = 17.
b
Donor blood group.
c
n = 90.
step, all other assay steps were executed in the same
manner within each experiment. The established protocol
is described in this section.
RBC and Serum Preparation
RBCs. Reagent blood group A, B, or AB RBCs were
gathered from healthy individuals or ABOi KT donors at
Kyungpook National University Hospital in Daegu and
Maryknoll Medical Center in Busan, Republic of Korea.
Sera. Sera were obtained from group AB (as a negative
control) and other healthy individuals or ABOi KT recipients
( Table 1). If the screening test for RBC alloantibodies was
positive, samples were not included. Samples were diluted
in isotonic saline.
Informed consent was obtained from all healthy individuals
and patients before this study. Throughout this report, the
blood groups of used RBCs and serum are expressed as
RBCs and serum (eg, A cells and B serum).
Column Agglutination Technique (CAT)
Titrations were performed using the polyspecific low ionic
strength saline (LISS)/Coombs card (Bio-Rad Laboratories,
Inc, Hercules, CA) according to the method described by
the manufacturer. Briefly, 25 µL of each serially diluted
serum and 50 µL of the prepared RBCs were added to
the gel card microcolumns. After incubation at 37°C for 15
minutes, the gel cards were centrifuged. The titer endpoint
was the reciprocal of the highest dilution demonstrating 1+
or weak positivity.
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Whereas FC is relatively simple to perform and yields
semiquantitative results with only 1 tube per sample,
this application has not been widely practiced; the
FC protocol is neither optimized nor standardized. In
particular, the Seebach protocol does not seem to be
highly sensitive. Based on 2 reports, the positivity rate
of the anti-ABO IgG among healthy individuals was very
low (only 5 individuals tested positive among the 56
individuals in the 2 study groups)8 and the measured
levels of ABO antibodies were lower than those of
the tube indirect antigloblulin test (IAT) performed
simultaneously in the study.9
Table 1. Characteristics of the Cohort Individuals
Science
1,000
Figure 1
0
200
400
1,000
0
R3
0
200
Time (102.40 s)
400
1,000
Time gate
1,000
1,000
SSC
Data acquisition and analysis of Flow ABO Ab. The time gate
was set to include red blood cell (RBC) events only on the timedependent plot. The singlet RBC gate was set around singlet
RBCs to exclude RBC agglutinates on the forward scatter (FSC)/
side scatter (SSC) plot of the gated RBC events. Mean fluorescence intensity (MFI) is the geometric mean for the main peak on
the anti–immunoglobulin G (IgG) fluorescein isothiocyanate (FITC)
histogram of the gated singlet RBC events. MFIs were obtained
individually for samples and negative controls and used to calculate a sample/control MFI ratio. A cells and B serum were used.
0
1,000
800
800
Control Singlet RBC
gate
600
400
400
200
200
0
0
200
400
600
800
Sample
600
0
1,000
0
200
400
600
800
1,000
MFI ratio
Counts
120
90
M1
60
7.3
30
=
M2
575.0
575.0
=
7.3
7.3
0
100
101
102
103
104
anti-IgG FITC
Measurement of Anti-ABO IgG by Flow
Cytometry (Flow ABO Ab)
Sample and 2 negative control sera were diluted to 1:10
strength. They were always run simultaneously with
undiluted sera to prepare for the high ABO antibody titers.
RBC fixation. Each 20 µL of EDTA whole blood was
washed once 3.0 mL of phosphate-buffered saline (PBS)
(×1) and fixed in 3.0 mL of 0.1% paraformaldehyde at 4°C
for 20 minutes. Then, RBCs were washed three times with
3.0 mL of PBS and resuspended in 1.4 mL of PBS at a
RBC concentration of approximately 0.8% (∼67,000 RBCs/
µL). Before testing, RBCs from the third party, neither
from recipients nor from donors, were prepared as a pool
from 3 individuals of the same ABO group as the ABOi KT
donor. RBCs were defined to be fixed based on the first
experiment described in the Results section.
