I M M U N O H E M AT O L O G Y
Serological studies of piperacillin antibodies
Regina M. Leger, Patricia A. Arndt, and George Garratty
BACKGROUND: Penicillin-induced immune hemolytic
anemia (IHA) is associated with immunoglobulin G
antipenicillin detected by testing penicillin-coated red
blood cells (RBCs). Antibodies to piperacillin, a semi-
synthetic penicillin, would be expected to react similarly;
however, antipiperacillin can be detected by testing in
the presence of the drug. Piperacillin is commonly used
in combination with tazobactam, which causes nonim-
munologic protein adsorption onto RBCs. In six cases
of piperacillin-induced IHA, reactivity with piperacillin-
coated RBCs was not similar to reactivity of antipenicil-
lin with penicillin-coated RBCs.
STUDY DESIGN AND METHODS: Antipiperacillin was
tested against piperacillin-coated RBCs prepared using
different pH buffers. Plasma from blood donors and
sera/plasma from patients were tested with piperacillin-
coated, penicillin-coated, and uncoated RBCs. Hapten
inhibition studies were performed using different con-
centrations of piperacillin. Donors’ plasma were tested
in the presence of piperacillin; sera from patients with
IHA were tested in the presence of tazobactam.
RESULTS: Piperacillin required high pH for binding to
RBCs. Agglutination of piperacillin-coated RBCs was
observed in 91 percent of donors’ and 49 percent of
patients’ plasma and was inhibited by piperacillin. In
contrast to patients with IHA due to piperacillin, donors’
plasma tested in the presence of piperacillin did not
react. Tazobactam antibodies were not detected.
CONCLUSION: A high percentage of donors’ and
patients’ plasma contain an antibody to piperacillin or a
chemically related structure detected by testing with
piperacillin-coated RBCs. A diagnosis of piperacillin-
induced IHA should not be made solely on the reactivity
of a patient’s plasma/serum with piperacillin- or
piperacillin/tazobactam-coated RBCs; testing in the
presence of piperacillin is more reliable.
S
everal cases of piperacillin-induced immune
hemolytic anemia (IHA) have been reported.1-5
Piperacillin is a semisynthetic penicillin; thus,
antibodies to piperacillin would be expected to
react similarly to antibodies to penicillin (e.g., serum and
eluate reactive with drug-coated red blood cells [RBCs]).
The earliest report implicating piperacillin as the cause
of anemia1 had no serologic testing or evaluation for
hemolysis. The report by Thickett and coworkers3 pro-
vided insufficient serologic details for the method used.
The other reports demonstrated that antipiperacillin can
be detected by testing in the presence of the drug (the
so-called “immune complex” method), sometimes in the
absence of reactivity with piperacillin-coated RBCs.2,4,5
Piperacillin is often used in combination with tazo-
bactam, a b-lactamase inhibitor, in the United States as
Zosyn (Wyeth, Philadelphia, PA) or in other countries as
Tazocin (Wyeth) for treating serious infections, for
example, in patients with cystic fibrosis. The combined
antibiotic is active against many Gram-positive and
Gram-negative bacteria, including Pseudomonas aerugi-
nosa. Cases of Zosyn-induced IHA have also been
reported,5-9 not all of which were tested for piperacillin
antibodies. Zosyn-coated RBCs can be reactive for two
reasons: 1) antibodies to piperacillin and 2) nonimmuno-
logic protein adsorption due to the tazobactam
component;4,10,11 although not reported, antibodies to
tazobactam must also be considered.
Nonimmunologic protein adsorption is thought to
occur when the RBC membrane is modified by the drug so
ABBREVIATIONS: AHG = antihuman globulin; BB = barbital
buffer; IHA = immune hemolytic anemia.
From the American Red Cross Blood Services, Southern
California Region, Pomona, California.
Address reprint requests to: Regina M. Leger, MSQA,
MT(ASCP)SBB, CMQ/OE, American Red Cross Blood Services,
Southern California Region, 100 Red Cross Circle, Pomona, CA
91768; e-mail: legerre@usa.redcross.org.
Received for publication February 6, 2008; revision
received May 21, 2008; and accepted May 25, 2008.
doi: 10.1111/j.1537-2995.2008.01852.x
TRANSFUSION 2008;48:2429-2434.
