Adner1968

Coombs'-Positive Hemolytic Disease in Malaria
MARVIN M. ADNER, M.D., LESLIE B. ALTSTATT, M.D., and
MARCEL E. CONRAD, M.D., F.A.C.P.
Washington, D. C.
Qrr^vrADv I n a stu(
ly of 131 soldiers evacu-
3UMMARY a t e d f r o m Vietnam with drug-re-
sistant Plasmodium falciparum malaria, 4 patients
were found with a positive direct antiglobulin
test of the immunoglobulin (Ig) G type. In three
patients the positive Coombs' tests seemed tem-
porally related to the administration of quinine
for relapsed malaria and were associated with
hemolysis. Two of these patients had a quinine-
related dermatitis, one developed blackwater
fever within hours after the initiation of quinine
therapy, and another had a panagglutinin in
quinine-free red cell eluates. The fourth patient
had compensated hemolysis and a positive direct
Coombs' test which seemed unrelated to quinine
therapy. The indirect Coombs' test was negative
in all subjects, and no antiquinine antibodies were
found in sera or red cell eluates from these pa-
tients.
Coombs'-positive hemolytic disease is an un-
usual complication of malarial infections. Clini-
cal observations in man suggest that quinine has
a causal role in the hemolytic reaction. The mech-
anism by which this drug-induced hemolysis oc-
curs is not known.
MALARIA FREQUENTLY CAUSES anemia of
greater severity than can be attrib-
uted to the destruction of parasitized red
blood cells. It has been postulated that an
autoimmune-type hemolysis occurs, but the
evidence to support this hypothesis is
largely indirect (1).
This report describes four soldiers with
Plasmodium falciparum malaria who had
a positive direct antiglobulin test. Three of
these patients developed an acute hemo-
lytic episode shortly after quinine therapy
was initiated.
SUBJECTS AND METHODS
The subjects of this study were four white
soldiers with a positive direct Coombs' test who
were evacuated from Vietnam to Walter Reed
General Hospital because of P. falciparum ma-
laria.
The direct antiglobulin tests were performed
with commercial Coombs' sera.* f These anti-
globulin sera gave negative reactions with a
red blood cell preparation containing 10%
reticulocytes. Red cell eluates were prepared
by ether extraction (Patient 2) (2) and by heat-
ing (Patients 1, 3, and 4) (3). The specificity
of the antibody in eluates from Patient 1 was
tested with two commercial red cell panels
(Identigen® J and Ten-Cell® panel f). The class
of protein in the eluate was determined by the
immunodiffusion method of Ouchterlony (4)
using rabbit antihuman immunoglobulin (Ig) G,
IgA, IgM, transferrin, and whole serum.J The
quinine concentration of red cell eluates of
Patient 1 was determined by a fluorometric
method (5). The gamma globulin neutraliza-
tion test (6) was performed with IgG prepared
by purification of human fraction II on
diethylaminoethanol cellulose.
Indirect Coombs' tests were performed with
a 4% suspension of pooled 0+ red cells (Spec-
trogen®f) and test sera prepared in the fol-
lowing manner: [1] red cells suspended in a 1:5
dilution of compatible fresh normal serum (di-
luent = triethanolamine-buffered saline, pH 7.3,
containing added calcium and magnesium) plus
test sera; [2] red cells suspended in the diluted
fresh normal serum containing 1, 10, 25, 50, or
100 mg/100 ml quinine sulfate plus test
sera; [3] red cells suspended in buffered
Received July 17, 1967; revision accepted Sep-
tember 12, 1967.
From the Department of Hematology, Walter
Reed Army Institute of Research, Washington,
D. C.
Requests for reprints should be addressed to Lt.
Col. Marcel E. Conrad, MC, USA, Department of
Hematology, Walter Reed Army Institute of Re-
search, Washington, D. C. 20012.
* Ortho Pharmaceutical Corp., Raritan, N. J.
f Spectra Biologicals, Inc., East Brunswick, N. J.
j Hyland Laboratories, Los Angeles, Calif.