DTT treatment. Dithiothreitol (DTT) was prepared as a 0.01
M solution in PBS. Aliquots were stored frozen and then
thawed at the time of testing. To destroy immunoglobulin
M (IgM) antibodies, 25 µL of serum and an equal volume of
DTT were incubated at 37°C for 30 minutes.11
Sensitization and Fluorescent Staining. To sensitize
RBCs with ABO antibodies, 3.0 µL of fixed RBCs was
incubated with 50 µL of the DTT-treated serum at 4°C for
30 minutes. RBCs were then washed 3 times with 3.0 mL
of isotonic saline precooled to 4°C using a temperature-
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controlled centrifuge set at 4°C. These RBCs were stained
with anti-human IgG dispensed in 20 µL aliquots at a 1:40
dilution into each tube at 4°C for 30 minutes. The antihuman IgG was a Fcγ-specific, fluorescein isothiocyanate
(FITC)–conjugated, and goat-origin F(ab)2 antibody
(Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA). After a single wash, 50 µL of isotonic saline
precooled to 4°C was added to each tube; RBCs were then
resuspended and ready for FC at 4°C in the dark.
Flow Cytometry. A FACSCalibur flow cytometer with CELLQuest Pro software was used, which was calibrated using
CaliBRITE beads and FACSComp software on a daily basis
(all from BD Biosciences, Franklin Lakes, NJ). Event count
or time was chosen as the collection criteria. The target
for the total event count of singlet RBCs was set at 20 000
events; the elapsed data acquisition time limit was set at
102.4 seconds. Collection stopped if acquisition exceeded
either criteria.
Two kinds of gates, time and singlet RBC, were used for
data acquisition and analysis (Figure 1); the time gate on
the time/side scatter (SSC) plot gated the events of singlet
RBCs or agglutinates, particularly excluding non-RBC
events in samples with severe agglutination/lysis. NonRBC events may represent the fragments of lysed RBCs
and appear at the terminal acquisition phase, as shown
in Figure 2 and Figure 3; the singlet RBC gate on the
forward scatter (FSC)/SSC plot gated singlet RBC events for
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FSC
150
Science
SSC
LISS
Figure 2
1,000
Low ionic strength saline (LISS) vs dithiothreitol (DTT). DTT
treatment prevented agglutination/lysis; LISS enhancement
worsened the situation. Hence, DTT treatment yielded a
higher mean fluorescence intensity (MFI) ratio than did LISS
treatment (52.5 and 30.3, respectively). A cells and B serum
were used. SCC indicates side scatter; IgG, immunoglobulin
G; FITC, fluorescein isothiocyanate; and FSC, forward scatter.
1,000
0
0
200
400
SSC
800
Counts
600
400
200
0
0
400
200
600
800
100
101
102
SSC
0
0
200
400
SSC
Counts
600
800
0
101
100
1,000
SSC
102
104
anti-IgG FITC
0
0
200
400
600
800
1,000
Time
800
600
600
SSC
1,000
800
SSC
1,000
400
400
200
200
0
0
200
400
600
800
0
1,000
100
101
Counts
10
8
6
4
2
0
M1
158.2
101
102
102
103
104
anti-IgG FITC
103
anti-IgG FITC
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103
1,000
100
284
1,000
M1
FSC
When optimizing each step, we considered the variation
yielding the highest MFI ratio (ie, the highest signal-tonoise ratio) as the best of all variations. We did so because
that variation has the highest discriminatory power
between negative and positive.
800
535.3
FSC
Expected MFI ratio = measured MFI ratio × dilution
(if undiluted, dilution = 1) [Equation 1]
600
104
10
8
6
4
2
0
M1
100
101
102
1,113.8
103
104
anti-IgM PE
In an analysis of the time/SSC plot, the elapsed
data acquisition times and the total event counts of
singlet RBCs were considered to be rough indices of
agglutination/lysis. Agglutination/lysis hinders RBCs
from passing through the flow cell readily, resulting in
diminished acquired RBC events per second, sparse dots
on the plot, and a prolonged elapsed time. In severe cases,
the 20 000 target for the total event count could not be
reached within the time limit of 102.4 seconds.
Statistical Analysis
Statistical analyses were performed using Microsoft
Excel 2007 (Microsoft Corporation, Redmond, WA) and
SPSS software, version 11 (SPSS, Inc, Chicago, IL).
A comparison of Flow ABO Ab and CAT was carried
out using a correlation coefficient or a McNemar test.
