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TRANSFUSION MEDICINE
Brief report
Immunogenicity of blood group antigens: a mathematical model corrected for
antibody evanescence with exclusion of naturally occurring and pregnancy-related
antibodies
Christopher A. Tormey1,2 and Gary Stack1,2
1Pathology and Laboratory Medicine Service, Veterans Affairs (VA) Connecticut Healthcare System, West Haven; and 2Department of Laboratory Medicine, Yale
University School of Medicine, New Haven, CT
Blood group antigen immunogenicity is a
crucial factor in red blood cell alloimmunization. Previous calculated estimates of
immunogenicity suffered from several key
shortcomings. To address these issues
we have (1) introduced a correction factor
for antibody persistence rates into traditional immunogenicity calculations,
(2) calculated immunogenicities only in
men to eliminate pregnancy-related anti-
bodies, and (3) excluded antibodies reactive only at room temperature to minimize
the contribution of naturally occurring
antibodies. With these corrections, we
have calculated the immunogenicities of
common blood group antigens using data
collected on clinically significant alloantibodies (n ⴝ 452) in a male patient population. We observed a 3- to 5-fold increase
in immunogenicity for some antigens (ie,
Jka, Cw, Lua) and smaller changes in others compared with traditionally calculated estimates. In addition, we have
calculated the transfusion-related immunogenicities of antigens traditionally associated with naturally occurring antibodies (eg, anti-Lea, -Leb, -M, and -P1). (Blood.
2009;114:4279-4282)
Introduction
The immunogenicity of a blood group antigen is an important
factor in determining whether a person transfused with red blood
cells expressing that antigen will develop the corresponding
alloantibody.1 The “Giblett equation” is a common method used in
estimating the immunogenicity of blood group antigens.2 This
calculation involves dividing the total number of antibodies of a
given specificity by the probability that an antigen-negative person
will be transfused with antigen-positive red blood cells. This value
is then normalized to the immunogenicity of the K antigen by
dividing by the corresponding calculation for K. To date, the
immunogenicities of antigens other than D and K have been based
largely on this calculated approach.2-7
These calculated estimates are subject to several potential
inaccuracies. For example, any factors that reduce antibody
detection will cause an erroneous reduction in the calculated
immunogenicity of the corresponding antigen. Antibody evanescence, that is, a decrease in antibody titers to below the limits of
detection, is one such factor. Evanescence is a particular problem
because alloantibody evanescence rates differ depending on the
antigenic specificity of the antibody.8-11 No prior study has
attempted to correct for this potential artifact.
Errors in the estimation of transfusion-related immunogenicity
may also result from the inclusion of naturally occurring and
pregnancy-related antibodies. Only transfusion-induced antibodies
should be counted for the calculation, otherwise immunogenicities
will be artifactually inflated. Moreover, because the circumstances
leading to the development of pregnancy-related antibodies are
different from those leading to transfusion-related antibodies, we
believe that they should be studied separately. However, to our
knowledge no prior study has excluded pregnancy-related antibodies from immunogenicity calculations. To eliminate naturally
occurring antibodies, most previous immunogenicity estimates
used a “shotgun” approach of excluding all anti-Lea, -Leb, -M, -N,
and -P1 antibodies, because they are often naturally occurring.2-4
Because of this shotgun exclusion, the transfusion-related immunogenicities of antigens such as Lea, Leb, M, N, and P1 are not known,
although these antigens can induce alloantibodies after transfusion.6 No prior study has used the selective approach of excluding
antibodies reactive at room temperature, which would eliminate
naturally occurring antibodies while permitting the calculation of
any antigen’s transfusion-related immunogenicity.
The goals of our study, therefore, were to develop an approach
to reduce these sources of error. To accomplish this we (1) corrected traditional immunogenicity calculations for antibody persistence, (2) eliminated pregnancy-related alloantibodies by studying
an exclusively male patient population, and (3) excluded antibodies
reactive only at room temperature to minimize the contribution of
naturally occurring antibodies. Our additional goal was to calculate
transfusion-related immunogenicities of antigens traditionally associated with naturally occurring antibodies (eg, anti-Lea, -Leb, -M,
-N, and -P1).
Submitted June 15, 2009; accepted August 8, 2009. Prepublished online as Blood
First Edition paper, August 27, 2009; DOI 10.1182/blood-2009-06-227793.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Methods
The number, specificities, and persistence rates of blood group alloantibodies documented in the paper transfusion records of 18 750 military veterans
between 1961 and 2006 at the Veterans Affairs Connecticut Healthcare
System (VACHCS) were described previously.8,12 We examined antibodies
4279
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4280
BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
TORMEY and STACK
in this dataset detected in male patients and that could be found on typical
commercial antibody screening panels.13 For immunogenicity calculations,
we used previously published antigen frequency data for white Europeans
because 80% of alloimmunized patients self-reported their race as white.12,14
Naturally occurring antibodies were defined as those that reacted only at
room temperature. Alloantibodies detected in female patients and autoantibodies were excluded. Immunogenicity calculations for the D antigen were
omitted because transfusions were matched for D at the VACHCS, causing
the number of anti-D to be artifactually underrepresented compared with all
other antibodies.