Volume 48, November 2008 TRANSFUSION 2429
LEGER ET AL.
that protein attaches to the surface nonimmunologi-
cally.12 This can result in a positive direct antiglobulin test
(DAT) and in “false-positive” indirect antiglobulin tests
(IATs) when RBCs coated with the drug in vitro are incu-
bated with normal plasma. Nonimmunologic protein
adsorption, which is independent of drug antibody for-
mation, may or may not be associated with hemolytic
anemia.12
Penicillin-induced IHA is usually associated with a
strongly positive DAT due to immunoglobulin (Ig)G and a
high-titer IgG antipenicillin in the patient’s serum and in
an eluate prepared from the patient’s RBCs, detected
by testing with penicillin-coated RBCs.12 Patients without
IHA can have low-titer antibodies to penicillin, presum-
ably due to exposure to penicillin in our environment;
therefore, it is more reliable when diagnosing IHA due to
penicillin to show that the antibody eluted from patient’s
DAT-positive RBCs is antipenicillin. Optimal coating of
RBCs with penicillin in vitro occurs at high pH, for
example, pH 10, but coating sufficient to detect significant
penicillin antibodies occurs at lower pH (e.g., pH 7-8).13
A review of six cases of antipiperacillin detected in our
laboratory by testing in the presence of piperacillin
revealed that the antibody reactivity with piperacillin-
coated RBCs was not similar to what is expected of
antipenicillin. Four of five tested samples were reactive
with piperacillin-coated RBCs; however, none of three
acid eluates were reactive, and pooled group AB inert
plasma used as a negative control directly agglutinated
the piperacillin-coated RBCs.
Conditions for coating RBCs with piperacillin and
the reactivity of normal plasma or sera against
piperacillin-coated RBCs were investigated. Also, sera
from patients with IHA who received Zosyn were tested for
antibody to the b-lactamase inhibitor component tazo-
bactam.
MATERIALS AND METHODS
Ethylenediaminetetraacetate (EDTA) plasma from
unlinked blood donors and plasma or sera from unlinked
random patients were obtained for testing with
piperacillin-coated RBCs. Sera from 13 patients with IHA
suspected to be induced by Zosyn were tested upon initial
investigation or retrieved from frozen storage.
Testing with drug-coated RBCs
Drug-coated RBCs were prepared by incubating solutions
of piperacillin (Lederle Laboratories, Pearl River, NY; or
American Pharmaceutical Partners, Inc., Schaumburg, IL),
penicillin G (Sigma-Aldrich Co., St Louis, MO), Zosyn
(Lederle), or tazobactam (Lederle) with group O RBCs for 1
hour at room temperature.12 The drug solutions were pre-
pared at 40 mg per mL in barbital buffer (BB; pH 9.8).
2430 TRANSFUSION Volume 48, November 2008
Piperacillin-coated RBCs were also prepared in
phosphate-buffered saline (PBS; pH 7.0-7.2 or pH 8.0) for
comparison studies. The ratio of drug solution to RBCs
used for the preparation of penicillin-coated RBCs (15:1,
e.g., 1.5 mL of drug solution plus 0.1 mL of RBCs) was
compared to the ratio used for preparation of
cephalosporin-coated RBCs (10:1, e.g., 1 mL of drug solu-
tion plus 0.1 mL of RBCs).12 For testing, 1 drop of 3 to 5
percent drug-coated or uncoated RBCs was incubated with
2 drops of plasma or serum for 1 hour at 37°C. After being
centrifuged and examined for agglutination, the cells were
washed and tested with polyspecific antihuman globulin
(AHG, Ortho Clinical Diagnostics, Raritan, NJ). Sera con-
taining antipenicillin or antipiperacillin, previously identi-
fied in our laboratory, were used as positive controls to
show that the RBCs were drug-coated. To determine the
immunoglobulin class of antibodies, testing was repeated
after incubation of the reactive plasma with 0.01 mol per L
dithiothreitol (DTT) or PBS.14
Hapten inhibition studies were performed by incu-
bating piperacillin (differing concentrations of 10, 1, and
0.1 mg/mL in PBS), penicillin (10 mg/mL), or PBS with an
equal volume of plasma for 1 hour at 37°C.12 Piperacillin-
coated RBCs were added and incubated for an additional
hour. Tests were examined for agglutination and then
carried on to the antiglobulin phase.
Testing in the presence of drug
Two drops of plasma plus 2 drops of a 1 mg per mL solu-
tion of the drug in PBS (or 2 drops of PBS) were incubated
with 1 drop of 5 percent group O ficin-treated RBCs for 1
hour at 37°C. After being examined for agglutination, the
RBCs were washed and tested with polyspecific AHG. A
previously identified antipiperacillin was used as a posi-
tive control.
