BRIEF REPORT

Acute autoimmune hemolytic anemia due to anti-Ena

autoantibody successfully treated with rituximab

Elena Nedelcu,1 Megan Desai,2 Jennifer Green,3 Kathleen M. Bensing,4 Austin Turner,2
David Head,2 and Pampee P. Young2

T
wo cases of autoimmune hemolytic anemia
BACKGROUND: Autoimmune hemolytic anemia (AIHA) due to anti-Ena have been previously
(AIHA) due to anti-Ena has been previously reported in reported to be associated with sudden massive
association with massive intravascular hemolysis, intravascular hemolysis, disseminated intravas-
disseminated intravascular coagulation, and fatal cular coagulation, and fatal outcome.1,2 In yet other stud-
outcomes. Here we report a case of successfully treated ies, the clinical picture of AIHA with anti-Ena is not fully
AIHA due to anti-Ena. specified.3-7 To our knowledge, there is no report of a suc-
CASE REPORT: A 69-year-old male with a past cessfully treated AIHA with anti-Ena specificity. Here we
medical history of cirrhosis due to nonalcoholic report a case of acute and severe AIHA due to anti-Ena auto-
steatohepatitis status post–orthotopic liver transplant antibody and discuss the clinical presentation, laboratory
presented with 1-month history of progressive anemia. At features, and therapy response.
presentation, his hemoglobin (Hb) was 5.6 g/dL,
hematocrit (Hct) 16%, reticulocytes 0.3%, direct bilirubin
(bili) 4 g/dL, lactate dehydrogenase 533 units/L
CASE REPORT
(reference, 125-220 units/L), and haptoglobin 254 mg/dL
(reference, 40-273 mg/dL). Blood bank testing revealed A 69-year-old male with a past medical history of cirrhosis
an autoantibody present in his plasma and a direct due to nonalcoholic steatohepatitis status post–orthotopic
antiglobulin test positive for immunoglobulin G (IgC) but liver transplant 18 months before presentation was trans-
negative for complement. He received 1 unit of an ferred from an outside hospital for 1-month history of pro-
incompatible blood group O phenotypically matched red gressive anemia. He had a history of multiple blood
blood cell unit. product transfusions in the distant past (although not
RESULTS: Over the course of the next 5 days, the Hb
and Hct decreased to 4.1 g/dL and 12%, respectively, ABBREVIATIONS: AIHA 5 autoimmune hemolytic anemia;
direct bili increased to 12.3 mg/day, reticulocytes slightly bili 5 bilirubin; FS 5 ficin-sensitive; GP(s) 5 glycophorin(s);
increased to 0.9%, and haptoglobin decreased to less IS 5 immediate spin.
than 8 mg/dL. Marrow study showed a hypercellular
From the 1University of California San Francisco, Department
marrow with erythroid hyperplasia. Additional workup
of Laboratory Medicine, San Francisco, California; the
performed at a reference laboratory identified an anti-Ena 2
Vanderbilt University Medical Center, Department of
autoantibody. He received prednisone and weekly
Pathology, Microbiology and Immunology, Nashville,
rituximab infusions and was monitored weekly. At the
Tennessee; the 3Vanderbilt University Medical Center,
2-month visit, Hb and Hct were 10 g/dL and 32%,
Department of Medicine, Division of Hematology, Nashville,
respectively.
Tennessee; and the 4Immunohematology Reference Laboratory,
CONCLUSION: Unlike two of the previously reported
BloodCenter of Wisconsin, Milwaukee, Wisconsin.
fatal cases of AIHA with anti-Ena specificity, this 69-year-
Address reprint requests to: Elena Nedelcu, MD, University
old male treated with weekly rituximab infusion
of California, San Francisco, 505 Parnassus, Room M501, San
underwent clinical recovery and significant anemia
Francisco, CA 94143; e-mail: Elena.Nedelcu@ucsf.edu.
improvement.
Received for publication March 30, 2017; revision received
August 15, 2017; and accepted August 21, 2017.
doi:10.1111/trf.14363
C 2017 AABB
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NEDELCU ET AL.