Fractional persistence rates for alloantibodies represent the ratio of
persistently detected antibodies of an indicated specificity to the total
antibodies of that specificity; rates were obtained from a previous study of
this patient population.8 We calculated antibody persistence rates only for
antibodies whose initial induction was documented at the study center to
obtain more accurate persistence rates.8 The persistence rates of antibodies
already present at the time of initial testing before transfusion at the study
center can never truly be known, because we cannot determine the total
number of antibodies induced before that initial testing.
Results and discussion
Of 540 total antibodies formed by male patients, 85.6% (462/540)
were directed against clinically significant antigens included in a
typical commercial panel (Table 1’s “Total antibodies”). Antibodies
likely to be naturally occurring, that is, those reactive only at room
temperature, were subtracted from the total of each corresponding
specificity to approximate those that were transfusion induced
(Table 1’s “Antibodies reacting at 37°C and/or antiglobulin phase”).
With this definition, transfusion-induced antibodies constituted
97.8% (452/462) of antibodies made by men.
The traditional immunogenicity calculation of Giblett assumes
that the total number of antibodies of a given specificity is related to
the immunogenicity of the corresponding antigen (Figure 1A).2
However, the number of antibodies of a given specificity is also
related to the persistence of that antibody over time. As such, we
introduced a correction factor into the Giblett equation for antibody
persistence to account for antibodies that were induced, but
disappeared, before they could have been detected. This modification consisted of dividing the number of antibodies detected for a
given specificity by the fractional persistence rate for that antibody
(Figure 1B). This ratio should yield the total number of antibodies
that would have been detected if persistence rates were 100%.
Antibody data from our study population were used to calculate
immunogenicities by both modified and traditional equations
(Table 1’s “Traditional antigen potency” and “Antigen potency
corrected for antibody persistence”). The largest correction was
observed for Jka, whose immunogenicity was approximately 5-fold
higher than that obtained with the original Giblett equation. As a
result, the relative potency of Jka rose from the tenth most potent
antigen in the uncorrected ranking to fourth. This corrected
estimate for Jka ranged between 2- and 26-fold higher than that
calculated in previous publications using the original Giblett
equation.2-5 Lua and Cw also demonstrated relatively large increases
in immunogenicity of approximately 4-fold. The potencies of C, c,
Fya, and S appeared to decrease slightly, whereas those of M, Leb,
and P1 appeared to increase slightly. The potencies of E and Lea
were essentially unchanged.
Given that most antibodies traditionally considered to be
naturally occurring (eg, anti-Lea, -Leb, -M, -N, and -P1) appeared to
be predominantly transfusion induced in men,12 we were able to
calculate transfusion-related immunogenicities for these antigens.
Based on the modified equation, Lea, Leb, P1, and M were among
the 10 most potent antigens overall with a potency rank order of
Lea ⬎ P1 ⬎ M ⬎ Leb. To our knowledge, the only other study to
calculate relative immunogenicities for Lewis, P1, and M antigens
was that of Winters et al.5 However, most antibodies with those
Table 1. Comparison of the immunogenicities of blood group antigens obtained with corrected and traditional calculations, showing key
data used in the calculation
Total
antibodies*
Antibodies reacting at 37°C and/or
antiglobulin phase*
Traditional antigen
potency†
Fractional persistence
rate‡
Antigen potency corrected for
antibody persistence§
Fold
change储
Jka
20
20
0.077
0.11
0.37
4.81
Lua
11
10
0.094
0.13
0.40
4.26
Cw
8
8
0.19
0.14
0.70
3.68
P1
21
18
0.074
0.33
0.12
1.62
Antigen
Leb
19
18
0.062
0.37
0.089
1.44
M
18
18
0.073
0.43
0.090
1.23
Lea
40
37
0.15
0.50
0.16
1.07
K
118
118
1.00
0.53
1.00
1.00
E
105
105
0.35
0.52
0.35
1.00
c
26
26
0.11
0.62
0.097
0.88
Fya
29
29
0.090
0.75
0.064
0.71
C
25
25
0.080
0.77
0.055
0.69
S
10
9
0.025
1.00
0.013
0.52
V
3
3
0.21
e
2
2
0.071
s
2
2
0.014
N
3
2
0.007
Fyb
1
1
0.005
Jkb
1
1
0.004
*Values were obtained from a previous study of this patient population.12
†Calculations were based on the traditional equation of Giblett.2 The potency of K was set at 1.00 and all other potencies were expressed relative to K.