Sera or plasma from four IHA patients with previously
identified antipiperacillin reactive in the presence of pip-
eracillin were retested as above with untreated RBCs in the
presence of 1 and 10 mg per mL piperacillin to determine
if reactivity by the “immune complex” method could be
inhibited. In parallel, the drug solutions were incubated
with the sera for 1 hour (as for the hapten inhibition
studies) before adding untreated RBCs.
Sera from 13 patients who were suspected of having
IHA due to Zosyn were tested as above in the presence of
tazobactam (Sigma-Aldrich) against untreated and ficin-
treated RBCs. Fresh normal serum was added as a source
of complement.
RESULTS
Optimal preparation of piperacillin-coated RBCs
One example of antipiperacillin (diluted 1 in 2) did not
react with piperacillin-coated RBCs prepared at pH 7 and

TABLE 1. Reactivity* of blood donors’ and random patients’ samples with piperacillin- and penicillin-coated
RBCs†
Donors’ samples
RBCs Number tested
Piperacillin-coated 100
Penicillin-coated 19
Uncoated 100
* Reactivity was agglutination in all samples except two donors’ samples that reacted weakly by antiglobulin test.
† RBCs treated in BB, pH 9.8.
pH 8. The antipiperacillin agglutinated (titer ⱖ 32) the
piperacillin-coated RBCs prepared at pH 9.8. Control
RBCs incubated in parallel in PBS at pH 7 or pH 8 or in BB
at pH 9.8 did not react. There was no significant difference
between the reactivity of one example of antipiperacillin
with RBCs coated at a 15:1 ratio (as used to prepare
penicillin-coated RBCs) compared to a 10:1 ratio (as used
to prepare cephalosporin-coated RBCs). The agglutinin
and AHG titers were 2 and 8 and 2 and 4, respectively, for
the two ratios. RBCs treated with piperacillin in pH 9.8 BB
at a 10:1 ratio were used for the rest of this study, consis-
tent with the preparation of piperacillin-coated RBCs in
our earlier case report.4
Drug-coated RBCs
The reactivity of donors’ and patients’ samples with
piperacillin- and penicillin-coated RBCs prepared at pH
9.8 is shown in Table 1. Nineteen donors’ plasma tested
with piperacillin-coated RBCs were also tested with
penicillin-coated RBCs. One donor’s plasma reacted with
both piperacillin- and penicillin-coated RBCs; 12 others
that reacted with piperacillin-coated RBCs did not react
with penicillin-coated RBCs; none reacted with penicillin-
coated RBCs only. The 5 patients’ samples tested with
penicillin-coated RBCs were selected based on reactivity
with piperacillin-coated RBCs. All reactivity in both sets of
samples was agglutination observed after the 37°C incu-
bation except for samples from 2 donors reacting weakly
by antiglobulin test only. The agglutination was weak (1+)
in 58 percent of the samples, moderate (2-3+) in 34
percent of the samples, and strong (4+) in 8 percent of the
samples with similar distribution in the two groups. An
additional 15 of 20 donors’ samples and 1 of 4 patients’
samples reacted with RBCs subsequently coated with an
alternative source of piperacillin sodium (Sigma-Aldrich)
in BB. The reactivity was agglutination after the 37°C incu-
bation (weak to 4+) as noted in the original set of data.
Reactivity of 12 donors’ plasma with piperacillin-
coated RBCs was abolished at 37°C and AHG phases after
DTT treatment, indicating the antibodies were IgM. The
dilution controls reacted 1+ to 4+ at 37°C.
Antibody reactivity in six of six donors’ plasma
against piperacillin-coated RBCs was completely inhib-
DETECTION OF PIPERACILLIN ANTIBODIES
Patients’ samples
Number positive Number tested Number positive
91 35 17
1 5 1
0 35 0
TABLE 2. Example of reactivity due to
nonimmunologic protein adsorption
Serum 37°C (agglutination) AHG
Plus Zosyn-coated RBCs* 0 1+
Plus piperacillin-coated RBCs 0 0
Plus tazobactam-coated RBCs 0 3+
* All RBCs were treated in BB, pH 9.8.
ited after incubation with 10 mg per mL piperacillin; incu-
bation with 1 or 0.1 mg per mL was less efficient, but
resulted in at least partial inhibition. Incubation with
10 mg per mL penicillin resulted in partial inhibition of
reactivity against piperacillin-coated RBCs in one of two
donors’ plasma and complete inhibition in the second
plasma.
