Download Diagnostic pitfalls of drug-induced immune hemolytic anemia

George Ga r r at t y
1935–201 4
Journal of Blood Group Serology and Molecular Genetics
V o l u m e 3 0, N u m b e r 2 , 2014
Immunohematology
Journal of Blood Group Serology and Molecular Genetics
Volume 30, Number 2, 2014
CO N TEN TS
43
Introduction to Immunohematology Special Edition on
Drug-Induced Immune Cytopenias
P.A. Arndt and R.M. Leger
44
Report
Drug-induced immune hemolytic anemia: the last 30 years
of changes
P.A. Arndt
55
Report
Drug-induced immune thrombocytopenia: incidence, clinical
features, laboratory testing, and pathogenic mechanisms
B.R. Curtis
66
Report
Drugs that have been shown to cause drug-induced immune
hemolytic anemia or positive direct antiglobulin tests: some
interesting findings since 2007
G. Garratty and P.A. Arndt
80
Case Report
Diagnostic pitfalls of drug-induced immune hemolytic anemia
A. Salama and B. Mayer
85
Report
How we investigate drug-induced immune hemolytic anemia
R.M. Leger, P.A. Arndt, and G. Garratty
95
Report
Drug-induced immune neutropenia/agranulocytosis
B.R. Curtis
102
Announcements
105
A dv ertisements
109
Instructions
for
Authors
Editor-in- Chief
E d i t o r ia l B o a r d
Sandra Nance, MS, MT(ASCP)SBB
Philadelphia, Pennsylvania
Patricia Arndt, MT(ASCP)SBB
Thierry Peyrard, PharmD, PhD
Barbara J. Bryant, MD
Mark Popovsky, MD
Lilian Castilho, PhD
S. Gerald Sandler, MD
Pomona, California
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Cynthia Flickinger, MT(ASCP)SBB
Philadelphia, Pennsylvania
Paris, France
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Washington, District of Columbia
Martha R. Combs, MT(ASCP)SBB
Durham, North Carolina
Jill R. Storry, PhD
New York City, New York
Joyce Poole, FIBMS
Christine Lomas-Francis, MSc
Lund, Sweden
Geoffrey Daniels, PhD
Bristol, United Kingdom
David F. Stroncek, MD
Bristol, United Kingdom
Dawn M. Rumsey, ART(CSMLT)
Anne F. Eder, MD
Nicole Thornton
Norcross, Georgia
Bethesda, Maryland
Washington, District of Columbia
Bristol, United Kingdom
Brenda J. Grossman, MD
Senior M edi cal Editor
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Ralph R. Vassallo, MD
Christine Lomas-Francis, MSc
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New York City, New York
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Marion E. Reid, PhD, FIBMS
Philadelphia, Pennsylvania
A s s o c iat e M e d i c a l E d i t o r s
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Baltimore, Maryland
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Philadelphia, Pennsylvania
M olecul ar Editor
Margaret A. Keller
Philadelphia, Pennsylvania
Immunohematology is published quarterly (March, June, September, and December) by the
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O n O ur C ov er
George Garratty was my boss, my mentor, and, later, my friend. He instilled in each of his staff the requirement to
associate clinical findings with laboratory and serologic findings and that these aspects contributed to optimal care
of patients with immune hemolytic anemias. In his preface to Immunobiology of Transfusion Medicine, published in
1994, he said, “Avoiding the immune destruction of circulating cells such as erythrocytes, leukocytes, and platelets is
one of the major goals of transfusion medicine. The in vivo and in vitro reactions involved in these immune reactions
provide easily studied human models for complement- and macrophage-mediated cell destruction, autoimmunity, and
drug-induced immune destruction of cells. Although complement-mediated cell destruction is understood quite well,
many aspects of the more common extravascular destruction of blood cells are still not understood. Over the last four
decades, we have learned that important factors such as immunoglobulin class and subclass, complement-activating
ability of the antibody, quantity of cell-bound antibody and complement components, affinity of the antibody, and
activity of the mononuclear phagocyte system all play a role, but there are still many anomalies between the observed
in vivo destruction and our in vitro results. We need to reconcile these differences before in vitro assays can be
improved and to forecast accurately the survival of transfused cellular components.” We miss his presence among us.
Sandra Nance, MS, MT(ASCP)SBB
Senior Director, Immunohematology Reference Laboratory (IRL)/
National Reference Laboratory for Blood Group Serology (NRLBGS), American Red Cross Biomedical Services
Introduction to Immunohematology
Special Edition on Drug-Induced
Immune Cytopenias
This issue of Immunohematology on drug-induced
immune cytopenias (hemolytic anemia, thrombocytopenia,
and neutropenia) was organized and guest edited by the late
Dr. George Garratty. Dr. Garratty started writing regular
reviews for Immunohematology on drug-induced immune
hemolytic anemia (DIIHA) and positive direct antiglobulin
tests in 1985. His first review covered “some controversies,
unusual aspects, or subjects that have not been thoroughly
discussed before” and included a table with 45 drugs. The
second review 4 years later concentrated on mechanisms and
ended with the statement “[i]t seems to me that in 1989 we
still have more questions than answers.” In his 1994 review,
Dr. Garratty updated information on current theories about
DIIHA and included a table with 76 drugs. In 2004, about 100
drugs had been associated with DIIHA, but a large number of
DIIHAs were associated with cephalosporins so they were the
emphasis of the review that year. With regard to mechanisms
of DIIHA, Dr. Garratty stated that “personally I do not think
that, in 2004, we have any better explanations tha[n] we did
in 1994!” The most recent review in 2007 was by Dr. Garratty
and Pat Arndt and included three tables: the first table
contained information on 108 drugs associated with drugdependent antibodies, the second table listed 17 drugs possibly
or probably associated with drug-independent antibodies
(autoantibodies), and the third table showed 9 drugs that had
been shown to cause nonimmunologic protein adsorption.
This issue contains the final installment of this series of
reviews by Dr. Garratty. The review by Dr. Garratty and Pat
Arndt updates the three previous tables and summarizes new
information on drugs currently associated with DIIHA. In
addition, in this issue, there are three others articles on DIIHA.
The article by Pat Arndt is a written version of her Sally Frank
Award Lecture given at the 2012 AABB meeting; it covers some
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
of the changes seen with DIIHA during the last 30 years. The
article by Gina Leger, Pat Arndt, and Dr. Garratty describes
in detail how Dr. Garratty’s laboratory investigates DIIHA;
there are differences in how laboratories perform testing for
DIIHA (e.g., gel vs. tube; untreated vs. enzyme-treated red
blood cells), which could affect the results. The article by Dr.
Abdulgabar Salama and Dr. Beate Mayer covers some of the
pitfalls and errors they have observed that occur both clinically
and in the laboratory that can result in misdiagnosis or lack
of serologic evaluation for DIIHA. Rounding out the druginduced cytopenias are two thorough reviews by Dr. Brian
Curtis on drug-induced immune thrombocytopenia (DIIT)
and drug-induced immune neutropenia (DIIN). Some of the
same drugs are found in association with DIIHA, DIIT, and
DIIN, but usually not in the same patients. Other similarities
can be seen among these three different types of drug-induced
immune cytopenias.
Unfortunately, Dr. Garratty passed away during the final
preparation of this issue. His reviews and other publications
on DIIHA have been a valuable source of information for
clinicians and laboratory scientists for almost 40 years. It is
fitting that this issue of Immunohematology is published as a
tribute to him.
Patricia A. (Pat) Arndt
Regina M. (Gina) Leger
Immunohematology Research Laboratory
American Red Cross Blood Services
Southern California Region
100 Red Cross Circle
Pomona, CA 91768
43
Report
Sally Frank Memorial Award and Lectureship*
Drug-induced immune hemolytic anemia:
the last 30 years of changes
P.A. Arndt
Drug-induced immune hemolytic anemia (DIIHA) is a rare
condition that occurs primarily as a result of drug-induced
antibodies, either drug-dependent or drug-independent. Drugdependent antibodies can be detected by testing drug-treated
red blood cells (RBCs) or untreated RBCs in the presence of
a solution of drug. Drug-independent antibodies react with
untreated RBCs (no drug added) and cannot be distinguished
from warm autoantibodies. Many changes have occurred during
the last 30 years, such as which drugs most commonly cause
DIIHA, the optimal testing methods for identifying them, and
the theories behind the mechanisms by which they react. This
article reviews the major changes in DIIHA since the early 1980s
involving the immune complex mechanism, cephalosporins,
nonimmunologic protein adsorption, and penicillins. Because
serologic results associated with DIIHA can mimic those
expected with autoimmune hemolytic anemia or hemolytic
transfusion reactions, DIIHA may go undetected in some cases.
Immunohematology 2014;30:44–54.
Key Words: immune hemolytic anemia, drugs, antibodies,
red blood cells, IgG, complement
It was a case of being at the right place at the right time.
Shortly after my graduation in 1983 from the Specialist in
Blood Banking (SBB) program at the American Red Cross in
Los Angeles, there was an opening for a research associate in
Dr. George Garratty’s research laboratory, and I was hired. Dr.
Garratty’s main research focus was the immune hemolytic
anemias. My coworkers in the research laboratory in the 1980s
were Nina Postoway and Sandy Nance. Sandy taught me how
to perform flow cytometric analyses on red blood cells (RBCs)
and the monocyte monolayer assay (MMA). Nina taught me
how to perform the enzyme-linked antiglobulin test for direct
antiglobulin test (DAT)-negative autoimmune hemolytic
anemia (AIHA) workups and also how to do drug-induced
immune hemolytic anemia (DIIHA) workups. Nina said to
expect that most, if not all, DIIHA workups would be negative.
She was right…at first…and then things started to change. I
worked in Dr. Garratty’s research laboratory at the Red Cross
in Southern California for a little over 30 years and during that
*Presented at the 2012 AABB Annual Meeting
44
time saw many changes relating to DIIHA. This presentation
will review some of those changes.
Background
The incidence of DIIHA is unknown, but Dr. Garratty
suggested that it may be about 1 in a million.1 Causes of DIIHA
can be classified into two broad categories: drug-induced
antibodies and nonimmunologic protein adsorption (NIPA).
Drug-induced antibodies can be further subdivided into drugdependent antibodies and drug-independent antibodies. The
first reported drug-dependent antibody was an antibody
to stibophen (an anthelmintic drug) in 1956,2 and the first
described drug-independent antibodies were autoantibodies
induced by methyldopa (an antihypertensive drug) in 1966.3,4
The first drug noted to cause NIPA was cephalothin (an
antibiotic); positive DATs were first reported in 1967,5,6 and
positive indirect antiglobulin tests (IATs), in 1971.7
The next few paragraphs summarize knowledge with
regard to DIIHA in the early 1980s, using the 1980 textbook
Acquired Immune Hemolytic Anemias by Petz and Garratty8 as
a reference. At that time, 32 drugs had been reported to cause
immune hemolytic anemia (IHA) or positive DATs. The Petz
and Garratty book8 described four mechanisms: (1) immune
complex, (2) drug adsorption, (3) NIPA, and (4) AIHA.
Immune Complex
This mechanism was said to occur when drugs and drug
antibodies (IgG or IgM) combined to form immune complexes,
which were then nonspecifically adsorbed onto RBCs and
activated complement. Most drugs that caused DIIHA reacted
by this mechanism: prototype drugs were quinidine (an
antiarrhythmic drug) and phenacetin (a nonsteroidal antiinflammatory drug [NSAID]). Typical characteristics of this
mechanism were (1) a previously sensitized patient needed
to take only a small amount of the drug; (2) as a result of
complement activation, patients had acute intravascular
hemolysis (hemoglobinemia and hemoglobinuria) and often
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
2012 Sally Frank Award Lecture: DIIHA
renal failure; (3) the DAT was positive, often with C3 only;
and (4) in vitro reactions (agglutination, hemolysis, and
sensitization) could only be demonstrated by incubating a
mixture of the patient’s serum, the drug, and test RBCs (the
“immune complex” method).
Drug Adsorption
This mechanism was used to describe the few drugs that
bound firmly to RBC membranes; antibodies to these drugs
reacted with the RBC-bound drug. Penicillin (an antibiotic) was
the prototype drug for this mechanism. Typical characteristics
of penicillin-induced DIIHA were (1) the patients had received
large doses of penicillin intravenously, e.g., 10 million units
daily for one or more weeks; (2) the extravascular hemolysis
developed over the course of 7–10 days, and hemolysis of
decreasing severity could persist for several weeks after the
penicillin was stopped; (3) the DAT was strongly positive for
IgG, and sometimes C3 was also present; and (4) in vitro tests
of the patient’s serum and eluate with drug-treated RBCs were
positive (serum IgG antibody titer usually ≥1000).
Nonimmunologic Protein Adsorption
This mechanism occurred when drugs modified the
RBC membrane so that plasma proteins were adsorbed
nonimmunologically. Cephalothin (trade name Keflin) was the
prototype drug for this mechanism. Characteristics of NIPA
included the following: (1) patients taking cephalothin had a
positive DAT (using antisera directed to albumin, complement,
fibrinogen, immunoglobulins, etc.) but no hemolytic anemia,
and (2) in vitro, cephalothin-treated RBCs incubated in
normal plasma were coated with numerous proteins (the IAT
was positive using various antisera as above), but those same
RBCs incubated with an eluate were typically nonreactive
because protein levels in the eluate were too low.
Autoimmune Hemolytic Anemia
This mechanism occurred when the drug induced
an autoantibody that reacted with normal RBCs (no drug
added), similar to reactivity seen with autoantibodies found
in idiopathic IgG warm AIHA. The prototype drug for this
mechanism was methyldopa (trade name Aldomet). The
characteristics of methyldopa-induced autoantibodies were
(1) 10 to 36 percent of patients developed a strongly positive
DAT (IgG ± C3) after taking methyldopa for 3 to 6 months
(this appeared to be dose-dependent), and the DAT could
take up to 2 years to become negative after methyldopa was
stopped; (2) about 0.8 percent of patients gradually developed
extravascular hemolytic anemia, which resolved after cessation
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
of methyldopa; and (3) the patient’s serum and eluate both
contained an autoantibody serologically indistinguishable
from other IgG warm autoantibodies. One proposed mechanism was that methyldopa caused an alteration of the
immune system.9 Proof that methyldopa caused AIHA came
only from clinical observations.
Changes Since the Early 1980s
The Research Laboratory
In 1990, Nina and Sandy left the research laboratory to
pursue other transfusion medicine careers, and in 1993, Gina
Leger joined our laboratory from the Immunohematology
Reference Laboratory. It was my turn to teach her most of the
standard tests, and by then, DIIHA workups had become very
interesting. So she, too, was in the right place at the right time.
The Immune Complex Mechanism
The immune complex mechanism was proposed in the
1950s to 1960s to explain drug-induced thrombocytopenia
(i.e., drug antibodies bind with the drug and then the drug–
antibody complex is adsorbed nonspecifically by platelets) and
was then later extended to apply to RBCs.10,11 Criticisms of this
mechanism started appearing in the 1970s to 1980s. Some
findings that do not support the immune complex mechanism
include the following: (1) drug–antibody immune complexes
(e.g., RBC-bound or in plasma) have never been demonstrated
experimentally; (2) some drug-dependent antibodies bind to
their target cells by their Fab domain, e.g., quinidine antibodies
with platelets12,13 and tolmetin antibodies with RBCs14; if the
binding was nonspecific, one would expect binding through
both the Fab and the Fc domains; (3) drug-dependent
antibodies appear to be highly specific for certain cell lines,
e.g., either platelets or RBCs (this would not be expected if
immune complexes were attaching nonspecifically to cells); (4)
some drug-dependent antibodies react with specific antigens
on target cells, e.g., anti-quinidine with glycoprotein Ib/IX or
IIb/IIIa on platelets15 (see Table 1 for reported specificities of
drug-dependent antibodies reactive with RBCs); and (5) many
patients have been described with antibodies reacting by more
than one mechanism, e.g., drug-independent autoantibodies
plus drug-dependent antibodies reacting by the drug
adsorption or immune complex method (reviewed in Petz and
Garratty36 and in Garratty37).
Some alternative hypotheses to the immune complex
mechanism have been proposed to explain observations with
drug-induced antibodies. Ackroyd38 proposed that for one type
of drug-induced thrombocytopenia, the drug and cell formed
45
P.A. Arndt
Table 1. Reported blood group specificities of drug antibodies;
specificities varied from simple (e.g., anti-e) to broad (e.g., antibody
reactive with all cells except null cells)
Blood group
Drugs (and earliest reference to note specificity)
Rh
catechins, diclofenac,17 glafenine,16 hydrochlorothiazide,18
ibuprofen,19 moxalactam,16 nomifensine,20 piperacillin,21
quinine,22 rifampin,23 streptomycin,24 sulindac,25 teicoplanin,26
tolmetin27
I
dexachlorpheniramine maleate,28 fluorouracil,29 metrizoic
acid,30 nitrofurantoin,28 nomifensine,31 rifampin,28 thiopental32
Lutheran
elliptinium,16 rifampin,16 piperacillin,33 sulfamethoxazole33
P
elliptinium,16 meglumine iothalamate,16 nomifensine31
Kell
glafenine,16 trimethoprim34
MNS
streptomycin, sulfamethoxazole
Kidd
chlorpropamide35
H
sulfamethoxazole34
16
24
33
a loose complex, and the antibody was specific for only the
combined drug–cell antigen. This drug–cell complex has been
referred to as a “neoantigen.” Another type of neoantigen could
occur if a drug induced a chemical change to a membrane to
create a new antigen (changed membrane only).39–41 To explain
why some patients with DIIHA have antibodies working
by more than one mechanism, e.g., the immune complex
method in addition to autoantibodies, Habibi42 suggested
a single mechanism whereby the formation of a drug–cell
conjugate initiates the immune response. Mueller-Eckhardt
and Salama39 proposed a unifying hypothesis similar to the
proposals of Ackroyd38 and Habibi.42 They proposed that
the drug (or drug metabolite) first interacts with the cell
membrane; this drug–membrane structure then provokes
the production of two types of antibodies: drug-dependent or
drug-independent antibodies.39 Figure 1 shows how it might
be possible to have antibodies to (1) drug alone, (2) drug plus
membrane (antibody reacts with both components), (3) drug
plus membrane (antibody reacts mainly with the membrane),
or (4) combinations of the first three.37 Additionally, it has
been proposed that a drug associated with immune cytopenia
may react with the antibody first, modifying the antibody to
improve its fit with a membrane epitope.40,43
Conclusion
The immune complex mechanism is used as a hypothesis
to explain the reactions seen with certain drugs in vivo and
in vitro. This theory still explains the serologic and clinical
findings (e.g., RBC-bound complement, intravascular hemolysis, and renal failure), but there has been no experimental
evidence to prove the presence of immune complexes and, as
46
Fig. 1 A drawing to represent the proposed unifying hypothesis
of drug-induced antibody reactions,37 based on suggestions by
Habibi42 and Mueller-Eckhardt and Salama.39 Drug binds to a cell
membrane (loosely or firmly), and drug-induced antibodies could
react with (1) drug only, (2) drug + membrane, (3) mainly membrane,
or (4) a combination of the first three. Antibodies to drug only are
characteristic of the drug adsorption mechanism (e.g., penicillin).
Antibodies to drug + membrane produce in vitro reactions typical
of the immune complex mechanism (e.g., quinidine). Antibodies to
mostly membrane result in an autoantibody type of reaction typically
found in conjunction with a drug-dependent antibody.
shown above, there is evidence to dispute this mechanism.
When describing this mechanism, this uncertainty can
be indicated by adding quotes around the words “immune
complex” or by using the phrase “so-called immune complex
mechanism.” It is more accurate to describe the method used to
detect these antibodies as testing in the presence of a solution
of drug instead of the immune complex method.
Cephalosporins
The cephalosporins are a class of ß-lactam antibiotics
grouped into generations based on their antimicrobial properties. In the 1970s to early 1980s, DIIHA was described only
a handful of times as being associated with first and second
cephalosporins (cephalothin, cefazolin, and cefamandole;
reviewed in Garratty44). These cephalosporins, like penicillin
(another ß-lactam antibiotic), formed covalent bonds with RBC
membranes; thus, drug-treated RBCs could be prepared in the
laboratory. The characteristics of these early cephalosporin
DIIHA cases were (1) the DAT was positive for IgG, sometimes
also C3; (2) the serum and RBC eluate reacted with drugtreated RBCs; (3) preincubation of the cephalosporin with
the antibody caused inhibition of reactivity with drug-treated
RBCs; and (4) in vivo hemolysis was extravascular. Thus, up to
the mid-1980s there was a general assumption that antibodies
to cephalosporins reacted by the drug adsorption mechanism,
and the best way to detect them was with drug-treated RBCs.
Then things started to change.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
2012 Sally Frank Award Lecture: DIIHA
Cefotaxime
In 1987, Salama et al.45 described a patient with immune
complex–mediated intravascular hemolysis caused by an
antibody to cefotaxime (a third-generation cephalosporin).
The DAT was positive for C3 only, the serum was nonreactive
with drug-treated RBCs but did react by the immune complex
method (serum plus RBCs in the presence of a solution of
drug), and preincubation of drug with the antibody did not
cause inhibition of its reactivity.
Ceftriaxone
A year later, our laboratory reported, in an abstract at
the 1988 AABB meeting, a fatal case of DIIHA attributable to
ceftriaxone (another third-generation cephalosporin).46 The
clinical and serologic characteristics were those associated
with the immune complex mechanism. The DAT was positive
for C3 only, and the serum reacted only by the immune complex
method (serum plus RBCs in the presence of a solution of
drug); drug-treated RBCs were nonreactive.
the absence of the in vitro drug was primarily detected in
patients with DIIHA caused by cefotetan but was found with
some other cephalosporins as well. Our current approach
for detection of drug-dependent antibodies of any kind is as
follows: (1) if the drug antibody has been well studied, use
the previously described method(s) for detection, or (2) if the
drug has not been previously described or well studied, test
by both methods: (a) serum and RBC eluate with drug-treated
RBCs (and untreated RBCs) and (b) serum with untreated or
enzyme-treated RBCs in the presence of soluble drug (or saline
as a dilution control).47
Table 2. Reasons for serum to be reactive in drug-dependent
antibody tests when drug has not been added in vitro
(e.g., controls)
Alloantibody
Autoantibody, two types
1. Autoantibody alone
a. Idiopathic
Cefotetan
In 1989, there was one AABB abstract and, in 1990, there
were four AABB abstracts describing DIIHA (one fatality)
attributable to a second-generation cephalosporin, cefotetan
(reviewed by Garratty44). These antibodies reacted by more
than one method: all five reacted with cefotetan-treated RBCs,
three of four reacted in the presence of a solution of cefotetan,
and two of four reacted with untreated RBCs (no cefotetan
added). Table 2 lists reasons for reactivity of serum in tests
when the drug was not added in vitro. In cases of DIIHA
caused by cefotetan, we believe this reactivity with untreated
RBCs is a result of drug-independent autoantibodies that
are found in combination with drug-dependent antibodies
(possibly represented by the “mainly membrane” epitope in
Fig. 1).
Thus, it was clear by the late 1980s that the best way to
detect cephalosporin antibodies was not always by testing
drug-treated RBCs. Since then, there have been numerous
reports of DIIHA caused by different cephalosporins.
Table 3 summarizes how these cephalosporin antibodies
were detected. Antibodies to first- and second-generation
cephalosporins were primarily detected using drug-treated
RBCs with the exception of cefotetan-dependent antibodies,
which were detected equally by two methods: drug-treated
RBCs and in the presence of a solution of drug. Antibodies
to third-generation cephalosporins were primarily detected
by testing in the presence of a solution of drug (ceftriaxone
antibodies are only detected by this method). Reactivity in
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
b. Drug-independent antibody (e.g., methyldopa or fludarabine)
2. Autoantibody found in the presence of drug-dependent antibody
Circulating drug or drug plus antibody immune complexes present in the
blood sample
Presence of drug in reagent (e.g., low-ionic-strength saline [LISS])
Table 3. Drug-induced immune hemolytic anemia caused by
cephalosporins: summary of methods used and results (number
positive/number tested) in references in which data on methods
were given
Reference
Drug-treated
RBCs
Serum + RBCs
+ drug
No in vitro
drug
Cephalothin
5
5/5
1/1
0/5
Cefazolin
2
2/2
0/1
0/2
Cephalexin
1
1/1
0/0
?
Cefoxitin
4
4/4
1/2
0/3
Cefamandole
1
1/1
0/0
0/1
Cefotetan
31
31/31
20/22
12/25
Cefuroxime
2
2/2
0/2
0/2
Cefotaxime
4
3/4
3/4
1/4
Cephalosporin
First-generation
Second-generation
Third-generation
Ceftazidime
4
2/4
3/4
0/4
Ceftriaxone
28
0/12
28/28
5/19
Ceftizoxime
4
2/4
3/4
2/4
Cefixime
1
1/1
1/1
0/1
RBCs = red blood cells.
47
P.A. Arndt
Case Studies
In the 1970s, the most commonly seen drug-induced
antibodies in Dr. Garratty’s laboratory were caused by
methyldopa and penicillin (Table 4). This scenario changed
in the 1980s, and for the last several decades, cephalosporins,
particularly cefotetan and ceftriaxone, have been the most
common causes of DIIHA.48 Two cases illustrate common
features of DIIHA caused by ceftriaxone or cefotetan.
Table 4. Causes of drug-induced immune hemolytic anemia seen
in Dr. Garratty’s laboratories during two different 10-year periods
Number (percentage) of cases
Drug
1969–78
Methyldopa
29 (67%)
Penicillin
10 (23%)
2003–12
Ceftriaxone
17 (19%)
Cefotetan
24 (28%)
Piperacillin
32 (37%)
Platinum-based chemotherapies
Others
Total
6 (7%)
4 (9%)
8 (9%)
43
87
Patient 1 was a 10-year-old boy with juvenile idiopathic
arthritis who had previously received ceftriaxone. He was
admitted to the emergency room with fever and vomiting. At
1409 hours, his hemoglobin (Hb) was 10.9 g/dL. Ceftriaxone
(tradename Rocephin) was started at 1542 hours. At 1615
hours, his blood pressure dropped and a change in consciousness
was noted; therefore, the ceftriaxone was stopped, but it was
then restarted at 1745 hours. At 1825 hours, his Hb had
plummeted to 0.5 g/dL; transfusions were unsuccessfully
attempted, and he was pronounced dead at 1834 hours. Postmortem samples were sent to our laboratory to determine
whether the patient had fatal DIIHA caused by ceftriaxone.
RBC-bound IgM and C3 were detected on the patient’s RBCs
(no IgG or IgA). Ceftriaxone-treated RBCs cannot be prepared
in vitro (we and others have tried several methods without
success), so the only way to test for ceftriaxone antibodies is in
the presence of a solution of ceftriaxone. The patient’s serum
plus ceftriaxone caused direct agglutination (2+) of untreated
RBCs; the phosphate-buffered saline (PBS) dilution control
was nonreactive. The patient’s serum plus ceftriaxone caused
strong agglutination (4+) of ficin-treated RBCs; when fresh
normal serum as a source of complement was added, there
was also hemolysis and the antiglobulin test was positive
(3+), presumably because of the anti-C3 component of the
polyspecific antiglobulin sera. The patient’s serum plus PBS also
48
caused agglutination (2+) of ficin-treated RBCs. This reactivity
without adding the drug in vitro was presumably because of
the presence of ceftriaxone in the patient’s circulation at the
time that the blood sample was drawn but could also have
been the result of autoantibody. Two ways to distinguish an
autoantibody (which should be persistent) from circulating
drug (which would be transient; the time it takes for a drug to
clear the circulation depends on the half-life of the drug and
the patient’s renal status) are to (1) obtain a new blood sample
from the patient after the drug has cleared (e.g., a few days after
the drug is stopped); an autoantibody would be expected to
still react but a drug antibody should no longer react; and (2)
dialyze the sample to remove the drug.47,49
Thus, Patient 1 had fatal DIIHA caused by ceftriaxone.