prothrombin time (PT) (15.8 sec; reference, 11.0-14.6 sec)

and partial thromboplastin time (PTT) (47.0 sec; reference,
23.8-32.0 sec) were both prolonged.
Right knee arthrocentesis demonstrated prosthetic
joint infection for which the patient underwent irrigation
and debridement and received antibiotics with resultant
resolution of fevers and improved knee pain and mobility.
Liver biopsy was negative for transplant rejection. Blood
bank testing revealed an ABO discrepancy with forward
typing as A and reverse typing as O blood group, as well as
a plasma panagglutinin reacting 2 to 31 with most reagent
RBCs. He received 1 unit of incompatible phenotypically
matched O D1 RBCs. Since his historical blood type was
known to be A, the extrareactivity causing his ABO dis-
crepancy was thought to be due to his plasma panaggluti-
Fig. 1. Marrow aspirate smear showing moderately hypercellu- nin. Repeat DAT at our institution before any blood
lar marrow with trilineage hematopoiesis; erythroid hyperpla- transfusion was positive (11) with anti-immunoglobulin
sia; no increase in blasts; no abnormal myeloid, lymphoid, or (Ig)G and no complement fixation noted.
plasma cell infiltrates; and no significant dysplasia. The review of his peripheral blood smear collected
before any RBC transfusion revealed leukopenia with
transfused in the past 3 months before this admission) and marked anemia with numerous spherocytes, decreased
was known to have blood group AD1 and with a negative polychromasia, no nucleated RBCs, and adequate PLT
red blood cell (RBC) antibody screen. Blood bank testing count and morphology. As the etiology of his anemia was
performed at the outside institution revealed an autoanti- unclear a marrow (BM) biopsy was performed to evaluate
body in his plasma and weakly positive direct antiglobulin the possibility of an underlying primary BM failure or a
test (DAT). Alloantibodies against major RBC antigens clonal process and showed erythroid hyperplasia and no
were ruled out. The patient’s RBCs were phenotyped and evidence of malignancy (Fig. 1). A complete blood count
found to be negative for the K, Jkb, and N antigens and pos- at time of this biopsy showed a WBC count of 4.7 3 109/L,
itive for C, c, E, e, Fya, Fyb, Jka, M, S, and s antigens. Hb level of 5.2 g/dL, and PLT count of 134 3 109/L. Over
On arrival, the patient reported profound fatigue, right the course of 5 days, the Hb and Hct decreased to
knee swelling and pain necessitating a cane for walking, 4.1 g/dL and 12%, respectively; direct bili increased to
and associated fevers and night sweats. His vital signs were 12.3 mg/dL; and reticulocytes increased only to 0.9%.
notable for mild tachycardia (heart rate (HR) 98), hyper- Over 2 more days the haptoglobin decreased to less than
tension (blood pressure (BP) 150/76), and intermittent 8 mg/dL. Since his reticulocytopenia was persistent, a
fever (temperature (T) max 38.88C). The patient was alert, second BM analysis was performed and showed similar
oriented, and in no acute distress with scleral pallor and findings to the ones noted in the prior biopsy including a
right knee effusion with limited range of motion noted on moderately hypercellular marrow with erythroid hyper-
physical examination. His white blood cell (WBC) count plasia with atypia consistent with stress erythropoiesis.
was 2.7 3 109/L (reference, 3.9 3 109-10.7 3 109/L), hemo- The complete blood count at time of second BM biopsy
globin (Hb) 5.6 g/dL (reference, 14.0-18.1 g/dL), hemato- showed a WBC count of 5.7 3 109/L, Hb level of 6.0 g/dL,
crit (Hct) 16% (reference, 41%-49%), reticulocytes 0.3% and PLT count of 154 3 109/L. In addition, the peripheral
(reference, 0.5%-1.8%), platelet (PLT) count 136 3 109/L blood smear showed RBC agglutination.
(reference, 135 3 109-371 3 109/L), total bilirubin (bili) During this time, over a total of 10 days, he received a
5.5 mg/dL (reference, 0.2-1.2 mg/dL), direct bili 4.0 mg/dL total of 15 RBC group O phenotypically matched least-
(reference, 0.2-1.2 mg/dL), lactate dehydrogenase 533 incompatible units. His Hct varied between 12 and 28%,
units/L (reference, 125-220 units/L), haptoglobin 254 mg/ but reticulocytes consistently remained less than 1%. The
dL (reference, 40-273 mg/dL), and ferritin 5007 ng/mL (ref- relationship between the laboratory values and adminis-
erence, 24-336 ng/mL). Liver function tests were notable tered therapy is depicted in Fig. 2. Due to the presence of a
for elevations in aspartate aminotransferase (AST) (50 persistent panagglutinin and worsening hemolysis despite
unit/L; reference, 5-10 units/L), alanine aminotransferase RBC transfusions, additional workup was performed at a
(ALT) (97 units/L; reference, 0-55), and alkaline phospha- reference laboratory. The DAT was positive with IgG (11)
tase (735 units/L; reference, 40-150 units/L). The patient’s and no complement fixation noted. At the reference labora-
creatinine was mildly elevated (1.35 mg/dL; reference, tory, initial testing showed an eluate (Gamma Elu-Kit II,
0.72-1.25 mg/dL) above his baseline (1.1 mg/dL) and uri- Immucor) weakly reactive with all cells tested. The patient’s
nalysis demonstrated 5 RBCs/high power field (hpf). His serum reacted strongly (41) at all phases of antibody