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400
0
the final analysis. The amount of ABO antibodies binding to
the RBCs was measured as the mean fluorescence intensity
(MFI) ratio (sample MFI/control MFI). Each MFI was obtained
on the anti-IgG FITC histogram of the gated singlet RBCs.
When sera were diluted for sensitization, the measured
MFI ratio was multiplied by the dilution used to obtain the
expected MFI ratio of the original (ie, undiluted) sera:
104
Time
150
600
200
Simultaneous measurement of anti-ABO immunoglobulin M
(IgM) and immunoglobulin G (IgG). Severe agglutination/lysis
occurred. No clear narrow peak was obtained on the anti-IgG
fluorescein isothiocyanate (FITC) or anti-IgM phycoerythrin
(PE) histogram. The anti-human IgM PE was Fc5µ-specific and
donkey-origin F(ab)2 antibodies (Jackson ImmunoResearch
Laboratories, Inc, West Grove, PA). A cells and O serum were
used. SSC indicates side scatter.
103
1,000
800
Figure 3
120.2
anti-IgG FITC
1,000
400
1,000
0
FSC
200
800
M1
1,000
DTT
0
600
Time
20
Science
Table 2. Results of Experiments to Optimize Each Assay Step in Flow ABO Aba
Variation
MFI Ratio
3.9
6.4
Experiment B - DTT Treatment and Sensitization Temperature
Experiment C - LISS Enhancement
LISS
4.6
No LISS
3.5
Experiment D - LISS Enhancement vs. DTT Treatment
30.0
52.5
34.5
0
1
2
3
4
1.1
1.0
2.4
8.4
6.3
Experiment F - Temperature of Washing Solution
37°C
4°C
1.2
1.7
Experiment G - Individual vs. IgG and IgM
Anti-IgG FITC only
IgG FITC
FL2
Anti-IgM PE only
FL1
IgM PE
Anti-IgG FITC and Anti-IgM PE
IgG FITC
IgM PE
39.8
1.0
1.3
6.5
45.6
3.9
RBCs, red blood cells; DTT, dithiothreitol; LISS, low ionic strength saline; IgG, immunoglobulin G; IgM, immunoglobulin M; FITC, fluorescein isothiocyanate; FL2 fluorescence
parameter 2; PE, phycoerythrin; FL1, fluorescence parameter 1.
a
Each experiment was performed individually. Therefore, a comparison of MFI ratios is only proper within each experiment, rather than between experiments. A cells and O
serum were used for the individual vs. IgG and IgM tests; A cells and B serum were used for all other experiments.
Statistical significance was defined as P<.05. Some results
are expressed as mean (standard deviation, SD).
Results
Optimization of Each Assay Step of Flow
ABO Ab
RBC Fixation, DTT Treatment, and Sensitization
Temperature. Fixed RBCs yielded higher MFI ratios
and prevented agglutination/lysis more efficiently
than unfixed RBCs ( Table 2). Agglutination/lysis was
apparently diminished following the DTT treatment,
whereas IgG, nearly intact, was detected more
sensitively than with no treatment. A sensitization
temperature of 4°C was more favorable than 37°C when
the DTT treatment was combined.
LISS Enhancement. At sensitization, 25 µL of serum
and an equal volume of low ionic strength saline (LISS)
as a potentiator were incubated with RBCs. This LISS
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enhancement induced more agglutination/lysis, although
it yielded higher MFI ratios than when isotonic saline
replaced LISS ( Table 2; Figure 2).
LISS vs DTT. Treatment with DTT prevented
agglutination/lysis and yielded higher MFI ratios than
did the LISS enhancement (Figure 2). Diluting the stock
solution of DTT in LISS (not in PBS; rather, introducing
LISS and DTT) revealed little additional benefit ( Table 2).
Repeat Washing and Temperature of the Washing
Solution. Three turned out to be the optimal number of
washes performed after sensitization ( Table 2). During
the washes after sensitization, the washing solution at 4°C
retained the RBC-bound ABO antibodies better than that
at 37°C.
Measurements of IgM and IgG. Anti-IgM phycoerythrin
(PE) and anti-IgG FITC were added to a single tube to
measure the anti-ABO IgM and IgG simultaneously. This
experiment induced severe agglutination/lysis because
the sera could not be pretreated with DTT (Figure 3).