Although DIIHA caused by ceftriaxone occurs in adults, it is
usually more acute and severe in children. Often in children,
the time from receipt of drug to reaction is 1 hour or less
and the nadir Hb is 5 g/dL or less. Twelve of 18 reported
ceftriaxone-induced fatalities were in children. Typically,
patients with DIIHA caused by ceftriaxone have a history
of previous ceftriaxone therapy and signs of intravascular
hemolysis (hemoglobinemia, hemoglobinuria, and renal
failure). One study performed to determine the prevalence of
ceftriaxone antibodies in 64 pediatric patients with HIV or
sickle cell disease detected antibodies in 8 patients (12.5%); 1
patient had fatal DIIHA minutes after receiving ceftriaxone.50
Serologic results in DIIHA caused by ceftriaxone include
the following: (1) positive DAT (C3, sometimes C3 plus IgG)
and (2) drug-dependent antibody (primarily IgM) that can
only be detected in the presence of ceftriaxone (reactions
include agglutination, hemolysis, and positive antiglobulin
test). About two-thirds of the patients’ samples we have tested
reacted without the addition of in vitro ceftriaxone; this was
most likely because of the presence of circulating drug in the
blood samples.51 There have been three reports of DIIHA
caused by ceftriaxone in which antibodies were only detected
by a gel method using ex vivo samples (e.g., urine from people
receiving ceftriaxone).52–54
Patient 2 had cefotetan-induced IHA; details of this case
were previously reported in Immunohematology.55 In brief, a
woman received two doses of cefotetan (trade name Cefotan)
at the time of a cesarean section. Five days later, she had
signs of IHA (Hb = 8.7 g/dL, reticulocytes = 10.5%, lactate
dehydrogenase [LDH] = 503 U/L, haptoglobin <6 mg/dL) and
a positive DAT (IgG and C3). Her serum contained a hightiter drug-dependent antibody that reacted with cefotetantreated RBCs (hemolysin titer = 128, agglutination titer =
1024, and IAT titer = 128,000) and a weak autoantibody
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
2012 Sally Frank Award Lecture: DIIHA
that reacted with untreated RBCs (IAT titer = 1). An eluate
prepared from her RBCs reacted with cefotetan-treated RBCs
(3+ IAT); the last wash control was weakly reactive (1+ IAT),
which is not an unusual finding in cefotetan-induced IHA.56
The serum antibody also reacted when tests were performed
in the presence of a solution of cefotetan (1 mg/mL) but to a
much lower titer (IAT titer vs. untreated RBCs = 512) than
with cefotetan-treated RBCs (prepared using cefotetan at
a concentration of 40 mg/mL). It is possible that cefotetan
antibodies are reacting with weakly cefotetan-coated RBCs
and not by the immune complex mechanism when tested in
the presence of soluble cefotetan.56 This woman had received
cefotetan 3 years earlier at the time of another cesarean section
and at that time had signs of IHA after 13 days (Hb = 5 g/dL,
reticulocytes = 15%, haptoglobin <6 mg/dL). Serum drawn
just before the current surgery demonstrated preexisting anticefotetan (IAT titer vs. cefotetan-treated RBCs = 512). Thus, it
was likely that she had two episodes of cefotetan-induced IHA:
a primary response 3 years earlier (which was not recognized
at the time) and then a later secondary response.
Cefotetan is commonly used prophylactically with surgery,
e.g., cesarean sections. The hemolytic anemia becomes
apparent 1–2 weeks after receipt of the drug, and the patients
return to the emergency room where the differential diagnosis
includes sepsis and they are sometimes given more cefotetan
with disastrous results.57,58 Only one dose of cefotetan can
result in dramatic hemolysis; this could be explained by prior
environmental exposure (e.g., in food or water), which primes
the immune system.59 The hemolytic anemia sometimes lasts
longer than expected; this could be because cefotetan has been
shown to remain RBC-bound for a median of 67.5 days.60
In cefotetan-induced IHA, the Hb drops to a mean of about
5 g/dL, signs of intravascular hemolysis are often present
(hemoglobinemia, hemoglobinuria, and renal failure), and
fatalities occur.61 Patients with anti-cefotetan should be warned
to never receive cefotetan again. Can these patients receive
other cephalosporins? Serologic studies of anti-cefotetan with
other cephalosporins showed little in vitro cross-reactivity,
but there are no data to determine whether these in vitro data
relate to in vivo reactivity.62
Serologic results in cefotetan-induced IHA include the
following: (1) the DAT is positive (usually both IgG and C3 are
present); (2) serum contains a drug-dependent antibody that
may react to very high titers with, and completely hemolyze,
cefotetan-treated RBCs; (3) the serum drug-dependent
antibody also reacts with untreated RBCs in the presence
of a solution of cefotetan; (4) serum may also contain a lowtiter drug-independent autoantibody (enhanced by sensitive
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
methods, e.g., ficin, polyethylene glycol [PEG]); and (5) an
eluate reacts strongly with cefotetan-treated RBCs; the last
wash control is often reactive as well.63 Some other issues
that arise when testing cefotetan-treated RBCs are that (1)
cefotetan causes NIPA, which can lead to positive IATs when
testing undiluted serum, and (2) about 80 percent of normal
sera contain low-titer IgM anti-cefotetan agglutinins. Testing
patients’ and control sera at a 1/100 dilution should overcome
both of these problems when testing cefotetan-treated RBCs47;
patients with DIIHA caused by cefotetan have very high titer
antibodies (median IAT titer = 16,00056) that would not be
missed by testing serum at a 1/100 dilution.
Conclusion
In the early 1980s, it was generally well accepted that
cephalosporin antibodies, like penicillin antibodies, would
be expected to cause extravascular hemolysis in vivo and
that cephalosporin antibodies would be detected using drugtreated RBCs. Today we know that cephalosporin antibodies
can be associated with quite severe (e.g., fatal) intravascular
hemolysis in vivo and that these antibodies may react with
drug-treated RBCs, or in the presence of a solution of drug, or
by both methods.
Nonimmunologic Protein Adsorption
In the 1960s to 1980s, NIPA was described with
cephalosporins, diglycoaldehyde, suramin, and cisplatin.
There were no recognized cases of hemolytic anemia; thus,
the assumption was that NIPA only caused positive DATs
and IATs, not decreased RBC survival. Then things started to
change.
ß-L actamase Inhibitors
In 1985, clavulanic acid was reported to cause positive
DATs (45% of 23 patients) as a result of NIPA.64 In 1992, a
high incidence of positive DATs (39%) was reported in
59 patients taking Unasyn (a combination of ampicillin
and sulbactam).65 None of the patients in either of these
reports showed signs of hemolytic anemia. Clavulanic acid
(clavulanate) and sulbactam are both ß-lactamase inhibitors
used in combination with ß-lactam antibiotics (e.g., ampicillin
and ticarcillin). In 1998, our laboratory reported that both
sulbactam and clavulanate caused NIPA.66 In this same report,
we also described 3 patients who had temporal relationships
of drug administration (after receipt of either Unasyn, which
contains sulbactam, or Timentin, which contains clavulanate)
with positive DATs and hemolytic anemia; no drug-dependent
antibodies were detected. In 2000, we showed that sulbactam49
P.A. Arndt
and clavulanate-treated RBCs, after incubation with normal
plasma, showed increased reactivity with monocytes in an
MMA,67 an in vitro method used to predict decreased RBC
survival in vivo.68 A third ß-lactam antibiotic, tazobactam,
has also been associated with NIPA,69 positive MMAs,67 and
hemolytic anemia, possibly as a result of NIPA in a patient
with high plasma IgG level.70
Conclusion
In the early 1980s, NIPA was thought to be a cause of
positive DATs and IATs but not hemolytic anemia. Today,
there is in vitro and in vivo evidence that NIPA may also be
the cause of hemolytic anemia.
Penicillins
Numerous cases of DIIHA as a result of the ß-lactam
antibiotic penicillin have been reported since the 1960s.
Characteristics included the following: (1) the DAT was
positive for IgG with or without C3, (2) the patient’s serum and
an eluate contained an IgG anti-penicillin that reacted with
penicillin-treated RBCs (serum titer ≥1000), (3) incubation of
the anti-penicillin with soluble penicillin inhibited reactivity
with penicillin-coated RBCs, and (4) the hemolysis was
extravascular in vivo. These characteristics also applied
to other drugs in the penicillin family (e.g., ampicillin,
amoxicillin, nafcillin, and ticarcillin). Thus, it was assumed
that the best way to detect antibodies to penicillins was to test
drug-treated RBCs. Because some people without hemolytic
anemia had low-titer IgM penicillin antibodies in their serum,
it was important when diagnosing DIIHA caused by penicillin
to demonstrate high-titer IgG anti-penicillin in the patient’s
serum or anti-penicillin in an eluate from the patient’s RBCs.
But then in the 1990s things started to change.
Piperacillin
In 1994, Johnson et al.21 reported the first serologically
well-documented case of DIIHA caused by piperacillin, a
semisynthetic penicillin. The DAT was positive (IgG and C3),
the serum reacted with both piperacillin-treated RBCs and
with untreated RBCs in the presence of soluble piperacillin, and
an RBC eluate was nonreactive by both methods. In addition,
the piperacillin antibody had relative anti-e specificity. In
2002, our laboratory reported two patients with DIIHA caused
by piperacillin; one of the patients’ samples (serum and eluate)
reacted only in the presence of soluble piperacillin (piperacillintreated RBCs were nonreactive).71
In 2008, our laboratory reported that the best way to
detect piperacillin antibodies was by the immune complex
50
method; this differed from the best way to detect antibodies
to other penicillins, which was by testing drug-treated RBCs.72
The immune complex method was recommended because
the sera of 91 percent of 100 normal donors and 49 percent of
35 random patients directly agglutinated piperacillin-treated
RBCs; two donors’ plasma reacted weakly by antiglobulin test
only. Samples from donors were nonreactive when testing
ficin-treated RBCs in the presence of piperacillin (the immune
complex method). Antibodies to piperacillin found in donors
were determined to be IgM, and reactivity with piperacillintreated RBCs could be inhibited by preincubation with
piperacillin or penicillin. In contrast, four patients with DIIHA
caused by piperacillin had piperacillin antibodies reactive
by the immune complex method that were not inhibited by
preincubation with solutions of piperacillin.72
In addition to piperacillin-treated RBCs, we have found
sera from donors and random patients to directly agglutinate
some other drug-treated RBCs; Table 5 shows data from our
laboratory. It is interesting to ponder why this occurs. We
know that in the United States, our food sources are exposed
to drugs (e.g., antibiotics added to cattle feed). Small amounts
of drugs can also be detected in tap water because drugs may
be discarded down the drain or sewer or excreted in patients’
urine and unfortunately water treatment does not remove drug
residue. There is also an increasing environmental presence
of some drugs (e.g., platinum) in the air, soil, and water as a
byproduct of automobile catalytic converters.73 There may be
other environmental sources. Thus, we may all be exposed to
drugs for decades without knowing it. The presence of drug
antibodies in normal donors and random patients is one
reason why it is important to test normal sera as a control
when testing drug-treated RBCs.72,73
Table 5. Percentage of agglutinins reacting with drug-treated red
blood cells found in our laboratory when screening blood donors’
and random patients’ sera*
Drug
Blood donors
Random patients
5%
6%
Cephalothin59
39%
NT
Ticarcillin
33%
NT
Penicillin (unpublished results)
66
Cefotetan (and unpublished results)
80%
78%
Piperacillin72
91%
49%
Oxaliplatin73
16%
4%
Cisplatin
7%
NT
93%
60%
59
73
Meropenem
74
*It is interesting to note that, for some drugs, fewer patients than donors
have antibodies.
NT = not tested.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
2012 Sally Frank Award Lecture: DIIHA
Thus, because many normal donors and random patients
may react with piperacillin-treated RBCs, which can confuse
the interpretation of a DIIHA workup, testing with piperacillintreated RBCs is not the best way to detect piperacillin
antibodies. Luckily, piperacillin antibodies found in patients
with DIIHA also react by the immune complex method (serum
plus RBCs in the presence of a solution of drug), making this
the preferred method of testing. Table 6 lists the different
penicillins associated with DIIHA and how the antibodies
were detected. Piperacillin was the only one with reactivity by
all three methods; the reactivity seen when no in vitro drug
was added was attributable to the presence of the circulating
drug at the time the patient’s sample was collected.
Table 6. Drug-induced immune hemolytic anemia caused by
penicillins: summary of methods used and results (number positive/
number tested) in reviewed references in which data on methods
were given
Reference
Drug-treated
RBCs
Serum + RBCs
+ drug
No in vitro
drug
Penicillin
15
15/15
1/1
0/10
Ampicillin
4
4/4
0/1
0/4
Nafcillin
3
3/3
0/1
0/3
Ticarcillin
2
2/2
0/0
1/2
Amoxicillin
2
2/2
0/0
0/2
Piperacillin
18
4/5
17/17
11/15
Drug
RBCs = red blood cells.
Case Study
In the 1990s, cefotetan was the single most common drug
seen by our laboratory to cause DIIHA.48 In the last decade,
this has changed so that now piperacillin is the single most
common drug to cause DIIHA seen in our laboratory (Table 4).
The following case illustrates some features of DIIHA caused
by piperacillin.
Patient 3 had cystic fibrosis and had undergone bilateral
lung transplantations. Laboratory data indicated that he
was exhibiting hemolysis—the Hb dropped from 11.9 to 6.4
g/dL, the LDH was 1014 U/L, and the total bilirubin was
elevated. The patient’s DAT was positive (3+ IgG only); his
serum was reactive with all cells tested by saline, low-ionicstrength saline (LISS), PEG, and gel IAT methods (1–3+); and
an eluate from his RBCs reacted 2–3+ by PEG-IAT with all
RBCs, including the patient’s ethylenediaminetetraacetic acid
(EDTA) glycine acid–treated RBCs. Adsorptions removed
the serum antibody, and no underlying alloantibodies were
detected. Thus, serologically it appeared that the patient
had a warm autoantibody but the patient’s physician did not
think that he had warm AIHA (the hemolytic anemia was too
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
severe). The patient had been started on Zosyn (a combination
of piperacillin and tazobactam) 2 days before the start of
hemolysis and the positive serology. Thus, the Zosyn was
stopped, the patient was transfused with 4 units of RBCs, and
samples were sent to our laboratory for a DIIHA workup.
The two components of Zosyn are piperacillin, a ß-lactam
antibiotic, and tazobactam, a ß-lactamase inhibitor. We
believe that when a drug consists of multiple components,
each component should be tested separately if possible to
avoid misinterpretations.47 Piperacillin and tazobactam can
both be obtained commercially (Sigma Chemical Co., St.
Louis, MO). Numerous cases of DIIHA caused by piperacillin
antibodies have been reported, but there have been no reports
of antibodies to tazobactam. Tazobactam does cause NIPA,69,70
but there are no commonly available serologic tests to prove
NIPA as a cause of DIIHA. Thus, in the laboratory, we usually
only test for antibodies to piperacillin. Piperacillin-treated
RBCs can be prepared using a high-pH buffer,72 but we do
not use them when testing patients’ sera for the presence of
piperacillin antibodies because (1) plasma from donors and
random patients without IHA agglutinate piperacillin-treated
RBCs,72 (2) piperacillin antibodies in patients with DIIHA do
not have high titers so testing a dilution (as is done in cefotetan
antibody workups) will not help (unpublished results), and (3)
sera from some patients with DIIHA caused by piperacillin
are nonreactive with piperacillin-treated RBCs.71 Thus, our
preferred method for testing sera for piperacillin antibodies in
patients with IHA is to test RBCs in the presence of a solution
of piperacillin. Patient 3’s serum reacted weakly (1+ IAT) with
untreated RBCs in the presence of piperacillin; the PBS control
was negative. The patient’s serum caused strong agglutination
(3+) with ficin-treated RBCs in the presence of piperacillin; the
IAT was also positive (3+). The control of the patient’s serum
(obtained 1 day after the Zosyn was stopped) plus PBS also
reacted weakly (1+ IAT) with ficin-treated RBCs, presumably
because of the presence of the circulating drug. No in vitro
hemolysis was observed, even in tests with fresh normal
serum added.
Thus, Patient 3, with cystic fibrosis, had DIIHA caused
by piperacillin. There are several other reports of piperacillininduced IHA in patients with cystic fibrosis who receive
antibiotics for lung infections.75 This patient’s initial serology
was consistent with warm AIHA, but the clinical picture did not
fit (the hemolysis was too severe). Some patients with DIIHA
caused by piperacillin have been noted to have intravascular
hemolysis, and some fatalities have been reported. Most
patients with DIIHA caused by piperacillin have received
the combination of piperacillin plus tazobactam (tradename
51
P.A. Arndt
Zosyn in the United States). Typical expected serologic results
in DIIHA are a positive DAT with a nonreactive eluate, but this
patient had a strongly reactive eluate. This has been observed
in some other cases of DIIHA caused by piperacillin and
with other drugs (e.g., cefotetan),56 usually when the drug is
present in the patient’s circulation. Several days after the drug
is stopped, the apparent autoantibody in the serum and in an
RBC eluate should no longer be detectable.
Serologic results in DIIHA caused by piperacillin include
(1) a positive DAT, usually with both anti-IgG and anti-C3, and
(2) reactivity of serum plus RBCs in the presence of soluble
piperacillin (agglutination and positive IAT). Seventy percent
of the patients we studied with DIIHA caused by piperacillin
presented with apparent warm autoantibodies (some with
autoanti-e specificity)76; these were most likely piperacillin
antibodies reacting with the circulating drug present in the
patients’ sera. This result has been reported by others.77
Conclusion
Up through the 1980s, it was assumed that antibodies to
the penicillins could only be detected by testing drug-treated
RBCs and that these patients had primarily extravascular
hemolysis. Penicillin antibodies in sera had high titers (≥1000),
and RBC eluates reacted with penicillin-treated RBCs.
Now we know that antibodies to at least one semisynthetic
penicillin, piperacillin, react with both drug-treated RBCs and
in the presence of soluble drug. In patients we studied with
DIIHA caused by piperacillin, serum antibodies had low to
moderate titers (50% of titers were less than 20; unpublished
results), and eluates were often nonreactive when tested with
piperacillin-treated RBCs.76 Anti-piperacillin in sera from
patients we studied with DIIHA caused by piperacillin also
had low titers when tested in the presence of a solution of
piperacillin (median titers were 4 with untreated RBCs and 32
with ficin-treated RBCs; unpublished results); 50 percent of
eluates reacted in the presence of piperacillin.76
Closing Remarks
In closing, it is important to remember that DIIHA is rare.
Millions of doses of these drugs are given to patients without
IHA occurring. Drug-dependent antibodies, when they are
present, may react by multiple methods: (1) drug-treated
RBCs, (2) untreated or enzyme-treated RBCs in the presence
of a solution of drug, or (3) untreated RBCs (no drug added
in vitro). NIPA may be a cause of DIIHA; the only proof is
temporal (e.g., if a patient redevelops hemolytic anemia after
rechallenge with the drug). Finally, DIIHA may be confused
52
with AIHA49 (e.g., see Patient 3) or hemolytic transfusion
reactions (e.g., if a patient receives drug and transfusion
around the same time) and thus we are most likely missing
some cases (e.g., see Patient 2).
Acknowledgments
I would like to acknowledge and thank the blood bankers
who encouraged or inspired me to learn more about blood
banking and attend an SBB program: LaVonna Hasz, Ron
Simpson, Ira Shulman, and Claire McGrath. I would also
like to acknowledge and thank my coworkers in the Research
Laboratory: Nina Postoway, Sandy Nance, Gina Leger, and of
course, my boss and mentor for over 30 years, George Garratty.
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25.DeCoteau J, Reis MD, Pinkerton PH, Ward J, Coovadia
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26. Colluccio E, Villa MA, Villa E, et al. Immune hemolytic anemia
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27. Weitekamp LA, Johnson ST, Fueger JT, Clark JR, Aster RH.
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28. Duran-Suarez JR, Martin-Vega C, Argelagues E, Massuet L,
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29.Sandvei P, Nordhagen R, Michaelsen TE, Wolthuis K.
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33.Arndt PA, Revilla J, Leger RM, Garratty G. Studies on
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solution. Transfusion 2012;52:844–8.
35. Sosler SD, Behzad O, Garratty G, Lee CL, Postoway N, Khomo
O. Acute hemolytic anemia associated with a chlorpropamideinduced apparent auto anti-Jka. Transfusion 1984;24:206–9.
36.Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed.
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37. Garratty G. Immune hemolytic anemia associated with drug
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emphasis on the role of drug metabolites. Transfus Med Rev
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40.Shulman NR, Reid DM. Mechanisms of drug-induced
immunologically mediated cytopenias. Transfus Med Rev
1993;7:215–29.
41.Aster RH. Drug-induced immune thrombocytopenia: an
overview of pathogenesis. Semin Hematol 1999;36(Suppl 1):
2–6.
42.Habibi B. Drug induced red blood cell autoantibodies codeveloped with drug specific antibodies causing haemolytic
anaemias. Br J Haematol 1985;61:139–43.
43.Bougie DW, Wilker PR, Aster RH. Patients with quinineinduced immune thrombocytopenia have both “drugdependent” and “drug-specific” antibodies. Blood 2006;108:
922–7.
44.Garratty G. Immune cytopenia associated with antibiotics.
Transfus Med Rev 1993;7:255–67.
45.Salama A, Göttsche B, Schleiffer T, Mueller-Eckhardt C.
‘Immune complex’ mediated intravascular hemolysis due to
IgM cephalosporin-dependent antibody. Transfusion 1987;27:
460–3.
46.Garratty G, Postoway N, Schwellenbach J, McMahill PC. A
fatal case of ceftriaxone (Rocephin)-induced hemolytic anemia
associated with intravascular immune hemolysis. Transfusion
1991;31:176–9.
47. Leger RM, Arndt PA, Garratty G. How we investigate druginduced immune hemolytic anemia. Immunohematology
2014;30:85–94.
48. Arndt PA, Garratty G. The changing spectrum of drug-induced
immune hemolytic anemia. Semin Hematol 2005;42:137–44.
49. Johnson ST, Fueger JT, Gottschall JL. One center’s experience:
the serology and drugs associated with drug-induced immune
53
P.A. Arndt
hemolytic anemia—a new paradigm. Transfusion 2007;47:
697–702.
50. Quillen K, Lane C, Hu E, Pelton S, Bateman S. Prevalence of
ceftriaxone-induced red blood cell antibodies in pediatric
patients with sickle cell disease and human immunodeficiency
virus infection. Pediatr Infect Dis J 2008;27:357–8.
51.Arndt PA, Leger RM, Garratty G. Serologic characteristics
of ceftriaxone antibodies in 25 patients with drug-induced
immune hemolytic anemia. Transfusion 2012;52:602–12.
52.Meyer O, Hackstein H, Hoppe B, Göbel FJ, Bein G, Salama
A. Fatal immune haemolysis due to a degradation product of
ceftriaxone. Br J Haematol 1999;105:1084–5.
53. Seltsam A, Salama A. Ceftriaxone-induced immune haemolysis: two case reports and a concise review of the literature.
Intensive Care Med 2000;26:1390–4.
54.Kim S, Song KS, Kim HO, Lee HM. Ceftriaxone induced
immune hemolytic anemia: detection of drug-dependent
antibody by ex-vivo antigen in urine. Yonsei Med J 2002;43:
391–4.
55.Arndt PA. Practical aspects of investigating drug-induced
immune hemolytic anemia due to cefotetan or ceftriaxone—a
case study approach. Immunohematology 2002;18:27–32.
56.Arndt PA, Leger RM, Garratty G. Serology of antibodies to
second- and third-generation cephalosporins associated with
immune hemolytic anemia and/or positive direct antiglobulin
tests. Transfusion 1999;39:1239–46.
57. Garratty G, Leger RM, Arndt PA. Severe immune hemolytic
anemia associated with prophylactic use of cefotetan in
obstetric and gynecologic procedures. Am J Obstet Gynecol
1999;181:103–4.
58. Perkins J. Fatal drug-induced immune hemolytic anemia due
to cefotetan: a case study. Asian J Transfus Sci 2008;2:20–3.
59.Arndt P, Garratty G. Is severe immune hemolytic anemia,
following a single dose of cefotetan, associated with the
presence of “naturally-occurring” anti-cefotetan (abstract)?
Transfusion 2001;41(Suppl):24S.
60. Davenport RD, Judd WJ, Dake LR. Persistence of cefotetan on
red blood cells. Transfusion 2004;44:849–52.
61. Viraraghavan R, Chakravarty AG, Soreth J. Cefotetan-induced
haemolytic anaemia. A review of 85 cases. Adverse Drug React
Toxicol Rev 2002;21:101–7.
62.Arndt PA, Garratty G. Cross-reactivity of cefotetan and
ceftriaxone antibodies, associated with hemolytic anemia,
with other cephalosporins and penicillin. Am J Clin Pathol
2002;118:256–62.
63.Arndt PA, Leger RM, Garratty G. Reactivity of “last wash”
elution controls in investigation of cefotetan antibodies
(abstract). Transfusion 1999;39(Suppl):47S.
64.Williams ME, Thomas D, Harman CP, Mintz PD, Donowitz
GR. Positive direct antiglobulin tests due to clavulanic acid.
Antimicrob Agents Chemother 1985;27:125–7.
65.Lutz P, Dzik W. Very high incidence of a positive direct
antiglobulin test (+ DAT) in patients receiving Unasyn®
(abstract). Transfusion 1992;32(Suppl):23S.
54
66.Garratty G, Arndt PA. Positive direct antiglobulin tests
and haemolytic anaemia following therapy with betalactamase inhibitor containing drugs may be associated with
nonimmunologic adsorption of protein onto red blood cells. Br
J Haematol 1998;100:777–83.
67. Arndt P, Garratty G. Can nonspecific protein coating of RBCs
lead to increased RBC destruction? Drug-treated RBCs with
positive antiglobulin tests due to nonspecific uptake give
positive monocyte monolayer assays (abstract). Transfusion
2000;40(Suppl):29S.
68. Arndt PA, Garratty G. A retrospective analysis of the value of
monocyte monolayer assay results for predicting the clinical
significance of blood group alloantibodies. Transfusion
2004;44:1273–81.
69. Arndt PA, Leger RM, Garratty G. Positive direct antiglobulin
tests and haemolytic anaemia following therapy with the betalactamase inhibitor, tazobactam, may also be associated with
non-immunologic adsorption of protein onto red blood cells.
Vox Sang 2003;85:53.
70.Broadberry RE, Farren TW, Bevin SV, et al. Tazobactaminduced haemolytic anaemia, possibly caused by nonimmunological adsorption of IgG onto patient’s red cells.
Transfus Med 2004;14:53–7.
71. Arndt PA, Garratty G, Hill J, Kasper M, Chandrasekaran V.
Two cases of immune haemolytic anaemia, associated with
anti-piperacillin, detected by the ‘immune complex’ method.
Vox Sang 2002;83:273–8.
72.Leger RM, Arndt PA, Garratty G. Serological studies of
piperacillin antibodies. Transfusion 2008;48:2429–34.
73.
Leger RM, Garratty G. Antibodies to oxaliplatin, a
chemotherapeutic, are found in plasma of healthy blood
donors. Transfusion 2011;51:1740–4.
74.Arndt PA, Leger RM, Garratty G. IgM agglutinins to
meropenem, a beta-lactam antibiotic, are found in plasma
of healthy blood donors and random patients (abstract).
Transfusion 2012;52(Suppl):26A.
75.Mayer B, Yürek S, Salama A. Piperacillin-induced immune
hemolysis: new cases and a concise review of the literature.
Transfusion 2010;50:1135–8.
76.Arndt PA, Leger RM, Garratty G. Serological characteristics
of anti-piperacillin in seventeen patients with druginduced immune hemolytic anemia (abstract). Transfusion
2010;50(Suppl):37A.
77. Johnson ST. Warm autoantibody or drug dependent antibody?
That is the question! Immunohematology 2007;23:161–4.
Patricia A. Arndt, MS, MT(ASCP)SBB, Senior Research Associate,
American Red Cross Blood Services, Southern California Region,
100 Red Cross Circle, Pomona, CA 91768.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Report
Drug-induced immune thrombocytopenia:
incidence, clinical features, laboratory testing,
and pathogenic mechanisms
B.R. Curtis
Drug-induced immune thrombocytopenia (DIIT) is a relatively
uncommon adverse reaction caused by drug-dependent antibodies
(DDAbs) that react with platelet membrane glycoproteins only
when the implicated drug is present. Although more than 100
drugs have been associated with causing DIIT, recent reviews of
available data show that carbamazepine, eptifibatide, ibuprofen,
quinidine, quinine, oxaliplatin, rifampin, sulfamethoxazole,
trimethoprim, and vancomycin are probably the most frequently
implicated. Patients with DIIT typically present with petechiae,
bruising, and epistaxis caused by an acute, severe drop in platelet
count (often to <20,000 platelets/µL). Diagnosis of DIIT is
complicated by its similarity to other non–drug-induced immune
thrombocytopenias, including autoimmune thrombocytopenia,
posttransfusion purpura, and platelet transfusion refractoriness,
and must be differentiated by temporal association of exposure
to a candidate drug with an acute, severe drop in platelet
count. Treatment consists of immediate withdrawal of the
implicated drug. Criteria for strong evidence of DIIT include (1)
exposure to candidate drug–preceded thrombocytopenia; (2)
sustained normal platelet levels after discontinuing candidate
drug; (3) candidate drug was only drug used before onset of
thrombocytopenia or other drugs were continued or reintroduced
after resolution of thrombocytopenia, and other causes for
thrombocytopenia were excluded; and (4) reexposure to the
candidate drug resulted in recurrent thrombocytopenia. Flow
cytometry testing for DDAbs can be useful in confirmation of
a clinical diagnosis, and monoclonal antibody enzyme-linked
immunosorbent assay testing can be used to determine the
platelet glycoprotein target(s), usually GPIIb/IIIa or GPIb/IX/V,
but testing is not widely available. Several pathogenic mechanisms
for DIIT have been proposed, including hapten, autoantibody,
neoepitope, drug-specific, and quinine-type drug mechanisms. A
recent proposal suggests weakly reactive platelet autoantibodies
that develop greatly increased affinity for platelet glycoprotein
epitopes through bridging interactions facilitated by the drug is a
possible mechanism for the formation and reactivity of quininetype drug antibodies. Immunohematology 2014;30:55–65.