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 ACUTE AIHA WITH ANTIGENS SPECIFICITY

Fig. 2. Laboratory values (Hb, indirect bili, haptoglobin, and reticulocytes %) in relationship with therapy administered (blood
transfusions, steroids, and rituximab).

identification testing (immediate spin [IS], 378C, and indi- positive” control and mixed-field reactions were not noted.
rect antihuman globulin). The patient’s serum failed to Had the patient been En(a–), then we would have antici-
react with the same antibody identification panel after pated mixed field. These findings suggest that the patient
0.1% ficin treatment of the reagent RBCs, indicating the RBCs were not En(a–) and that the antibody was an auto
presence of a ficin-sensitive (FS) antigen. The patient’s anti-Ena-FS. Trypsin testing was not performed.
serum was tested against five examples of En(a–) reagent The patient’s plasma was treated with 2-
RBCs as well as a Dantu1 cell, which has altered glyco- mercaptoethanol to determine whether the antibody had
phorin (GP)A and B. Two of the En(a–) cells were negative an IgM component. This testing showed reduction of the
at all phases of reactivity. The Dantu1 cell was negative at IS and 378C reactivity indicating the antibody was both
IS and 378C but still reactive at antihuman globulin. IgM and IgG in nature.
These results taken together indicated that the patient Treatment for his AIHA included 1 mg/kg prednisone
had an antibody directed against GPA or a component of daily for 3 weeks before slow tapering, followed by
GPA. Differential alloadsorptions using R1R1, R2R2, and rr 375 mg/m2 rituximab weekly for 4 weeks and a 1-mg folic
reagent RBCs were performed on the patient’s serum to acid tablet by mouth daily. His clinical picture and labora-
remove the autoantibody reactivity and determine the tory values were monitored weekly. His anemia improved
presence of underlying alloantibodies. No alloantibodies rapidly with the use of rituximab. At a 2-month return
were detected in the patient’s alloadsorbed serum. To final- visit, his Hb and Hct were 10 g/dL and 32%, respectively.
ize the testing, reagent RBCs were treated with neuramini- He was clinically stable and laboratory variables for
dase to determine that the patient’s antibody was truly hemolysis had resolved.
directed against GPA and not the Pr antigen sites on the GP.
Anti-Pr is characteristically a cold autoantibody directed
DISCUSSION
against the Pr (protease sensitive) antigens located on the
a
GPs. Anti-Pr has also been known to cause severe hemo- Anti-En is an immune antibody directed against high-
lytic anemia.6,7 An eluate prepared from the patient’s RBCs incidence Ena antigen present on GPA also known as
reacted with all cells tested using a saline indirect antiglob- CD235. GPA is a Type I transmembrane sialoglycoprotein
ulin test (IAT). The eluate was tested against an En(a–) that traverses the RBC membrane one time and carries
reagent RBC and was found to be negative, confirming the M and N antigens. Antibodies against GPA antigens,
anti-Ena specificity. Neuraminidase-treated RBCs were such as anti-M and anti-N, may be naturally occurring, do
positive when tested with the eluate showing that the elu- not usually react at 378C, and are known to rarely cause
ate did not have anti-Pr specificity.8 The patient’s RBCs hemolysis; therefore, they are not considered clinically
were tested with anti-Ena from the corresponding serum of significant.9 In contrast to the anti-M and anti-N, anti-
the En(a–) reagent RBC. Although the patient had been bodies against the Ena part of GPA are made by very rare
transfused, his RBCs tested equally as strong as a “normal individuals who lack this antigen. Anti-Ena is a collective

