I M M U N O H E M AT O L O G Y
Streamlining ABO antibody titrations for monitoring
ABO-incompatible kidney transplants
R. Sue Shirey, Wei Cai, Robert A. Montgomery, Vishesh Chhibber, Paul M. Ness, and Karen E. King
BACKGROUND: We have monitored ABO antibody
titers in 53 ABO-incompatible kidney transplants (INKTs)
using a time-consuming, conventional test tube (CTT)
method that included a 30-minute room temperature
(RT) phase, followed by incubation for 30 minutes at
37°C and conversion to the anti-human globulin (AHG)
phase. Our studies have indicated that AHG ABO anti-
body titers are critical for clinical management, but RT
titers do not supplement clinical decision making.
Therefore, we assessed AHG titers by two methods: 1)
a revised test tube (TT) method without RT and 2) an
anti-immunoglobulin G (IgG) gel microcolumn (IgG gel)
method with a goal of streamlining ABO antibody
titrations.
STUDY DESIGN AND METHODS: Fifty frozen samples
from our INKT collection with anti-A and/or anti-B AHG
titers of 2 to 512 were titrated by revised TT method
with 30 minutes at 37°C and conversion to AHG and
by IgG gel method with 15 minutes at 37°C and
centrifugation.
RESULTS: The titers using the revised TT and IgG gel
methods had 64 and 52% concordance, respectively,
with CTT AHG titers. Neither the revised TT AHG titers
nor the IgG gel titers varied by more than one standard
dilution from the CTT AHG titers, which is within accept-
able limits for titration techniques.
CONCLUSIONS: The revised TT and IgG gel titers are
comparable to the CTT AHG titers. The IgG gel method
offers the best titer turnaround time, eliminating 45
minutes of incubation time alone. Implementation of this
technique would benefit ABO INKT patients by provid-
ing titer results in a more timely manner.
_2478 631..634
I
t has been shown that the ABO barrier in renal trans-
plantation can be circumvented by pretreatment of
the recipient with a combination of immunosup-
pression and apheresis to reduce the ABO antibody
concentrations permitting successful engraftment of an
ABO-incompatible kidney transplant (INKT).1-5 The trans-
fusion medicine laboratory plays a critical role in these
cases by monitoring ABO antibody titers during the pre-
treatment regimen as well as after transplantation.5
Our ABO INKT program began in 1999, and to date we
have monitored ABO antibody titers in 53 ABO INKT
patients using a time-consuming conventional test tube
(CTT) method that included a 30-minute room tempera-
ture (RT) incubation phase followed by incubation for 30
minutes at 37°C with subsequent conversion to the anti-
human globulin (AHG) test phase.5 Our experience has
indicated that the AHG titer values are critical for the clini-
cal management of ABO INKT patients and that RT titers
offer no additional data that affects clinical decisions.5 We
have consistently relied on the AHG titer values for the
clinical management of ABO INKT patients.5 By eliminat-
ing the RT phase, we proposed that titer turnaround times
could be improved, provided that the alternative method
yielded ABO antibody AHG titers comparable to AHG
titers determined by the conventional method. The goal
of this study was to streamline ABO antibody titer
ABBREVIATIONS: AHG = anti-human globulin; CTT =
conventional test tube; IgG gel = IgG gel microcolumn;
INKT = incompatible kidney transplant(s); RT = room
temperature; TT = test tube.
From Transfusion Medicine and the Department of Surgery,
Johns Hopkins Hospital, Baltimore, Maryland.
Address reprint requests to: R. Sue Shirey, Transfusion Medi-
cine, Johns Hopkins Hospital, Carnegie Room 667, 600 North
Wolfe Street, Baltimore, MD 21287-6667; e-mail: sshirey@
jhmi.edu.
Received for publication July 15, 2009; revision received
August 27, 2009; and accepted August 27, 2009.
doi: 10.1111/j.1537-2995.2009.02478.x
TRANSFUSION 2010;50:631-634.
Volume 50, March 2010 TRANSFUSION 631
SHIREY ET AL.

determinations for monitoring ABO INKT patients by Revised TT titration method

assessing AHG titers using the following titration Titrations were performed on each selected sample
methods: 1) a revised TT method where the RT incubation according to the CTT titration method described above,
phase was eliminated and 2) an anti-IgG gel microcolumn except the 30-minute RT incubation test phase was
(IgG gel) method. omitted.