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No DTT
37°C
1.1
4°C1.4
DTT37°C 1.3
4°C2.2
LISS only
DTT only
DTT diluted in LISS
MFI Ratio
Experiment E - No. of Times Washing After Sensitization
Experiment A - Fixed vs Unfixed RBCs
Unfixed RBCs
Fixed RBCs
Variation
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Determination of optimal initial dilution of negative controls and samples.
In this experiment, low ionic strength
saline (LISS)–enhanced sensitization
and its volume were assumed to belong
to serum diluents together with isotonic
saline; sera were not treated with dithiothreitol (DTT). A cells with AB sera or B
serum were used. RBCs indicates red
blood cells; S/CO, sample/cutoff; and
MFI, mean fluorescence intensity.
1,000
Entire events
100
4.0
2.2
1.1
Mean of
negatives
1.2
3.2
4.8
2.5
3.4
1 in 4
1 in 8
10
Undiluted 1 in 2
This finding suggests that anti-ABO IgM and IgG should
be measured individually ( Table 2).
Initial Sample Dilution. It is important to determine whether
the sera should be diluted, particularly when the sera have
borderline reactivity. We compared 4 dilutions to determine
the best dilution for the negative controls and samples:
undiluted, 1 in 2, 1 in 4, and 1 in 8. Group A RBCs were
sensitized with the following sera of each dilution: 6 AB
sera, and 1 B serum with anti-A of borderline reactivity.
In this experiment, the sample/cutoff (S/CO) ratio, rather
than the MFI ratio, was considered to be an index for the
discriminatory power in comparing the dilutions. For each
dilution, a cutoff value (the average MFI for 6 AB sera + [3
× SD ]) and a sample value (the average MFI for duplicates
of 1 B serum) were obtained. The S/CO ratio is the sample
value divided by the cutoff value. A higher S/CO ratio was
regarded as a more favorable dilution for discriminating
between negative and positive. Combined with the dilution,
2 data analysis methods, entire events and singlet RBCs,
were also compared (Figure 4). The entire events variable
represents all RBC events, including RBC agglutinates
passing through the time gate only (irrespective of the singlet
RBC gate). Of all 8 cases, the 1-in-2 dilution and analysis of
singlet RBCs yielded the highest S/CO ratio of 4.8.
The last experiment showed that the best initial dilution of
serum was 1 in 2; Table 2 shows that the DTT treatment
was more effective than the LISS enhancement. Because
the DTT treatment dilutes the sera to 1 in 2, the DTT
treatment of undiluted sera was finally adopted.
Determination of Cutoff MFI Ratio. The Flow ABO Ab
of the established protocol was performed in 30 healthy
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1 in 4
1 in 8 Undiluted 1 in 2
nontransfused males from group AB ( Table 1). Each serum
sensitized RBCs of a single individual selected randomly
from group A, B, or AB. The average MFI ratio for these
30 samples was 1.03 (0.12). Therefore, the cutoff (ie, the
average MFI ratio + [3 × SD ]) was 1.39.
Is the Optimal Temperature for IgG (4°C)
Due to IgM?
The anti-ABO IgG sensitized the RBCs more optimally at
4°C than at 37°C. This may pose the question of whether
this result occurs due to the effect of increased IgM
binding at 4°C. Two possible mechanisms underlying this
effect can be hypothesized, namely, cross-reaction of the
secondary antibody (anti-IgG FITC) with the RBC-bound
IgM and agglutination of RBCs by IgM. We sensitized O
cells with group-AB serum containing no ABO antibodies
but instead containing cold agglutinins to verify that
neither of these mechanisms is responsible. Cold
agglutinins are IgM autoantibodies against I antigens in
the RBCs of all adults.
Regarding the first mechanism mentioned in the previous
paragraph, RBC-bound cold agglutinins did not affect the
MFI for staining with the anti-IgG FITC, as shown on the
anti-IgG FITC/anti-IgM PE plot in Figure 5. Also, the MFI
value for anti-IgG FITC staining of DTT-treated serum, in
which cold agglutinins were destroyed, was similar to that
of the untreated serum ( Table 3).