‡Fractional persistence rates represent the ratio of persistently detected antibodies of the indicated specificity to total antibodies of that specificity, as calculated in a
previous study of this same patient population.8
§Values were obtained according to the modified immunogenicity calculation presented in Figure 1B and described in ⬙Results and discussion.⬙ Blood group specificities
associated with 3 or less total antibodies were excluded from the modified immunogenicity calculation due to potential errors associated with small sample size.
储Fold change was calculated as follows: ⬙Antigen potency corrected for antibody persistence⬙ divided by ⬙Traditional antigen potency.⬙
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Figure 1. Equations for calculating blood group antigen immunogenicity. (A) The
traditional Giblett immunogenicity calculation.2 (B) The modified immunogenicity calculation.
specificities in that study were naturally occurring.5 As such, the
relevance of their calculations to transfusion-associated immunogenicity is questionable. Several other studies have addressed this
issue by excluding all anti-M, -P1, -Lea, and -Leb antibodies.2-4
However, that approach prevented the calculation of the immunogenicities of their corresponding antigens, and did not exclude
antibodies of other specificities that may have been naturally
occurring. Our approach of calculating antigen immunogenicities
only for antibodies reactive at 37°C and/or antiglobulin phase
avoided these earlier pitfalls.
Because our patient population included only alloimmunized
men, the immunogenicity calculations excluded pregnancy-related
antibodies. Although many pregnancy-induced antibodies are clinically significant, the circumstances of their induction are sufficiently different from those of transfusion-induced antibodies to
merit their separate study. For example, the route of exposure, the
volume of antigen-positive red blood cells (antigenic load), and
perhaps host immune status may differ in the settings of pregnancy
and transfusion. However, it should be noted that by studying only
men, we may have introduced a sex-related bias to immunogenicity
estimates.
IMMUNOGENICITY OF BLOOD GROUP ANTIGENS
4281
Despite our efforts to reduce errors associated with immunogenicity estimates, the possibility for inaccuracy remains. For example, even though we attempted to exclude naturally occurring
antibodies, some may have been inadvertently included if they
reacted at 37°C and/or antiglobulin phase. Warm-reactive, naturally occurring anti-E, -Lua, -Cw, -M, -N, and -P1 have been
reported.6,14,15 In theory, our approach would also exclude transfusion-induced antibodies reactive only at room temperature, such as
newly formed antibodies before isotype switching from immunoglobulin M to immunoglobulin G.6 Rare examples of antibodies
typically considered clinically significant, but that react only at
room temperature, such as anti-D, -E, and -Fya, would also be
excluded.6,14-16 Because only 2% of the antibodies in our study
reacted at room temperature alone, and most of these have
specificities known to be naturally occurring,6,14,15 the number of
such antibodies would be very low.
Inaccurate antibody persistence rates are another potential
source of error. Unfortunately, antibody numbers for many specificities were likely too small to yield accurate results. Moreover,
accurate persistence rates require regular antibody testing at
defined intervals, which was not possible with a retrospective
study.8 In addition, antibody evanescence is potentially subject to
variation over time, because serologic detection techniques and
reagents changed over the course of the data collection period.
Even with these shortcomings, our modified approach should
provide an improved theoretical framework for future calculations.
In summary, we have modified the calculation of antigen
immunogenicity to correct for several potential errors. Specifically,
we have corrected the Giblett equation for antibody persistence and
have excluded pregnancy-related and naturally occurring antibodies from the calculation. The effect of these corrections was most
evident for Jka, whose immunogenicity was previously underestimated because of the high evanescence rate of anti-Jka. We have
also reported the transfusion-related immunogenicities of antigens
traditionally associated with naturally occurring antibodies, several
of which ranked among the top 10 most immunogenic antigens.
Acknowledgment
This material is the result of work supported with resources and the
use of facilities at the VACHCS.
Authorship
Contribution: C.A.T. and G.S. designed the project, collected and
reviewed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Christopher A. Tormey, Department of
Laboratory Medicine, Yale University School of Medicine, 333
Cedar St, PO Box 208035, New Haven, CT 06520; e-mail:
[email protected].
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2009 114: 4279-4282
doi:10.1182/blood-2009-06-227793 originally published
online August 27, 2009
Immunogenicity of blood group antigens: a mathematical model
corrected for antibody evanescence with exclusion of naturally
occurring and pregnancy-related antibodies
Christopher A. Tormey and Gary Stack
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