Section 3D Specific Forms of Anemia: Hemolytic Anemias

Chapter
Drug-Induced Hemolytic Anemia

13 Oreofe O. Odejide, MD, MPH

Etiology and Epidemiology generation of antibodies that are directed against epitopes on
drugs and/or their metabolites6 or a combination of drug and
Drug-induced hemolytic anemia (DIHA) is an uncommon but
RBC membrane proteins, ultimately resulting in hemolysis.
important diagnosis. It is characterized by immune-mediated
A population of drug-dependent antibodies reacts with
destruction of red blood cells (RBC), resulting in an abrupt
drug alone. These antibodies can be detected in vitro, using
drop in hemoglobin levels after exposure to the implicated
drug-coated RBCs. This has been well-described with penicil-
drug. It has an estimated incidence of one case per million of
lin and cefotetan.7,8 When cefotetan is administered to some
the population.1 The true incidence is likely higher, as mild
patients, it covalently binds to proteins on the RBC membrane,
cases are probably not fully investigated, and it can be fre-
resulting in formation of IgG antibodies directed only against
quently mistaken for warm autoimmune hemolytic anemia.2,3
the drug epitope. The immunoglobin-coated RBCs then
Since the earliest reports of DIHA in the 1950s, more than a
undergo Fc-mediated extravascular hemolysis by splenic
hundred drugs have been implicated as causes of immune
macrophages.
hemolysis.4 As the landscape of drugs causing hemolytic
Another population of antibodies reacts with neoantigens,
anemia has grown, the drugs commonly associated with hemo-
variably composed of part drug and part RBC membrane.
lytic anemia have also changed. Methyldopa, a frequently used
These antibodies react in vitro when the serum of a patient
antihypertensive medication in the 1960s and 1970s, was the
with DIHA by this mechanism is mixed with the implicated
most common drug causing hemolytic anemia, representing
about 67 percent of cases in a series.5 Currently, antimicrobials,
Table 13.1 Examples of drugs that have been reported to cause DIHA
specifically, second- and third-generation cephalosporins, are
the most common causes of DIHA.5 Drug-dependent Drug-independent Nonimmune
antibody antibody protein
mechanism mechanism adsorption
Pathophysiology mechanism
There are two widely accepted mechanisms of immune destruc-
tion of RBCs in DIHA. These are based on the type of anti- Acetaminophen Cladribine Carboplatin
bodies induced by the putative drug, namely1 drug-dependent Acyclovir Fludarabine Cephalothin
antibody mechanism and2 drug-independent antibody mech- Amphotericin B Levodopa Cisplatin
anism. The antibodies involved in DIHA are of the IgM and
IgG classes. A third mechanism of drug-induced hemolysis Cefazolin Mefenamic acid Clavulanate
recently described is nonimmune protein adsorption Examples Cefotetan Methyldopa Oxaliplatin
of drugs that have been reported to cause DIHA through the Cefoxitin Procainamide Sulbactam
three mechanisms are displayed in Table 13.1 (See Chapter 12,
Table 12.3 for a more comprehensive list). Ceftazidime Tazobactam
Ceftriaxone
Drug-Dependent Antibody Mechanism Cefuroxime
Immune hemolysis by drug-dependent antibodies is the most Cephalexin
common mechanism of DIHA. Drug-dependent antibodies
Ciprofloxacin
require the implicated drug to be present to demonstrate
reactivity against RBCs in vitro. Drugs may bind firmly to Diclofenac
RBC membrane proteins forming covalent bonds, may be Penicillin
loosely bound to the RBC membrane, or may exist free in
Piperacillin
plasma. These associations result in immunogenicity and