Key Words: drug-dependent platelet antibodies, druginduced immune thrombocytopenia
Drug-induced immune thrombocytopenia (DIIT) is an
unusual disorder that occurs after exposure to prescription
medications, herbal remedies, foods, and nutritional
supplements, in which affected individuals produce drugdependent antibodies (DDAbs) that bind and bring about
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
destruction of their platelets.1,2 Drug-induced bone marrow
suppression can cause thrombocytopenia through decreased
platelet production, and platelet destruction by drug-induced
autoantibodies is a form of DIIT. However, in these forms of
drug-induced thrombocytopenia, DDAbs are not typically
involved. Heparin-induced thrombocytopenia (HIT) is
another DIIT that occurs in 1 to 5 percent of patients receiving
heparin. Patients affected by HIT develop antibodies against
complexes of heparin and platelet factor 4 (PF4) that stimulate
platelet activation through platelet Fc receptor engagement,
resulting in clinically significant thrombosis. The pathogenesis
of HIT is different from that of DIIT caused by nonheparin
drugs, and the thrombocytopenia in HIT usually does not
reach levels that result in bleeding symptoms; consequently,
HIT will not be further discussed, but readers are referred to
numerous excellent, recent, in-depth reviews on the subject.3–5
The focus of this review will be on DIIT, in which patients
develop drug-dependent platelet antibodies targeting platelet
membrane glycoproteins (GPs) that cause clinically significant
thrombocytopenia primarily by clearance of antibody-coated
platelets in the reticulo-endothelial system (RES).
Incidence and Drugs Most Frequently Implicated
The exact frequency of DIIT is not known, but the overall
incidence is estimated to be 10 cases per million population
per year.6 Higher incidence is reported in specific patient
populations, including 25 percent of critically ill patients,7
the elderly,8 and patients taking the drugs quinine and
sulfamethoxazole-trimethoprim who were reported to be at
higher risk of developing DIIT—38 and 26 cases per million
exposed, respectively.9 Thrombocytopenia associated with the
GPIIb/IIIa inhibitors, abciximab, eptifibatide, and tirofiban
occurs in approximately 1 to 2 percent of patients exposed to
these drugs and as many as 10 percent of patients exposed to
abciximab for the second time.10
Recently, several groups have mined data from available
sources to determine those drugs most frequently associated
55
B.R. Curtis
with DIIT.8,11–14 Reese et al.11 used published case reports,
reference laboratory testing of patients’ sera for DDAbs over a
13-year period, and data mining of 41 years’ worth of the U.S.
Food and Drug Administration’s Adverse Event Reporting
System (AERS) to identify those drugs most frequently
implicated in causing DIIT. They identified 1468 drugs
suspected of causing thrombocytopenia, and 102 of these
were evaluated by all three methods. Only 23 of the 102 drugs
demonstrated a causal association with thrombocytopenia by
all three methods (Table 1). In a separate analysis focused on
children (<18 years old) of the same published case reports
and DDAb test data over a 5-year period (2008–2012), 34
drugs showed a causal association with thrombocytopenia
(Table 1).12 Garbe et al.14 used active surveillance of patients
in 50 German hospitals (Berlin Case-Control Surveillance
Study) to identify drugs that might cause DIIT. They
identified 90 cases of acute immune thrombocytopenia, in
which a drug was possibly associated. A total of 85 different
drugs were involved, and 31 drugs were determined to be
definitely (n = 7) or probably (n = 24) related to the patients’
thrombocytopenia. Tirofiban, abciximab, sulfamethoxazoletrimethoprim, and flu vaccine were the most frequently
implicated drugs. Arnold et al.13 analyzed data from the same
case reports database used by Reese et al.,11 combined with
a search of MEDLINE and EMBASE, of more than 72 years
of DIIT case reports for only those who had been tested for
DDAb by two or more laboratories. Using these criteria and
four independent assessors, only 16 drugs met the criteria for
a definite association with DIIT (Table 1).
Analysis of the most current data (2012) from our
reference laboratory (Platelet and Neutrophil Immunology
Lab, BloodCenter of Wisconsin, Milwaukee, WI) of all serum
tests for DDAbs from patients with suspected DIIT showed
that 101 different drugs were tested during this period (Table
2). Vancomycin (n = 127), piperacillin-tazobactam (n = 34),
sulfamethoxazole-trimethoprim (n = 43), cefepime (n = 24),
and ceftriaxone (n = 15) were the five most frequently tested
drugs, and there were 18 drugs for which DDAbs against
Table 1. Drugs frequently associated with drug-induced immune thrombocytopenia from literature sources
Reese et al.11
Arnold et al.13
Reese et al.12
Garbe et al.14
abciximab
acetaminophen
amiodarone
ampicillin
carbamazepine
eptifibatide
ethambutol
haloperidol
ibuprofen
irinotecan
naproxen
oxaliplatin
phenytoin
piperacillin
quinidine
quinine
ranitidine
rifampin
simvastatin
sulfisoxazole
tirofiban
sulfamethoxazole-trimethoprim
valproic acid
vancomycin
abciximab
carbamazepine
ceftriaxone
eptifibatide
heparin*
ibuprofen
mirtazapine
oxaliplatin
penicillin
quinidine
quinine
rifampin
sulfamethoxazole-trimethoprim
suramin
tirofiban
vancomycin
acetaminophen
aminosalicylic acid
carbamazepine
ceftriaxone
dactinomycin
diazoxide
glucagon
hepatitis B vaccine
HPV vaccine
imipramine
inamrinone
isotretinoinb
lamivudineb
lamotrigine
levetiracetam
Lupinus termis bean
methylphenidate
oxyphenbutazone
phenobarbital
phenytoin
quetiapine
quinine
rifampin
rituximab
sodium stibogluconate
spironolactone
sulfamethoxazole-phenazopyridine
sulfamethoxazole-trimethoprim
sulfasalazine
sulfasoxazole
sulfathiazole
tolmetin
tranilast
vancomycin
abciximab
amlodipine
benzylpenicillin
carbamazepine
ceftriaxone
cefuroxime
ciprofloxacin
citalopram
clopidogrel
diclofenac
eptifibatide
furosemide
ibuprofen
indapamide + perindopril
influenza vaccine
lorazepam
melperone
nitrofurantoin
phenoxymethylpenicillin
pneumococcal vaccine
poliomyelitis vaccine
ranitidine
risperidone
spironolactone
tamoxifen
terazosin
tirofiban
trimethoprim/sulfamethoxazole
valproate
vitamin B12
*Heparin was excluded from the other reports because its pathogenic mechanism differs significantly from nonheparin drugs.
56
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune thrombocytopenia
platelets were detected using a flow cytometry assay (Table
2). Carbamazepine, eptifibatide, ibuprofen, quinidine, quinine,
oxaliplatin, rifampin, sulfamethoxazole, trimethoprim, and
vancomycin are all represented in data from both Tables 1
and 2, suggesting that these 10 drugs are the most frequently
associated with DIIT caused by DDAbs. Although the drugs
listed in Tables 1 and 2 are the most frequently reported, it
should be pointed out that there are numerous other drugs1
and even herbal remedies,15–17 foods,17–20 and beverages17,21,22
that have been implicated in DIIT. Illicit drug use has also been
associated with DIIT. Cocaine and heroin are sometimes cut
or mixed with drugs known to cause DIIT, like quinine23,24 or
levamisole.25 Testing for DDAbs can be helpful in making a diagnosis in this setting and should include testing with quinine
to determine whether it has been added as an adulterant.
The rates of thrombocytopenia observed with chemotherapeutic agents are quite high, ranging from 21 to 70
percent.26,27 Bone marrow suppression is the major cause
of thrombocytopenia associated with this class of drugs.28
However, DIIT has been reported in a number of cases involving
various chemotherapeutic compounds.29–32 Oxaliplatin, a
widely used platinum compound for the treatment of colorectal
cancer, has been implicated in an increasing number of DIIT
cases.26,29,32–34
The incidence data just described are useful in identifying
drugs that are the top candidates for consideration in
a thrombocytopenic patient with compatible temporal
association and could be useful in hastening diagnosis and
treatment. Clearly, good medical practice requires that the
clinical criteria established for DIIT, together with laboratory
confirmation, be applied to all patients suspected of DIIT,
including exclusion or inclusion of all drugs to which the patient
has been recently exposed. Another issue worth mentioning
about the identification of drugs causing DIIT is that to
have good available data, physicians, pharmacists, testing
laboratories, and investigators must regularly report identified
cases of DIIT. Unfortunately, when an accurate diagnosis is
made, the case is often not reported to national databases (e.g.,
AERS) or is not published in the medical literature. It is my
opinion that this is partly the result of the increasing difficultly
clinicians and investigators face trying to publish a single
clinical case report of a novel finding such as DIIT because of
the reluctance of scientific journal editors. This trend continues
at the risk that investigators will no longer bother to submit
such cases, and this important information will be lost.
Clinical Features, Diagnosis, and Treatment
Patients with DIIT typically present with petechiae,
bruising, and epistaxis caused by an acute, often-severe
drop in platelet count. When thrombocytopenia is severe
(i.e., ≤20,000 platelets/µL), mucosal bleeding can occur
Table 2. Drugs (n = 101) tested for drug-dependent platelet antibodies in patient sera 2011–12*
acetaminophen
albuterol
allopurinol
amiodarone
amlodipine
amoxicillin-clavulanic acid (1)‡
argatroban
aspirin
atorvastatin
aztreonam
bumetanide
carbamazepine (2)‡
carvedilol
cefazolin
cefepime (24)†, (1)‡
ceftazidime (3)‡
ceftriaxone (15)†, (2)‡
cefuroxime
celecoxib
cephalexin
ciprofloxacin (1)‡
citalopram
clindamycin
clonazepam
clopidogrel
cyclosporine
digoxin
diltiazem
diphenhydramine
divalproex
eptifibatide (2)‡
escitalopram
esomeprazole
ezetimibe
famotidine
fentanyl
fluconazole
fluoxetine
furosemide (1)‡
gabapentin
gentamicin
glimepiride
haloperidol
hydralazine
hydrochlorothiazide
ibuprofen
imipenem-cilastatin
ipratropium
ketorolac
lansoprazole
levetiracetam
levofloxacin
linezolid
lisinopril
loratadine
lorazepam
losartan
meropenem
metformin
metoprolol
metronidazole
milrinone
nafcillin (1)‡
naproxen
nifedipine
quetiapine
omeprazole
oxaliplatin (3)‡
oxycodone-acetaminophen
pantoprazole
penicillin
phenobarbital (2)‡
phenytoin (2)‡
piperacillin-tazobactam (34)† (5 piperacillin)
pravastatin
prednisone
quetiapine
quinidine
quinine (1)‡
rifampin (1)‡
rosuvastatin
sertraline
sildenafil
simethicone
simvastatin
spironolactone (3)‡
sulfamethoxazole-trimethoprim (22)†‡ (11/22
sulfamethoxazole or 6/22 trimethoprim only)
tacrolimus
tobramycin
triamterene
valacyclovir
valganciclovir
vancomycin (127)†, (30)‡
voriconazole
*Drugs (n = 101) for which sera from patients with suspicion of drug-induced immune thrombocytopenia were tested in flow cytometry assay by Platelet &
Neutrophil Immunology Lab, BloodCenter of Wisconsin, Milwaukee.
†Five most frequently tested drugs (number of patient samples tested).
‡Drugs (n = 18) for which drug-dependent antibodies (DDAbs) were detected (number of DDAbs detected).
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
57
B.R. Curtis
in the gastrointestinal or genitourinary tracts (so-called
wet purpura). In extreme cases, intracranial or pulmonary
hemorrhage resulting in fatalities has been reported. Platelet
counts usually fall rapidly from normal or near-normal
levels to nadirs in the single digits within several hours of
a repeat drug exposure. For patients receiving a drug for
the first time, 5 to 7 days of exposure is usually required for
sensitization. Patients previously sensitized can experience
rapid drops in platelet counts within 1 to 2 hours of repeat
exposure. DIIT is a diagnosis of exclusion, i.e., the exclusion
of the many other causes of thrombocytopenia. Therefore, the
diagnosis is complex and is often mistaken for other immune
platelet disorders such as immune thrombocytopenia (ITP),35
posttransfusion purpura (PTP),36 or platelet transfusion
refractoriness (PTR),37 especially for patients receiving
multiple medications.
ITP usually presents as isolated thrombocytopenia most
commonly occurring in response to an unknown stimulus
and is caused by nondrug autoantibodies that bind and
destroy autologous platelets. ITP can also develop secondarily
to other autoimmune disorders, viral infections, and certain
drugs (e.g., procainamide and gold salts)6; however, in this
form of DIIT, true autoantibodies develop, and also do not
require the presence of drug for their detection in laboratory
tests. Distinguishing DIIT from ITP can be quite challenging;
however, it is important to do so to prevent patients with DIIT
from undergoing unnecessary, invasive treatments.38
PTP is a rare disorder, occurring approximately 5 to 10
days after a blood transfusion, in which there is an acute,
severe drop in platelet count very similar in timing and
severity of the thrombocytopenia that occurs in DIIT. PTP
is distinguishable from DIIT by its association with a blood
transfusion rather than medication, but this distinction can be
more difficult when a patient receives both. Another difference
is that platelet-specific antibodies, most often targeting human
platelet alloantigen (HPA)-1a, are often detected in the sera of
patients with PTP, whereas DDAbs specific for the suspected
drug, and not HPA antibodies, are detected in DIIT.
PTR develops most often in oncology patients, and occurs
when posttransfusion platelet increments do not achieve
expected levels despite multiple platelet transfusions. Although
the majority of PTR is attributable to nonimmune causes (e.g.,
bone marrow suppression, fever, sepsis, etc.), immune forms
caused by class I HLA antibodies and, rarely, platelet-specific
antibodies also occur. Distinguishing thrombocytopenia
caused by DIIT from that caused by PTR can be particularly
58
challenging. A major complicating factor is that many patients
at risk of developing PTR also receive drugs reported to
have caused DIIT, so a detailed review of platelet counts and
timing of drug exposures is critical in this setting. Testing
of a patient’s serum for HLA and platelet-specific antibodies
can be helpful in identifying possible immune causes of PTR.
However, discontinuing candidate drugs, when possible, with
careful observation for a rise in platelet count over the course
of 3 to 4 days may be required in exceptionally difficult cases.
Clinical criteria that have been established to implicate a
suspected drug include the following39:
1. Exposure to the candidate drug preceded thrombocytopenia.
2. Recovery from thrombocytopenia was complete and
sustained after discontinuing candidate drug.
3. Candidate drug was the only drug used before the
onset of thrombocytopenia, or other drugs were
continued or reintroduced after discontinuation of
the candidate drug with a sustained normal platelet
count.
4. Other causes for thrombocytopenia were excluded.
5. Reexposure to the candidate drug resulted in recurrent
thrombocytopenia.
Evidence is considered Definite if criteria 1, 2, 3, and 4 are
met; Probable if criteria 1, 2, and 3 are met; Possible if criterion
1 only is met; and Unlikely if criterion 1 is not met.
For patients receiving chemotherapy who exhibit
thrombocytopenia, it is common to attribute this complication
to suppression of megakaryocytopoiesis, a recognized side
effect of these classes of drugs. However, as mentioned
previously, DIIT has been reported to develop from exposure
to these drugs. Accordingly, it is important to consider DIIT
in patients undergoing chemotherapy who have a sudden,
isolated drop in platelet levels.
Once a clinical diagnosis of DIIT is established, treatment
consists of immediate withdrawal of the implicated drug, and
platelet counts should be monitored until they reach normal,
sustained levels. Patients with severe thrombocytopenia or
hemorrhage may require platelet transfusions.40 Treatment
with corticosteroids has not been shown to be an effective
treatment in DIIT.6,41 Platelet levels typically begin to recover
in 3 to 4 days, as the drug is cleared from the circulation,
and attain normal levels by 5 to 7 days after stoppage of the
offending drug.1 It is possible for drug clearance and resolution
of thrombocytopenia to be prolonged in patients with renal
impairment.6
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune thrombocytopenia
Laboratory Testing for Drug-Dependent Platelet
Antibodies
Detection of serum antibodies that react with normal
platelets only in the presence of the suspected drug is helpful
in confirming a clinical diagnosis of DIIT. Several different
methods have been used during the years for detection of
DDAbs,6,13,42 but use of flow cytometry with intact platelets is
one of the most sensitive assays currently in use.29,42–44 A typical
assay involves incubation of serum with group O platelets and
buffer or drug in the well of a microtiter plate, followed by
washing of the platelets to remove unbound immunoglobulins.
The test well incubated with buffer is washed in buffer, and
the well with drug is washed with buffer containing drug,
usually at the therapeutic concentration. Platelets are then
incubated with a combination of fluorescent-labeled antiimmunoglobulin reagents: fluorescein-isothiocyanate–labeled
anti-human immunoglobulin (Ig) G and phycoerythrinlabeled anti-human IgM. Fluorescent events are acquired on
a flow cytometer with simple forward scatter and side-scatter
gates set to identify platelets. Stronger median fluorescence
intensity values obtained with samples in the presence of drug
compared with those without drug are considered positive
for DDAbs (Fig. 1). Sera from normal, healthy individuals
previously tested and shown not to have DDAbs and sera from
A
patients previously shown to have drug-dependent antibodies
should be tested in each assay as important negative and
positive controls, respectively. Although this testing is available
in the United States,45 unfortunately, it is not more widely
available because it requires an experienced laboratory with
access to sufficient rare control samples and a large inventory
of various pharmaceuticals. There has been a recent call to
create broader access to laboratory testing.13
Several issues encountered with flow cytometry testing for
DDAbs include insolubility of some drugs, non-DDAbs present
in patient sera, and the fact that some DDAbs recognize only
drug metabolites. Because most drugs have affinity for albumin
in circulation, drugs that are poorly soluble in buffer can often
be adequately solubilized in a buffer containing 5 percent
bovine serum albumin.42 Non-DDAbs, e.g., autoantibodies
and class I HLA antibodies, are easily identified because they
bind to platelets in both the presence and absence of drug,
and strong non-DDAbs can mask the presence of a DDAb
in testing. Absorption of sera with platelets in the absence of
drug before testing will remove non-DDAbs and reveal DDAbs
present. DDAbs are encountered that show reactivity only with
metabolites of the native drug.46,47 In these cases, metabolites
can sometimes be obtained from pharmaceutical companies or
other commercial sources, and as a last resort, urine collected
from someone having taken the drug for several days can be
B
MFI = 453.2
MFI = 4.1
Sulfamethoxazole
MFI = 4.4
Buffer
Platelet Events
Platelet Events
Buffer
Sulfamethoxazole
Normal Serum
Patient Serum
MFI = 4.1
Fluorescence Intensity
Fluorescence Intensity
Fig. 1 Fluorescence histograms from immunofluorescence detection of IgG drug-dependent platelet antibodies by flow cytometry assay.
Normal isolated platelets were incubated with sera and washed, and platelet-bound antibodies were detected with fluorescent anti-human
IgG. (A) Normal serum incubated with platelets in the presence of either sulfamethoxazole (black histograms) or buffer (white histograms)
shows low IgG fluorescence, indicating no antibodies are present. (B) Serum from a patient with suspected drug-induced thrombocytopenia
after exposure to sulfamethoxazole-trimethoprim incubated with platelets showing high IgG fluorescence (MFI = 453.2) only with
sulfamethoxazole, indicating the presence of sulfamethoxazole drug-dependent antibodies. Median fluorescence intensity (MFI) values are
shown for each histogram.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
59
B.R. Curtis
used as a source of metabolites for testing after adjusting the
pH and salt concentration.
Enzyme-linked immunosorbent assays (ELISAs) like the
modified antigen-capture ELISA (MACE)43 and immunoblotting procedures48–50 are primarily used to identify the
platelet GPs that DDAbs target. In the MACE, platelet
sensitization with patient’s serum and drug is the same as
that just described for flow cytometry. Sensitized platelets
are then lysed in detergent, the platelet GP–drug–antibody
complexes are captured in a microtiter plate well with specific
monoclonal antibodies, and DDAbs are detected with enzymelabeled anti-human IgG reagents. The majority of DDAbs
bind to GPIIb/IIIa,29,42,43,51 the major fibrinogen receptor, or
GPIb/IX/V,52,53 a receptor for von Willebrand factor on
platelets. There has been a single report of a carbimazoledependent platelet antibody targeting platelet endothelial cell
adhesion molecule-1 (PECAM-1/CD31).54 Flow cytometry
assays using platelets from patients with the platelet function
disorders Glanzmann thrombasthenia (platelets lack GPIIb/
IIIa) and Bernard-Soulier syndrome (platelets lack GPIb/IXV)
have also been used successfully to identify the platelet GP
targets of DDAbs.29,55
An in vivo mouse model was recently developed in
which normal human platelets and human serum containing
strong quinine-dependent platelet antibodies are transfused
into nonobese diabetic/severe combined immunodeficient
(NOD/scid) mice and allowed to circulate. Rapid clearance of
platelets occurred after intraperitoneal injection of quinine.56
This model might be useful in detecting low-affinity DDAbs
that wash off platelets and go undetected in conventional
laboratory tests or those induced by drug metabolites, which
the authors showed could be produced by the mice after
intraperitoneal injection of the native drug.56
The clinical sensitivity and specificity of the different assays
is unknown because it is extremely difficult to obtain the large
numbers of samples that would be required both from patients
with a clinical diagnosis of DIIT and from patients taking the
drugs who did not exhibit thrombocytopenia. In a single study
of 59 patients exposed to vancomycin, 29 of 31 who met the
criteria for vancomycin-induced thrombocytopenia tested
positive for vancomyin-dependent platelet antibodies by flow
cytometry, and all 25 who did not develop thrombocytopenia
gave negative results. In addition, 10 samples from patients not
exposed to vancomycin but with quinine-dependent antibodies
tested negative for vancomycin DDAbs, and only 1 of 451 sera
from normal healthy subjects never exposed to vancomyin
had weak IgM-positive reactivity.51 Altogether, these results
indicate that the flow cytometry assay had high sensitivity
60
(93.5%) and specificity (99.8%) for detection of DDAbs. It has
been suggested that tests for DDAbs are not practical for use in
clinical decision making because the testing is available in only
a handful of laboratories in the world, DDAbs are not always
detected, and there are claims of suboptimal test turnaround
times. However, it certainly can be argued that for cases with
a difficult diagnosis that knowing the results of DDAb testing,
even within 2 to 3 days, is probably more clinically useful than
continuing to expose a patient with undiagnosed DIIT to the
offending drug.
Pathogenic Mechanisms
DDAbs that are the cause of DIIT are exquisitely unique in
their ability to bind to specific platelet membrane GPs only in
the presence of the drug that induced them. In fact, the drug
is so critical to the reactivity of these antibodies that, during
the performance of DDAb assays, they will easily and rapidly
wash off the platelets if drug is not present throughout testing.
Several mechanisms have been proposed to explain the
formation of these most interesting antibodies (Fig. 2).6,57–60
Hapten Mechanism
Haptens are low-molecular-weight (usually <5000
daltons) molecules that are not capable of eliciting an immune
response unless they are coupled to a larger carrier protein.
Drugs such as penicillin and some cephalosporin drugs when
covalently linked to cell surface proteins can elicit drugspecific antibodies. This mechanism is well described for
DDAbs targeting red blood cells that cause immune hemolytic
anemia.61 Although it is a potential mechanism to explain
DIIT, attempts in our laboratory to detect DDAbs with drugcoated platelets have not been successful to date, and there are
no known reports in which hapten-induced antibodies have
been detected in DIIT.1,59
Autoantibody Mechanism
There are reports of patients who exhibited acute,
severe thrombocytopenia after exposure to specific drugs,
but no DDAbs can be detected in their serum. However,
autoantibodies that are not dependent on drug can be detected
in these patients’ sera, suggesting that the drug induced a true
autoantibody, but this is very difficult to prove.62
Neoepitope Mechanism
The most popular mechanism espoused for DDAb
formation is the neoepitope mechanism (Fig. 2). It is well
established that most drugs in circulation make low-affinity
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune thrombocytopenia
Drug
Drug-Induced Antibody
1
6
Platelet Glycoprotien
Fab Antibody Fragment
Drug
3
5
2
4
Fig. 2 Proposed mechanisms for drug-induced antibody formation and platelet destruction. (1) Hapten-dependent antibody: Covalent
binding of drug/hapten to platelet membrane protein(s) induces antibody specific to drug/hapten. (2) Autoantibody: Drug induces formation
of a true platelet autoantibody. (3) Fiban-type drug-dependent antibody: Drug binding to GPIIb/IIIa induces neoepitopes that are recognized
by drug-dependent antibody. (4) Drug-specific antibody: Antibody recognizes mouse components of mouse/human chimeric Fab fragments
of drug bound to platelet GPIIb/IIIa. (5) Quinine-type drug-dependent antibody: Drug and platelet glycoprotein together form a combinatorial
epitope recognized by drug-dependent antibody. (6) Quinine-type drug-dependent antibody: Drug induces antibody that binds more tightly
to platelet glycoprotein in the presence of a soluble drug. Figure created by Rachel Kubsh.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
61
B.R. Curtis
interactions with many different proteins in the plasma,
especially albumin, but also probably platelet GPs, e.g.,
GPIIb/IIIa. These interactions are speculated to induce
conformational changes in the protein structures that elicit
DDAbs in certain individuals. However, when drug is cleared
from the circulation and the conformation of the platelet
protein reverts to its native state, DDAbs, although still present,
can no longer bind and destroy the platelets (Fig. 2). It is
proposed that the platelet GPIIb/IIIa inhibitors, tirofiban and
eptifibatide, small-molecule fibrinogen receptor antagonists
(fibans), induce DDAbs by this mechanism.6,63 Fibans are a
widely used class of drugs capable of preventing restenosis
in patients undergoing percutaneous transluminal coronary
angioplasty (PTCA) procedures. Interestingly, an unusual
aspect of fiban-dependent antibodies is that they can occur
naturally in approximately 2 percent of normal people.63,64
Drug-Specific Mechanism
Abciximab (Reopro) is a chimeric (mouse-human)
monoclonal antibody Fab fragment specific for GPIIIa used
primarily in PTCA procedures to prevent thrombus formation.
Like the previously mentioned fibans, it blocks interactions
of fibrinogen with GPIIb/IIIa to prevent platelet aggregation.
Approximately 1 to 2 percent of patients receiving abciximab
exhibit acute thrombocytopenia within a few hours of receiving
the drug.10 Although abciximab binds tightly to platelets, it does
not cause thrombocytopenia by macrophage clearance in the
RES because it lacks the immunoglobulin Fc; rather, affected
patients make drug-specific antibodies that appear to recognize
murine sequences in the complimentary determining region
3 (CDR3) region of abciximab (Fig. 2).65,66 Like DIIT induced
by fibans, some patients affected with abcixmab-induced
immune thrombocytopenia also have preexisting antibodies in
their serum.66 Many normal, healthy people never exposed to
abciximab also have IgG antibodies that react with abciximabcoated platelets65,67; however, these antibodies recognize the
cleaved end of the Fab fragment that is created when the drug
is treated with papain. It is unknown whether Fab-reactive
antibodies seen in healthy people can cause DIIT should these
individuals ever be exposed to the drug.
Quinine-Type Drug Mechanism
The classic DDAbs induced by drugs such as quinine,
quinidine, sulfamethoxazole, and vancomycin attach tightly
to platelets only in the presence of the sensitizing drug, and
most often target GPIIb/IIIa or GPIb/IX/V. These antibodies
bind their epitopes by their Fab domains,68 indicating that
their reactivity with platelets is not the result of drug–
62
antibody immune complexes engaging platelet Fc receptors,
as is the case with heparin-PF4 antibodies that cause HIT.
Early mechanisms proposed to explain platelet destruction by
quinine-type DDAbs were (1) the previously described hapten
mechanism; however, quinine-type antibodies still react with
platelets in the presence of large concentrations of soluble drug
that should block their binding if they were produced by this
mechanism; (2) drug-induced conformational changes in the
GPs that are targeted by DDAbs; and (3) compound epitopes
consisting of both drug and platelet protein form that are
recognized by antibodies. However, none of these proposed
mechanisms have been proven.