MATERIALS AND METHODS Anti-IgG gel titers

CTT titration method
A CTT titration method was used to determine ABO anti-
body titers in potential recipients of ABO INKTs as out-
lined in the AABB Technical Manual.6 Serial dilutions of
the patients’ plasma samples were prepared in saline, and
4% saline-suspended donor-type indicator red blood cells
(RBCs) were added to each dilution. Titrations were incu-
bated at ambient (22-25°C) RT followed by incubation for
30 minutes at 37°C with subsequent conversion to the
AHG test phase using monospecific anti-IgG (Gamma
Clone, Immucor, Inc., Norcross, GA). Agglutination was
scored using the Marsh7 scoring system. The titer end-
point was the reciprocal of the highest dilution yielding
macroscopic score 3 (i.e., weak) agglutination, rather than
the more commonly used 1+ titration endpoint.8
The CTT ABO antibody titers were determined at a
time proximate to the ABO-incompatible kidney trans-
plantation and included parallel titration of a known
control sample. After titration, plasma aliquots were
frozen at -65°C.
For this study, 50 frozen samples from our ABO INKT
collection with anti-A and/or anti-B CTT AHG titers
ranging from 2 to 512 were selected (Table 1). Since an
AHG titer of 16 or less is required for ABO-incompatible
kidney transplantation, 18 of the 50 (36%) samples
selected had anti-A and/or anti-B titers of 16. ABO anti-
body titers were determined on these 50 samples by the
revised TT titration method and the anti-IgG gel titration
method described below.
Titrations were performed using anti-IgG gel cards (Micro
Typing Systems, MTS, Ortho Clinical Diagnostics, Raritan,
NJ) by the method described by Josephson and cowork-
ers.9 Serial dilutions of each sample were prepared as
described above. Twenty-five microliters of each plasma
dilution and 50 mL of 0.8% indicator RBCs prepared in MT
Diluent 2 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+.
RESULTS
Table 2 compares TT AHG titer results with and without a
30-minute RT incubation phase preceding 37°C incuba-
tion and antiglobulin tests. Thirty-two (64%) of AHG titers
without RT incubation were identical to conventional
AHG titers that included RT incubation designated CTT,
while 18 (36%) of 50 varied by one dilution with seven
(14%) reacting one tube higher and 11 (22%) reacting one
tube lower than the conventional method. No AHG titer
result varied more than one standard dilution from the
corresponding CTT AHG titer result.
Table 3 compares the IgG gel titers to the CTT AHG
titers that included a RT test phase. Twenty-six (52%) of
the 50 samples had IgG gel titers identical to the CTT AHG
titers, while 24 (48%) varied by one standard dilution with

TABLE 2. Comparison of AHG titer results by TT

methods with and without a RT incubation phase
Revised TT titers without (RT) phase†
TABLE 1. Summary of the titers for 50 samples
CTT AHG Identical to >CTT <CTT
selected for this study*
titers* Number CTT AHG titer AHG titer AHG titer
Number of samples
2 1 1
CTT AHG titers† Anti-A‡ Anti-B Anti-A/anti-B§ Total 4 3 3
2-8 6 2 2 10 8 6 4 2
16 12 5 1 18 16 18 13 2 3
32-64 9 3 12 32 7 4 1 2
128-256 7 2 9 64 5 3 2
512 1 1 128 6 3 2 1
Total 34 13 3 50 256 3 1 1 1
512 1 1
* Titers determined using group A1B indicator RBCs.
† CTT AHG titrations that included a RT incubation phase. Total 50 32 (64%) 7 (14%) 11 (22%)
‡ Anti-A titers determined with group A1 or group A2 indicator * CTT AHG titers = titrations that included a 30-minute incuba-
cells based on the kidney transplant donor blood type. tion phase at RT.
§ Samples from group O recipients of kidney transplants from † Revised TT titers = TT titrations performed without the con-
group A1B kidney donors. ventional 30-minute RT incubation phase.