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 Section 3D: Specific Forms of Anemia: Hemolytic Anemias
A. Drug-dependent Antibody B. Drug-dependent Antibody
Mechanism: drug is bound to Mechanism: antibody is directed
RBC and antibody is directed against neoantigen variably
against drug epitope e.g. composed of drug and RBC
penicillin membrane proteins e.g. ceftriaxone
drug and RBCs. Ceftriaxone is an example of a drug that
has been shown to result in generation of this population of
drug-dependent antibodies.8 Unlike penicillin, which forms
firm covalent bonds with RBC membrane proteins, ceftriaxone
attaches loosely to the RBC membrane, forming a neoantigen
composed of both drug and membrane proteins. The neoanti-
gen then results in generation of antibodies, which can fix
complement, resulting in intravascular hemolysis.
Drug-Independent Antibody Mechanism
In this mechanism, antibodies are capable of reactivity in vitro
without addition of any drug. Therefore, this is identical to the
generation of RBC autoantibodies. The clinical findings are
indistinguishable from idiopathic warm autoimmune hemo-
lytic anemia, except for resolution of hemolysis associated with
discontinuation of the implicated drug. The exact mechanism
by which drug-independent antibodies are formed is poorly
understood. Some of the proposed mechanisms include
molecular mimicry, immune dysregulation, and drug adsorp-
tion causing altered RBC membrane antigens.9,10 It is note-
worthy that not every patient who forms drug-independent
antibodies eventually develops immune hemolytic anemia.
Examples of drugs in this category are methyldopa and fludar-
abine. Methyldopa results in the production of RBC autoanti-
bodies in about 20 percent of patients receiving the drug, but
only about 1 percent of patients go on to develop hemolytic
anemia.11
Nonimmune Protein Adsorption
It has been described as far back as the 1970s that adminis-
tration of some drugs may result in proteins binding to RBC
membranes nonimmunologically.12 This was initially thought
to be an exclusively in vitro process causing positive direct
antiglobulin tests, with no in vivo implication of hemolysis.
Recent evidence, however, suggests that a few cases of non-
immunological adsorption of IgG to RBC membranes result in
hemolytic anemia.13,14 Positive monocyte monolayer assay
testing, indicative of shortened RBC survival in vivo, in non-
immunological protein adsorption has also provided further
evidence that this mechanism results in hemolysis.15 Examples
of drugs in this category include cephalothin, tazobactam, and
oxaliplatin.
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Figure 13.1 Drug induced hemolytic anemia
mechanisms
C. Drug-independent Antibody
Mechanism: drug results in
generation of RBC auto-
antibodies e.g. methyldopa
Clinical Presentation
Patients with DIHA present with symptoms related to anemia
and hemolysis. The severity of symptoms varies with the
degree of anemia and the rapidity with which it develops.
Patients develop symptoms within minutes to days after expos-
ure to the implicated drug. Associated symptoms include
pallor, fatigue, and dyspnea. In more severe cases, patients
may develop lethargy, confusion, hypotension, syncope, and
death. Jaundice and scleral icterus may also be present second-
ary to circulating unconjugated bilirubin from hemolyzed
RBCs. In cases with massive intravascular hemolysis, patients
will develop dark-colored urine due to hemoglobinuria and
may also develop oliguria or anuria indicative of renal failure.
There are key differences in DIHA between pediatric and
adult populations. Children often develop symptoms very
shortly after receiving the offending drug. The majority of
children develop acute intravascular hemolysis in less than
1 hour after receipt of the offending drug. There is a steep
decline in hemoglobin concentration, with a nadir 5 g/dL
reported in more than 70 percent of children.16 Accordingly,
severe symptoms and fatalities are quite common. Several cases
exemplifying this have been described in ceftriaxone-induced
hemolytic anemia.16–18 Conversely, in adults, hemolysis often
develops hours to days after receiving the offending drug, and
decline in hemoglobin and resultant symptoms tend to be
milder. Consequently, there are fewer fatalities.
Most patients with hemolysis secondary to DIHA have had
prior exposure to the offending drug without symptoms. The
prior exposure to the drug induces the development of circulat-
ing antibodies, which then result in hemolysis during subse-
quent exposure to the drug. An important exception to this is
cefotetan, which is the most common cause of DIHA. In a
review of 85 cases of cefotetan-induced hemolytic anemia by
the Food and Drug Administration, only 18 percent of patients
had a history of prior receipt of cefotetan.19 In addition, cefote-
tan antibodies have been identified in the plasma of individuals
without prior exposure to cefotetan.7 This phenomenon has
been explained by the postulation that extensive use of prophy-
lactic antibiotics in cattle and chicken feed in the United States
may result in primary immunization of individuals who con-
sume these animals or their products as part of their diet, with
subsequent development of anticefotetan antibodies.7