33
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34 ADNER, ALTSTATT, AND CONRAD Annals of
Internal Medicine
FIGURE 1. Laboratory values during the clinical course in Case 1 (see text).
saline containing 1, 10, 25, 50, or 100 mg/100
ml quinine sulfate plus test sera; [4] red cells
suspended in the diluted fresh normal serum
plus test sera that had been incubated with
equal volumes of quinine sulfate for 30 min
at 37 C; [5] red cells incubated in buffered
saline containing 50 mg/100 ml quinine sulfate
(1 part cells/20 parts quinine solution) for 2
hr at 37 C, washed once in saline, and resus-
pended in the diluted fresh normal serum plus
test sera.
The pooled 0+ red cells were treated with
trypsin by the method of Morton and Pickles
(7). The trypsinized cells and test sera were
then prepared as described above [1 to 5]. De-
tection of incomplete antibodies was performed
according to the method of Dacie and Lewis
(8).
CASE REPORTS
PATIENT 1
This 21-year-old soldier developed P. falci-
parum malaria in Vietnam during October
1965. (See Figure 1 for clinical course.) Sub-
sequently, he had five malarial relapses that
were treated with quinine sulfate and chloro-
quine. In March 1966 he became febrile with
parasitemia (2,500/mm8
) and was treated with
quinine sulfate. After 3 days of therapy the
symptoms of malaria and the parasitemia dis-
appeared. However, the hematocrit, which was
38% at the time of institution of quinine
therapy, fell to 25% by the sixth day of treat-
ment. The patient had a positive direct anti-
globulin test, decreased plasma hemoglobin-
binding capacity (6 mg/100 ml), and hyper-
bilirubinemia (1.3 mg/100 ml, indirect fraction;
0.2 mg/100 ml, direct fraction); and a bone
marrow aspirate revealed a marked normo-
blastic erythroid hyperplasia. Therapeutic
blood levels of quinine had been attained
(7 mg/liter), and this drug was continued for
a total of 14 days of therapy. During this period
the hematocrit stabilized at 30% with 10%
reticulocytes, and the direct red cell anti-
globulin test remained positive. After dis-
continuation of the quinine therapy the
hematocrit increased to normal levels, the
reticulocyte count decreased, and the direct
antiglobulin test became negative. During the
next 5 months the patient had no malarial
relapses nor evidence of hemolytic disease.
Methemoglobin reduction test was normal 3
months after the hemolytic episode.
PATIENT 2
This 34-year-old soldier developed malaria
in November 1965 and had two relapses during
the next 60 days. (See Figure 2 for clinical
course.) Each episode responded to quinine,
and the patient developed a mild pruritic, ery-
thematous, macular eruption during the second

Volume 68, No. 1
January 1968
COOMBS-POSITIVE HEMOLYTIC DISEASE IN MALARIA
35
FIGURE 2. Laboratory values during the clinical course in Case 2 (see text).
relapse. Chloroquine produced clinical improve-
ment of a third relapse, but parasitemia per-
sisted. On February 17, 1966, parasitemia in-
creased, fever occurred, and quinine therapy
was started. Four hours later the patient de-
veloped hemoglobinuria. The hematocrit de-
creased from 38 to 16%, the total serum bili-
rubin increased to a peak level of 18 mg/100
ml (indirect bilirubin, 9.2 mg/100 ml), and the
direct Coombs' test was positive. The patient
had marked depletion of multiple coagulation
factors. The plasma quinine level was 6.5 mg/
liter. He became oliguric. Quinine therapy was
stopped, and treatment with pyrimethamine,
chloroquine, sulfadiazine, heparin, cortico-
steroids, and osmotic diuretics was initiated.
Renal function improved, and the rate of
hemolysis decreased. The Coombs' test became
negative on February 21, but the hematocrit
remained at 20% without reticulocytosis. A
bone marrow aspirate showed marked megalo-
blastic changes, and antimalarial drug therapy
was stopped; we believed that the megaloblastic
changes were caused by the antifolic action
of pyrimethamine. Reticulocytosis recurred
spontaneously, and the hematocrit became nor-
mal. During the next 3 months the patient had
no malarial relapses or evidence of hemolytic
anemia. Methemoglobin reduction test was nor-
mal 3 months after the hemolytic episode.