Regarding the second mechanism, in the “4°C and no
DTT” scenario ( Table 3), which maximized the binding
of cold agglutinins to RBCs, the value obtained after
analyzing entire events including RBC agglutinates was
similar to that of the negative control. The resulting MFI
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1
286
Singlet RBCs
Sample
4.0 S/C ratio
Cutoff
MFI Ratio
Figure 4
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SSC
0
0
200
400
600
800
Agglutinates
600
400
200
0
103
102
101
200
400
600
800
100
1,000
100
150
102
103
104
150
M1
0
10
0
10
1
M1
11.8
10
2
182.1
0
10
3
10
4
100
101
102
103
104
anti-IgM PE
anti-IgG FITC
Comparison of Sensitivity Between Flow ABO Ab and
CAT. The sera used in this experiment were diluted to have
borderline reactivity of anti-ABO IgG. Sera from 17 patients
were stored before or during pretransplant conditioning
regimens ( Table 1). Original sera were titrated by CAT
using the donor’s RBCs. Then, these sera were diluted:
1) 1 in [the determined CAT titer]; and 2) 1 in [2× the CAT
titer]. All 34 samples of diluted sera (n = 17 and n = 17,
respectively) were further tested by Flow ABO Ab, using
the third party’s RBCs. The measured MFI ratio, which
exceeded the previously defined cutoff value of 1.39, was
assigned as being qualitatively positive. The positivity rate
of Flow ABO Ab (23/34 [68%]) tended to be higher than
101
anti-IgG FITC
that of CAT (17/34 [50%]), although this difference was not
significant ( Table 4; P = .18).
Correlation of the Measured Levels of ABO Antibodies
Between Flow ABO Ab and CAT. In the 17 undiluted
recipient sera mentioned previously, the MFI ratio of
Flow ABO Ab was highly correlated with the CAT titer (r =
0.843, P <.001, Figure 6). An equation for the first degree
between 2 variables (MFI ratio and CAT titer) is as follows:
log10 (MFI ratio) = 1.027 × log10 (CAT titer) + 0.082
[Equation 2]
Linearity of MFI Ratios. We theorized that a MFI ratio
would reflect the level of ABO antibodies in the serum.
To verify this, we compared the expected MFI ratios
according to Equation 1 among 3 dilutions of 17 sera as
described herein: undiluted, 1 in the CAT titer, and 1 in the
2× CAT titer (Figure 7). The expected MFI ratios were not
Table 3. The Effect of IgM (Cold Agglutinins)–Bound RBCs to Anti-IgG FITCa
Entire Events
Singlet RBCs
MFI (Anti-IgG FITC) MFI (Anti-IgG FITC)
Variation
Cold Agglutinin
Negative Control
MFI Ratio
Cold Agglutinin
Negative Control
MFI Ratio
37°C No DTT
DTT
17.1
18.4
16.1
15.7
1.1
1.2
9.6
9.6
9.3
9.7
1.0
1.0
10.8
10.8
0.9
0.9
4°C
No DTT
16.9
18.6
DTT 14.5 19.2
0.9
9.7
0.89.4
IgM, immunoglobulin M; IgG, immunoglobulin G; RBCs, red blood cells; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; DTT, dithiothreitol.
O cells and AB serum containing cold agglutinin were used.
a
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Counts
FSC
Assay Performance Verification
1,000
104
1,000
0
ratio was almost identical to that of the singlet RBC
analysis (0.8 and 0.9, respectively). This finding suggests
that the MFI ratio for anti-IgG FITC is not affected by RBC
agglutinates induced by IgM.
800
Time
anti-IgM PE
The effect of immunoglobulin M (IgM) (cold agglutinins)–
bound red blood cells (RBCs) to anti–immunoglobulin G (IgG)
fluorescein isothiocyanate (FITC) staining. No cross-reaction
of anti-IgG FITC to IgM was observed. O cells and AB serum
containing cold agglutinins were used. SCC indicates side
scatter; FSC, foward scatter; and PE, phycoerythrin.