Diagnostic Evaluation/Laboratory Findings
Accurate diagnosis of DIHA requires the documentation of
hemolysis associated with drug therapy and confirmation by
serologic testing. It is essential to obtain a detailed history of all
medications received 2 weeks before the evidence of hemolysis.
It is particularly important to pay attention to medications
administered as prophylaxis prior to surgeries, as these medi-
cations may be overlooked even though they play a significant
role in DIHA. For example, many cases of cefotetan-induced
hemolytic anemia are associated with prophylaxis for surgery.
Laboratory Findings
The laboratory findings are consistent with evidence of hemo-
lytic anemia, with a mean hemoglobin drop of 6.65 g/dL and a
mean nadir of 5.2 g/dL reported in 85 cases.19 The reticulocyte
count is elevated, as well as levels of lactate dehydrogenase,
total and indirect bilirubin, while the serum haptoglobin is
decreased. Peripheral blood smear usually demonstrates spher-
ocytosis in severe cases. Red blood cell indices may reveal an
elevated mean corpuscular volume, reflective of the degree of
reticulocytosis and an elevated mean corpuscular hemoglobin
concentration, indicative of spherocytosis. In cases of drug-
dependent antibodies against part-membrane and part-drug
(e.g., ceftriaxone), massive intravascular hemolysis can occur
because of complement fixation. Laboratory studies in these
situations may show hemoglobinuria and elevated blood urea
nitrogen and creatinine, reflecting acute renal failure.
Direct Antiglobulin Test (DAT) also called Direct Coombs
test: This should be positive in all cases of DIHA. It is the most
reliable laboratory finding in DIHA. If this is negative, a diagnosis
of DIHA is highly unlikely. In drug-dependent antibody-mediated
cases, where antibodies react only with drug-treated RBCs (e.g.,
penicillin or cefotetan), immunoglobulin (IgG) is detected on
patients’ RBCs, with or without complement (C3). On the other
hand, for cases in which drug-dependent antibodies react with
neoantigens composed of part-RBC membrane and part-drug
(e.g., ceftriaxone), DAT testing detects C3, with or without IgG
on the RBCs. Although IgG is the most common immunoglobu-
lin class associated with DIHA, IgM can also be involved. Quinine
has been shown to induce IgM antibody production, which readily
activates the complement pathway resulting in intravascular hem-
olysis.20 DAT testing in this situation typically detects only C3 and
not IgM on the surface of RBCs. While DAT is a very sensitive test
in diagnosing DIHA, it has limited specificity. Other immune
processes such as autoimmune hemolytic anemia and acute and
delayed hemolytic transfusion reactions are also characterized by
positive DATs (See Chapter 12, figure 12.3).
Indirect Antiglobulin Test (IAT) also called Indirect
Coombs test: In drug-dependent antibody-mediated DIHA, the
IAT could either be positive or negative. The serum of patients
with drug-dependent antibodies may test positive with reagent
RBCs because significant amounts of residual drug or drug-
antibody complexes may still be present at the time of testing.
DIHA through the drug–independent antibody mechanism
results in positive IAT, as the antibodies present in the patient’s
serum should be reactive with reagent RBCs regardless of whether
there is residual drug present (See Chapter 12, figure 12.3).
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Chapter 13: Drug-Induced Hemolytic Anemia
Elution: An elution is commonly performed after a posi-
tive DAT to identify the antibody-coating RBCs. The anti-
bodies are characterized by testing the eluate against reagent
RBCs. The eluted antibodies fail to react with reagent RBCs in
cases of drug-dependent DIHA because the offending drug is
not present. This is an important distinguishing feature from
warm autoimmune hemolytic anemia, where a positive DAT is
typically accompanied by a positive elution reaction. Further
testing of the eluate with drug-coated RBCs or reagent RBCs in
the presence of the offending drug demonstrates reactivity,
confirming the diagnosis of hemolytic anemia mediated by
drug-dependent antibodies. In cases of drug-independent
DIHA, the eluate demonstrates reactivity against reagent RBCs
despite absence of the offending drug because the mechanism
of hemolysis involves true autoantibodies.
Treatment and Prognosis
Discontinuation of the implicated drug is the primary treatment
for DIHA. Due to rapid clearance of drugs from the plasma,
drug-dependent antibodies can cause no further harm. There-
fore, the hemolytic anemia usually resolves soon after stopping
the drug. Despite resolution of symptoms, patients may have a
persistently positive DAT for months following the hemolytic
episode. Steroids are not indicated for treatment except in cases of
drug-independent antibody-mediated DIHA (e.g., methyldopa,
fludarabine), in which generated antibodies are true autoantibo-
dies. In situations in which hemolysis is severe and persistent
despite discontinuation of the drug and steroid administration,
other treatments used in warm autoimmune hemolytic anemia
such as intravenous immunoglobulin (IVIG) or immunosup-
pressive agents (e.g., rituximab, azathioprine, cyclophospha-
mide) should be considered. Supportive management such as
transfusion of RBCs and intravenous fluid infusion are often
necessary depending on the degree of anemia and decrease in
circulatory volume. Dialysis may be required in the event of renal
failure. Exposure to the implicated drug must be avoided in the
future, as repeated exposure could result in severe, potentially
fatal hemolysis.
Case Study
A 70-year-old man with recently diagnosed colon adenocarci-
noma was admitted for a right hemicolectomy. The patient
received perioperative prophylaxis with 2 g of cefotetan intra-
venously. The surgical procedure was uncomplicated. On post-
operative day 4, he was noted to be jaundiced. He also
complained of fatigue and dizziness on attempts of trying to
get up from his bed. Laboratory workup revealed a five-point
drop in his hemoglobin (6.2 g/dL from a preoperative value of
11 g/dL). Reticulocyte count was elevated at 8.5 percent, lactate
dehydrogenase was 1291 IU/L, total bilirubin was 14.6 mg/dL
(direct bilirubin 5.9 mg/dL), and haptoglobin was less than
8 mg/dL. Physical exam revealed jaundice, scleral icterus, and
an abdominal incision with no evidence of bleeding.
A peripheral blood smear was obtained and displayed in
Figure 13.2. DAT was positive and demonstrated both anti-IgG
and anti-C3. The eluate did not demonstrate reactivity with
reagent RBCs. It however demonstrated anti-cefotetan reactivity
when incubated with cefotetan-coated RBCs.
97
 Section 3D: Specific Forms of Anemia: Hemolytic Anemias