The mechanisms by which quinine-type DDAbs form
and react with platelets have been intensely studied in the
laboratory of Dr. Richard Aster at the BloodCenter of Wisconsin
in Milwaukee. Using chimeric human/rat GPIIIa and targeted
mutants of human GPIIIa, they identified a 17–amino acid (aa
50–66) stretch spanning the hybrid and plexin-semaphorinintegrin (PSI) domains of GPIIIa that was recognized by all
three antibodies only in the presence of quinine.50 Substitution
in human GPIIIa of only three amino acids (Ala50, Arg62, or
Asp66) abolished DDAb reactivity, as did reduction of wildtype GPIIIa with 2-mercaptoethanol, indicating that disulfide
linkages are also important in formation of this epitope.
Burgess et al.52 previously identified a peptide sequence
(aa 282–293) in GPIbα that was also required for reactivity
of a single quinine DDAb, and studies by Asvadi et al.69
demonstrated that amino acid residues Arg110 and Gln115 in
GPIX were critical for binding of six quinine DDAbs.
Later studies again by the Aster group49 showed that 14
quinine DDAbs, including the three originally studied, all
recognized a small 29-kD recombinant fragment of GPIIIa
that included the three amino acids (Ala50, Arg62, or Asp66)
previously shown to be critical for DDAb binding. Additional
studies with blocking monoclonal antibodies and overlapping
mutant GPIIIa constructs showed that as many as eight
different overlapping epitopes were recognized by the 14
different quinine DDAbs, leading this group to propose that
DDAbs induced by quinine, and presumably other drugs,
do not recognize a single distinct epitope on a GP. This
result further indicates that mechanisms proposing DDAbs
recognize drug-induced conformational changes elsewhere in
platelet membrane GPs are not viable explanations for DDAb
formation.49
In other studies, the Aster lab59 found both the classic
drug-dependent platelet-reactive antibodies and drug-specific
antibodies in plasma from 6 of 7 patients who exhibited DIIT
after exposure to quinine. DDAbs reacted with GPIIb/IIIa
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune thrombocytopenia
or GPIb/IX/V on platelets only in the presence of quinine.
Drug-specific antibodies bound directly to quinine that was
covalently linked to human serum albumin, and this reactivity
was inhibited by excess soluble quinine. Despite recognizing
different targets, both types of antibodies were identical in
their drug specificity, and this suggests that both are produced
against a similar antigen as part of the same B-cell immune
response. Based on these findings, the authors proposed a
novel model in which patients have autoantibodies with only
weak reactivity for platelet GP epitopes in the absence of drug,
but that this reactivity is significantly enhanced through
bridging interactions by drug binding between both the
protein and antibody; the resultant sandwich entraps a drug at
the antigen–antibody interface and greatly increases antibody
binding energy for the GP autoepitope (Fig. 2).6,59 Two quininedependent mouse monoclonal antibodies recently developed
by Aster’s group with specificity for epitopes on human GPIIb
will be valuable tools to help further elucidate the complex
mechanisms responsible for DIIT.70
Summary
DIIT is a relatively uncommon, but often-undiagnosed,
disorder that can occur after exposure to numerous drugs.
Several recent large analyses of data from multiple sources
indicate that the drugs carbamazepine, eptifibatide, ibuprofen,
quinidine, quinine, oxaliplatin, rifampin, sulfamethoxazole,
trimethoprim, and vancomycin are most often implicated.
Diagnosis is complex and involves excluding other causes of
thrombocytopenia and ascertainment of the patient’s complete
medication history. Laboratory testing of patient’s serum for
DDAbs can be very useful in confirming a clinical diagnosis
and, with broader access, will be used more often. The hallmark
of treatment is immediate cessation of the offending drug(s)
and platelet transfusions for patients with severe bleeding
symptoms. DDAbs usually target GPIIb/IIIa and GPIb/IX/V
on the platelet membrane surface. Several mechanisms have
been proposed to explain the development and reactivity of
DDAbs, and a recent hypothesis suggests that many are weakly
reactive platelet autoantibodies that develop greatly increased
affinity for platelet GP epitopes through bridging interactions
facilitated by the drug. Data obtained from studies using newly
developed drug-dependent monoclonal antibodies and mouse
models of DIIT should yield important new knowledge about
this fascinating disorder.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Acknowledgments
I wish to acknowledge the technical assistance of Ms.
Rachel Kubsh in preparation of Figure 2.
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57.Aster RH. Drug-induced immune cytopenias. Toxicology
2005;209:149–53.
58.Aster RH, Curtis BR, McFarland JG, Bougie DW. Druginduced immune thrombocytopenia: pathogenesis, diagnosis,
and management. J Thromb Haemost 2009;7:911–8.
59. Bougie DW, Wilker PR, Aster RH. Patients with quinine-induced
immune thrombocytopenia have both “drug-dependent” and
“drug-specific” antibodies. Blood 2006;108:922–7.
60. Aster RH, Curtis BR, Bougie DW. Thrombocytopenia resulting
from sensitivity to GPIIb-IIIa inhibitors. Semin Thromb
Hemost 2004;30:569–77.
61. Ries CA, Rosenbaum TJ, Garratty G, Petz LD, Fudenberg HH.
Penicillin-induced immune hemolytic anemia. Occurrence of
massive intravascular hemolysis. JAMA 1975;233:432–5.
62.Aster RH. Can drugs cause autoimmune thrombocytopenic
purpura? Semin Hematol 2000;37:229–38.
63.Bougie DW, Wilker PR, Wuitschick ED, et al. Acute
thrombocytopenia after treatment with tirofiban or eptifibatide
is associated with antibodies specific for ligand-occupied
GPIIb/IIIa. Blood 2002;100:2071–6.
64.Bednar B, Cook JJ, Holahan MA, et al. Fibrinogen receptor
antagonist-induced thrombocytopenia in chimpanzee and
rhesus monkey associated with preexisting drug-dependent
antibodies to platelet glycoprotein IIb/IIIa. Blood 1999;94:
587–99.
Notice to Readers
All articles published, including communications and
book reviews, reflect the opinions of the authors and do
not necessarily reflect the official policy of the American
Red Cross.
65.Curtis BR, Swyers J, Divgi A, McFarland JG, Aster RH.
Thrombocytopenia after second exposure to abciximab
is caused by antibodies that recognize abciximab-coated
platelets. Blood 2002;99:2054–9.
66.Curtis BR, Divgi A, Garritty M, Aster RH. Delayed
thrombocytopenia after treatment with abciximab: a distinct
clinical entity associated with the immune response to the
drug. J Thromb Haemost 2004;2:985–92.
67.Christopoulos C. Platelet surface IgG in patients receiving
infusions of Fab fragments of a chimaeric monoclonal antibody
to glycoprotein IIb-IIIa. Clin Exp Immunol 1994;98:6–11.
68.Christie DJ, Mullen PC, Aster RH. Fab-mediated binding
of drug-dependent antibodies to platelets in quinidine- and
quinine-induced thrombocytopenia. J Clin Invest 1985;75:
310–14.
69.
Asvadi P, Ahmadi Z, Chong BH. Drug-induced
thrombocytopenia: localization of the binding site of GPIXspecific quinine-dependent antibodies. Blood 2003;102:
1670–7.
70.Bougie DW, Birenbaum J, Rasmussen M, Poncz M, Aster
RH. Quinine-dependent, platelet-reactive monoclonals mimic
antibodies found in patients with quinine-induced immune
thrombocytopenia. Blood 2009;113:1105–11.
Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet &
Neutrophil Immunology Lab, Blood Research Institute, BloodCenter
of Wisconsin, PO Box 2178, Milwaukee, WI 53201-2178.
Attention:
State Blood Bank Meeting Organizers
If you are planning a state meeting and would like copies
of Immunohematology for distribution, please send request,
4 months in advance, to [email protected]
For information concerning the National Reference
Immunohematology is on the Web!
Laboratory for Blood Group Serology, including the American
www.redcross.org/about-us/publications/
immunohematology
Rare Donor Program, contact Sandra Nance, by phone at
(215) 451-4362, by fax at (215) 451-2538, or by e-mail at
[email protected]
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For more information, send an e-mail to
[email protected]
65
Report
Drugs that have been shown to cause druginduced immune hemolytic anemia or positive
direct antiglobulin tests: some interesting
findings since 2007
G. Garratty and P.A. Arndt
This review updates new findings in drug-induced immunehemolytic anemia (DIIHA) since the 2007 review in
Immunohematology by these authors. Twelve additional drugs
have been added to the three tables listing drugs associated
with drug-dependent antibodies, drugs associated with
drug-independent antibodies, and drugs associated with
nonimmunologic protein adsorption. Other updated findings
include (1) piperacillin is currently the most commonly
encountered cause of DIIHA, (2) new data on blood group
specificity of drug-dependent antibodies, (3) drug-dependent
antibodies detected in healthy donors, (4) DIIHA associated with
transplantation, and (5) DIIHA associated with chemotherapeutic
drugs. Immunohematology 2014;30:66–79.
Key Words: drugs, immune hemolytic anemia, autoantibody,
drug-dependent antibody, nonimmunologic protein adsorption
Dr. Garratty has published five previous reviews
on drug-induced immune hemolytic anemia (DIIHA) in
Immunohematology; the last one in 2007 was coauthored by
Pat Arndt.1–5 This issue is devoted to drug-induced immune
cytopenias (red blood cells [RBCs], platelets, and white blood
cells [WBCs]). If one reads the DIIHA reviews previously
published in Immunohematology, you will get a view of how
many drugs were found to be responsible over each 5-year
period (1985–2007), which ones were the most common
causes of DIIHA, and what concepts were most commonly
suggested to explain various types of DIIHA. The reviews
by Arndt6 and Leger et al.7 in this issue cover many of these
areas in great detail and add new data that were not fully
covered in the previous Immunohematology reviews. Some
other recent reviews by Garratty can update you further.8–12
For readers who do not have access to these publications,6–12
we will summarize the most important information that has
emerged in the last 10 years, with emphasis on the period
since 2007.
66
Drugs Causing DIIHA
In our 2007 review, we included tables containing 125
drugs that we believed had reasonable evidence of causing
DIIHA.5 Indisputable evidence would require the following
findings:
1. A well-defined hemolytic anemia (HA).
2. A temporal relationship to drug therapy and the start
of the HA.
3. A positive direct antiglobulin test (DAT) after drug
therapy (preferably with a negative DAT result
preceding therapy).
4.Results of testing for drug antibodies (e.g., using
drug-treated RBCs or testing serum in the presence
of soluble drug7,8). These results must be accompanied
by pertinent controls.7,8 Laboratory tests cannot
prove that autoantibodies are drug-induced, but it
is important to show that apparent autoantibodies
are truly autoantibodies and not drug-dependent
antibodies that are reacting without the in vitro
addition of drug because enough drug is present in
the patient’s plasma or serum.5–7,13
5. Patient responds hematologically after drug cessation.
Unfortunately, this is not easy to define, as patients
are often simultaneously given steroids.
Since 2007, we have added seven new drugs to Table 1
(drug-dependent
antibodies):
hydrocortisone
(2008),
pemetrexed (2008), cimetidine (2010), etoricoxib (2013),
iomeprol (2013), puerarin (2013), and vincristine (2013).
Of these 115 drugs, five (carbromal, cefpirome, furosemide,
methadone, and norfloxacin) may have caused positive
DATs but no HA. Twenty-seven (23%) drugs were associated
with drug antibodies reacting with drug-treated RBCs; 44
(38%) drugs were associated with antibodies reacting in the
presence of soluble drug; 41 (36%) drugs were associated
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
DIIHA: update since 2007
with antibodies detected by both methods. Fifty (43%) drugs
were associated with in vitro reactions against untreated cells
without any drug being added. These could be associated with
drug-independent antibodies, but we believe that many may
have been caused by drug or drug complexes being present
in the patient’s plasma or serum (see footnote in Table 1). It
will probably be no surprise that not all of the drugs listed in
Table 1 satisfy 100% of our criteria for perfection. Hence, we
can only say that there is reasonable evidence that these drugs
were the cause of DIIHA.
We added four new drugs to Table 2 that appeared to cause
autoimmune hemolytic anemia (drug-independent antibodies):
rituximab (2003), alemtuzumab (2007), weidean (2009), and
bendamustine (2013). All but one of the drugs in Table 2
(chaparral) may have caused a DAT-positive autoimmune HA
(AIHA). It should be noted that 16 of 21 drugs in this group
were classified as needing more evidence to support that they
belong in this group. One drug (cephaloridine) was added to
the nonimmunologic protein adsorption group (Table 3); this
was observed in 1967 but was unfortunately left off our 2007
Table 3.
Classification of Drug Antibodies
There are two main types of drug antibodies: drugdependent antibodies and drug-independent antibodies.
Drug-dependent antibodies need the drug to be present for
in vitro reactions; the drug can be bound to test RBCs or soluble
drug can be added to patient’s plasma or serum (source of
antibody) and allogeneic group O RBCs; after 37°C incubation,
the tests are inspected for lysis, direct agglutination, and by
the indirect antiglobulin test (IAT). Enzyme-treated RBCs
should be used in addition to untreated RBCs.7 Test results
are only valid when appropriate controls are used (see Leger et
al.7 for complete details). Salama’s group does not advise using
enzyme-treated RBCs169 because of problems with nonspecific
reactions, but we believe these can usually be overcome with
experience and pertinent controls.
Drug-independent antibodies react without any drug
being added in vitro. The results are indistinguishable from
RBC autoantibodies. It is thought that such drugs affect the
immune system so that true autoantibodies and sometimes
AIHA are produced. There are no special in vitro tests to define
that a particular drug caused the AIHA. The diagnosis rests on
the physician stopping the drug and finding that the anemia
resolves, but the serology (e.g., DAT) can remain positive for
some time longer.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Positive DATs and DIIHA Caused by Nonimmune
Protein Adsorption
Drug-induced positive antiglobulin tests (e.g., DATs) and
sometimes HA can occur with no drug antibodies involved,
and yet RBCs are destroyed by macrophages in the spleen
and liver. Thus, the drug interaction with the RBCs does not
involve drug-induced antibodies, but the shortened life of the
RBCs is by a cellular immune mechanism. The drugs that can
be involved in this mechanism are listed in Table 3. These
drugs can change the RBC membrane so that proteins (e.g.,
immunoglobulins, complement, albumin, etc.) attach to the
RBCs, leading to positive antiglobulin tests. This may involve
the patient’s RBCs in vivo (positive DAT), or may occur in
vitro (positive IAT) when the patient’s serum or plasma is
incubated with drug-coated RBCs (e.g., when testing for drug
antibodies). This phenomenon was first observed with the
first-generation cephalosporin, cephalothin, and was later
found to occur with other cephalosporins (e.g., cefotetan).
It was termed the “membrane modification mechanism” or
“nonimmunological protein adsorption (NIPA).” It was thought
for many years to be an interesting phenomenon that could
interfere with interpretation of laboratory tests but had no
clinical significance; then β-lactamase inhibitors, which cause
NIPA, were shown to possibly cause DIIHA.162,167 Other drugs
(see Table 3) can also cause NIPA and DIIHA (e.g., cisplatin
and oxaliplatin). The involvement of NIPA is sometimes
hard to define, as drug antibodies (e.g., anti-oxaliplatin)
may also be involved.101 The nonimmunologically adsorbed
immunoglobulin (Ig) G (and possibly C3) can react with
receptors on macrophages, even though it is not an antibody
to the drug or the RBCs. The most useful test to indicate that
NIPA is occurring is to show that albumin is present on the
RBCs by testing with anti-human albumin by the antiglobulin
test.7 Such antisera have to be standardized carefully in-house,
as no U.S. Food and Drug Administration–licensed reagent is
available.
New Findings Since Our Last Review in
Immunohematology (2007)
In the period covered by our 2007 review (2005–2007),
the most common drugs to cause DIIHA in patients’ samples
submitted to our laboratory were cefotetan, ceftriaxone,
and piperacillin, in that order. For the period 2008–2013,
piperacillin, rose to number 1, followed by cefotetan and
ceftriaxone (Table 4). We are starting to see a small increase in
DIIHA caused by drugs in the platinum family.
67
G. Garratty and P.A. Arndt
Table 1. Drugs associated with cases of IHA or positive DAT, or both, in which drug-dependent antibodies were detected*
Drug (alternative name)
Aceclofenac
Reference
Therapeutic category
Number of references
(single [year] vs.
multiple [<5, <10,
or ≥10])
14
NSAID
Single (1997)
15,16
NSAID
Multiple (<10)
Acyclovir
17
Antiviral
Single (2003)
Aminopyrine
18
NSAID
Single (1961)
Amoxicillin
19
Antimicrobial
Multiple (<5)
Amphotericin B
20
Antimicrobial
Multiple (<5)
Ampicillin
21
Antimicrobial
Multiple (<10)
Antazoline
22
Antihistamine
Multiple (<5)
Aspirin
23
Analgesic, antipyretic,
anti-inflammatory
Single (1984)
Azapropazone (Apazone)
24
Anti-inflammatory,
analgesic
Multiple (<5)
Buthiazide (Butizide)
25
Diuretic, antihypertensive
Single (1984)
Carbimazole
26
Antithyroid
Multiple (<5)
Carboplatin‡
27
Antineoplastic
Multiple (<10)
Carbromal
28
Sedative, hypnotic
Single (1970)
Catechin [(+)-Cyanidanol-3]
(Cianidanol)
29
Antidiarrheal
Multiple (≥10)
Cefamandole
30
Antimicrobial
Single (1985)
Cefazolin
31
Antimicrobial
Multiple (<10)
Cefixime
32
Antimicrobial
Single (2000)
Cefotaxime‡
33
Antimicrobial
Multiple (<5)
34–38
Antimicrobial
Multiple (≥10)
Cefoxitin‡
39
Antimicrobial
Multiple (<10)
Cefpirome
40
Antibacterial
Single (2005)
Ceftazidime
41
Antimicrobial
Multiple (<10)
Ceftizoxime
42
Antimicrobial
Multiple (<5)
Ceftriaxone‡
43,44
Antimicrobial
Multiple (≥10)
Cefuroxime
45
Antibacterial
Multiple (<5)
Acetaminophen (Paracetamol)
Cefotetan‡
Cephalexin
Method detecting serum antibody
HA
Positive
DAT
Drugcoated
RBCs
†
†
**
¶
**
†
46
Antimicrobial
Multiple (5)
¶
Antimicrobial
Multiple (≥10)
¶
Chloramphenicol
50
Antibacterial
Multiple (<5)
Chlorinated hydrocarbons
51
Insecticides
Multiple (<10)
Chlorpromazine
52
Antiemetic, antipsychotic
Multiple (<10)
Chlorpropamide‡
53,54
Antidiabetic
Multiple (<10)
Cimetidine‡
55
Antiulcerative
Single (2010)
Ciprofloxacin
56
Antibacterial
Multiple (<10)
57,58
Antineoplastic
Multiple (<10)
Cloxacillin
59
Antibacterial
Single (1980)
Cyclofenil
60
Gonad-stimulating
principle
Multiple (<5)
Cisplatin (Cisdiaminodichloroplatinum)
68
Not
reported
Reactive
without
drug added
in vitro
†
47–49
Cephalothin‡
Serum
+ drug
+ RBCs
**
**
¶
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
DIIHA: update since 2007
Table 1. Drugs associated with cases of IHA or positive DAT, or both, in which drug-dependent antibodies were detected* (continued)
Drug (alternative name)
Number of references
(single [year] vs.
multiple [<5, <10,
or ≥10])
Method detecting serum antibody
Positive
DAT
Serum
+ drug
+ RBCs
Reference
Therapeutic category
Cyclosporin (Cyclosporine)
61
Immunosuppressant
Multiple (<5)
Dexchlorpheniramine maleate
(Chlorpheniramine)
62
Antihistaminic
Single (1981)
63–66
NSAID
Multiple (≥10)
Diethylstilbestrol (Stilboestrol)
67
Estrogen
Multiple (<5)
Dipyrone
68
NSAID
Multiple (<5)
Erythromycin‡
69
Antimicrobial
Multiple (<5)
Etodolac
70
NSAID
Single (2000)
†
Etoricoxib
71
NSAID
Single (2013)
†
Ethambutol
17
Antibacterial
Single (2003)
Fenoprofen
72
NSAID
Single (1988)
Fluconazole
17
Antifungal
Single (2003)
Fluorescein
73
Injectable dye
Single (1993)
Fluorouracil
74
Antineoplastic
Multiple (<10)
Furosemide
40
Diuretic
Multiple (<5)
75,76
Analgesic
Multiple (<5)
Hydralazine
77
Antihypertensive
Single (1977)
Hydrochlorothiazide‡
78
Diuretic
Multiple (<10)
9-Hydroxy-methyl-ellipticinium
(elliptinium acetate)
79
Antineoplastic
Multiple (<5)
Hydrocortisone
80
Glucocorticoid
Single (2008)
Diclofenac‡
Glafenine (Glaphenine)
Ibuprofen
81
NSAID
Multiple (<5)
Imatinib mesylate
82
Antineoplastic
Multiple (<5)
Insulin
83
Antidiabetic
Multiple (<5)
Iomeprol
84
Radiopaque medium
Single (2013)
Isoniazid
85
Antimicrobial
Multiple (<10)
Latamoxef (Moxalactam)
75
Antimicrobial
Single (1985)
Levofloxacin (Ofloxacin)
86
Antibacterial
Multiple (<5)
Mefloquine‡
87
Antimicrobial
Multiple (<5)
Melphalan
88
Antineoplastic
Single (1967)
6-Mercaptopurine
89
Antineoplastic
Single (2000)
Methadone
90
Analgesic
Multiple (<5)
Methotrexate
91
Antineoplastic,
antirheumatic
Multiple (<5)
Metrizoic acid
92
Radiopaque medium
Single (1991)
Minocycline
93
Antibacterial
Single (1994)
Nabumetone
94
Anti-inflammatory,
analgesic
Single (2003)
Nafcillin‡
95
Antimicrobial
Multiple (<10)
Naproxen
96
Anti-inflammatory,
analgesic, antipyretic
Multiple (<5)
Nitrofurantoin
97
Antibacterial
Single (1981)
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
HA
Drugcoated
RBCs
Not
reported
†
Reactive
without
drug added
in vitro
**
**
**
**
†
†
**
**
**
†
69
G. Garratty and P.A. Arndt
Table 1. Drugs associated with cases of IHA or positive DAT, or both, in which drug-dependent antibodies were detected* (continued)
Method detecting serum antibody
Reference
Therapeutic category
Number of references
(single [year] vs.
multiple [<5, <10,
or ≥10])
Nomifensine§
98
Antidepressant
Multiple (≥10)
Norfloxacin
99
Antimicrobial
Single (1999)
Oxaliplatin‡
100, 101
Antineoplastic
Multiple (≥10)
p-Aminosalicylic acid (PAS) (paraaminosalicylsäure)
102
Antimicrobial
Multiple (<10)
Pemetrexed
103
Antineoplastic
Multiple (<5)
Penicillin G‡
104, 105
Antimicrobial
Multiple (≥10)
106
NSAID
Multiple (≥10)
17
Anticonvulsant,
antiarrhythmic
Single (2003)
Piperacillin‡
107
Antimicrobial
Multiple (≥10)
**
Probenecid‡
108
Uricosuric
Multiple (<5)
**
Propyphenazone
109
NSAID
Single (1998)
Puerarin
110
Chinese herb
Multiple (<5)
Pyrazinamide
17
Antibacterial
Single (2003)
Drug (alternative name)
Phenacetin‡ (Acetophenetidin)
Phenytoin (Fenitoine)
Pyrimethamine (Pirimetamine)
17
Antimicrobial
Multiple (<5)
Quinidine
111
Antiarrhythmic,
Antimicrobial
Multiple (≥10)
Quinine
106
Antimicrobial
Multiple (<10)
Ranitidine
112
Antiulcerative
Multiple (<5)
Rifabutin
17
Antibacterial
Single (2003)
113–115
Antibacterial
Multiple (≥10)
116
Antimicrobial
Multiple (<5)
Rifampin‡ (Rifampicin)
Stibophen
HA
Positive
DAT
Drugcoated
RBCs
Serum
+ drug
+ RBCs
†
¶
Not
reported
Reactive
without
drug added
in vitro
**
**
**
**
Streptokinase
117
Thrombolytic
Single (1989)
Streptomycin
118–120
Antimicrobial
Multiple (<10)
Sulfasalazine
121
Anti-inflammatory
Multiple (<5)
Sulfisoxazole
17
Antibacterial
Single (2003)
Sulindac
122
Anti-inflammatory
Multiple (<10)
**
Suprofen
123
NSAID
Single (1989)
**
Tartrazine
124
Colorant
Single (1979)
Teicoplanin
125
Antimicrobial
Multiple (<5)
Temafloxacin§
126
Antimicrobial
Multiple (<5)
Teniposide
127
Antineoplastic
Single (1982)
Tetracycline
128
Antimicrobial
Multiple (<10)
Thiopental sodium
129
Anesthetic
Single (1985)
Ticarcillin‡
130
Antimicrobial
Multiple (<5)
Tolbutamide
131
Antidiabetic
Multiple (<5)
Tolmetin‡
132
NSAID
Multiple (≥10)
Triamterene
133
Diuretic
Multiple (<5)
Trimellitic anhydride
134
Used in prep of resins,
dyes, adhesives, etc.
Multiple (<5)
70
**
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
DIIHA: update since 2007
Table 1. Drugs associated with cases of IHA or positive DAT, or both, in which drug-dependent antibodies were detected* (continued)
Drug (alternative name)
Reference
Therapeutic category
Number of references
(single [year] vs.
multiple [<5, <10,
or ≥10])
Trimethoprim and
Sulfamethoxazole‡
135
Antibacterial
Multiple (<5)
Vancomycin
17
Antibacterial
Single (2003)
Vincristine
136
Antineoplastic
Single (2013)
Zomepirac
137
NSAID
Single (1983)
Method detecting serum antibody
HA
Positive
DAT
Drugcoated
RBCs
Serum
+ drug
+ RBCs
Not
reported
Reactive
without
drug added
in vitro
IHA = immune hemolytic anemia; DAT = direct antiglobulin test; HA = hemolytic anemia; RBCs = red blood cells; NSAID = nonsteroidal anti-inflammatory
drug.
* When a drug antibody is indicated to be reactive by two methods, e.g., vs. drug-treated RBCs and when serum + drug + RBCs are mixed together, not all
cases necessarily had drug antibodies reactive by both methods. Using ampicillin, for example, four reported antibodies reacted with drug-treated RBCs and
were either nonreactive (n = 1) or not tested (n = 3) by the serum + drug + RBCs method, and two antibodies reacted when serum + drug + RBCs were
tested but were nonreactive with drug-treated RBCs.
† One or more samples only positive or strongest reactions seen with ex vivo (urine or serum) or metabolite.
‡ Cases of DIIHA or positive DAT caused by these drugs have been identified in Dr. Garratty’s laboratory.
§ No longer manufactured.
¶ Associated with nonimmunologic protein adsorption (NIPA).
**One or more samples positive, possibly owing to the presence of circulating drug or drug-antibody immune complexes.
Table 2. Drugs associated with cases of IHA or positive DAT or both in which only drug-independent antibodies (autoantibodies) were
detected
Reference
Therapeutic category
Number of references
(single [year] vs. multiple
[<5, <10, or ≥10])
Alemtuzumab
138
Antineoplastic; immunosuppressant
Multiple (<5)
Bendamustine
139
Antineoplastic
Multiple (<5)
Captopril
140
Antihypertensive
Multiple (<5)
Drug (alternative name)
Chaparral
141
Herbal
Single (1980)
Cimetidine
142
Antiulcerative
Multiple (<10)
Cladribine (2-chloro-deoxyadenosine)
143
Antineoplastic
Multiple (<10)
Fenfluramine
144
Anorexic
Single (1973)
Fludarabine*
145,146
Antineoplastic
Multiple (≥10)
Interferon
147
Antineoplastic, antiviral
Multiple (≥10)
Interleukin-2
148
Antineoplastic
Multiple (<5)
Ketoconazole
149
Antifungal
Single (1987)
Lenalidomide
150
Immunomodulatory
Single (2006)
Levodopa (L-dopa)
151
Antiparkinsonian
Multiple (≥10)
Mefenamic acid
152
NSAID
Multiple (≥10)
Mesantoin (Mephenytoin)
153
Anticonvulsant
Single (1953)
Methyldopa*
154
Antihypertensive
Multiple (≥10)
Nalidixic acid
155
Antibacterial
Multiple (<10)
Procainamide*
156,157
Antiarrhythmic
Multiple (<10)
Rituximab
158
Antineoplastic
Single (2003)
Tacrolimus
159
Immunosuppressant
Multiple (<10)
Weidean
160
Chinese herbs
Single (2009)
HA
Positive DAT
More
evidence
needed
IHA = immune hemolytic anemia; DAT = direct antiglobulin test; HA = hemolytic anemia; NSAID = nonsteroidal anti-inflammatory drug.