1,000
SSC
Figure 5
Science
FlowABO Ab
MFI Ratio
Correlation of the measured levels of ABO antibodies between
Flow ABO Ab and column agglutination technique (CAT) in ABOincompatible kidney transplantation (ABOi KT) (N = 17). The red
horizontal line signifies the cutoff mean fluorescence intensity
(MFI) ratio to discriminate between negative and positive by
Flow ABO Ab. The 2 green vertical lines signify the target CAT
titers (ie, 8 or 16) of the pretransplant conditioning regimens. See
Table 1 for the characteristics of recipients and their donors.
A cells/O serum
B cells/O serum
B cells/A serum
AB cells/A serum
AB cells/B serum
AB cells/O serum
1,000
Figure 6
100
10
Cutoff
1
1
log(MFI ratio) = 1.027log(titer) + 0.082
r = 0.843
P = <.001
Target
10
100
1,000
significantly different between 2 dilutions of any pairs.
This finding supports our expectations.
Positivity Rates of Flow ABO Ab Among Healthy
Individuals. Flow ABO Ab (for IgG) was performed among
30 patients from group A and 30 healthy individuals from
group B (Table 1). Positivity rates for anti-B among group
A and anti-A among group B were 100% (Figure 8). The
percentages of cases in which the total event counts of
singlet RBCs were less than 10 000 (ie, half the target
count) were 43% and 30%, respectively. This occurred
probably due to severe agglutination/lysis by the high titers
of anti-ABO IgG. In these cases, data acquisition was more
efficient with sera diluted to 1 in 10 than with undiluted
sera. The MFI ratios tended to increase according to age
among individuals from group A and those from group B;
this finding did not reach statistical significance (P = .16
and P = .06, respectively).
Table 4. Comparison of Sensitivity Between
Flow ABO Ab and CAT in the 34 Serum
Samples Diluted to Have Borderline Reactivity
of Anti-ABO IgGa
Column Agglutination Technique
Flow ABO Ab
Dilution 1
Dilution 1 in
in CAT Titer
2× CAT Titer
(Positive), No. (Negative), No.
Total, No. (%)
Positive
Negative
Total
13
4
17
23 (68%)
11 (32%)
34 (100%)
10
7
17
CAT, column agglutination technique; IgG, immunoglobulin G.
a
P = .18, per McNemar testing.
288
Lab Medicine Fall 2012 | Volume 43, Number 6
Discussion
Flow ABO Ab showed a higher positivity rate (68%) in 34
borderline samples than did CAT (50%), although CAT
had the advantage of using polyspecific antiglobulin with
the capacity to detect IgM and IgG. This finding clearly
suggests that the sensitivity of Flow ABO Ab is comparable
to that of CAT.
A comparison of MFI ratios among undiluted or diluted
sera verified that the MFI ratios reflect the levels of ABO
antibodies in the serum (Figure 7). In human leukocyte
antigen (HLA) cross-matches by FC, MFI ratios are also
acceptable for exacting a quantitative assessment of
the level of HLA antibodies. To quantify immunoglobulin
molecules bound to RBCs, another approach using
molecules of equivalent soluble fluorochrome (MESF)
actually calibrates the instrument by converting the
measured fluorescence to an equivalent value base on
a set of fluorescence standards.12
However, the nearly significant difference of the expected
MFI ratios between undiluted sera and 1 in 2× CAT titer
diluted sera (P>.049) suggests that sera with relatively high
levels of ABO antibodies should be appropriately diluted
for enhanced accuracy. In contrast, sera with relatively low
levels of ABO antibodies may not require dilution because
discrimination of positive from negative is more essential
than titration.
Also, our protocol represents a great improvement in
assay sensitivity compared to the Seebach protocol for
the anti-ABO IgG. The Seebach protocol demonstrated
positivity rates of anti-B among individuals in group A
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CAT titer
Science
P = 0.050
Figure 7
10,000
MFI Ratio, Expected
Comparisons of expected mean fluorescence intensity (MFI)
ratios among 3 dilutions (undiluted, 1 in CAT titer, and 2× CAT
titer) of 17 sera (averages: 138.6, 488.2, and 533.8, respectively).