PATIENT 3
This 24-year-old soldier developed malaria
in November 1965. The initial episode and
relapses were treated with quinine, pyrimeth-
amine, and chloroquine. The patient was ad-
mitted to Walter Reed General Hospital during
the third relapse. A pruritic, erythematous,
urticarial eruption developed and persisted on
quinine therapy after other drugs were dis-
continued. The patient developed a mild ane-
mia with a positive direct Coombs' test, reticu-
locytosis, indirect hyperbilirubinemia, and de-
creased plasma hemoglobin-binding capacity
(20 mg/100 ml). No parasites were present in
thick peripheral blood smears. Heterophil and
cold agglutinin studies were normal. Methemo-
globin reduction test was normal. The skin rash
and the hemolytic anemia improved markedly
within 1 day after cessation of quinine therapy.
The patient had no relapses during the next
3 months of observation.
PATIENT 4
This 24-year-old soldier was evacuated from
Vietnam in November 1965 after an initial
malarial infection that responded to 3 days of
chloroquine therapy. On admission to Walter
Reed General Hospital the patient was asymp-
tomatic, and a thick smear of blood showed no

Internal Medicine
parasites. The splenic tip was palpable; the
hematocrit was 38% with 1% reticulocytes. The
white blood count was 9,700 cells/mm* with a
differential count that showed 47 neutrophils,
2 bands, 1 metamyelocyte, 33 lymphocytes, 3
monocytes, and 12 eosinophils. Stool specimens
contained no ova or parasites. During January
1966 the patient had a malarial relapse that
responded to 2 weeks of quinine therapy. Dur-
ing the next 3 months the patient had per-
sistent eosinophilia but no parasitemia. A bone
marrow aspirate showed mild normoblastic ery-
throid hyperplasia, eosinophilia, and malarial
merozoite and ring forms. The hematocrit was
49%, reticulocyte count, 1.4%, and the direct
Coombs' test, strongly positive. An autologous
^Cr red blood cell survival showed a half-life
of 21 days (normal half-life, 26 to 35 days).
Methemoglobin reduction test was normal. The
patient remained asymptomatic until July 1966.
Then he had a relapse of malaria that re-
sponded to combinational therapy with qui-
nine, pyrimethamine, and sulfadiazine. Sub-
sequently, eosinophilia, evidence of hemolysis,
and parasitemia have not been found.
SPECIAL LABORATORY STUDIES
Blood specimens from 131 patients evac-
uated from Vietnam because of P. falci-
parum malaria were examined. The in-
direct Coombs' test was negative in all
specimens, but the direct antiglobulin test
was positive in blood from four subjects.
In three (Patients 1, 3, and 4) the gamma
globulin neutralization test showed that
the positive reaction was caused by an im-
munoglobulin (Ig) G antibody on the sur-
face of the red blood cells. The positive
antiglobulin reaction was inhibited by in-
cubation of the Coombs' sera with an equal
volume of 1% IgG before the addition of
red blood cells. Concentrations of IgG less
than 0.1 to 0.4 mg/100 ml did not inhibit
the reaction. The gamma globulin neu-
tralization test was not used to study Pa-
tient 2.
Eluates of the Coombs'-positive red cells
from Patient 1 contained antibody activity.
These eluates showed a positive indirect
antiglobulin reaction when incubated with
any of the cells in two commercial red cell
panels. The panels included cord cells but
no Rh-null cells. An eluate was concen-
trated twentyfold and shown to contain
IgG and transferrin but not IgA or IgM.
Fluorometric analysis showed no quinine
in the concentrated eluate. Thus, Patient
1 had an IgG antibody on his red cells
that acted as a panagglutinin in the
absence of quinine. Eluates from the
Coombs'-positive red blood cells of Patients
2, 3, and 4 failed to show antibody activity.
Numerous serum samples from these
patients drawn during the periods when
the Coombs' tests were positive and the
following 2 to 3 months after the Coombs'
test became negative were tested for un-
usual antibody activity. In no instance was
there any evidence of abnormal antibody
activity against normal red cells and tryp-
sinized red cells suspended in saline or in
varying concentrations of quinine sulfate.
Incubation of the sera with varying con-
centrations of quinine did not bring out
any antibody activity against normal red
cells. The results of the laboratory studies
are summarized in Table 1.