*Cases of drug-induced immune hemolytic anemia or positive DAT caused by these drugs have been identified in Dr. Garratty’s laboratory.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
71
G. Garratty and P.A. Arndt
Table 3. Drugs associated with the detection of nonimmunologic protein adsorption onto RBCs
Reference
Therapeutic category
Number of references
(single [year] vs. multiple
[<5, <10, or ≥10])
35,36
Antimicrobial
Multiple (≥10)
Cephaloridine
48
Antimicrobial
Multiple (<5)
Cephalothin*
47,48
Antimicrobial
Multiple (≥10)
58
Antineoplastic
Multiple (<10)
Clavulanate potassium* (Clavulanic acid)
161,162
β-Lactamase inhibitor
Multiple (<5)
Diglycoaldehyde (INOX)
Drug (alternative name)
Cefotetan*
Cisplatin
163,164
Antineoplastic
Multiple (<5)
Oxaliplatin*
101
Antineoplastic
Multiple (≥10)
Sulbactam*
161,165
β-Lactamase inhibitor
Multiple (<5)
166
Antihelminthic, antiprotozoal
Single (1988)
167,168
β-Lactamase inhibitor
Multiple (<5)
Suramin
Tazobactam*
HA
Positive
DAT
Drug-dependent
antibody(ies)
also detected
RBCs = red blood cells; HA = hemolytic anemia; DAT = direct antiglobulin test; IHA = immune hemolytic anemia.
*Cases of drug-induced immune hemolytic anemia or positive DAT caused by these drugs have been identified in Dr. Garratty’s laboratory.
Table 4. Drug antibodies detected by the Research Lab at the
American Red Cross in Pomona, CA, from 1978 to 2013
Years
PlatinumPiperacillin based drugs Other drugs
Ceftriaxone
Cefotetan
1978–83
0
0
0
0
7
1984–89
2
0
0
0
4
1990–95
2
20
0
0
7
1996–2001
6
45
2
0
6
2002–07
7
15
6
3
7
2008–13
14
13
30
5
6
Total
31
93
38
8
37
Drug Antibodies Showing Blood Group Specificity
I (G.G.) have been interested in this area for more than
30 years. The first report (in 1977) concerned a streptomycin
antibody having Rh specificity.118 In 1981, Duran-Suarez et
al.97 described antibodies to three drugs with I specificity. In
1984, we described a chlorpropamide-induced autoanti-Jka.54
In 1983 and 1985, Habibi et al.129,170 and, in 1986, Salama
and Mueller-Eckhardt171 added more drugs showing this
phenomenon. The list has grown quite long (see Table 1 of
Arndt6), but this is still not a common finding. There are many
piperacillin antibodies showing blood group antigen specificity
that have been described since 1984.107,172 Most of these were
Rh specificities (especially anti-e or “relative” anti-e specificity).
Of 37 piperacillin antibodies we were sent to investigate, the
referring laboratories detected 8 anti-e, 1 anti-e–like antibody,
and 1 possible anti-f when testing serum without piperacillin
added in vitro (in vivo circulating piperacillin was most likely
72
present). Twenty-seven of these were tested with R1R1 and
R2R2 RBCs in the presence of in vitro added piperacillin; 16 of
27 (59%) were nonreactive (7) or weaker (9) with R2R2 RBCs
(e.g., e or relative e specificity), and 2 (8%) were weaker with
R1R1 RBCs (relative c specificity).172
In 2013, we reported results of tests from our own
laboratory during the last 30 years on 53 antibodies to 13
different drugs.172 We found no blood group specificity in
single examples of antibodies to acetaminophen, phenacetin,
probenecid, quinidine, teniposide, cefotaxime, cimetidine, or
diclofenac, four examples of anti-ceftriaxone, or ten examples of
anti-cefotetan. We found some blood group antigen specificities
associated with sulfamethoxazole (MNS and Lutheran) and
piperacillin (Rh and Lutheran). Some specificities only became
apparent when the antibodies were diluted.
Drug Antibodies Detected in Healthy Blood Donors
We have known for many years that healthy humans can
have drug antibodies in their plasma or serum. Low-titer, often
IgM, penicillin antibodies were detected in blood donors (5%)
or patients with DIIHA (6%). This finding did not confuse the
serologic diagnosis because patients with penicillin DIIHA
had very high titer (>1000), usually IgG, penicillin antibodies.
About 30 to 40 percent of blood donors were found to have
antibodies to cephalothin and ticarcillin. Problems were
encountered when it was noticed that 80 percent of donors
and patients with no DIIHA had cefotetan antibodies; luckily,
patients with DIIHA had very high titers of cefotetan antibodies
compared with patients with no HA. The most interesting
results in this area involved piperacillin. In 2008, we reported
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
DIIHA: update since 2007
that 91 percent of donors’ sera and 49 percent of random
patients’ sera, contained piperacillin antibodies reactive
against piperacillin-coated RBCs.173 Luckily, we determined
that antibodies to piperacillin (unlike antibodies to other
penicillins) react well by adding soluble drug solution to the
patients’ sera, and this reactivity correlated with the DIIHA.
We no longer use piperacillin-coated RBCs in screening tests.
A few other drug antibodies have been detected in healthy and
sick individuals with no DIIHA (oxaliplatin,174 cisplatin,174 and
meropenem175).
It is not known why some of the preceding drug antibodies
are found in healthy donors or why they sometimes appear
more commonly in healthy donors than in random patients. For
instance, 91 percent of donors and only 49 percent of random
patients have piperacillin antibodies; 16 percent of donors and
only 4 percent of patients have oxaliplatin antibodies.
It has been suggested that environmental factors may
be responsible for the production of antibodies. For instance,
automobile catalytic convertors release platinum into the
atmosphere and may explain the production of antibodies
to drugs that belong to the platinum family (oxaliplatin,
carboplatin, and cisplatin). Another major concept to explain
the production of antibodies to antibiotics in individuals with no
DIIHA is that, in the United States, antibiotics are routinely fed
to cattle and chickens, so we are continually exposed to drugs
such as the cephalosporins and penicillins. Many countries in
Europe have banned this practice mainly to prevent problems
with treatment resistance of many organisms.
DIIHA Associated With Transplantation
Many of the drugs used for transplantation are designed
to affect the immune system, so we should not be surprised to
see true AIHA develop sometimes. Trying to prove the cause
of HA or production of RBC autoantibodies (e.g., positive DAT
or IAT) is a difficult task, but DIIHA has to be considered.
Examples of drugs said to cause DIIHA after transplantation
are cyclosporine, tacrolimus, and the combination of
alemtuzumab, mycophenolate, and daclizumab. It is almost
impossible to prove that the HA is caused by one of these drugs,
so publications (e.g., Elimelakh et al.138) often hypothesize
that the combination of such drugs has caused the immune
aberrations. There are often no demonstrable drug-dependent
antibodies but often the presence of drug-independent
antibodies (autoantibodies). Some interesting points are
(1) some of the AIHAs have not been warm AIHA but are
associated with cold agglutinins, (2) there are interesting
associations with posttransplant lymphoproliferative disorder
(PTLD),159 and (3) the hemolytic anemia can develop long after
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
the transplant and long after therapy with the suspected drug
(e.g., up to 2 years).
DiGiuseppe et al.159 reported on a child who had a liver
transplant at 1 year old; he was successfully maintained on
cyclosporine for 4 years and then switched to tacrolimus,
which was increased from 2 mg to 8 mg twice daily. The
patient developed AIHA; his hemoglobin fell to 3.5 g/dL
after transfusion of one unit of RBCs, and he died. Postmortem examination revealed a clinically unsuspected PTLD.
Elimelakh et al.138 reported on cases of red cell aplasia (RCA)
and AIHA after immunosuppression with alemtuzumab (antiCD52), mycophenolate mofetil (MMF), and daclizumab (antiTac/CD25) in pancreas transplant patients. Data from a 2-year
period were reported for 357 pancreas transplants. AIHA was
detected in 16 patients (7 of these also had RCA; 3 had RCA
only). All were DAT+ (all but one had RBC-bound IgG + C3).
When MMF was discontinued in the RCA/AIHA group, seven
patients recovered from RCA, but only three also recovered
from the HA. In the AIHA-alone group (nine patients), MMF
was discontinued in two patients with no effect on the HA.
Other patients in this group were treated with steroids,
rituximab, intravenous IgG, splenectomy, or plasma exchange,
with remission seen in only two of the nine patients. The
authors suggested that a combination of the drugs may have
been involved in the AIHA. It is interesting that the RCA,
which is thought to be caused by autoantibody to early RBC
precursors, appeared to be caused by MMF.
Drug-Induced AIHA Associated With
Chemotherapeutic Drugs
Although several chemotherapeutics have been suggested
to cause DIIHA, similar problems to those of the transplant
associations make the diagnosis difficult. Often several drugs
are involved and no drug-dependent antibodies are detectable;
the antibodies are usually drug-independent and the patient
appears to have developed a true AIHA. The best examples
of such drugs are the purine analogs (e.g., fludarabine and
cladribine) used to treat patients with chronic lymphocytic
leukemia (CLL). Hemolytic anemia after fludarabine therapy
occurred in 14 of 66 (22%),176 9 of 52 (17%),177 5 of 36 (14%),178
and 5 of 104 (5%)146 patients. The analyses of patients with
CLL are more complex, with many more confounding factors
than the data accumulated in the 1970s on the prototype drug
(methyldopa), which inclues RBC autoantibodies and AIHA in
patients taking the drug for hypertension. Patients with CLL
are a very different group of patients, as CLL is known to be
associated with positive DATs and AIHA without any drug
involvement. Nevertheless, there are reports of exacerbation
73
G. Garratty and P.A. Arndt
of AIHA that was present before fludarabine therapy. There
are fewer reports of AIHA caused by fludarabine in de novo
CLL patients receiving fludarabine for the first time compared
with patients who have had multiple courses of alkylating
agents among whom the prevalence is about 20 percent. There
are reports of catastrophic hemolysis (some fatalities) after
fludarabine therapy.
In 2008, an interesting study of 777 patients with CLL
randomly assigned to receive chlorambucil (C) or fludarabine
(F) alone or with cyclophosphamide (FCy) was published.179
Fourteen percent of these patients had a pretreatment positive
DAT. Only 28 percent of patients with positive DATs had an
associated HA. Of 249 patients, those treated with F were
most likely to become DAT-positive. Patients treated with F
or C were twice as likely as those treated with FCy to have
AIHA. The authors concluded that a positive DAT was a good
prognostic indicator and that FCy combination therapy may
protect against AIHA. There is an excellent review on AIHA in
patients with CLL by Hamblin.180
In one publication, 300 patients with CLL taking
fludarabine, cyclophosphamide, and rituximab were followed.181
Nineteen (6.5%) developed HA. The authors considered these
to be AIHA even though 82 percent of the DATs were negative.
Such cases of DAT-negative AIHA have been reported in other
publications on fludarabine-induced HA.
Recently, there has been an interesting association
between fludarabine and bendamustine, a chemotherapeutic
drug that is also used for CLL and lymphoma. Several cases of
bendamustine-associated AIHA (involving drug-independent
autoantibodies) have been described. Goldschmidt et al.139
reported on five cases of AIHA in 31 CLL patients treated
with bendamustine; none of the non-CLL patients developed
AIHA; and two of five cases were DAT-negative. All five
cases had received fludarabine previously. One single case of
bendamustine-associated AIHA was DAT-negative and had
received no previous fludarabine.182 Although fludarabine is
a purine nucleoside and bendamustine is an alkylating agent,
it contains a purine-like benzimidazole ring. This might
explain why the bendamustine appeared to give a more severe
“secondary” response in patients who had sometimes had a
less severe HA caused by fludarabine.
Closing
As new therapeutic drugs appear, we will need to be aware
of their potential for inducing drug-dependent antibodies,
drug-independent antibodies, or NIPA. The diagnosis is not
always easy, and as stated in previous summaries,2,4 when
74
studying DIIHA, we typically end up with more questions
than answers!
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George Garratty, PhD, FRCPath, Scientific Director, and Patricia A.
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Region, 100 Red Cross Circle, Pomona, CA 91768.
79
Case R ep ort
Diagnostic pitfalls of drug-induced immune
hemolytic anemia
A. Salama and B. Mayer
Immune hemolytic anemia (IHA) is a rare complication of drug
administration. However, its true incidence remains obscure, as
there are a number of factors that may lead to misdiagnosis. The
clinical and serologic pictures are variable, and there is a great deal
of unawareness that certain drugs can cause IHA. Furthermore,
serologic results can be easily misinterpreted, resulting in a
wrong diagnosis. Immunohematology 2014;30:80–84.
Key Words: drug-induced antibody, immune hemolytic
anemia, drug-dependent antibody, autoantibodies, immune
hemolysis, hemoglobinuria, hemoglobinemia, intravascular
hemolysis
Although drug-induced immune hemolytic anemia
(DIIHA) is a well-known complication of drug administration,
a number of diagnostic pitfalls still occur on the patient’s side,
at patient bedside, and in laboratories.
The key to a correct diagnosis is a sophisticated history of
drug administration and sufficient clinical as well as serologic
experience in this field. As demonstrated in this issue, the list
of drugs implicated in DIIHA continues to grow, not only by a
variety of new drugs but also by known drugs. However, some
published data rely solely on temporal relationships between
hemolysis and drug administration without confirmation by
serologic testing. On the other hand, there is evidence that
DIIHA is far more common than has yet been estimated.
Ultimately, serologic testing is frequently inadequate or
even lacking. In addition, some cases remain unrecognized
or unreported. In this review, we attempt to highlight the
main errors leading to false diagnosis or a lack of diagnosis.
To provide clarity to such errors, we first describe a few
representative cases.
Representative Cases
Cases 1 and 2
A 54-year-old woman was admitted for hematuria and
anemia. She rapidly recovered during clinical observation,
presumably as a result of unintended discontinuation of
all medications. Serologic testing revealed an only C3dpositive direct antiglobulin test (DAT), and her plasma was
80
observed to be reddish. Based on these findings, DIIHA was
suspected. However, the patient was discharged from the
hospital without diagnosis. The patient was readmitted on
the following day with massive hemolysis, thrombocytopenia,
and shock, and subsequently died because of complications
resulting from massive hemolysis. From a clinical viewpoint,
an acute Evans syndrome was initially suggested. However,
we highly suspected a DIIHA. The patient was revealed
to have received at irregular intervals antihypertensive
drugs including nifedipine and a combination of buthiazide,
reserpine, rescinnamine, raubasine, and potassium chloride.
After several weeks of uninformative serologic testing, the
drugs were ingested by a volunteer. Serologic testing in the
presence of serum samples collected after drug ingestion (ex
vivo antigen) disclosed the buthiazide as the causative drug.1
In that study, we also described a second patient (Case 2) who
received an intramuscular injection containing a drug mixture
because of a prolapsed disk. The patient later experienced
shock and renal failure that could not be clinically explained.
We further suspected DIIHA, but serologic testing was
unsuccessful, even in the presence of ex vivo antigens of the
injected drugs. On repeat questioning of the patient several
months later, she declared that she had ingested nomifensine
(antidepressant) before the onset of lumbar pain. She had not
previously declared this owing to feelings of shame because of
her depression and therapy.
Case 3
A 79-year-old woman had abdominal pain and her urine
was reddish. Because of suspected hematuria and urinary
tract infection, treatment with an antibiotic drug was started;
subsequently her condition abruptly deteriorated, eventually
resulting in shock. The patient was immediately transferred
to the hospital, where urosepsis, shock, and hemolysis were
suspected. A blood sample was referred to our laboratory as all
crossmatched packed red blood cell (RBC) units were positive
at the hospital providing treatment. On the basis of this history,
we suspected DIIHA and advised an immediate transfusion.
Unfortunately, the patient died, and the question as to which
drug she had received before the development of abdominal
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Diagnostic pitfalls
pain and hematuria could not be answered. On further
investigation, evidence of vitamin E was found in her handbag.
The drug prescribed from her physician was, we believe,
harmless, as hemolysis was present before deterioration of her
condition. In an attempt to determine the causative drug, the
patient’s belongings were searched and more than 40 drugs
were found (Fig. 1). Thus, it was impossible to identify the
causative drug. Similar cases may remain unrecognized either
owing to DIIHA not being suspected or owing to inadequate
serologic testing or a lack in its implementation.
Fig. 1 Numerous drugs found in the possession of a patient who
died of drug-induced immune hemolytic anemia (Case 3).
Case 4
In 2010, we described a patient with fatal immune
hemolysis caused by specific antibodies to 5-fluorouracil
(5-FU).2 The patient had cholangiocarcinoma and was
receiving chemotherapy including oxaliplatin and 5-FU.
Before admission, the patient had been repeatedly treated (15
times) with the same combination of drugs without relevant
side effects. She experienced fatal hemolysis that was initially
concealed by a blood transfusion. Furthermore, serologic
findings (positive antibody screening test and positive C3dDAT) were misinterpreted as an in vitro phenomenon caused
by cold agglutinins. After thorough clinical and serologic
reinvestigation of the case, it became clear that hemolysis was
related to drug administration. Initially, oxaliplatin, a drug
frequently resulting in immune hemolysis, was suspected.
Only after dialysis of the patient’s serum and intensive
serologic retesting with several ex vivo metabolites was 5-FU
determined to be the cause of the hemolysis.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Pitfalls and Limitations
Pitfalls on the Patient’s Side
As described above in our case reports, information
concerning the patient plays a key role in clinical and serologic
aspects. The vast majority of patients are not informed about
symptoms of hemolysis and that drugs may, although rarely,
result in hemolysis.
The clinical picture of DIIHA is extremely variable,
and the affected patients are usually unable to recognize a
relationship between the drugs and secondary symptoms
of hemolysis, i.e., tachycardia or dyspnea caused by anemia,
abdominal pain, lumbar pain, and red urine (hemoglobinuria)
caused by intravascular hemolysis. Depending on the patient’s
information and the most prominent symptoms, the following
errors may occur before or after consideration of DIIHA as a
possible diagnosis:
1. Some patients may recover without further investigations,
with the diagnosis remaining unclear.
2. Isolated patients deny drug ingestion, i.e., antidepressant
drugs (Case 2), herbal remedies, or other drugs.
3.The dilemma commonly faced is that some patients
infrequently or simultaneously ingest various drugs;
therefore, it is sometimes impossible to identify what
drug(s) could have caused the hemolysis and drug testing is
not possible, even when DIIHA is suggested. We are aware
of several cases in which we were unable to identify the
causative drug because of these factors.
4. Some patients, as well as physicians, automatically exclude
long-term administered drugs that have not presented with
prior signs of complications.
5. It is largely unknown that drug administration does not
result in significant primary immunization within the
first 5 days if the patient has not previously received the
drug. Thereafter, immune reaction may occur at any time
during continuous or intermittent administration or after
reexposure of the causative drug.3
Pitfalls at Patient Bedside
The clue to correct diagnosis of DIIHA primarily remains
in the hands of the primary physician. If this physician is
not familiar with all aspects of acquired immune hemolytic
anemias, this may then result in numerous errors.
1. Unfortunately, many physicians are not familiar with
DIIHA. They are unaware that clinical manifestations
and pictures are variable. Furthermore, there remains
a lack of knowledge from a clinical or serologic
viewpoint concerning the difference between DIIHA by
81
A. Salama and B. Mayer
2.
3.
4.
5.
82
autoantibodies (aAbs) alone and that induced by drugdependent antibodies (ddAbs). These deficiencies are the
reasons behind the various failures at the clinical bedside
and in laboratories (see subsequent discussion).
Patients in addition to physicians seem to exclude drugs as
a cause of acute reactions when they have been tolerated
for a long time. Many physicians do, in fact, consider
DIIHA in many patients with hemolysis. However, they
seem to suspect, in general, only newly administered
drugs. Most frequently, patients are asked whether they
have altered their medications or received a new drug.
Detailed information including drug history, onset and
type of hemolysis, clinical picture, and course are rarely
documented.
Inconclusive patient or drug history may lead to confusion
with autoimmune hemolytic anemia (AIHA) of the warm
type, particularly if the drug has led to the production of
ddAbs or aAbs, leading to positive immunoglobulin G (IgG)
DAT. Only a few drugs are known to cause the production
of the latter antibodies alone (e.g., methyldopa, fludarabine),
and some drugs have been reported to cause both types
(e.g., diclofenac, cefotetan).4,5 It is unknown how many
cases of warm AIHA are in fact not idiopathic but related to
drugs. On physical examination, there are no characteristic
findings of AIHA related to drugs. Autoantibodies induced
by drugs are also serologically indistinguishable from
“idiopathic” autoantibodies. Unfortunately, hemolysis
related to drug-induced aAbs may persist for several weeks
or even months after discontinuation of the causative
drug. In such cases, recovery could be attributed to
treatment rather than to stopping the drug. Thus, these
cases remain unrecognizable as DIIHA.
On admission, almost all patients who exhibit immune
hemolysis automatically receive high doses (at least 0.5–
1.0 mg/kg body weight) of steroids. In the case of DIIHA,
an improvement caused by intentional or unintentional
cessation of the causative drug might be attributed to
treatment and not to discontinuation of the drug(s). The
incidence of these cases remains obscure.
Confusion with acute hemolytic transfusion reactions is
also relatively common. Some patients may receive drugs
that cause acute hemolysis before or simultaneously with
blood transfusion. In most of these cases, a hemolytic
transfusion reaction (HTR) will primarily be suspected
rather than DIIHA (Case 4). The correct diagnosis can
only be established if laboratory testing does not confirm
either HTR or AIHA. This result is supported by several
reported2,6–11 and unreported cases.
6. A fatal failure is the administration of the causative drug
during the hemolytic attack. This failure may occur as
long as the true diagnosis is unclear or when true AIHA
has been suggested. Most dangerous is when the main
symptom of the underlying disease is similar to that of
intravascular hemolysis. We were aware of some patients
who received diclofenac because of chronic lumbar pain
and were administered the drug again after development
of acute lumbar pain related to massive intravascular
hemolysis rather than to the chronic affliction.
7. Infrequently, only secondary symptoms of DIIHA may
initially be significant, leading to misdiagnosis or no
diagnosis at all. During observation, the causative drugs
are often automatically ceased. If these patients do not
exhibit complications, they may quickly recover and be
discharged without a final diagnosis (see described cases).
Pitfalls in the Laboratory
Similar to the situation at the clinical bedside, many
serologists are not well informed about all the serologic
and clinical aspects of DIIHA. This makes correct testing
and interpretation of serologic findings often impossible.
Unfortunately, to date, there remain no standard tests or
guidelines to confirm serologic findings by certified laboratories
similar to those for blood cell antigens and antibodies. These
deficiencies represent risks of pitfalls leading to false-positive
and false-negative results, misdiagnosis, or no diagnosis.
False-Positive Results
1. Use of enzyme-treated RBCs may result in nonspecific
agglutination in the presence of abnormal patient’s
serum but not in the presence of normal serum. Indeed,
the samples are not identical, and in vitro–treated RBCs
are sometimes more susceptible to patient’s serum
samples than to normal serum samples. We have not
observed a reaction between specific ddAbs and enzymetreated RBCs, and not with native cells. However, some
investigators have found that the use of enzyme-treated
RBCs can be important for enhancing the detection
of ddAbs. In these cases, it is indispensable to use an
appropriate negative control (saline instead of the drug; if
both react similarly, then a ddAb is not present).
2. Use of in vitro–treated RBCs may result in nonspecific
reactions (nonimmunologic protein adsorption) similar
to those observed by using enzyme-treated RBCs. This
phenomenon has been described for different drugs
including β-lactamase inhibitors, platinum, and some
cephalosporins.12
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Diagnostic pitfalls
False-Negative Results
Different reasons may lead to false-negative results.
1. Testing of other drugs but not the causative drug may
occur in cases in which a number of drugs are suspected
or when only an innocent drug is suspected (see described
cases).
2. Some laboratories still use test systems requiring washing
procedures. Because the vast majority of drugs do not
bind tightly to RBCs, ddAbs may escape detection if the
wash solution used does not contain the causative drug or
metabolites (1 mg/mL).13
3. Some ddAbs recognize, in the presence of the drug,
RBC antigens and do not react with cells lacking the
corresponding antigen.14–17 This phenomenon must be
considered by using different or pooled RBCs and, most
importantly, the patient’s RBCs.
4. Some ddAbs react with RBCs only in the presence of
drug metabolites and not with the native drug (Cases 1
and 2). Because drug metabolites are usually not available
or remain unknown, the use of ex vivo preparations, e.g.,
urine or serum from persons receiving the suspected
drug, is obligatory in all suspected cases that demonstrate
negative results in the presence of the native drug.14,15 In
such cases, we recommend the use of different ex vivo
preparations (urine from the patient and urine or serum
from different persons taking the drug) to include rare or
even private metabolites.
5. Some ddAbs may become, within a relatively short time,
undetectable in the patient’s serum. Thus, in such cases,
negative results do not exclude DIIHA. Unfortunately,
there is little information regarding this aspect. However,
we are aware of isolated cases in which the causative
antibody became undetectable within a 2-week period
or less of the event occurring, i.e., diclofenac-dependent
antibodies and one iohexol-dependent antibody. Initially,
these antibodies were detectable in the presence of the
drug or its metabolites.
Misdiagnosis
The most common misdiagnoses are as follows:
1. Warm AIHA in cases in which aAbs are detectable. In
addition to the phenomenon that drugs may lead to the
production of aAbs or ddAbs, some drugs also result in
the production of both types in the same patient. Thus,
the presence of aAbs does not exclude ddAbs. The latter
antibodies must be suspected in all cases in which
intravascular hemolysis abruptly develops. In all these
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
cases, a C3d-positive DAT (with or without IgG) is a
cardinal finding.3
2. Panagglutinating antibodies that may be attributed to
cold agglutinins of high thermal amplitude, agglutinating
IgM warm aAbs, unspecific reactions, or IgG warm
aAbs, sometimes with specificity. This phenomenon is
characteristic in many cases during the acute phase of
hemolysis and when the drug and its metabolite are still
present in the circulation.
3. Acute HTRs are primarily suspected in nearly all cases in
which blood transfusion is administered before or during
hemolysis.2,6–11 If serologic testing remains inconclusive, a
misdiagnosis will result even when alloantibodies are not
detectable.
4. Infrequently, toxic reactions might be suggested if ddAbs
escape serologic testing, e.g., contrast media.18
No Diagnosis
Some patients have a history of DIIHA but have not been
investigated at the appropriate stage. We are aware of several
cases in which DIIHA appears to have developed, but could
not be excluded or proven.
References
1. Salama A, Mueller-Eckhardt C, Kissel K, Pralle H, Seeger W.
Ex vivo antigen preparation for the serological detection of
drug-dependent antibodies in immune haemolytic anaemias.
Br J Haematol 1984;58:525–31.
2. Yürek S, Riess H, Kreher S, Dörken B, Salama A. Fatal immune
haemolysis due to antibodies to individual metabolites of
5-fluorouracil. Transfus Med 2010;20:265–8.
3. Salama A. Drug-induced immune hemolytic anemia. In: Sipes
IG, McQueen CA, Gandolfi AJ, eds. Comprehensive toxicology.
Vol 4. 1st ed. New York, NY: Pergamon, 1997:73–85.
4.Salama A. Drug-induced immune hemolytic anemia. Expert
Opin Drug Saf 2009;8:73–9.
5.Garratty G. Immune hemolytic anemia caused by drugs.
Expert Opin Drug Saf 2012;11:635–42.
6.Ahrens N, Genth R, Salama A. Belated diagnosis in three
patients with rifampicin-induced immune haemolytic anaemia.
Br J Haematol 2002;117:441–3.
7. Shirey R, Iding J, King KE, Ness PM. Drug-induced immune
hemolysis mimicking an acute hemolytic transfusion reaction
[abstract]. Transfusion 2005;45:100A–1A.
8.Shirey R, Yamada C, Ness PM, King KE. Drug-induced
immune hemolytic anemia imitating a severe delayed hemolytic
transfusion reaction [abstract]. Transfusion 2007;47:185A.
9. Stroncek D, Procter JL, Johnson J. Drug-induced hemolysis:
cefotetan-dependent hemolytic anemia mimicking an acute
intravascular immune transfusion reaction. Am J Hematol
2000;64:67–70.
83
A. Salama and B. Mayer
10. Leaf DE, Langer NB, Markowski M, Garratty G, Diuguid DL.
A severe case of cefoxitin-induced immune hemolytic anemia.
Acta Haematol 2010;124:197–9.
11.Gupta S, Piefer CL, Fueger JT, Johnson ST, Punzalan RC.
Trimethoprim-induced immune hemolytic anemia in a
pediatric oncology patient presenting as an acute hemolytic
transfusion reaction. Pediatr Blood Cancer 2010;55:1201–3.
12. Arndt P, Garratty G, Isaak E, Bolger M, Lu Q. Positive direct
and indirect antiglobulin tests associated with oxaliplatin can
be due to drug antibody and/or drug-induced nonimmunologic
protein adsorption. Transfusion 2009;49:711–18.