hemolytic anemia. The patient’s laboratory indices

2. There are several mechanisms of DIHA. The
Source : Lichtman MA, Shafer MS, Felgar RE, Wang N:
Lichtman’s Atlas of Hematology: http: //www.accessmedicine.com
Copyright © The McGraw-Hill Companies, Inc. All rights reserved.
Figure 13.2
3. The most important management of this patient is to
1. What is the most likely cause of the patient’s anemia?
2. What is the specific mechanism through which the
patient’s anemia occurred?
3. What is the appropriate management of the patient’s anemia?
Answers
1. The patient has cefotetan-induced hemolytic anemia. He
received cefotetan 4 days prior to the clinical detection of his
of anemia, reticulocytosis, elevated lactate dehydrogenase,
indirect hyperbilirubinemia, and low haptoglobin all
indicate that he has hemolytic anemia. The peripheral blood
smear shows spherocytosis and polychromasia. His positive
DAT and lack of reactivity of the eluate with reagent RBCs
are highly suggestive of DIHA. The reactivity of the eluate
with cefotetan-coated RBCs confirms the diagnosis of
cefotetan-induced hemolytic anemia.
mechanism outlined in this case study is most
consistent with drug-dependent antibody-mediated
hemolytic anemia. Drug-dependent antibodies
classically result in positive DAT, while the eluate does
not react with reagent RBCs. The eluate will, however,
react when drug is present. In this case, the eluate
demonstrated reactivity when incubated with cefotetan-
coated RBCs, confirming that the antibodies were
indeed drug-dependent.
discontinue cefotetan (if still being administered) and
avoid further exposure to cefotetan. Given the severity
of the patient’s anemia and symptoms, supportive
management with RBC transfusions is also necessary.
Steroid administration is not required because this is
not a case of DIHA mediated by drug-independent
antibodies.

anemia. N Engl J Med. 1985; 313 immunohematologic reactions to

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