The expected MFI ratios were not significantly different between
2 dilutions of any pairs (via paired t test). This finding suggests
that the MFI ratio linearly reflects the level of ABO antibodies
P = 0.091
1,000
100
10
Cutoff
1.0
Measured
MFI ratio
×1
Undiluted
Measured
MFI ratio
× CAT titer
1 in CAT titer
Measured
MFI ratio
× 2 × CAT titer
1 in 2 × CAT titer
Diluted
to accomplish this aim are already available, including
DTT treatment,11 RBC fixation,8 and a secondary antibody
consisting of fragment antigen bindings (Fab fragments)
instead of F(ab)26. The former 2 ways were introduced in
our protocol. However, the Fab fragments method was not
used due to lack of availability from most manufacturers.
Our protocol is different from the Seebach protocol
in that sera are treated with DTT, RBCs are fixed with
0.1% paraformaldehyde instead of Karnovsky buffer
containing formaldehyde and glutaraldehyde (because
paraformaldehyde is a popular reagent in laboratories),
sensitized RBCs are washed 3 times instead of once,
IgM antibodies are not measured by FC, and the time
gate is used to include RBC events only and to monitor
agglutination/lysis.
A B
1,000
1,000
Group B
100
Anti-A MFI Ratio
Anti-B MFI Ratio
Group A
y = 6.317x – 127.72
r = 0.265
P = 0.160
10
Cutoff
1
0
20
40
Age, y
60
80
y = 6.145x – 155.37
r = 0.346
P = 0.060
100
10
Cutoff
1
0
20
40
60
80
Age, y
Figure 8
Measurements of Flow ABO Ab among the 30 blood group A (part A) and the 30 blood group B (part B) healthy individuals. Anti-ABO
immunoglobulin G (IgG) was determined to be positive in all tested individuals. MFI indicates mean fluorescence intensity.
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289
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0.1
and anti-A among those in group B to be 1/28 (4%) and
4/28 (14%), respectively.8 In contrast, our protocol yielded
30/30 (100%) in both groups (Figure 8). Our finding is
consistent with that of another report,13 in which tube
LISS-IAT with 2-mercaptoethanol treatment for IgG
yielded a titer of 2 or more in all 712 blood donors of
group A, B, or O. Our results showed that the levels of
ABO antibodies tended to increase with age, although
not in a significant manner. Another study4 also reported
that variations between sera of different individuals far
exceeded age-related changes of the agglutination titer.
FC is a single cell-detecting technique. Therefore,
agglutination/lysis by ABO antibodies themselves or
secondary antibodies should be minimized or prevented
for detection of ABO antibodies by FC. Several methods
P = 0.4.03
Science
For ABOi KT, the pretransplant target level of ABO
antibodies by CAT is generally accepted as less than or
equal to a titer of 8 through 16, to which an equivalent MFI
ratio by Flow ABO Ab is 10.2 through 20.8, according to
Equation 2. The actually measured MFI ratios for the 3 sera
of CAT titer 8 were 1.7, 8.2, and 27.2, and those for the CAT
titer 16 were 13.4, 19.3, 23.2, and 31.4, being distributed
in a wide range (Figure 6). Therefore, the target MFI ratio
needs to be newly defined using more samples with the
target CAT titer. Also, each laboratory should define its
own target MFI ratio because the MFI ratio is subject
to variation between laboratories. Future studies are
necessary to correlate our results with a clinical outcome
to decide whether the CAT or the Flow ABO Ab method is
preferable for discordant cases.
Only the IgG isotype is accepted as responsible for graft
rejection by ABO antibodies in ABOi KT.1,14,15 FC is not
appropriate to measure the IgM isotype due to severe
agglutination/lysis; a simpler hemagglutination technique
can be used instead. Nevertheless, we may develop another
FC protocol for IgM in future studies, if sufficiently high initial
dilution of sera can be used to sensitize the RBCs.
In summary, fixed RBCs should be sensitized with DTTtreated sera at 4°C to measure the levels of anti-ABO IgG
by FC. Remaining hemagglutination and hemolysis should
be monitored on a time-dependent plot. Only singlet RBCs
should be analyzed, excluding RBC agglutinates. Diluted
sera should be run simultaneously with undiluted sera to
prepare for the high antibody titers. We suggest that our
protocol is an additional effective tool for measuring antiABO IgG. Further studies are needed to clarify whether this
method is preferable to CAT for a correlation with a clinical
outcome. LM
290
Lab Medicine Fall 2012 | Volume 43, Number 6
Acknowledgments
This research was supported by Kyungpook National
University Fund 2012, 201213900000.