13. Salama A, Berghöfer H, Mueller-Eckhardt C. Detection of celldrug (hapten)-antibody complexes by the gel test. Transfusion
1992;32:554–6.
14.Salama A, Mueller-Eckhardt C. Rh blood group-specific
antibodies in immune hemolytic anemia induced by
nomifensine. Blood 1986;68:1285–8.
15. Salama A, Mueller-Eckhardt C. The role of metabolite-specific
antibodies in nomifensine-dependent immune hemolytic
anemia. N Engl J Med 1985;313:469–74.
16.Salama A, Kroll H, Wittmann G, Mueller-Eckhardt C.
Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drugdependent antibodies. Br J Haematol 1996;95:640–4.
17. Bougie D, Johnson ST, Weitekamp LA, Aster RH. Sensitivity to
a metabolite of diclofenac as a cause of acute immune hemolytic
anemia. Blood 1997;90:407–13.
18. Mayer B, Leo A, Herziger A, Houben P, Schemmer P, Salama
A. Intravascular immune hemolysis caused by the contrast
medium iomeprol. Transfusion 2013;53:2141–4.
Abdulgabar Salama, MD, Medical Director (corresponding author),
and Beate Mayer, MD, Immunohematology Reference Laboratory,
Institute of Transfusion Medicine, Charité—Universitätsmedizin
Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
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I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Report
How we investigate drug-induced immune
hemolytic anemia
R.M. Leger, P.A. Arndt, and G. Garratty
Drugs are a rare cause of immune hemolytic anemia, but an
investigation for a drug antibody may be warranted if a patient
has definitive evidence of immune hemolysis, other more common
causes of hemolysis have been excluded, and there is a good
temporal relationship between the administration of a drug and
the hemolytic event. Drug antibodies are either drug-dependent
(require drug to be in the test system) or drug-independent
(reactive without drug present in the test). Drug-dependent
antibodies are investigated by testing drug-treated red blood cells
(RBCs) or by testing RBCs in the presence of a solution of drug.
Drug-independent antibodies are serologically indistinct from
idiopathic warm autoantibodies and cannot be defined or excluded by serologic testing. Nonimmunologic protein adsorption,
caused by some drugs, is independent of antibody production but
may also cause immune hemolytic anemia. Serologic methods for
testing for drug antibodies are presented, and observations from
more than 30 years of this laboratory’s experience are discussed.
Immunohematology 2014;30:85–94.
Key Words: immune hemolytic anemia, drugs, antibodies,
drug-treated red blood cells, testing in presence of drug,
penicillin, cephalosporin, antibiotics
Dr. Garratty’s Immunohematology Research Laboratory
at the American Red Cross in Southern California has
investigated drug-induced immune hemolytic anemia (DIIHA)
for 35 years. When contacted to investigate a possible DIIHA,
we ask a series of questions:
• Does the patient have hemolytic anemia (e.g., decreased
hemoglobin or hematocrit, increased reticulocytes,
increased indirect bilirubin and lactate dehydrogenase,
decreased haptoglobin)?
•Does the patient have a positive direct antiglobulin
test (DAT)? Is there IgG or C3 or both present on the
patient’s red blood cells (RBCs)? The DAT is used to
establish an immune etiology for a hemolytic anemia.
For DIIHA the DAT should be positive for IgG or C3 (or
both) close to the time of the observed hemolysis. If not
tested until weeks later, the DAT may be weak or even
negative, especially if the patient has been transfused
with multiple RBC units.1
• Was an eluate tested and what were the results? The
typical prompt for pursuing DIIHA in a patient with
hemolytic anemia is a positive DAT and nonreactive
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
eluate. Some recent findings, however, show that an
eluate may be reactive while the patient is still receiving
the drug (e.g., piperacillin, cefotetan).
• What drug(s) is (are) the patient taking now or recently?
Have any been implicated in causing DIIHA? It is
important to obtain a complete medication history with
dates of administration. This includes antibiotics the
patient may have received for a surgery in the previous
2 to 3 weeks; such medications might only appear
on the surgical record and not on a listing of current
medications.
And most importantly:
•Is there a temporal relationship between the drug
administration and the hemolytic anemia? If the
hemolysis began before the drug was administered, then
there is no temporal relationship. In some situations,
there may not be any prior administration of the drug
in question (e.g., cefotetan), while in others, the patient
may have received the drug previously without any
obvious adverse effects (ceftriaxone, piperacillin).
In summary, because DIIHA is rare, the time and effort
of an investigation should only be undertaken when the
patient has definite evidence of a hemolytic anemia and a good
temporal relationship to treatment with a particular drug
exists. When this has been established, the clinician should
consider stopping the drug, and specimens should be collected
for an investigation.
The approach and methods described in this article have
been used in our laboratory for several decades.2 Additional
information gleaned from our experience is added here.
Types of Drug-Induced Antibodies
There are two types of drug-induced antibodies: drugdependent and drug-independent. Serologically, we can only
determine the presence of drug-dependent antibodies. Drugdependent antibodies are subdivided into those that react with
drug-treated RBCs (e.g., antibodies to penicillin and some
cephalosporins) and those that react with untreated RBCs
in the presence of a solution of the drug (e.g., antibodies to
85
R.M. Leger et al.
ceftriaxone and quinine). Some drug-dependent antibodies
react both with drug-treated RBCs and in the presence of
a soluble drug; others react by only one of these methods.
Drug-independent antibodies (e.g., autoantibodies induced
by methyldopa and fludarabine) have serologic reactivity
without the drug being present and are indistinguishable from
idiopathic IgG warm autoantibodies.
Nonimmunologic Protein Adsorption
Some drugs modify the RBC membrane and cause
nonimmunologic adsorption of protein (NIPA), resulting
in positive direct and indirect antiglobulin tests.3 NIPA is
independent of antibody production, but may be a cause of
hemolytic anemia.4 Drugs that cause NIPA include some
cephalosporins, platinum-based chemotherapies (cisplatin,
carboplatin, and oxaliplatin), and β-lactamase inhibitors
(clavulanic acid, sulbactam, and tazobactam). NIPA should be
suspected when a patient’s plasma or serum and most normal
plasma or sera react in an indirect antiglobulin test with
drug-treated RBCs, but the eluate from the patient’s RBCs is
nonreactive with the drug-treated cells. When testing RBCs
treated with a drug known to cause NIPA, the patient’s serum
and the controls (negative and positive) are tested both undiluted
(for detection of a hemolysin or a directly agglutinating drug
antibody) and at a dilution of 1 in 20 (to avoid NIPA in the
antiglobulin test). Normal sera diluted 1 in 20 generally do not
contain enough protein for NIPA to be detected.
To verify that NIPA has occurred, we test the patient’s
RBCs (DAT) or the drug-treated RBCs after incubation with
plasma (indirect antiglobulin test) using antihuman albumin
(Sigma-Aldrich, St. Louis, MO).5 The antihuman albumin is
not licensed for RBC indirect antiglobulin testing, so it must
be standardized for this purpose. To determine the appropriate
dilution of antihuman albumin, doubling dilutions prepared in
phosphate-buffered saline (PBS) are tested against untreated
and cephalothin-treated RBCs after incubation at 37°C with
normal plasma.5 The amount of protein that adsorbs to the
RBCs increases with time (e.g., 2 hours vs. 30 minutes). When
new dilutions of antihuman albumin are prepared, expected
reactivity with cephalothin-treated and -untreated RBCs
(after incubation with normal plasma) is verified.
presence of a soluble drug). It is preferable to have a sample
from when in vivo hemolysis was first observed. If the patient’s
serum or plasma appears to have an autoantibody, it is also
beneficial to have a sample collected 2 to 3 days after the drug
was stopped. This second sample helps to determine whether
the observed autoantibody reactivity is truly autoantibody
or caused by a drug antibody reacting with a circulating
drug present in the sample obtained while the patient is still
receiving the drug. This apparent autoantibody is observed
quite often with antibodies to piperacillin. Once the drug has
cleared from the patient’s circulation (e.g., about 1–2 days after
the drug is stopped), this apparent autoantibody reactivity
disappears, whereas true autoantibody would persist.
Initial immunohematology testing should be completed
before beginning drug studies. The presence or absence of
alloantibodies and autoantibodies and eluate results will
guide the selection of RBCs used for the drug studies and the
methods to be used. Autoantibodies will need to be removed
by adsorption before performing tests for drug antibodies for
the same reason they are removed by adsorption to detect the
presence of other alloantibodies.
We prepare eluates from the patient’s EDTA RBCs using
a rapid acid elution kit (e.g., Gamma Elu-Kit II, Immucor,
Norcross, GA). To wash the RBCs in preparation for the
elution, we substitute cold (4°C) low-ionic strength saline
for the kit wash solution. In 1995, we showed that washing
RBCs with the low-ionic kit wash solution can result in falsepositive eluates when high-titer antibodies are present.6 Cold
PBS can also be substituted but may be less efficient for lower
affinity antibodies. The original study on false-positive eluates
began with detection of anti-D in the eluate from D– RBCs
after adsorption with a polyclonal anti-D. Coincidental to that
research, anti-cefotetan was detected in acid eluates prepared
from maternal and cord RBCs that were collected many
weeks after the mother received cefotetan. In retrospect, we
realized that these eluates (prepared using the low-ionic kit
wash solution) probably gave false-positive results because
of nonspecific adsorption of high-titer anti-cefotetan to the
RBCs during the washing for the eluate preparation. This was
later confirmed with in vitro studies when the mother’s anticefotetan was adsorbed and eluted from untreated RBCs when
the kit wash solution was used but not when cold PBS was
used.6
Sample From Patient
Sample of Drug
We request at a minimum 5 mL of EDTA whole blood
and one or two 10-mL clot tubes (serum is required to detect
hemolysis or complement activation, e.g., for testing in the
86
We also request a sample of the drug under investigation
unless we already have it on hand (e.g., powder [1-g vial],
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Approach to investigating DIIHA
tablets, or capsules [2–4]; the drug should not be dissolved
before sending). We have not been successful using drugs in
a pediatric oral suspension formula because they hemolyze
RBCs.5,7
Although it would seem important to use the particular
drug that a patient received, it is optimal to use pure drug
rather than a commercial tablet, capsule, or suspension
product. These commercial formulations contain inert ingredients that can make it difficult to dissolve the drug and test
with RBCs. It is also possible that antibodies detected in drug
studies are directed against one of these inert ingredients
rather than the drug itself.8 Initial testing can be performed
with the particular drug the patient received, but positive
results should be confirmed using pure drug to ensure correct
interpretation. Drugs administered by intravenous injection
are free of excipients. Some pure drugs are available from
chemical companies (e.g., Sigma-Aldrich).
Some drugs are combinations of two drugs (e.g.,
Zosyn is a combination of piperacillin plus tazobactam).
Tazobactam causes NIPA, which can create a problem with
the interpretation of results, so it is preferable to test with the
separate components.
Test Methods
If the suspected drug has previously been reported as a
cause for DIIHA (see tables in Garratty and Arndt9), it is best
to follow the techniques used in the relevant report. If the drug
has never been reported or sufficient serologic details are not
provided, testing the patient’s serum and an eluate from the
patient’s RBCs against drug-treated RBCs and testing the
patient’s serum against untreated and enzyme-treated RBCs
in the presence of the soluble drug is recommended. Many
more drug antibodies are detected by testing in the presence of
soluble drug than by testing drug-treated RBCs (see Table 1 in
Garratty and Arndt9).
We perform all our DIIHA investigations in tubes. Salama
et al.10 described using the gel test for detecting drug antibodies.
We have done limited testing in the presence of soluble drug by
the gel test (ID-Micro Typing System, Pompano Beach, FL)
using buffered gel and anti-IgG, -C3d gel cards and comparing
the results with tube tests. Anti-ceftriaxone could be identified
by the gel test for 9 of 11 examples tested; the presence of anticeftriaxone could not be discerned for two examples. Some
agglutination and hemolysis detected by tube test was not
detected by the gel test, and some antiglobulin tests with the
PBS substituted for the drug were stronger by gel test (e.g., 2+
by gel vs. negative or ½+ by tube test).11
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Table 1. Preparation of drug-treated RBCs
Variable
Penicillin-treated RBCs
Cephalosporin-/other-treated
RBCs
RBCs, group O
Fresh, <7 days old
Fresh, <7 days old
Amount of drug
600 mg
400 mg
0.1 M sodium barbital
(pH 9.6–9.8)
Phosphate-buffered
saline (pH 7.1–7.4)
Volume buffer
15 mL
10 mL
Volume packed RBCs
1 mL
1 mL
Room temperature
37°C
1 hour
1 hour
Buffer (pH)
Temperature of
incubation
Time of incubation
RBCs = red blood cells.
Preparing Drug Solutions
The solution of drug must be isotonic for use with RBCs.
Dissolving drugs in PBS, pH 7.1 to 7.4, is the preferred method.
Some drugs are readily soluble in PBS, others are somewhat
soluble, and still others are insoluble. The Merck Index is an
excellent source for looking up the solubility of a drug.12 For
drugs that are somewhat soluble, we incubate the solution at
37°C for about 10 to 15 minutes and mix vigorously on a vortex
mixer; the solution is then centrifuged, and the supernatant is
transferred to a clean tube. Even if the drug is not completely
soluble, sufficient drug may be present for an antibody to be
detected. Tablets can be crushed using a mortar and pestle,
taking care to tease apart and remove as much of the outer
coating as possible. Drug or inert ingredients that are not
dissolved can damage the RBCs used in the test, so efforts to
remove particulate matter or find a better medium for solution
are needed. Drugs that are insoluble in PBS are problematic.
A lower pH or solvent (e.g., alcohol) may be helpful, but the
solution would need to be diluted in PBS for an appropriate
pH and isotonicity to be used with RBCs. Drugs that are
supplied as an aqueous solution may also need to be diluted in
PBS before adding to RBCs. Dissolving drugs in an albumin
solution has been suggested,13 but we have found that 6 percent
albumin decreases the binding of some drugs (e.g., cefotetan,
cephalothin) to RBCs.14 Thus, albumin should only be used
if the drug is insoluble in water and PBS; perhaps 1 percent
albumin in PBS, as used in drug-induced thrombocytopenia
investigations, would be a better alternative than 6 percent
albumin.15
Some drugs are not stable in solution. We have found
that anti-cefotetan titers were dramatically reduced when the
solution of cefotetan was just 2 hours old compared with tests
87
R.M. Leger et al.
with a fresh solution for both 40 and 1 mg/mL preparations.
Unless the stability of a particular drug in solution has been
verified by reproducible titers over time, these solutions need
to be used as soon as possible after preparation both for
treating RBCs and for testing in the presence of the drug. We
have found 1 mg/mL solutions of ceftriaxone in PBS to be
stable up to 5 days11 and piperacillin to be stable up to 1 week
(unpublished observations) when stored at 4°C.
Preparing Drug-Treated RBCs
For preparing most drug-treated RBCs, we start with a
drug concentration of 40 mg/mL in PBS. This concentration
originated from early studies for optimally preparing penicillinand cephalothin-treated RBCs.16 It is interesting to note one
cannot make penicillin-coated RBCs with the concentration of
drug found in vivo (Garratty, unpublished data).
Optimal binding of penicillin G to RBCs occurs with 600
mg of penicillin G in 15 mL of high-pH buffer (40 mg/mL),
such as barbital, 0.1 M (pH 9.6–9.8) incubated with 1 mL of
RBCs at room temperature for 1 hour.16,17 After treating, the
RBCs are washed three to four times with PBS or until no
hemolysis is visible. Increased hemolysis can occur if treating
is extended beyond 1 hour.
Cephalosporin-treated RBCs are prepared slightly
differently: 400 mg of the drug is dissolved in 10 mL of PBS,
pH 7.3 (for 40 mg/mL), and incubated with 1 mL of RBCs at
37°C for 1 hour.2 So 40 mg/mL of drug is used but at a 10:1
ratio of drug solution to RBCs (vs. 15:1 for penicillin). Also,
binding of cephalosporins is greater at 37°C than at room
temperature (the opposite of penicillin).16 Originally, pH 10.0
buffer was used to prepare cephalosporin-treated RBCs, but a
high pH is not required for optimal coating.16 In fact, a lower
pH (e.g., pH 6–7) decreases NIPA that occurs when some
cephalosporin- and other drug-treated RBCs are incubated
with plasma or serum (i.e., during testing).18,19 When testing a
drug that we have not previously tested or for which there is no
previous report, we typically use the cephalosporin treatment
method, unless the drug is in the penicillin family. The
preparation of penicillin-treated and cephalosporin-treated
RBCs is compared in Table 1.
Some drugs require a different buffer (e.g., borate for
nafcillin20,21) or incubation at 4°C (e.g., for erythromycin7,22).
Occasionally, a drug is only available in aqueous solution at a
lower concentration or contains an ingredient that damages
RBCs (e.g., hemolysis) during treatment. This takes a trial and
error approach, testing different concentrations, temperatures,
and time of treatment incubation. Chemotherapeutic drugs in
88
aqueous solution, such as oxaliplatin and carboplatin, have
been successfully used at 1 or 5 mg/mL in PBS, respectively,
to treat RBCs. Cimetidine, which was supplied as an aqueous
solution containing alcohol, hemolyzed RBCs when prepared
at 40 mg/mL at both 37°C and room temperature, but was used
successfully at 15 mg/mL in PBS with a room temperature
treatment incubation.23
For whatever method is used, another aliquot of RBCs is
prepared under the same conditions in the appropriate buffer
or PBS (without the drug) to serve as the control untreated
RBCs. These untreated RBCs are tested in parallel with the
drug-treated RBCs. The volume of drug-treated and untreated
RBCs prepared can be scaled down as long as the appropriate
ratio is kept constant (e.g., dissolving 100 mg of cephalosporin
in 2.5 mL of PBS and adding 0.25 mL of packed RBCs
maintains the 40 mg/mL concentration with a 10:1 ratio).
Some drug-treated (e.g., penicillin, cephalothin, cefotetan)
and untreated control RBCs can be stored for a short time
(a few days) at 4°C, with Alsever’s solution added; however,
reactivity of treated RBCs will weaken over time. These RBCs
can also be stored frozen in liquid nitrogen. Other drug-treated
RBCs might not store well. For example, cimetidine-treated
RBCs were nonreactive after storage overnight at 4°C.23
Testing Drug-Treated RBCs
Drug-treated RBCs are tested in parallel with the control
untreated RBCs by tube test. Two sets of tubes are labeled for
each sample to be tested. For the patient, serum (or plasma)
and an eluate and last wash are tested. Negative controls
include normal sera or plasma (pooled or several individual
sera) and PBS. A positive control is tested whenever available.
If the drug under investigation is known to cause NIPA, the
patient’s serum and the normal sera or plasma are also tested
at a dilution of 1 in 20, and the positive control is tested at a
dilution of 1 in 20 or greater.
Two drops of each sample and control are tested with one
drop of the drug-treated or control untreated RBCs (3–5%
suspension in PBS). The tubes are incubated at 37°C for 1 hour
and then centrifuged and examined for hemolysis (if serum
was tested) and agglutination. The RBCs are washed four times
with PBS, two drops of antihuman globulin are added, and the
tubes are centrifuged and examined for agglutination. If the
patient’s plasma is tested, testing with anti-IgG is sufficient;
if patient’s serum is tested, we use polyspecific antihuman
globulin to also detect complement activation.
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Approach to investigating DIIHA
Interpretation of Results
Test results with drug-treated RBCs are often
straightforward. Results definitive for a drug antibody are
(1) reactivity of the patient’s serum and eluate with the drugtreated RBCs and no reactivity with the untreated RBCs, (2)
no reactivity of the normal sera or plasma and PBS controls,
and (3) reactivity of the positive control, if available, with
only the drug-treated RBCs (see expected results for a drug
antibody that reacts with drug-treated RBCs in Table 2).
Reactivity can be hemolysis, direct agglutination, or a positive
indirect antiglobulin test, or a combination of these results. In
cases of DIIHA caused by high-dose intravenous penicillin,
the anti-penicillin is detectable in an eluate prepared from
the patient’s RBCs. Theoretically, this should be true for other
drug antibodies reactive with drug-treated RBCs, but eluates
in these cases have not always been reactive.
For some drugs (e.g., cefotetan, oxaliplatin), the results can
be confusing and potentially misleading unless the appropriate
controls described previously are included. Table 3 shows the
interpretation of various results with drug-treated RBCs.
No reactivity of the patient’s serum and eluate together
with a positive result for the positive control indicates the
absence of a drug antibody. No reactivity of the patient’s
serum and eluate without a positive control, however, does not
exclude a drug antibody and can only be interpreted that the
drug antibody was not detected; without a positive control,
it is unknown whether the drug was truly bound to the test
RBCs. The report must state that a positive control was not
tested. No reactivity of the patient’s serum and eluate and no
reactivity of the positive control is an invalid test; there may be
no drug bound to those RBCs or the positive control may have
deteriorated.
Reactivity of the patient’s serum and eluate with both the
drug-treated and untreated RBCs indicates the presence of
alloantibody, autoantibody, or circulating drug or drug–antidrug complexes. Additional work is required. If an alloantibody
is present, new drug-treated RBCs that are negative for the
appropriate antigen(s) need to be tested. If autoantibody is
Table 2. Expected results when drug antibody reacts with drugtreated RBCs
Drug-treated RBCs
Untreated RBCs
60 min at
37°C
AHG
60 min at
37°C
AHG
Patient’s serum
+/0
+
0
0
Patient’s serum diluted 1 in 20*
+/0
+
0
0
Eluate
+/0
+
0
0
Last wash
0
0
0
0
Normal sera (pooled or 4–6
individuals)
0
0/+*
0
0
Normal sera diluted 1 in 20*
0
0
0
0
+/0
+
0
0
0
0
0
0
Sample tested
Positive control
PBS
*If drug causes nonimmunologic protein adsorption, normal sera will react at
the antiglobulin test; a 1 in 20 dilution should not react.
AHG = antihuman globulin; PBS = phosphate-buffered saline; RBCs = red
blood cells.
Table 3. Interpretation of tests with drug-treated RBCs
Drug-treated RBCs
Patient’s
Patient’s serum serum 1/20
Untreated RBCs
Eluate
Last wash
Normal sera/plasma
Normal sera/
plasma 1/20
PBS
Positive control Patient’s serum
Interpretation
+
NT
+
0
0
NT
0
+
0
Drug antibody
+
+
+
0
+
0
0
+
0
Drug antibody
+
NT
+
0
0
NT
0
NA
0
Drug antibody
0
NT
0
0
0
NT
0
+
0
No drug antibody detected
0
NT
0
0
0
NT
0
NA
0
No drug antibody detected;
?drug bound to RBCs; no
positive control
+ (AGT only)
0
0
0
+ (AGT only)
0
0
+ at ≥1/20*
0
NIPA but no drug antibody
detected
+ direct aggl,
+ or 0 AGT
0
0
0
with some sera, + direct
aggl, + or 0 AGT
0
0
+
0
Not DIIHA; environmental
exposure?
NT
+ /0
0
0
NT
0
+
+
Alloantibody? Autoantibody?
Drug–anti-drug? needs
additional testing
+
*When NIPA occurs, the positive control should be shown to react at dilution ≥1 in 20.
RBCs = red blood cells; PBS = phosphate-buffered saline; NT = not tested; NA = not available; AGT = antiglobulin test; NIPA = nonimmunologic protein
adsorption; aggl = agglutination; DIIHA = drug-induced immune hemolytic anemia.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
89
R.M. Leger et al.
present, the serum can be adsorbed and retested. Circulating
drug or drug–anti-drug complexes will also be removed by
adsorption. Alternatively, the patient’s serum can be dialyzed
to remove circulating drug. If the reactivity is caused by
circulating drug or drug–anti-drug complexes, the reactivity
should disappear after the patient is no longer receiving the
drug and the drug has cleared from the circulation.
If the normal sera or plasma reacts with the drug-treated
RBCs but does not react with the untreated RBCs, there are two
likely explanations. First, if the reactivity is at the antiglobulin
phase only, the reactivity is probably attributable to NIPA.
Several individual sera should be retested both undiluted and
diluted 1 in 20; the 1 in 20 dilution should be nonreactive (the
protein content should be too low to be a problem). For drugs
that cause NIPA, most or all normal sera will react with the
drug-treated RBCs; however, the strength can vary from weak
to strong among sera.
If the normal sera or plasma causes direct agglutination of
the drug-treated RBCs, but not the untreated RBCs, a low-titer
antibody to the drug could be present. Antibodies to several
drugs (e.g., penicillin, cephalothin, cefotetan, piperacillin,
meropenem, oxaliplatin) have been detected in plasma from
blood donors and patients who do not have hemolytic anemia.24
Widespread use of antibiotics in agriculture and environmental
contamination have been suggested as the source of exposure to
many drugs.25,26 Most of these antibodies are IgM and low titer,
but occasionally these antibodies react in sera diluted greater
than 1 in 20, especially anti-cefotetan. This agglutination can
be anywhere from 1+ to 4+ in strength. The presence of these
antibodies in the plasma of healthy individuals underscores
the importance of testing normal sera or plasma against drugtreated RBCs. It is imperative that the presence of a drug
antibody should not be determined on the basis of a single test
of undiluted patient’s plasma without sufficient normal sera
or plasma (either pooled or several individual sera) tested in
parallel. Anti-cefotetan found in normal sera has reacted up
to a dilution of 1 in 100, so in tests against cefotetan-treated
RBCs, the patient’s plasma should be strongly reactive, at least
at a dilution of 1/100 for identification of anti-cefotetan as a
cause of DIIHA (unpublished observations). Titration of the
patient’s plasma against drug-treated RBCs, in parallel with
normal sera, may help to distinguish a low-titer antibody
in normal sera from a high-titer antibody that is causing
hemolysis (see subsequent discussion).
Even though piperacillin is a semisynthetic penicillin,
we do not recommend testing piperacillin-treated RBCs
because plasma from a high percentage of blood donors
(91%) and patients (49%) directly agglutinated piperacillin90
coated RBCs.25 In contrast to cefotetan antibodies, piperacillin
antibodies in patients with DIIHA do not react to high titers
with piperacillin-treated RBCs (unpublished observations).
Thus, testing in the presence of a soluble drug is more reliable
for detecting clinically significant antibodies to piperacillin.
Hapten Inhibition to Prove Specificity
If positive results for a drug antibody are obtained when
testing drug-treated RBCs without a positive control or no
previous report, we would attempt hapten inhibition to prove
the specificity of the antibody. Inhibition of reactivity occurs
when the antibody has combined with the hapten of the same
specificity as the subsequently added antigen. Dilutions of the
patient’s serum incubated with different concentrations of the
drug are tested against the drug-treated RBCs. Alternatively, a
dilution of the patient’s serum that reacts 2+ can be selected for
testing with different concentrations of drug. For example, two
drops of plasma are incubated with two drops of drug solution
(10 mg/mL, 1 mg/mL, and 0.1 mg/mL) for 60 minutes at
37°C for the inhibition phase. The control against which the
inhibition tests are compared is two drops of plasma plus two
drops of PBS (substituted for the drug). One drop of 3 to 5
percent drug-treated RBCs is added after the inhibition phase
and incubated for 60 minutes at 37°C. Inhibition by the soluble
drug is shown by a weaker reaction (partial inhibition) or no
reaction (complete inhibition) with the drug-treated RBCs. As
shown in Table 4, a solution of oxaliplatin added to the plasma
inhibited direct agglutination of oxaliplatin-treated RBCs,
thus proving the specificity of the antibody. In this example,
the antibody reactivity of the 1 in 2 dilution of plasma was
too strong (3½+ with the PBS control) to be inhibited until
Table 4. Inhibition test: reactivity of plasma (diluted) plus solution
of oxaliplatin or PBS versus oxaliplatin-treated red blood cells
Plasma #1
1 in 2 dilution
Plasma #1
1 in 5 dilution
1
3+s
1+
2.5
3+
±
5
2+
0
3+s
2+
Partial inhibition with 5
mg/mL of drug; proof
of specificity of antioxaliplatin would have
been missed if only tested
with 1 mg/mL of drug
Complete inhibition with 5
mg/mL of drug and partial
inhibition with 2.5 and 1
mg/mL of drug
Solution added
Oxaliplatin (mg/mL)
PBS
Interpretation
PBS = phosphate-buffered saline; s = strong.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Approach to investigating DIIHA
the higher concentration of drug (5 mg/mL) was added for
the inhibition; when the serum was diluted further so that the
PBS control yielded a 2+ reaction, the inhibition was clear even
with the lower 1 mg/mL concentration. If the serum plus drug
reacts more strongly than the respective dilution of serum
plus PBS control, then the drug antibody works preferentially
when testing untreated RBCs in the presence of drug. Drug
antibodies that react only by testing in the presence of a
solution of drug (e.g., ceftriaxone) should not be inhibited. IgG
antibodies are more difficult to inhibit than IgM antibodies of
the same titer.27 Drugs with a common chemical core might
demonstrate cross-reactivity in hapten inhibition tests (e.g.,
some anti-piperacillin can be inhibited by penicillin).25
Testing in the Presence of Soluble Drug
Antibodies to many drugs are detected by testing untreated
RBCs in the presence of a solution of the drug. Piperacillin and
some of the second- and third-generation cephalosporins react
by this method; anti-ceftriaxone has been detected only by
testing in the presence of drug.