DISCUSSION
The anemia of malaria is more profound
than can be caused by the destruction of
parasitized erythrocytes. The evidence that
an immune-type hemolysis may be respon-
sible for the disproportionate anemia has
been summarized by Zuckerman (1). Al-
though most attempts to demonstrate ab-
normal red cell antibodies have been un-
successful, there are a few reports of a
positive Coombs' test in malarious patients
(9-11). Heidelberger and Mayer (12)
showed that incubation of malarial sera
with normal human erythrocytic stromal
antigen resulted in the fixation of comple-
ment. In addition, the sera of rats infected
with Plasmodium berghei contain hemag-
glutinins against trypsinized normal rat
erythrocytes (13, 14).
Evidence for immune hemolysis in ma-
laria is largely indirect. Autoagglutination
and microspherocytosis of nonparasitized

Volume 68, No. 1
January 1968
COOMBS'-POSITIVE HEMOLYTIC DISEASE IN MALARIA
37
TABLE 1. Summary of Studies of Four Patients with Malaria with Positive Coombs' Tests
Patient Evidence of Hemolysis Quinine
Therapy
at Time of
Hemolysis
Direct
Coombs'
Test
Gamma Globulin
Neutralization
Test
Antibody Activity
Eluate Serum
1
Rapidly developing anemia
Decreased plasma hemo-
globin-binding capacity
Reticulocytosis
Hyperbilirubinemia
Normoblastic erythroid
hyperplasia of bone marrow
+ +++ + + 0
2
Rapidly developing anemia
Absent plasma hemoglobin-
binding capacity
Hyperbilirubinemia
Hemoglobinuria
+ +++ (-)• 0 0
3
Anemia
Decreased plasma hemoglobin-
binding capacity
Reticulocytosis
Hyperbilirubinemia
+ ++ + 0 0
4
Decreased red cell
autosurvival
Erythroid hyperplasia
of bone marrow
0 +++ + 0 0
* Study not performed.
red cells have been observed in blood speci-
mens from malarious patients with acute
intravascular hemolysis (15). Transfusion
of labeled red blood cells from a patient
with blackwater fever had a shortened sur-
vival in a normal recipient. Conversely,
normal erythrocytes are prematurely de-
stroyed in malarious subjects (16). Marked
splenic erythrophagocytosis of nonparasi-
tized erythrocytes is frequently found in
malaria (17). These observations suggest
that the red blood cell is sensitized in ma-
laria, perhaps in a manner that is not usu-
ally demonstrated by available techniques.
Although quinine therapy cannot be im-
plicated in every case of blackwater fever,
the frequent association of hemolytic crisis
and the initiation of quinine therapy can-
not be disregarded (18-20). Quinine causes
a dose-related anemia in rabbits in vivo
and makes red blood cells more susceptible
to in vitro hemolysis (21, 22). However, the
administration of quinine to many patients
with malaria and the relative rarity of
blackwater fever make it unlikely that the
hemolysis is caused solely by a direct toxic
effect of quinine upon erythrocytes. Qui-
nine-associated hemolytic anemia has been
reported only in pregnant or malarious pa-
tients. This may indicate that quinine acts
upon previously sensitized or damaged
erythrocytes to accelerate or precipitate he-
molysis. Glucose 6-phosphate dehydrogenase
deficiency has been suggested as a precipi-
tating factor, but our patients showed no
evidence of this enzymatic defect (23). The
possibility that this is an immune response
to quinine is suggested by the usual occur-
rence of blackwater fever in patients after
multiple courses of quinine therapy and
the positive Coombs' test in certain pa-
tients with intravascular hemolysis. Ad-
ditional credence for this hypothesis is
obtained from previous reports of a

Internal Medicine
quinine-dependent complement-fixing anti-
body in the sera of patients receiving qui-
nine therapy (24, 25). The failure to dem-
onstrate a similar antibody in our patients
indicates that this is a complex problem
that requires further investigation.
ACKNOWLEDGMENT
We wish to acknowledge the helpful assist-
ance of Lt. Col. Kevin G. Barry, MC, USA,
Maj. Paul F. Gilliland, MC, USA, and Capt.
Max M. Inman, MC, USA, Department of Me-
tabolism, Walter Reed Army Institute of Re-
search, who made the clinical information and
specimens reported in this study available to us.
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3. LANDSTEINER, K., MILLER, C. P.: Serological
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