As shown in Table 2, both Zosyn- and tazobactam-
coated RBCs prepared at pH 9.8 reacted with normal
serum due to nonimmunologic protein adsorption. The
weaker reactivity with the Zosyn-coated RBCs can be
explained by the lower relative concentration of the tazo-
bactam component in the 40 mg per mL solution of drug
used to coat the RBCs (Zosyn is 89% piperacillin, 11%
tazobactam). When the serum was retested at a 1-in-20
dilution, the Zosyn-coated RBCs did not react and the
tazobactam-coated RBCs were only microscopically reac-
tive. Pooled group AB inert plasma reacted similarly.
Testing in the presence of drug
None of nine donors’ samples that reacted with the
piperacillin-coated RBCs reacted with enzyme-treated
RBCs in the presence of piperacillin. In contrast, antipip-
eracillin from all six patients with piperacillin-induced
IHA strongly agglutinated enzyme-treated RBCs in the
presence of the drug solution.
Unlike the inhibition by piperacillin observed when
testing plasma from donors or patients without IHA
against piperacillin-coated RBCs, antipiperacillin
detected by the “immune complex” method from four
patients with piperacillin-induced IHA was not inhibited
when incubated in the presence of either a 1 or a 10 mg
per mL solution of piperacillin. Rather, reactivity was
enhanced when the greater concentration of drug was
added to the test system.
Volume 48, November 2008 TRANSFUSION 2431
LEGER ET AL.
No evidence of an antibody to tazo-
bactam was detected in the sera from 13 TABLE 3. Summary of findings when testing plasma/serum for
patients with IHA suspected to be due
to Zosyn. None of the sera reacted with Group
untreated RBCs in the presence of Patients with IHA associated
tazobactam; three sera weakly reacted with piperacillin
with ficin-treated RBCs with or without Blood donors
Random patients
tazobactam added. Detection of anti-
bodies to piperacillin with piperacillin-
coated RBCs and in the presence of
piperacillin in samples from patients with piperacillin-
induced IHA, healthy blood donors, and random patients
is summarized in Table 3.
DISCUSSION
A high percentage of plasma or sera from blood donors
(91%) and patients (49%) tested in this study contained an
antibody to piperacillin (or a chemically related structure)
detected with piperacillin-coated RBCs. These antibodies
were IgM and could be inhibited by both piperacillin and
penicillin, but had low cross-reactivity with penicillin-
coated RBCs. In earlier studies using an unusual sensitive
passive hemagglutination technique, Levine and col-
leagues15,16 found that penicillin antibodies could be
detected in most unselected sera; most of these were IgM
and were easily neutralized by benzylpenicilloyl, the
major haptenic determinant of the penicillin molecule. In
other studies with methods normally used by immunohe-
matologists, penicillin antibodies were found in only 3
percent of samples from blood donors.17-19
Although penicillin was shown to inhibit antipiper-
acillin in two donors’ plasma, the antipiperacillin in these
two samples did not react with penicillin-coated RBCs.
Thus, the specificities do not appear to be identical, but
may reflect antibody formation to a portion(s) of the peni-
cillin nucleus common to the two drugs (Fig. 1). Epitopes
available for antibody interaction with the drug that is
bound to RBCs may be more limited compared to
epitopes available in a solution of the drug.
Many sera from donors or patients react with RBCs
coated with b-lactam antibiotics (e.g., penicillin,
cefotetan, piperacillin). This suggests that we are all
exposed to b-lactams or closely related chemicals in our
environment (e.g., possibly added to cattle feed, present
in milk). It is unclear why more donors’ plasma than
patients’ plasma/sera reacted with piperacillin-coated
RBCs. There was no apparent difference between fresh
and frozen stored EDTA samples or between plasma or
serum samples (data not shown). It is possible that the
difference between blood donors and patients may just
reflect the difference in immune response or status
between healthy versus sick individuals. This has been
illustrated recently in three publications that challenge
the old statistic that 80 percent of D- individuals make
2432 TRANSFUSION Volume 48, November 2008
piperacillin antibodies
Testing in the presence
Piperacillin-coated RBCs of piperacillin
Positive or negative Positive
Positive or negative Negative
Positive or negative Negative
Penicillanic acid
Penicillin G
Piperacillin
Fig. 1. Chemical structures for penicillanic acid (the b-lactam
building block of the penicillins), penicillin G, and piperacillin
(modified from Budavari20).
anti-D when transfused with D+ RBCs. All three of these
reports21-23 show that the older statistic applies to healthy
individuals but only 20 to 30 percent of patients make
anti-D. The patients were not those known to be espe-
cially immunosuppressed. An immune response to the D
antigen, however, is not completely analogous to the
current study, because all patients would have presum-
ably been exposed to b-lactam antibiotics similarly to
healthy blood donors.