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NEDELCU ET AL.

term that describes antibodies to high-prevalence anti- she developed acute renal failure and disseminated
gens that are on GPA. The GYPA*Null variant represents intravascular coagulation leading to ischemic bowel with
En(a–) individuals completely lacking GPA and therefore perforation and death.1 The second reported case is of a
also phenotyped as M– and N–. Other En(a–) variants are 40-year-old male with a history of relapsed Hodgkin’s lym-
due to single-nucleotide polymorphisms or hybrid GP phoma and multiple episodes of immune thrombocytope-
rearrangements that cause altered expression of the anti- nic purpura who developed AIHA with warm autoantibody
gens on GPA. Antibodies to these variants have been with anti-Ena specificity; thus he was diagnosed with Evans
assigned categories according to the effect that proteases syndrome (AIHA and immune thrombocytopenic pur-
have on GPA. Anti-EnaTS refers to an antibody that does pura).2 He was treated with 1 mg/kg prednisone daily with
not react with trypsin-treated RBCs; trypsin cleaves GPA temporary improvement in his PLT count and then later
at Amino Acid 39. Anti-EnaFS refers to an antibody with IVIG for worsening AIHA. Eventually, he received
directed toward epitopes that are FS and trypsin resistant. incompatible blood, and immediately after the transfusion
Anti-EnaFS are directed at determinants around Amino began, the patient became pulseless despite cardiopulmo-
Acids 46 to 56 of GPA. Anti-EnaFR refers to an antibody nary resuscitation. In both cases, the authors recognize the
directed to determinants that are ficin resistant. EnaFR acute AIHA development in contrast to the typical slowly
site occurs within Amino Acids 62 to 72 of GPA. The latter developing AIHA cases. Also in contrast with typical AIHA
antibody has been found in En(a–) individuals. Autoanti- in which the hemolysis is extravascular, these cases were
bodies with Ena specificity are usually of the anti-EnaFS associated with intravascular massive hemolysis. Like in
type. It has been reported that anti-EnaFS occurs in 1.6% our case, no alloantibodies were identified, describing only
of warm autoantibody cases.10,11 the anti-Ena as the sole culprit for the sudden and massive
These antibodies are known to be clinically signifi- hemolysis. In contrast to our case, the marrow status and
cant and can cause severe hemolytic transfusion reactions response is not addressed in the previously published
and hemolytic disease of the newborn and fetus. Less is reports and the presence of reticulocytopenia is not men-
known about the anti-Ena autoantibody. Two prior reports tioned. Therefore, the marrow status of the previously pub-
have been published, and in both cases, the antibody lished cases is not known.
caused massive IgG-mediated hemolysis with fatal out- We suggest that the association of panagglutinin reac-
come.1,2 In other reports of patients with warm AIHA, the tivity and worsening hemolysis with persistent reticulocy-
RBC eluates showed reactivity consistent with autoanti- topenia should raise the question of the rare anti-Ena
Ena; however, the clinical severity of the AIHA in these antibody and trigger further investigation and the rapid
patients was not described and the distinction between initiation of immunosuppressive. Continuation of transfu-
autoanti-Ena and anti-Wrb is not fully made.3-5 Therefore, sion therapy, even of phenotypically matched blood but
it is difficult to definitively assess the clinical severity of an positive for Ena antigen, might lead to worsening hemoly-
anti-Ena from prior literature. sis and should be considered with caution. Rituximab
Besides the rarity and the potential for severe clinical therapy, however, is a safer alternative known to have a
outcome, other challenging aspects of the anti-Ena good clinical response between 51.5 and 93% in warm
include the panagglutinin or a warm autoantibody reac- AIHA with various antibody specificities.12-16 Prospective
tivity pattern that masks the presence of alloantibodies, if studies show an even better clinical response ranging
any are present. The DAT may be negative or only weakly from 71.4% to 100%,17-19 and a Phase III randomized clini-
positive due to the sudden and extensive hemolysis. The cal trial sets the response rate at 75%.20
recognition of hemolysis may be delayed in cases of iron The utility of anticomplement therapy, such as eculi-
deficiency anemia or conditions associated with marrow zumab, for warm AIHA is unknown, although its use in
suppression, including chemotherapy administration. In cold agglutinin disease and in isolated severe cases of
addition to the diagnostic problems, these cases have sig- refractory warm AIHA with a DAT positive for both IgG
nificant therapeutic challenges as routine RBC transfusion and complement have been reported.21,22 Our patient’s
leads to further hemolysis of the infused units and rapidly DAT was negative for complement, similar to a majority of
worsening clinical picture. Even if quickly recognized, the prior case reports (although a confounding factor is
therapy may be challenging as En(a–) units are extremely the concomitant anti Wrb or anti-dl). As such, we would
difficult to find and transfusion of En(a–) blood might not not predict a therapeutic role for anticomplement drugs,
be beneficial if acute hemolysis has already developed. such as eculizumab, for such patients.
Two of the previously reported cases associated with Autoantibodies in warm AIHA are generally nonspe-
anti-Ena acute AIHA had a fatal outcome. The first case cific but specificities linked to the Rh, Kell, Kidd, Duffy, and
was of a 71-year-old female with a history of immune Diego blood group systems have been reported.23,24 There
thrombocytopenic purpura who developed sudden mas- are currently no data regarding the treatment of acute
sive intravascular hemolysis and was treated with intrave- AIHA with anti-Ena specificity. Although it is recognized
nous steroids and multiple RBC transfusions; however, that warm AIHA has particular transfusion challenges,25