632 TRANSFUSION Volume 50, March 2010

 STREAMLINING ABO ANTIBODY TITERS

pretreatment regimens and the point at which antibody

TABLE 3. Comparison of CTT AHG titers that levels have been sufficiently reduced to permit successful
included a RT test phase to the titers determined
using anti-IgG gel cards
engraftment.4,5,10 After transplantation, ABO antibody
IgG gel titers†
levels must continue to be monitored to detect possible
CTT AHG* Identical to >CTT <CTT antibody rebound that may signal antibody-mediated
titers Number CTT AHG titer AHG titer AHG titer rejection.
2 1 1 Since the ABO INKT program was launched in our
4 3 2 1
hospital, the transfusion medicine laboratory has rou-
8 6 4 2
16 18 13 3 2 tinely provided both RT and AHG titer values on each
32 7 2 3 2 sample. However, our data, as well as other reports, indi-
64 5 3 2
cate that RT titer results are not necessary.3-5,10,11 The
128 6 1 2 3
256 3 2 1 option of omitting RT titer reports offered us the opportu-
512 1 1 nity to improve turnaround time, provided that the modi-
Total 50 26 (52%) 12 (24%) 12 (24%) fication did not significantly affect the AHG titer results. In
* CTT AHG titers = titrations that included a 30-minute room addition, we wanted to examine a method more rapid
incubation phase at RT. than TT techniques for determining ABO antibody titers
† IgG gel titers = titrations performed using anti-IgG micro-
column gel method. using anti-IgG gel cards.9
This study shows that excluding the RT phase had
little effect on the AHG titers, since the revised TT method
yielded results comparable to AHG titers by the conven-
TABLE 4. Comparison of revised TT AHG titer tional method that included a RT phase. The TT AHG titer
results with the IgG gel titers results varied no more than one standard dilution, which
IgG gel titers†
is within the acceptable limits for titration methods.6 ABO
Identical to >Revised <Revised
Revised TT revised TT TT AHG TT AHG antibody titers determined by the IgG gel method showed
AHG titer* Number AHG titer titer titer 52% concordance with CTT AHG titer results and 48%
2 1 1 varied no more than one standard dilution, results similar
4 3 2 1 to those reported by Josephson and colleagues.9
8 6 6
16 18 16 2 Although antibody titers have been notoriously diffi-
32 7 6 1 cult to standardize, we found good correlation between
64 5 5 the two methods under study and the CTT AHG titers with
128 6 4 2
256 3 2 1 variations no greater than plus or minus one standard
512 1 1 dilution.6,8,12 Generally, titer values are considered accept-
Total 50 43 (86%) 5 (10%) 2 (4%) able if duplicate titers are within two standard dilutions.6
* Revised TT AHG titer = titers determined using a revised TT Titration variability may have been diminished in this
method without a RT incubation phase. study, because our titer endpoint for TT titration methods
† IgG gel titer = titration using anti-IgG microcolumn gel method.
was the reciprocal of the highest dilution demonstrating
weak agglutination, rather than the traditional 1+ titer
endpoint.6,8 AuBuchon and coworkers8 have reported that
12 (24%) one dilution higher and 12 (24%) one dilution using a weak-positive titer endpoint may reduce titration
lower than the CTT AHG titer. No IgG gel titer results variability.
varied more than one standard dilution from the CTT It is interesting to note that when the revised TT and
AHG titer. IgG GEL titers are compared, 86% of titer values were iden-
Table 4 compares the AHG titer results using the tical. The most likely explanation for this high concordance
revised TT method where the RT test phase was omitted to is that these titers were performed contemporaneously on
the IgG gel titers. Forty-three (86%) of the titer results were thawed samples by one technologist, while the conven-
identical and 7 (14%) varied by one standard dilution. Five tional titer results were determined on fresh samples at the
(10%) were one dilution greater than the TT-AHG titer, and time of submission by several different technologists.6,8,12
two (4%) were one tube lower than the TT AHG titer. No This study shows that both the revised TT and the IgG
IgG gel titer varied more than one standard dilution from gel are acceptable alternative methods for monitoring
the revised TT AHG titers. ABO antibody titers. However, the IgG gel method offers
the best turnaround time requiring only 15 minutes of
incubation at 37°C and eliminates the tedious reading of
DISCUSSION
TT agglutination reactions. Because the gel reactions are
Monitoring ABO antibody titers in potential recipients of stable, batch titrations can be easily accommodated by
ABO INKTs is critical for determining the effectiveness of IgG gel.

Volume 50, March 2010 TRANSFUSION 633

SHIREY ET AL.
As a result of this study, our laboratory has imple-
mented ABO antibody titer determinations by the IgG gel
method, which has cut our turnaround time in half and
has enabled us to provide titer values in a more timely
manner for patients in the ABO INKT program.
CONFLICT OF INTEREST
The authors have no conflict of interest.
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