For testing a patient’s sample in the presence of soluble
drug, we prepare a 1 mg/mL solution of the drug in PBS.
For example, 10 mg of powder, capsule contents, or crushed
tablet are dissolved in 10 mL of PBS. As described above, if
the drug is not completely dissolved, the solution is centrifuged
and the supernate is transferred to a clean tube. The drug
solution should be approximately pH 7 to be compatible with
RBCs (i.e., adjusted to pH 6–8, if necessary). The 1 mg/mL
concentration used for testing in the presence of soluble drug
has no correlation to the therapeutic in vivo concentration of
the drug. However, this concentration has been successfully
used for decades to detect most drug antibodies.
Patient’s serum is incubated in the presence of the soluble
drug, with and without the addition of fresh normal serum
as a source of complement, and tested with untreated and
enzyme-treated RBCs (see details in Table 5). Serum, rather
than plasma, is the preferred specimen for this testing for the
observation of hemolysis; this also allows for the addition of
fresh normal serum as a source of complement. Testing with
enzyme-treated RBCs and the addition of a complement source
may increase the sensitivity of the test, especially for hemolysis.
Pooled RBCs (e.g., R1R1 and R2R2 RBCs) are prepared at a
heavier concentration (6 to 10 percent); an aliquot of these
RBCs are enzyme-treated. The heavier RBC suspension
enhances detection of hemolysis. Because of the heavier RBC
suspension, more thorough washing for the antiglobulin test
may be accomplished if performed manually rather than using
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Table 5. Testing in the presence of a drug solution
1. Label two sets of tubes for untreated and enzyme-treated RBCs:
• Serum + drug
• Serum + PBS
• Serum + complement + drug
• Serum + complement + PBS
• Complement + drug
• Complement + PBS
• Positive control (if available) + drug*
• Positive control (if available) + PBS
2. Place 2 drops of serum, complement source, drug, and PBS in the
appropriate tubes
3. Add 1 drop of untreated or enzyme-treated RBCs (6–10% suspension)
4. Mix and incubate at 37°C for 1 hour
5. Centrifuge and examine for hemolysis and agglutination
6. Wash RBCs four times and add polyspecific antihuman globulin;
examine for agglutination
7. Add IgG-coated RBCs to nonreactive tests
*Positive control need not be tested with both untreated and enzyme-treated
RBCs.
RBCs = red blood cells; PBS = phosphate-buffered saline.
an automated cell washer. Antigen-negative RBCs are selected
if alloantibodies are present. If autoantibodies are present, the
serum is adsorbed with allogeneic RBCs before testing (any
alloantibodies can also be adsorbed out).
The complement source is serum from two or more
individuals, screened for reactivity with enzyme-treated RBCs,
pooled, aliquoted, and frozen at –55°C or less on the same day
that it was collected.28 To show that the complement source
has hemolytic ability, the complement source is added to serum
containing a hemolytic antibody (e.g., anti-I) that has been
inactivated by incubation at 56°C for 30 minutes (if necessary)
and tested with antigen-positive RBCs. Complement source
prepared in this manner can be stored at –55°C or less for
1 year.
Interpretation of Results
Hemolysis, agglutination, or positive indirect antiglobulin
tests may occur together or separately. The expected results for
a positive test for drug antibody are reactivity in the tests with
patient’s serum plus drug and no reactivity in the respective
control tests of patient’s serum plus PBS or the tests with
complement source but no patient’s serum. The tests with
serum plus PBS serve as controls for the respective tests with
serum plus drug solution (see Table 6). No reactivity in any of
the tests indicates no drug antibody is present.
There are times when results are not so straightforward
(see Table 7). The PBS control test(s) may be reactive. This can
be because of autoantibody, alloantibody, or circulating drug
or drug–anti-drug complexes. A positive result in the test with
drug and a significantly weaker result in the corresponding
91
R.M. Leger et al.
Table 6. Expected results when drug antibody reacts in the
presence of a soluble drug
Untreated RBCs
Enzyme-treated RBCs
60 min at
37°C
AHG
60 min at
37°C
AHG
Patient’s serum + drug
+/0
+/0
+
+
Patient’s serum + PBS
0
0
0
0
Patient’s serum + C + drug
+/0
+/0
+/H
+
Patient’s serum + C + PBS
0
0
0
0
Positive control + drug
+/0
+/0
+
+
Positive control + PBS
0
0
0
0
C + drug
0
0
0
0
C + PBS
0
0
0
0
Sample tested
RBCs = red blood cells; AHG = antihuman globulin; PBS = phosphatebuffered saline; C = complement source; H = hemolysis.
test with PBS indicate drug antibody is present. Equivalent
reactivity of patient’s serum in both the drug and PBS tests
may be caused by alloantibody or autoantibody. Adsorbing
the serum to remove alloantibody or autoantibody and drug
or drug–anti-drug complexes, or dialyzing the serum to
remove circulating drug, may resolve the problem. If the
reactivity is caused by circulating drug or drug–anti-drug
complexes, the reactivity should disappear after the patient is
no longer receiving the drug and the drug has cleared from
the circulation. For piperacillin, ceftriaxone, and cefotetan, the
reactivity should disappear about 1 to 2 days after the drug is
stopped; other drugs may have a longer elimination half-life.
The addition of fresh normal serum (complement) can
sometimes enhance agglutination of a drug antibody tested
against enzyme-treated RBCs. This phenomenon is not
completely understood. For one patient who probably had
a circulating drug that resulted in positive antiglobulin tests
in the PBS controls, direct agglutination of enzyme-treated
RBCs in the presence of ceftriaxone plus fresh normal serum
(complement source) provided the distinguishing reactivity for
a ceftriaxone antibody.11 The controls of complement plus drug
and complement plus PBS should be negative; the complement
plus drug test ensures the complement source by itself does
not cause complement activation in the presence of the drug
or react with the test RBCs (with or without drug). If the
complement plus drug test is positive at the antiglobulin test
and the complement plus PBS test is negative, NIPA may be
Table 7. Interpretation of various example tests in the presence of a soluble drug
Untreated RBCs
Test
Enzyme-treated RBCs
37°C
AHG
37°C
AHG
Interpretation
Patient’s serum + drug
0
0
3+
1+
Positive (drug antibody)
Patient’s serum + PBS
0
0
0
0
Patient’s serum + C + drug
0
0
4+
1+
Patient’s serum + C + PBS
0
0
0
0
Patient’s serum + drug
2+H/3+
3+
4+H
—
Patient’s serum + PBS
0
0
0
0
Patient’s serum + C + drug
2+H/3+
3+
4+H
—
Patient’s serum + C + PBS
0
0
0
0
Patient’s serum + drug
0
1+s
4+
3+
Patient’s serum + PBS
0
m+
±
1+
Patient’s serum + C + drug
0
1+
4+
2+
Patient’s serum + C + PBS
0
0
1+
±
Patient’s serum + drug
3+s
2+
2+H/4+
4+
Patient’s serum + PBS
0
0
3+
1+
Patient’s serum + C + drug
2+s
2+
2+H/4+
4+
Patient’s serum + C + PBS
0
0
3+
1+
Patient’s serum + drug
0
0
4+
4+
Patient’s serum + PBS
0
0
4+
4+
Patient’s serum + C + drug
0
1+
4+
4+
Patient’s serum + C + PBS
0
1+
4+
4+
Positive (drug antibody)
Positive (drug antibody) and circulating drug or drug–anti-drug complexes
Positive (drug antibody) and circulating drug or drug–anti-drug complexes
Not interpretable as a result of autoantibody or alloantibody; adsorb and retest
RBCs = red blood cells; AHG = antihuman globulin; PBS = phosphate-buffered saline; C = complement; H = hemolysis; — = no cells left; m+ = micro.
92
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Approach to investigating DIIHA
occurring. If both the complement plus drug and complement
plus PBS tests are positive and the patient’s serum plus drug or
PBS (without complement added) was not informative, the test
should be repeated with a new source of complement.
If negative results are obtained using this method but the
patient’s history warrants further evaluation, use of a different
concentration of drug (e.g., saturated solution18), wash for the
antiglobulin test using a solution of drug (e.g., 1 mg/mL29), or
testing with drug metabolites substituted for the drug solution
(see later discussion) should be considered.
Titration of Drug Antibodies
Antibody titration against drug-treated RBCs or in the
presence of drug is of interest when investigating a new drug
antibody. Antibody titration against drug-treated RBCs should
be performed if normal serum also reacted with the drugtreated RBCs for a correct interpretation. For agglutination
and indirect antiglobulin test titers, dilute the serum in PBS.
For a hemolysin titer, dilute the serum in fresh normal serum
(complement source) and read the test only for hemolysis. The
titer is the reciprocal of the last dilution that reacts 1+ (e.g., a
serum that reacts 2+ at the dilution 1 in 128, 1+ at 1 in 256,
and ± at 1 in 512 has a titer of 256).
Test Metabolites of Drug
If there is convincing history incriminating a particular
drug and the DAT is positive but the drug workup is not
informative, testing the patient’s serum and eluate against
metabolites of the drug should be considered. Salama et al.30
reported detection of antibodies to buthiazide and nomifensine
only when ex vivo antigen (i.e., serum from volunteers after
ingestion of the drugs) was tested.30 Antibodies to some
nonsteroidal anti-inflammatory drugs also have required
testing in the presence of a urine metabolite for detection.31
Uncharacterized metabolites can be obtained from serum
or urine of a patient or volunteer after taking the drug. The
serum or urine is collected before the drug is taken and at
various times after ingestion and is substituted for the drug
solution in testing in the presence of a soluble drug. Previous
reports may be helpful to determine the appropriate time(s) of
collection. For diclofenac, the morning void after an evening
dose of diclofenac (150 mg) was successful.32 The urine is
centrifuged, and the supernatant is adjusted to pH 6.0 to 8.0, if
necessary. Purified metabolites are sometimes available from
drug manufacturers.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Dialysis to Remove Drug From Patient’s Sample
When it is suspected that drug is present in a patient’s
sample collected when the patient was still receiving the drug
or shortly thereafter, the blood sample can be dialyzed to
remove the drug. The sample is placed in dialysis tubing (e.g.,
with a 12,000 to 14,000 molecular weight cutoff [MWCO]) and
dialyzed in PBS overnight at 4°C.31 Alternatively, Slide-A-Lyzer
Dialysis Cassettes (Thermo Scientific, Rockford, IL), 2,000
MWCO, 0.5- to 3.0-mL capacity, work well. A large volume of
PBS dialysate and two to three changes of the PBS in 24 hours
will drive the diffusion of drug out of the sample. The dialyzed
sample is tested with and without drug added. No reactivity
in the test without drug added (e.g., in the PBS control) and
reactivity in the test with soluble drug added confirms the
presence of a drug-dependent antibody.
Unexpected Observations
It should be obvious from the preceding discussion that
interpreting results of tests for drug antibodies is not always
easy. We have encountered three serologic problems when
investigating drug antibodies: (1) normal plasma (e.g., from
blood donors) directly agglutinates some drug-treated RBCs
(e.g., cefotetan,19 piperacillin,25 oxaliplatin26); (2) some drugs
cause nonimmunologic adsorption of protein, resulting in
positive indirect and direct antiglobulin tests3; and (3) drug
that is still circulating in a patient’s plasma when the sample
is collected can result in positive tests without adding drug
in vitro (e.g., when testing the serum plus PBS control in
the presence of drug).11 This latter problem has been evident
in many cases of antibodies to piperacillin; this is most
disconcerting because serologists are misled to interpret the
reactivity as warm autoantibody, especially if the eluate is
also reactive.33,34 Unfortunately, some of these patients with
antibodies to piperacillin are treated for warm autoimmune
hemolytic anemia and not immediately taken off the drug that
is causing the hemolysis.
Another unexpected result is reactivity of the last wash
control for eluates when testing cefotetan-treated RBCs.
Increasing the number of washes (e.g., 8 to 12) before preparing
the eluate does not eliminate the problem.19
In summary, the serologic methods described here
have been successful in identifying most drug antibodies.
Including the appropriate controls will help to ensure correct
interpretation of results. As new drugs come into use, we will
continue to learn.
93
R.M. Leger et al.
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23. Arndt PA, Garratty G, Brasfield FM, Vemuri SL, Asuncion DJ.
Immune hemolytic anemia due to cimetidine: the first example
of a cimetidine antibody. Transfusion 2010;50:302–7.
24.Arndt PA, Leger RM, Garratty G. IgM agglutinins to
meropenem, a beta-lactam antibiotic, are found in plasma
of healthy blood donors and random patients (abstract).
Transfusion 2012;52(Suppl):26A.
25.Leger RM, Arndt PA, Garratty G. Serological studies of
piperacillin antibodies. Transfusion 2008;48:2429–34.
26.
Leger RM, Garratty G. Antibodies to oxaliplatin, a
chemotherapeutic, are found in plasma of healthy blood
donors. Transfusion 2011;51:1740–4.
27. White JM, Brown DL, Hepner GW, Worlledge SM. Penicillininduced haemolytic anaemia. BMJ 1968;3:26–9.
28. Garratty G. The effects of storage and heparin on the activity of
serum complement, with particular reference to the detection
of blood group antibodies. Am J Clin Pathol 1970;54:531–8.
29.Salama A, Göttsche B, Schleiffer T, Mueller-Eckhardt C.
‘Immune complex’ mediated intravascular hemolysis due to
IgM cephalosporin-dependent antibody. Transfusion 1987;
27:460–3.
30. Salama A, Mueller-Eckhardt C, Kissel K, Pralle H, Seeger W.
Ex vivo antigen preparation for the serological detection of
drug-dependent antibodies in immune haemolytic anaemias.
Br J Haematol 1984;58:525–31.
31. Johnson ST, Fueger JT, Gottschall JL. One center’s experience:
the serology and drugs associated with drug-induced immune
hemolytic anemia—a new paradigm. Transfusion 2007;47:
697–702.
32.Sachs UJH, Santoso S, Röder L, Smart E, Bein G, Kroll H.
Diclofenac-induced antibodies against red blood cells are
heterogeneous and recognize different epitopes. Transfusion
2004;44:1226–30.
33. Johnson ST. Warm autoantibody or drug-dependent antibody?
That is the question! Immunohematology 2007;23:161–4.
34. Bandara M, Seder DB, Garratty G, Leger RM, Zuckerman JB.
Piperacillin-induced immune hemolytic anemia in an adult
with cystic fibrosis. Case Rep Med 2010;2010:161454.
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
(corresponding author), Research Associate II, Patricia A. Arndt, MS,
MT(ASCP)SBB, Senior Research Associate, and George Garratty,
PhD, FRCPath, Scientific Director, American Red Cross Blood
Services, Southern California Region, 100 Red Cross Circle, Pomona,
CA 91768.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Report
Drug-induced immune neutropenia/
agranulocytosis
B.R. Curtis
Neutrophils are the most abundant white blood cell in blood
and play a critical role in preventing infections as part of the
innate immune system. Reduction in neutrophils below an
absolute count of 500 cells/μL is termed severe neutropenia or
agranulocytosis. Drug-induced immune neutropenia (DIIN)
occurs when drug-dependent antibodies form against neutrophil
membrane glycoproteins and cause neutrophil destruction.
Affected patients have fever, chills, and infections; severe
infections left untreated can result in death. Treatment with
granulocyte colony-stimulating factor can hasten neutrophil
recovery. Cumulative data show that severe neutropenia or
agranulocytosis associated with exposure to nonchemotherapy
drugs ranges from approximately 1.6 to 15.4 cases per million
population per year. Drugs most often associated with
neutropenia or agranulocytosis include dipyrone, diclofenac,
ticlopidine, calcium dobesilate, spironolactone, antithyroid
drugs (e.g., propylthiouracil), carbamazepine, sulfamethoxazoletrimethoprim, β-lactam antibiotics, clozapine, levamisole,
and vancomycin. Assays used for detection of neutrophil
drug-dependent antibodies (DDAbs) include flow cytometry,
monoclonal antibody immobilization of granulocyte antigens,
enzyme-linked
immunosorbent
assay,
immunoblotting,
granulocyte agglutination, and granulocytotoxicity. However,
testing for neutrophil DDAbs is rarely performed owing to its
complexity and lack of availability. Mechanisms proposed for
DIIN have not been rigorously studied, but those that have been
studied include drug- or hapten-induced antibody formation
and autoantibody production against drug metabolite or protein
adducts covalently attached to neutrophil membrane proteins.
This review will address acute, severe neutropenia caused by
neutrophil-reactive antibodies induced by nonchemotherapy
drugs—DIIN. Immunohematology 2014;30:95–101.
Key Words: drug-dependent neutrophil antibodies, druginduced immune neutropenia, agranulocytosis
Drug-Induced Immune Neutropenia
Neutrophils (polymorphonuclear leukocytes [PMNs]) are
a type of granulocyte and are the major class of white blood
cells (WBCs) in human peripheral blood. The circulation of
a healthy human adult contains 4500 to 10,000 WBCs/µL
with approximately 60 percent or more being neutrophils.1
A healthy adult produces 1 × 1011 neutrophils each day, each
of which survives about 6 to 8 hours in the circulation.2
Neutrophils play an important role in the innate immune
response and are critical to host defense against infectious
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
disease. They circulate in the blood until recruited by
chemical signals to sites of infection, where they function as
phagocytes that engulf and kill extracellular pathogens with
their microbicidal arsenal. Macrophages also contribute
significantly to this process. Because of their important role in
preventing infections, disorders that result in a significant lack
of neutrophils put the host at risk of severe illness and even
death. A reduction of neutrophils (segmented and band cells)
in the blood circulation to less than an absolute neutrophil
count (ANC) of 1000 to 1500/μL is considered neutropenia.
An ANC less than 500/μL is considered severe neutropenia
or agranulocytosis, and individuals with an ANC less than
100/μL are at severe risk of morbidity and mortality from
infections.3,4 The terms “agranulocytosis” and “neutropenia”
are often used interchangeably, but some argue that use of the
term “agranulocytosis” should be reserved for conditions in
which the bone marrow cannot make sufficient granulocytes,
and “neutropenia” when just neutrophils fall below normal
levels.5 For the purposes of this review, the terms will be used
synonymously, with agranulocytosis representing cases of
severe neutropenia in which the ANC is less than 100/μL.
Like thrombocytopenia and anemia, neutropenia can be
induced after exposure to myriad drugs6 and results from either
decreased production or increased destruction of neutrophils.
Decreased production is usually a consequence of exposure
to chemotherapeutic drugs causing immune suppression
of bone marrow myeloid precursors. Idiosyncratic or acute
neutropenia resulting from increased neutrophil destruction is
commonly caused by adverse reactions to nonchemotherapy
drugs.3,4,7
Clinical Features, Diagnosis, and Treatment
Drug-induced immune neutropenia (DIIN) is typically
suspected when a sudden severe drop in neutrophils (ANC
<500/μL) occurs shortly after repeat exposure to a drug or
5 to 7 days after first exposure. Patients’ symptoms include,
but are not limited to, fever, chills, nonspecific sore throat,
and myalgia or arthralgia. Diagnosis is difficult because
patients often are asymptomatic before severe neutropenia
95
B.R. Curtis
is discovered, usually from a blood count performed for
another reason.8 Without treatment, patients can experience
severe infections, septicemia, and septic shock that can lead to
death in 2 to 10 percent of cases.4,7 In DIIN, the bone marrow
typically shows normal or mild hypocellularity with decreased
or absent myeloid precursor cells, or it can be hypercellular
with neutrophilic maturation arrest.8
Criteria for drug imputability are the same as those applied
in drug-induced immune thrombocytopenia or anemia: (1)
exposure to the candidate drug preceded neutropenia, (2)
recovery from neutropenia was complete and sustained after
discontinuing candidate drug, (3) candidate drug was the only
drug used before the onset of neutropenia or other drugs were
continued or reintroduced after discontinuation of candidate
drug with a sustained neutrophil count, and (4) other causes
of neutropenia were excluded.
Treatment of patients with DIIN begins with immediate
withdrawal of the implicated drug. Further treatment consists
of supportive care for complications such as fever and
infections, which may include analgesics and antibiotics for
severe infections (ANC <100/μL) and antifungal agents for
fungal infections. Treatment with recombinant granulocyte
colony-stimulating factor (G-CSF) in severe infections can
hasten neutrophil recovery and shorten neutropenic fever, but
is more often used in neutropenia induced by chemotherapy.4,7,9
Granulocyte transfusions are typically not required in DIIN,
and the risk–benefit is not favorable.9
Incidence and Drugs Most Frequently Implicated
In 1980, the International Agranulocytosis and Aplastic
Anemia Study (IAAAS), a population-based case-controlled
surveillance program, was coordinated, which involved Israel
and seven regions of Europe.10 The most recent analysis of
these data shows that the frequency of agranulocytosis in
patients receiving nonchemotherapy drugs is approximately
5 cases/million population per year.11 Cumulative data
from IAAAS, case reports, and clinical trials show that
agranulocytosis associated with drug exposure ranges
from approximately 1.6 to 15.4 cases per million population
per year.4,7,9,11–13 Drug-associated neutropenia frequency
increases with age, being highest in people older than 65
years, and the incidence is also higher in women.11 Drugs
most often associated with neutropenia include dipyrone
(metamizole), diclofenac, ticlopidine, calcium dobesilate,
spironolactone, antithyroid drugs (e.g., propylthiouracil),
carbamazepine, quinine, sulfamethoxazole-trimethoprim,
β-lactam antibiotics, clozapine, levamisole, and vancomycin
96
(Table 1).11,14 Neutropenia incidence is higher for a select
number of drugs.
Propylthiouracil is used to treat hyperthyroidism (Graves
disease) by decreasing the levels of thyroid hormone. Mild
leukopenia (WBCs <4000/μL) occurs in 12 percent of adults
and 25 percent of children receiving proplylthiouracil,15 but
severe neutropenia occurs less frequently (0.31%).16
A careful review of a 7-year period of 114 patients receiving
home intravenous vancomycin therapy in New Mexico found
12 percent developed vancomycin-induced neutropenia and
3.5 percent had drops in ANC to 500/μL or less.17
The anthelmintic agent levamisole has been known to
cause agranulocytosis or neutropenia in exposed patients
since the 1970s,18 primarily in individuals using cocaine
adulterated with levamisole,19,20 but also when used as adjuvant
chemotherapy in patients with colorectal cancer.21 Drug
Enforcement Administration (DEA) reports in 2009 showed
69 percent of seized cocaine was contaminated with as much
as 10 percent levamisole.22 Neutropenia has been reported
to occur in as many as 13 percent of patients exposed to
levamisole.22 In cancer trials, agranulocytosis associated with
levamisole showed dose dependence, with agranulocytosis
developing in 3.1 percent of patients receiving 2.5 mg/kg for
2 consecutive days every week compared with 0.1 percent
receiving the same dosage for 3 consecutive days but every
other week.22
Clozapine-induce neutropenia occurs in about 1
percent of patients during the first 3 months of treatment.3
Clozapine, a dibenzodiazepine, is an atypical antipsychotic
medication used in the treatment of schizophrenia resistant to
conventional neuroleptics. Clozapine is perhaps the drug most
often associated with agranulocytosis or neutropenia, and it
carries five black box warnings alerting physicians to that risk
and several other adverse effects of the drug. However, the
mechanism responsible for severe neutropenia after exposure
to clozapine is not immune mediated, but is the result of ATP
depletion through formation of an active nitrenium metabolite
that causes apoptotic destruction of neutrophils.5,23
Whereas the overall incidence of drug-induced
neutropenia or agranulocytosis has been examined, the
incidence of specifically DIIN is not well defined. This is
primarily related to the difficulty in distinguishing druginduced neutropenia caused by myelosuppression and direct
neutrophil cytotoxicity from immune mechanisms, and poor
sensitivity and availability of laboratory testing for detection
of drug-dependent neutrophil antibodies.8 Table 1 lists those
drugs that have most often been reported to cause DIIN.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune neutropenia
Table 1. Nonchemotherapy drugs reported to be associated with drug-induced neutropenia
Stroncek et al. 8*
Berliner et al.14†
Tesfa et al. 5†
aminopyrine
carbamazepine
alimemazine
cefepime
aminosalicylic acid
carbimazole
calcium dobesilate
ceftriaxone
BloodCenter of Wisconsin‡
amodiaquine
cephalosporins
cefepime
ciprofloxacin
amoxicillin
chloramphenicol
clozapine
clindamycin
ampicillin
methimazole
etanercept
ibuprofen
aprindine
penicillins
fluconazole
levetiracetam
carbimazole
sulfonamides
furosemide
piperacillin-tazobactam
cefotaxime
thiouracil
infliximab
quetiapine
ceftazidime
valproic acid
IVIG
sulfamethoxazole-trimethoprim
cefuroxime
ketoconazole
tacrolimus
cephradine
lamotrigine
vancomycin
chloral hydrate
mianserin
venlafaxine
chlorpropamide
metamizole
chloroquine
methimazole
clozapine
olanzapine
dicloxacillin
pyrithyldione
diclofenac
quinidine
dimethyl aminophenazone
quinine
flecainide
rifampicin
gold thiomalate
rituximab
Ibuprofen
rituximab
levamisole
spironolactone
mercuhydrin
sulfamethoxazole-trimethoprim
methimazole
sulfasalazine
nafcillin
triazole metamizole
oxacillin
ticlopidine
penicillin
vancomycin
phenytoin
zidovudine
procainamide
propyphenazone
propylthiouracil
quinidine
quinine
sulfapyridine
sulfathiazole
sulfafurazole
*Drugs reported to be associated with drug-induced immune neutropenia.
†Drugs reported to be highly associated with agranulocytosis.
‡Drugs in 2012 for which sera from patients with suspicion of drug-induced immune neutropenia were tested for antibodies in a flow cytometry assay by
Platelet & Neutrophil Immunology Lab, BloodCenter of Wisconsin, Milwaukee. No drug-dependent antibodies involving these drugs were detected in any of
the patients’ sera.
IVIG = intravenous immunoglobulin G.
Laboratory Testing for Drug-Dependent Neutrophil
Antibodies
As just mentioned, unlike for drug-induced immune
thrombocytopenia and anemia, laboratory testing for neutroI M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
phil drug-dependent antibodies (DDAbs) is not as productive,
and testing for neutrophil antibodies is not widely available.
The reasons for this are that neutrophil antibody testing
is technically complex, labor-intensive, and expensive. In
addition, the inability to maintain the integrity of neutrophils
97
B.R. Curtis
for testing by storage at 4°C or by cryopreservation requires
that cells be isolated from fresh blood each day. This demands
the regular availability of large numbers of blood donors. For
these reasons, it is critical that granulocyte antibody and
antigen testing be performed by an experienced laboratory
using appropriate controls. When neutrophil DDAb testing is
performed, methods used are very similar to those used for
testing of platelet DDAbs with substitution of freshly isolated
neutrophils for platelets. A typical assay consists of incubation
of isolated neutrophils with patient’s serum in the presence or
absence of the implicated drug, followed by washing of cells
incubated with drug with washes containing drug and those
not incubated with drug, just with buffer. Neutrophils are then
incubated with fluorescent or enzyme-labeled anti-human
immunoglobulin (Ig)G or anti-human IgM for detection
of bound DDAbs. Flow cytometry for immunofluorescent
detection of DDAbs (Fig. 1) and monoclonal antibody
immobilization of granulocyte antigens (MAIGA) assay for
enzyme-linked immunosorbent assay (ELISA) detection and
determination of the neutrophil glycoproteins targeted by
DDAbs are methods in current use.20,24–27 Other methods that
have been used are immunoblotting, granulocyte agglutination
test (GAT), and granulocytotoxicity.8 Despite the availability of
these various methods, neutrophil DDAbs, with the exception
A
of those induced by quinine,8,25,26,28,29 are rarely detected. The
paucity of neutrophil DDAbs detected suggests that currently
used tests lack sensitivity or that mechanisms responsible
for drug-induced neutropenia often do not involve antibody
formation (see next section on Pathogenic Mechanisms).
Like some platelet and red blood cell DDAbs,30,31
neutrophil DDAbs that can only be detected in the presence of
drug metabolites have also been reported.32 Another issue is
that, in the small number of cases in which patient sera have
been tested, often the sera were tested without the addition
of drug so that only nondrug antibodies were detected. The
presence of non-DDAbs in suspected cases of DIIN suggests a
drug-induced autoantibody mechanism is responsible for the
patient’s neutropenia. In vitro assays demonstrating DDAbs
that inhibit the growth of myeloid progenitor cells have also
been reported.33
The neutrophil glycoprotein targets of DDAbs have not
been well studied for most drugs. Stroncek et al.,28 using
immunoprecipitation, showed that a quinine-dependent
antibody targeted the neutrophil-specific protein CD177
and another unidentified glycophosphatidylinositol-linked
membrane glycoprotein. Meyer et al.27 found a cefotaxime
DDAb targeted CD11b and CD35 and a metamizole DDAb
directed against CD16.