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 ACUTE AIHA WITH ANTIGENS SPECIFICITY

specificity of the autoantibody might not matter as no 12. Narat S, Gandla J, Hoffbrand AV, et al. Rituximab in the treat-
patient or RBC survival benefit was demonstrated.26,27 ment of refractory autoimmune cytopenias in adults. Hae-
However, since fatal warm AIHA with anti-Ena specificity matologica 2005;90:1273-4.
was previously reported, rapid recognition and initiation of 13. Bussone G, Ribeiro E, Dechartres A, et al. Efficacy and safety
rituximab therapy may be lifesaving. of rituximab in adults’ warm antibody autoimmune haemo-
In conclusion, we describe here the clinical presenta- lytic anemia: retrospective analysis of 27 cases. Am J Hema-
tion and response to rituximab therapy of a 69-year-old tol 2009;84:153-7.
male who developed acute AIHA with anti-Ena specificity. 14. Dierickx D, Verhoef G, Van Hoof A, et al. Rituximab in auto-
Unlike two of the previously reported cases with fatal out- immune haemolytic anaemia and immune thrombocytope-
come, this patient achieved clinical recovery and anemia nic purpura: a Belgian retrospective multicentric study.
improvement with weekly rituximab infusion. J Intern Med 2009;266:484-91.
15. Pen~ alver FJ, Alvarez-Larra
n A, Dıez-Martin JL, et al. Rituxi-
mab is an effective and safe therapeutic alternative in adults
ACKNOWLEDGMENT with refractory and severe autoimmune hemolytic anemia.
Ann Hematol 2010;89:1073-80.
We thank VUMC Blood Bank staff and supervisors.
16. Maung SW, Leahy M, O’Leary HM, et al. A multi-centre ret-
rospective study of rituximab use in the treatment of
relapsed or resistant warm autoimmune haemolytic anae-
CONFLICT OF INTEREST mia. Br J Haematol 2013;163:118-22.
17. Zecca M, Nobili B, Ramenghi U, et al. Rituximab for the
The authors have disclosed no conflicts of interest.
treatment of refractory autoimmune hemolytic anemia in
children. Blood 2003;101:3857-61.
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