It is also not clear why piperacillin-coated RBCs
reacted more frequently than penicillin-coated RBCs in
either group. However, as can be seen in Fig. 1, the side
chains that confer the antimicrobial activity of penicillin G
and piperacillin are dramatically different. Penicillin is
optimally bound to RBCs at pH 10 but antipenicillin reacts
with RBCs treated with penicillin at pH values below 10
(e.g., 7 or 8).13 RBCs treated with piperacillin at pH 9.8
clearly reacted with antipiperacillin, whereas RBCs treated
at pH 7 or 8 did not react. However, the titer of the
antipiperacillin used for this study was lower than
the high-titer IgG antipenicillin used by Spath and
coworkers13 to demonstrate optimal conditions for
binding of penicillin to RBCs.
Four of the reported cases of piperacillin- or
piperacillin/tazobactam-induced IHA had evidence of
intravascular lysis, for example, hemoglobinuria.3,4,7,8 This
is consistent with other drug antibodies detected by

testing in the presence of a solution of drug, for example,
ceftriaxone. In one of our previous cases, attempts to
inhibit antipiperacillin reactive with piperacillin-coated
RBCs, with piperacillin, resulted in enhanced reactivity.4
In contrast, penicillin-induced IHA typically results in
extravascular lysis and the antibody is readily inhibited by
penicillin.
When patients are suspected of IHA due to Zosyn,
testing has been reported using Zosyn to coat RBCs or in
the presence of Zosyn.5-9 However, one cannot assume
that reactivity observed in either test method with Zosyn
is due to piperacillin antibodies; Zosyn (and Tazocin)
contains a second compound, tazobactam. Zosyn-
treated RBCs, especially if prepared at high pH, can
adsorb immunoglobulins and other proteins due to non-
immunologic protein adsorption (discussed above) with
reactivity detected in the IAT and antibodies to piperacil-
lin may not be present. This could be falsely interpreted
as indicating the presence of antibodies to Zosyn. Simi-
larly, because of the two drug components, antibodies to
either piperacillin or tazobactam, or both, may be the
cause of reactivity when testing in the presence of Zosyn.
Using pure piperacillin and tazobactam (both commer-
cially available) instead of Zosyn will avoid misinterpre-
tation of the results. Although we did not detect
antibodies to tazobactam in our series, the possibility
cannot be excluded.
Testing a pool of normal sera or plasma against drug-
treated RBCs is an important control for interpretation of
results. Direct agglutination of drug-coated RBCs by
normal sera, as observed here, indicates that agglutina-
tion by serum from a patient with IHA needs further
investigation. Some of the donors’ plasma tested with the
piperacillin-coated RBCs reacted 4+, so strength of reac-
tivity is not a reliable indicator of antibody significance.
Although we expect antibodies to drugs associated with
IHA to react to high titers with drug-coated RBCs, for
example, penicillin and cefotetan, this may not always be
the case. When the pool of normal sera is only reactive in
the IAT, nonimmunologic protein adsorption should be
suspected. We recommended using a 1-in-20 dilution of
normal sera and the patient’s serum, in parallel to undi-
luted sera, when testing drugs known to cause nonimmu-
nologic protein adsorption.12
In conclusion, the high percentage of plasma or
serum from donors or patients, with no IHA, that react
with piperacillin-coated RBCs indicates testing for piper-
acillin antibodies by this method is unreliable, especially
when antipiperacillin is not detected in an eluate
prepared from the patient’s DAT-positive RBCs. Addition-
ally, testing with Zosyn-coated RBCs does not differentiate
the presence of antipiperacillin from nonimmunologic
protein adsorption due to tazobactam. Testing in the pres-
ence of piperacillin is more reliable and is the method
we recommend for detecting clinically significant antipip-
DETECTION OF PIPERACILLIN ANTIBODIES
eracillin when investigating suspected cases of
piperacillin- or piperacillin/tazobactam-induced IHA.
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