B
MFI = 8.7
MFI = 69.8
Quinine
Buffer
Buffer
Neutrophil Events
Neutrophil Events
Quinine
Normal Serum
MFI = 8.5
Patient Serum
MFI = 9.9
Fluorescence Intensity
Fluorescence Intensity
Fig. 1 Fluorescence histograms generated from immunofluorescence detection of immunoglobulin G (IgG) drug-dependent neutrophil
antibodies by flow cytometry. Normal isolated neutrophils were incubated with sera and washed, and neutrophil-bound antibodies were
detected with fluorescent anti-human IgG. (A) Normal serum incubated with neutrophils in the presence of either quinine (black histograms)
or buffer (white histograms) shows low IgG fluorescence, indicating no antibodies are present. (B) Serum from a patient exposed to quinine
with drug-induced neutropenia incubated with neutrophils showing high IgG fluorescence (MFI = 69.8) only with quinine, indicating the
presence of quinine drug-dependent antibodies. Median fluorescence intensity (MFI) values are shown for each histogram.
98
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune neutropenia
Pathogenic Mechanisms
Mechanisms of drug-dependent antibody formation and
binding to neutrophils have not been rigorously studied, and
proposed mechanisms are largely unsubstantiated. However,
some of the mechanisms proposed to explain DDAbs formed
against platelets (see article in this issue of the journal)
probably also apply to those against neutrophils, but this has
not been proven. Greater study has been made of mechanisms
to explain nonimmunologic drug-induced neutropenia or
agranulocytosis. Proposed mechanisms of immunologic and
nonimmunologic drug-induced neutropenia include oxidation
of drugs by neutrophil reactive oxygen species (ROS), causing
the formation of toxic metabolites or haptens, cytotoxicity by
large granular T lymphocytes (T-LGL), disruptions of granulopoiesis and egress of neutrophils from the bone marrow,
hapten- or drug-specific antibodies, formation of drug-induced
autoantibodies, and genetic and epigenetic modifications that
predispose an individual to drug sensitivity.5,8 Mechanisms
specific for DIIN will be discussed next.
Drug Oxidation
It is essential that drugs undergo biotransformation,
primarily in the liver, into water-soluble metabolites that
can be excreted in the urine to prevent cellular accumulation
and toxicity.34 Biotransformation gives rise to reactive
intermediates and metabolites that can readily make
covalent linkages with cellular proteins, including neutrophil
membrane glycoproteins.22 It is also possible that neutrophil
enzymes such as myeloperoxidase (MPO) could be capable
of producing reactive drug metabolites.22 Once formed
and attached to neutrophil proteins, these molecules could
elicit drug-specific antibody production through a hapten
mechanism or by production of autoantibodies targeting
drug–protein adducts.5,22 Several drugs associated with a
significant incidence of agranulocytosis (e.g., methimazole,22
propylthiouracil,22 capatopril,22 levamisole,22 clozapine5,22)
have been reported to form drug–protein adducts in vitro,
although in contrast, structurally related drugs are rarely
associated with agranulocytosis.22
Hapten Mechanism
Haptens are low-molecular-weight (usually <5000 daltons)
molecules that are not capable of eliciting an immune response
unless they are coupled to a larger carrier protein. Drugs like
penicillin and some cephalosporin drugs when covalently
linked to cell surface proteins can elicit drug-specific/hapten
antibodies. This mechanism is well described for DDAbs
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
targeting red blood cells (RBCs), which cause immune
hemolytic anemia,35 but has not been proven to occur in druginduced thrombocytopenia.36 As mentioned in the previous
section, several drugs highly associated with agranulocytosis
do form drug–protein adducts with neutrophil membrane
glycoproteins that could elicit hapten-specific (drug-specific)
antibodies that destroy neutrophils.37 Hapten antibodies were
reported to cause DIIN in patients exposed to flecanide38 and
dipyrone.39
Immune Complex Mechanism
Fc-receptors (FcR) are a family of related protein molecules
expressed on various cells in the body that engage the Fc portion
of immunoglobulins (antibodies) and regulate activation
or inhibition of various cellular functions important in the
immune response, including phagocytosis, immunoglobulin
transport, and prevention of IgG catabolism.40 Resting
neutrophils express FcγRIIIb and FcγRIIa receptors and
express FcγRI receptors when induced by various neutrophilactivating stimuli.41 Therefore, neutrophils are capable of
binding immune complexes of DDAbs and drug, but this
mechanism has not been proven for DIIN.
Autoantibody Mechanism
Autoantibodies produced against platelets and RBCs have
been reported after exposure to gold salts and α-methyldopa,
respectively.42 In some cases of suspected DIIN, neutrophilreactive antibodies are detected that do not depend on the
presence of drug.20,43,44 It is possible that these non–drugdependent antibodies could be true autoantibodies induced
by the drug. Demonstration that serum from such patients
reacts with autologous neutrophils collected once their
ANCs normalize would be one way to confirm these are true
autoantibodies, but there are no known reports in which such
testing has been performed.
Summary
DIIN can occur in susceptible individuals 5 to 7 days after
first exposure to a drug. Those affected typically experience
acute, severe neutropenia or agranulocytosis (ANC <500/μL)
and symptoms of fever, chills, sore throat, and muscle and
joint pain. Although difficult to diagnosis, it is important to
identify DIIN because, if left untreated, mortality is as high as
10 percent in cases exhibiting severe infections. Idiosyncratic
drug-induced neutropenia or agranulocytosis occurs with
a frequency of approximately 1.6 to 15.4 cases per million
population per year, and is higher in the elderly and women.
99
B.R. Curtis
The classes of drugs most commonly involved in DIIN include
analgesics, antiarrhythmic agents, antibiotics, antimalarials,
and antithyroid agents. Laboratory testing for neutrophil
DDAbs as confirmation of DIIN is rarely performed owing to the
difficulty and low sensitivity of tests used and the lack of testing
availability. Flow cytometry, MAIGA, GAT, immunoblotting,
and granulocyte cytotoxicity methods have been used to
detect DDAbs. Some DDAbs target myeloid progenitor cells
and cause neutropenia by inhibiting neutrophil production.
Neutrophil glycoprotein targets that have been described
for DDAbs include CD11b, CD16, CD35, and CD177.
Mechanisms of drug-dependent antibody formation and
binding to neutrophils have not been rigorously studied,
and proposed mechanisms are largely unsubstantiated.
Mechanisms that have been proposed include destruction
of neutrophils by hapten/drug-specific antibodies, and autoantibodies that bind adducts of drug metabolites bound to
neutrophil membrane proteins formed after biotransformation
of drugs, although several of the other mechanisms of drugdependent antibody formation that have been described as
causative of drug-induced immune thrombocytopenia could
also possibly be involved in DIIN.
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247–53.
26. Gottschall JL, Neahring B, McFarland JG, Wu GG, Weitekamp
LA, Aster RH. Quinine-induced immune thrombocytopenia
with hemolytic uremic syndrome: clinical and serological
findings in nine patients and review of literature. Am J Hematol
1994;47:283–9.
27.Meyer O, Gaedicke G, Salama A. Demonstration of drugdependent antibodies in two patients with neutrophenia and
successful treatment with granulocyte-colony-stimulating
factor. Transfusion 1999;39:527–30.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
Drug-induced immune neutropenia
28.Stroncek DF, Shankar RA, Herr GP. Quinine-dependent
antibodies to neutrophils react with a 60-Kd glycoprotein on
which neutrophil-specific antigen NB1 is located and an 85-Kd
glycosyl-phosphatidylinositol-linked N-glycosylated plasma
membrane glycoprotein. Blood 1993;81:2758–66.
29. Hou M, Horney E, Stockelberg D, Jacobsson S, Kutti J, Wadenvik
H. Multiple quinine-dependent antibodies in a patient with
episodic thrombocytopenia, neutropenia, lymphocytopenia,
and granulomatous hepatitis. Blood 1997;90:4806–11.
30.Bougie DW, Benito AI, Sanchez-Abarca LI, Torres R,
Birenbaum J, Aster RH. Acute thrombocytopenia caused by
sensitivity to the glucuronide conjugate of acetaminophen.
Blood 2007;109:3608–9.
31. Cunha PD, Lord RS, Johnson ST, Wilker PR, Aster RH, Bougie
DW. Immune hemolytic anemia caused by sensitivity to a
metabolite of etodolac, a nonsteroidal anti-inflammatory drug.
Transfusion 2000;40:663–8.
32. Salama A, Schütz B, Kiefel V, Breithaupt H, Mueller-Eckhardt
C. Immune-mediated agranulocytosis related to drugs and
their metabolites: mode of sensitization and heterogeneity of
antibodies. Br J Haematol 1989;72:127–32.
33.
Pisciotta AV. Drug-induced agranulocytosis. Peripheral
destruction of polymorphonuclear leukocytes and their
marrow precursors. Blood Rev 1990;4:226–37.
34. Dekant W. The role of biotransformation and bioactivation in
toxicity. EXS 2009;99:57–86.
35. Ries CA, Rosenbaum TJ, Garratty G, Petz LD, Fudenberg HH.
Penicillin-induced immune hemolytic anemia. Occurrence of
massive intravascular hemolysis. JAMA 1975;233:432–5.
36.Aster RH. Drug-induced immune cytopenias. Toxicology
2005;209:149–53.
37.Uetrecht J. Idiosyncratic drug reactions: past, present, and
future. Chem Res Toxicol 2008;21:84–92.
38.Samlowski WE, Frame RN, Logue GL. Flecanide-induced
immune neutropenia. Documentation of a hapten-mediated
mechanism of cell destruction. Arch Intern Med 1987;147:
383–4.
39.Hargis JB, La Russa VF, Redmond J, Kessler SW, Wright
DG. Agranulocytosis associated with “Mexican aspirin”
(dipyrone): evidence for an autoimmune mechanism affecting
multipotential hematopoietic progenitors. Am J Hematol 1989;
31:213–15.
40. Masuda A, Yoshida M, Shiomi H, et al. Role of Fc receptors
as a therapeutic target. Inflamm Allergy Drug Targets 2009;8:
80–6.
41.Abramson JS, Wheeler JG. The neutrophil. New York, NY:
Oxford University Press; 1993.
42.Aster RH. Can drugs cause autoimmune thrombocytopenic
purpura? Semin Hematol 2000;37:229–38.
43. Murphy MF, Riordan T, Minchinton RM, et al. Demonstration
of an immune-mediated mechanism of penicillin-induced
neutropenia and thrombocytopenia. Br J Haematol 1983;
55:155–60.
44.Berkman EM, Orlin JB, Wolfsdorf J. An anti-neutrophil
antibody associated with a propylthiouracil-induced lupus-like
syndrome. Transfusion 1983;23:135–8.
Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet &
Neutrophil Immunology Lab, Blood Research Institute, BloodCenter
of Wisconsin, PO Box 2178, Milwaukee, WI 53201-2178.
Manuscripts
The editorial staff of Immunohematology welcomes manuscripts
pertaining to blood group serology and education for consideration
for publication. We are especially interested in case reports, papers
on platelet and white cell serology, scientific articles covering
original investigations, and papers on new methods for use in the
blood bank. For instructions for scientific articles, case reports,
and review articles, see Instructions for Authors in every issue of
Immunohematology or e-mail a request to [email protected].
Include fax and phone numbers and e-mail address with
all manuscripts and correspondence. E-mail all manuscripts
to [email protected]
Free Classified Ads and Announcements
Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.) without charge.
E-mail information to [email protected] or fax to (215) 451-2538
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Announcements
The Johns Hopkins Hospital Specialist in Blood Bank Technology Program
The Johns Hopkins Hospital was founded in 1889. It is located in Baltimore, Maryland, on the original founding site,
just 45 minutes from Washington, DC. There are approximately 1,000 inpatient beds and another 1,200 outpatient visits
daily; nearly 600,000 patients are treated each year.
The Johns Hopkins Hospital Transfusion Medicine Division is one of the busiest in the country and can provide
opportunities to perform tasks that represent the entire spectrum of immunohematology and transfusion medicine
practice. It provides comprehensive support to all routine and specialized areas of care for surgery, oncology, cardiac,
obstetrics, neonatal and pediatric, solid organ and bone marrow transplant, therapeutic apheresis, and patients with
hematological disorders to name a few. Our intradepartment immunohematology reference laboratory provides
resolution of complex serologic problems, transfusion management, platelet antibody, and molecular genotype testing.
The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is an onsite work-study, graduate-level
training program for certified medical technologists, medical laboratory scientists, and technologists in blood banking
with at least two years of full-time blood bank experience.
The variety of patients, the size, and the general intellectual environment of the hospital provide excellent opportunities
for training in blood banking. The program is a challenging one that will prepare competent and knowledgeable
graduates who will be able to effectively apply practical and theoretical skills in a variety of employment settings. The
Johns Hopkins Hospital Specialist in Blood Bank Technology Program is accredited by the Commission on Accreditation
of Allied Health Education Programs (CAAHEP). Please visit our Web site at http://pathology.jhu.edu/department/
divisions/transfusion/sbb.cfm for additional information.
Contact:Lorraine N. Blagg, MA, MLS(ASCP)CMSBB
Program Director
E-mail: [email protected]
Phone: (410) 502-9584
The Johns Hopkins Hospital
Department of Pathology
Division of Transfusion Medicine
Sheikh Zayed Tower, Room 3100
1800 Orleans Street
Baltimore, Maryland 21287
Phone (410) 955-6580
Fax (410) 955-0618
Web site: http://pathology.jhu.edu/department/divisions/transfusion/index.cfm
102
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Announcements, cont.
Masters (MSc) in Transfusion and Transplantation Sciences
at
The University of Bristol, England
Applications are invited from medical or science graduates for the Master of Science (MSc) degree in
Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October
2014 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also
be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized
jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of
Pathology and Microbiology. It includes:
• Scientific principles of transfusion and transplantation
• Clinical applications of these principles
• Practical techniques in transfusion and transplantation
• Principles of study design and biostatistics
• An original research project
Application can also be made for Diploma in Transfusion and Transplantation Science or a Certificate
in Transfusion and Transplantation Science.
The course is accredited by the Institute of Biomedical Sciences.
Further information can be obtained from the Web site:
http://ibgrl.blood.co.uk/MSc/MscHome.htm
For further details and application forms please contact:
Dr Patricia Denning-Kendall
University of Bristol
Paul O’Gorman Lifeline Centre
Department of Pathology and Microbiology
Southmead Hospital
Westbury-on-Trym, Bristol BS10 5NB, England
Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: [email protected].
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Announcements, cont.
104
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A dv e rtise m e nts
Reference and Consultation Services
Antibody identification and problem resolution
HLA-A, B, C, and DR typing
HLA-disease association typing
Paternity testing/DNA
IgA/Anti-IgA Testing
IgA and anti-IgA testing are available to do the
following:
• Identify IgA-deficient patients
• Investigate anaphylactic reactions
• Confirm IgA-deficient donors
F or
infor mation , c ontac t :
Mehdizadeh Kashi
at (503) 280-0210
Our ELISA for IgA detects protein to 0.05 mg/dL.
F or
additional infor mation c ontac t :
Cynthia Flickinger at (215) 451-4909
or e-mail:
or write to:
[email protected]
Tissue Typing Laboratory
or write to:
American Red Cross Biomedical Services
American Red Cross Biomedical Services
Musser Blood Center
Pacific Northwest Region
700 Spring Garden Street
3131 North Vancouver
Portland, OR 97227
Philadelphia, PA 19123-3594
ATTN: Cynthia Flickinger
CLIA licensed, ASHI accredited
CLIA licensed
Donor IgA Screening
National Reference Laboratory
for Blood Group Serology
• Effective tool for screening large volumes of donors
Immunohematology Reference Laboratory
AABB, ARC, New York State, and CLIA licensed
24-hour phone number:
(215) 451-4901
Fax: (215) 451-2538
American Rare Donor Program
24-hour phone number:
(215) 451-4900
Fax: (215) 451-2538
[email protected]
Immunohematology
Phone, business hours:
(215) 451-4902
Fax: (215) 451-2538
[email protected]
Quality Control of Cryoprecipitated–AHF
Phone, business hours:
(215) 451-4903
Fax: (215) 451-2538
• Gel diffusion test that has a 15-year proven track record:
Approximately 90 percent of all donors identified as
IgA deficient by this method are confirmed by the more
sensitive testing methods
F or
additional infor mation :
Kathy Kaherl
at (860) 678-2764
e-mail:
[email protected]
or write to:
Reference Laboratory
American Red Cross Biomedical Services
Connecticut Region
209 Farmington Ave.
Farmington, CT 06032
CLIA licensed
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105
Advertisements, cont.
National Platelet Serology Reference Laboratory
Diagnostic testing for:
•
•
•
•
•
Neonatal alloimmune thrombocytopenia (NAIT)
Posttransfusion purpura (PTP)
Refractoriness to platelet transfusion
Heparin-induced thrombocytopenia (HIT)
Alloimmune idiopathic thrombocytopenia purpura (AITP)
Medical consultation available
Test methods:
•
•
•
•
•
GTI systems tests
— detection of glycoprotein-specific platelet antibodies
— detection of heparin-induced antibodies (PF4 ELISA)
Platelet suspension immunofluorescence test (PSIFT)
Solid phase red cell adherence (SPRCA) assay
Monoclonal immobilization of platelet antigens (MAIPA)
Molecular analysis for HPA-1a/1b
For
further information, contact:
Platelet Serology Laboratory (215) 451-4205
Our laboratory specializes in granulocyte antibody detection
and granulocyte antigen typing.
Indications for granulocyte serology testing
include:
• Alloimmune neonatal neutropenia (ANN)
• Autoimmune neutropenia (AIN)
• Transfusion-related acute lung injury (TRALI)
Methodologies employed:
• Granulocyte agglutination (GA)
• Granulocyte immunofluorescence by flow cytometry (GIF)
• Monoclonal antibody immobilization of neutrophil antigens
(MAINA)
TRALI investigations also include:
• HLA (PRA) Class I and Class II antibody detection
For
further information, contact:
Cynthia Flickinger (215) 451-4909
[email protected]
Neutrophil Serology Laboratory (651) 291-6797
Sandra Nance (215) 451-4362
[email protected]
Randy Schuller (651) 291-6758
[email protected]
American Red Cross Biomedical Services
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
CLIA licensed
106
National Neutrophil Serology Reference Laboratory
American Red Cross Biomedical Services
Neutrophil Serology Laboratory
100 South Robert Street
St. Paul, MN 55107
CLIA licensed
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Advertisements, cont.
I M M U N O H E M ATO LO GY, Vo l u m e 3 0, N u m b e r 2, 2 014
107
Advertisements, cont.
Becoming a Specialist in Blood Banking (SBB)
What is a certified Specialist in Blood Banking (SBB)?
• Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology
(ASCP) Board of Certification (BOC) examination for the Specialist in Blood Banking.
• This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.
Individuals who have an SBB certification serve in many areas of transfusion medicine:
• Serve as regulatory, technical, procedural, and research advisors
• Perform and direct administrative functions
• Develop, validate, implement, and perform laboratory procedures
• Analyze quality issues preparing and implementing corrective actions to prevent and document nonconformances
• Design and present educational programs
• Provide technical and scientific training in transfusion medicine
• Conduct research in transfusion medicine
Who are SBBs?
Supervisors of Transfusion Services
Supervisors of Reference Laboratories
Quality Assurance Officers
Why become an SBB?
Professional growth
Executives and Managers of Blood Centers
Research Scientists
Technical Representatives
Job placement
LIS Coordinators
Consumer Safety Officers
Reference Lab Specialists
Job satisfaction
Educators
Career advancement
How does one become an SBB?
CAAHEP-accredited SBB Technology program or grandfather the exam based on ASCP education and experience criteria.
Fact: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam compared
to individuals who grandfather the exam. The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in
Blood Bank Technology Program.
Which approach are you more compatible with?
Contact the following programs for more information:
Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org
 Onsite or
: Online
Program
Contact Name
Phone Contact
E-mail Contact
Web Site
Blood Systems Laboratories
Marie P. Holub
602-996-2396
[email protected]
www.bloodsystemslaboratories.org
Program
Walter Reed Army Medical Center
William Turcan
301-295-8605
[email protected]
[email protected]
www.militaryblood.dod.mil/Fellow/default.aspx
American Red Cross, Southern California Region
Catherine Hernandez
909-859-7496
[email protected] www.redcrossblood.org/socal/communityeducation
ARC-Central OH Region
Joanne Kosanke
614-253-2740 ext. 2270
[email protected]
none
Blood Center of Wisconsin
Phyllis Kirchner
414-937-6271
[email protected]
www.bcw.edu
Community Blood Center/CTS Dayton, Ohio
Nancy Lang
937-461-3293
[email protected]
www.cbccts.org/education/sbb.htm
Gulf Coast Regional Blood Center
Clare Wong
713-791-6201
[email protected]
www.giveblood.org/services/education/sbb-distance-program
Hoxworth Blood Center, University of Cincinnati
Medical Center
Pamela Inglish
513-558-1275
[email protected]
www.grad.uc.edu
Indiana Blood Center
Jayanna Slayten
317-916-5186
[email protected]
www.indianablood.org
Johns Hopkins Hospital
Lorraine N. Blagg
410-502-9584
[email protected]
http://pathology.jhu.edu/department/divisions/transfusion/sbb.cfm
Medical Center of Louisiana
Karen Kirkley
504-903-3954
[email protected]
www.mclno.org/webresources/index.html
NIH Clinical Center Blood Bank
Karen Byrne
301-496-8335
[email protected]
www.cc.nih.gov/dtm
Rush University
Yolanda Sanchez
312-942-2402
[email protected]
www.rushu.rush.edu/cls
Transfusion Medicine Center at Florida Blood Services
Marjorie Doty
727-568-5433 ext. 1514
[email protected]
www.fbsblood.org
Univ. of Texas Health Science Center at San Antonio
Linda Myers
210-731-5526
[email protected]
www.sbbofsa.org
University of Texas Medical Branch at Galveston
Janet Vincent
409-772-3055
[email protected]
www.utmb.edu/sbb
University of Texas SW Medical Center
Lesley Lee
214-648-1785
[email protected]
www.utsouthwestern.edu/education/school-of-health-professions/
programs/certificate-programs/medical-laboratory-sciences/index.html
:
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:
:
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:
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:
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:
:
Revised May 2012
108
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Immunohematology
Instructions for Authors
I. GENERAL INSTRUCTIONS
Before submitting a manuscript, consult current issues of Immunohematology for style.
Number the pages consecutively, beginning with the title page.
II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH
LITERATURE REVIEW
A. Each component of the manuscript must start on a new page in the following
order:
1. Title page
2.Abstract
3.Text
4.Acknowledgments
5.References
6. Author information
7.Tables
8.Figures
B. Preparation of manuscript
1. Title page
a. Full title of manuscript with only first letter of first word capitalized (bold
title)
b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T. JONES,
J.H. BROWN, AND S.R. SMITH
c. Running title of ≤40 characters, including spaces
d. Three to ten key words
2.Abstract
a. One paragraph, no longer than 300 words
b. Purpose, methods, findings, and conclusion of study
3. Key words
a. List under abstract
4. Text (serial pages): Most manuscripts can usually, but not necessarily, be divided
into sections (as described below). Survey results and review papers may need
individualized sections
a. Introduction — Purpose and rationale for study, including pertinent
background references
b. Case Report (if indicated by study) — Clinical and/or hematologic data and
background serology/molecular
c. Materials and Methods — Selection and number of subjects, samples, items,
etc. studied and description of appropriate controls, procedures, methods,
equipment, reagents, etc. Equipment and reagents should be identified in
parentheses by model or lot and manufacturer’s name, city, and state. Do not
use patient’s names or hospital numbers.
d. Results — Presentation of concise and sequential results, referring to
pertinent tables and/or figures, if applicable
e. Discussion — Implication and limitations of the study, links to other studies; if
appropriate, link conclusions to purpose of study as stated in introduction
5. Acknowledgments: Acknowledge those who have made substantial contributions
to the study, including secretarial assistance; list any grants.
6.References
a. In text, use superscript, Arabic numbers.
b. Number references consecutively in the order they occur in the text.
7.Tables
a. Head each with a brief title; capitalize the first letter of first word (e.g., Table
1. Results of…) use no punctuation at the end of the title.
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b. Use short headings for each column needed and capitalize first letter of first
word. Omit vertical lines.
c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
8.Figures
a. Figures can be submitted either by e-mail or as photographs (5 ×7″ glossy).
b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of…), ending
with a period. If figure is submitted as a glossy, place first author’s name and
figure number on back of each glossy submitted.
c. When plotting points on a figure, use the following symbols if possible:
l l s s n n.
9. Author information
a. List first name, middle initial, last name, highest degree, position held,
institution and department, and complete address (including ZIP code) for all
authors. List country when applicable. Provide e-mail addresses of all authors.
III. EDUCATIONAL FORUM
A. All submitted manuscripts should be approximately 2000 to 2500 words with
pertinent references. Submissions may include:
1. An immunohematologic case that illustrates a sound investigative approach with
clinical correlation, reflecting appropriate collaboration to sharpen problem solving
skills
2. Annotated conference proceedings
B. Preparation of manuscript
1. Title page
a. Capitalize first word of title.
b. Initials and last name of each author (no degrees; all CAPs)
2.Text
a. Case should be written as progressive disclosure and may include the
following headings, as appropriate
i. Clinical Case Presentation: Clinical information and differential diagnosis
ii. Immunohematologic Evaluation and Results: Serology and molecular
testing
iii. Interpretation: Include interpretation of laboratory results, correlating
with clinical findings
iv. Recommended Therapy: Include both transfusion and nontransfusionbased therapies
v. Discussion: Brief review of literature with unique features of this case
vi. Reference: Limited to those directly pertinent
vii. Author information (see II.B.9.)
viii. Tables (see II.B.7.)
IV. LETTER TO THE EDITOR
A.Preparation
1. Heading (To the Editor)
2. Title (first word capitalized)
3. Text (written in letter [paragraph] format)
4. Author(s) (type flush right; for first author: name, degree, institution, address
[including city, state, Zip code and country]; for other authors: name, degree,
institution, city and state)
5. References (limited to ten)
6. Table or figure (limited to one)
Send all manuscripts by e-mail to [email protected]
109
Immunohematology
Instructions for Authors | New Blood Group Allele Reports
A. For describing an allele which has not been described in a peer-reviewed publication and for which an allele name or provisional allele name has been assigned by the
ISBT Working Party on Blood Group Allele Terminology (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/blood-groupterminology/blood-group-allele-terminology/)
B.Preparation
1. Title: Allele Name (Allele Detail)
ex. RHCE*01.01 (RHCE*ce48C)
2. Author Names (initials and last name of each (no degrees, ALL CAPS)
C.Text
1. Case Report
i. Clinical and immunohematologic data
ii. Race/ethnicity and country of origin of proband, if known
2. Materials and Methods
Description of appropriate controls, procedures, methods, equipment, reagents, etc. Equipment and reagents should be identified in parentheses by model or lot and
manufacturer’s name, city, and state. Do not use patient names or hospital numbers.
3.Results
Complete the Table Below:
Phenotype
Allele Name
Nucleotide(s)
Exon(s)
Amino Acid(s)
Allele Detail
References
e weak
RHCE*01.01
48G>C
1
Trp16Cys
RHCE*ce48C
1
Column 1: Describe the immunohematologic phenotype (ex. weak or negative for an antigen).
Column 2: List the allele name or provisional allele name.
Column 3: List the nucleotide number and the change, using the reference sequence (see ISBT Blood Group Allele Terminology Pages for reference sequence ID).
Column 4: List the exons where changes in nucleotide sequence were detected.
Column 5: List the amino acids that are predicted to be changed, using the three-letter amino acid code.
Column 6: List the non-consensus nucleotides after the gene name and asterisk.
Column 7: If this allele was described in a meeting abstract, please assign a reference number and list in the Reference section.
4. Additional Information
i. Indicate whether the variant is listed in the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/); if so, provide rs number and any population frequency information, if available.
ii. Indicate whether the authors performed any population screening and if so, what the allele and genotype frequencies were.
iii. Indicate whether the authors developed a genotyping assay to screen for this variant and if so, describe in detail here.
iv. Indicate whether this variant was found associated with other variants already reported (ex. RHCE*ce48C,1025T is often linked to RHD*DIVa-2)
D.Acknowledgments
E.References
F. Author Information
110
List first name, middle initial, last name, highest degree, position held, institution and department, and complete address (including ZIP code) for all authors. List country when
applicable.
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NON PROFIT
U.S. POSTAGE
PAID
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
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(Place Label Here)