To read this article online, scan
the QR code.
References
1. Ishida H, Miyamoto N, Shirakawa H, et al. Evaluation of immunosuppressive regimens in ABO-incompatible living kidney transplantation:
single center analysis. Am J Transplant. 2007;7:825-831.
2. Kumlien G, Wilpert J, Säfwenberg J, Tydén G. Comparing the tube
and gel techniques for ABO antibody titration, as performed in three
European centers. Transplantation. 2007;84:S17-S19.
3. Tanabe K. Interinstitutional variation in the measurement of anti-A/B
antibodies: the Japanese ABO-Incompatible Transplantation Committee
survey. Transplantation. 2007;84:S13-16.
4. Rieben R, Buchs JP, Flückiger E, Nydegger UE. Antibodies to histoblood group substances A and B: agglutination titers, Ig class, and IgG
subclasses in healthy persons of different age categories. Transfusion.
1991;31:607-615.
5. Kimura S, Yurugi K, Segawa H, et al. Rapid quantitation of
immunoglobulin G antibodies specific for blood group antigens A and B
by surface plasmon resonance. Transfusion. 2005;45:56-62.
6. Austin EB, McIntosh Y, Hodson C, Lee D. Anti-D quantification by
flow cytometry: an alternative to the AutoAnalyser? Transfus Med.
1995;5:203-208.
7. Roback JD, Barclay S, Hillyer CD. Improved method for fluorescence
cytometric immunohematology testing. Transfusion. 2004;44:187-196.
8. Stussi G, Huggel K, Lutz HU, Schanz U, Rieben R, Seebach JD.
Isotype-specific detection of ABO blood group antibodies using a novel
flow cytometric method. Br J Haematol. 2005;130:954-963.
9. Yung GP, Valli PV, Starke A, et al. Flow cytometric measurement
of ABO antibodies in ABO-incompatible living donor kidney
transplantation. Transplantation. 2007;84:S20-23.
10. Sundbäck M, Grufman P, Teller J, et al. Quantification of blood group
A and B antibodies by flow cytometry using beads carrying A or B
trisaccharides. Transplantation. 2007;84:S24-26.
11. Knight RC. Measuring IgG anti-A/B titres using dithiothreitol (DTT).
J Clin Pathol. 1978;31:283-287.
12. Bray RA and Gebel H. Clinical utility of flow cytometry in allogeneic
transplantation. In: Carey JL, Mcoy JP, Keren DF, eds. Flow Cytometry
in Clinical Diagnosis. 4th ed. Chicago, Illinois: American Society for
Clinical Pathology; 2007.
13. Mazda T, Yabe R, NaThalang O, Thammavong T, Tadokoro T.
Differences in ABO antibody levels among blood donors: a comparison
between past and present Japanese, Laotian, and Thai populations.
Immunohematology. 2007;23:38-41.
14. Shirey RS, Cai W, Montgomery RA, Chhibber V, Ness PM, King KE.
Streamlining ABO antibody titrations for monitoring ABO-incompatible
kidney transplants. Transfusion. 2010;50:631-634.
15. Tobian AAR, Shirey RS, Montgomery RA, et al. ABO antibody titer
and risk of antibody-mediated rejection in ABO-incompatible renal
transplantation. Am J Transplant. 2010;10:1247-1253.
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Depending on the method, different temperatures are
required to sensitize RBCs with anti-ABO IgM and IgG.
The adopted temperature and time parameters for CAT are
room temperature and 10 minutes for IgM and 37°C and
15 minutes for IgG2; those for FC are 4°C and 30 minutes
for IgM and IgG.8 Our experiment using cold agglutinins
verified that the increase in measurements of RBC-bound
IgG at 4°C is not due to the effect of RBC-bound IgM.
Although 4°C appears to expose ABO antigens more
strongly via conformation change, this temperature also
increases agglutination/lysis by IgM. Therefore, Flow ABO
Ab can use 4°C to determine the level of anti-ABO IgG
because DTT destroys IgM beforehand. However, CAT
should be performed at 37°C because agglutination/lysis is
severe at 4°C due to intact IgM.