Clinical Case Report

Direct antiglobulin test-negative autoimmune

hemolytic anemia associated with erythropoiesis
stimulating agent resistance in a CKD patient with
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IgG4-related disease
A case report
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Koichi Kitamura, MDa , Koichi Hayashi, MD, PhDb, Keiichi Iwanami, MD, PhDc, Toyomi Kamesaki, MD, PhDd,
Toshihiko Suzuki, MDa,*

Abstract
Rationale: Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (AIHA) is an important differential diagnosis for
erythropoiesis-stimulating agents-resistant renal anemia with hemolysis.
Patient concerns: An 82-year-old female with a past medical history of diabetes, end-stage kidney disease (estimated
glomerular filtration rate: 6.9 mL/min/1.73 m2), post-transcatheter aortic valve implantation, and IgG4-related retroperitoneal
fibrosis on prednisolone therapy was referred to our department for assessment of severe anemia. The patient underwent
immunohematological testing for serological evaluation.
Diagnoses: DAT-negative AIHA and IgG4-RD was established based on DAT-negative and immunohematological tests.
Interventions: Glucocorticoid was administered to the patient.
Outcomes: The patient’s condition including hemoglobin level was relieved.
Lessons: In chronic kidney disease, AIHA may contribute to the development of severe anemia and erythropoiesis-stimulating
agents-resistance, irrespective of the results of Coombs test. Reticulocytosis, undetectable haptoglobin and a fall in hemoglobinA1c
despite sustained hyperglycemia may be a clue to the diagnosis of hemolysis.
Abbreviations: AIHA = autoimmune hemolytic anemia, CKD = chronic kidney disease, DAT = direct antiglobulin test, ESA =
erythropoietin-stimulating-agents, IgG4-RD = IgG4-related disease, RBCs = red blood cells.
Keywords: AIHA, anemia associated with erythropoiesis stimulating agents resistance, CKD, IgG4-related disease

1. Introduction agent (ESA)-resistance developed. Further scrutinized evalu-

ation demonstrated a substantial number of RBC-bound IgG
Autoimmune hemolytic anemia (AIHA) is an acquired disorder
molecules sufficiently to cause hemolysis but less than the cutoff
characterized by the production of autoantibodies that bind
values for detection of DAT. In such immunologically perturbed
to the surface of circulating red blood cells (RBCs) and cause
milieu as IgG4-RD, DAT-negative AIHA should be weighed in
hemolysis.[1] Not all patients, however, present with positive
light of clinical suspicion.[1]
direct antiglobulin test (DAT, i.e., Coombs test) but negative
results are seen among 1% to 10% of the patients with AIHA.[2]
We report here a case with chronic kidney disease (CKD) and 2. Case presentation
IgG4-related disease (IgG4-RD), in which severe anemia with An 82-year-old female with the past medical history of diabe-
negative DAT and hemolysis and erythropoiesis stimulating tes, end stage kidney disease (estimated glomerular filtration

Informed consent was obtained from the patient and her family for publication of *Correspondence: Toshihiko Suzuki, Department of Nephrology, Endocrinology
this report. & Diabetes, Tokyo Bay Urayasu Ichikawa Medical Center, 3-4-32 Todaijima,
The authors have no funding and conflicts of interest to disclose. Urayasu, Chiba 279-0001, Japan (e-mail toshihikos@jadecom.jp).

All data generated or analyzed during this study are included in this published
article.
a
Department of Nephrology, Endocrinology and Diabetes, Tokyo Bay Urayasu
Ichikawa Medical Center, Chiba, Japan, b Department of Emergency and Critical
Care Medicine, St. Marianna University School of Medicine, Kanagawa, Japan,
c
Department of Rheumatology, Tokyo Bay Urayasu Ichikawa Medical Center,
Chiba, Japan, d Center for Community Medicine, Jichi Medical University, Tochigi,
Japan.
Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.
This is an open access article distributed under the Creative Commons
Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
How to cite this article: Kitamura K, Hayashi K, Iwanami K, Kamesaki T, Suzuki T.
Direct antiglobulin test-negative autoimmune hemolytic anemia associated with
erythropoiesis stimulating agent resistance in a CKD patient with IgG4-related
disease: A case report. Med Case Rep Study Protoc 2023;4:5(e00278).
Received: 28 January 2023 / / Accepted: 6 February 2023
http://dx.doi.org/10.1097/MD9.0000000000000278

1
 Kitamura et al. • Med Case Rep Study Protoc (2023) 4:5
rate: 6.9 mL/min/1.73 m2), post-transcatheter aortic valve
implantation, and IgG4-related retroperitoneal fibrosis on pred-
nisolone therapy was referred to our department for assessment
of severe anemia (Hb: 5.9 g/dL, Fig. 1A). Despite aggressive
treatment with transfusion and darbepoetin (60–120 µg/2 wk),
marked anemia with reticulocytosis (6.6%), undetectable hap-
toglobin and a dissociation between hemoglobin A1c and blood
glucose (3.9% vs 200 mg/dL) was observed. Neither lactate dehy-
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drogenase nor total bilirubin was elevated. Endoscopic evalua-
tion indicated no gastrointestinal tract bleeding. Immunological
findings revealed elevated IgG4 (264 mg/dL) but negative DAT.
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Her bone marrow aspirate and biopsy showed megaloblastic
erythroid hyperplasia without dysplasia. Erythrocyte enzyme
assays for glucose-6-phosphate dehydrogenase/pyruvate kinase
and flow cytometric analysis for paroxysmal nocturnal hemo-
globinuria were both negative. A transthoracic echocardiogram
showed no valvular abnormality.
Since DAT-negative AIHA was considered as a cause of ane-
mia, the patient’s sample was sent to the immunohematological
laboratory for serological evaluation. Thus, the number of RBC-
bound IgG molecules detected by flow cytometry was sufficient
enough to cause hemolysis but lower than the detection level
for DAT (106 molecules/RBC, Fig. 1A).[3] Other mechanisms
causing DAT-negative AIHA, including low-affinity IgG auto-
antibodies that are dissociated from RBCs during the washing
procedure and RBC-bound IgA or rare warm IgM autoantibod-
ies not detectable by the routine anti-human globulin reagent,
were eliminated. We finally diagnosed the patient as having
DAT-negative AIHA and IgG4-RD.
Figure 1. Laboratory data and their changes in response to steroid therapy. Various laboratory data on admission are illustrated (A). Temporal changes in the
data showed that prednisolone markedly ameliorated hemolytic anemia and responsiveness to ESA (B). AIHA = autoimmune hemolytic anemia, CKD = chronic
kidney disease, ESA = erythropoiesis stimulating agent, GI tract = gastrointestinal tract, HbA1c = hemoglobin A1c, IgG4-RD = IgG4-related disease, LDH =
lactate dehydrogenase, RBC = red blood cell.
2
Medicine Case Reports and Study Protocols
The patient received 1 mg/kg of oral prednisolone (Fig. 1B).
Two weeks later, her hemoglobin level was improved to 8.9 g/dL
along with a reduction in a reticulocyte count and elevated hap-
toglobin levels. She took 30 mg/d of prednisolone for the next
2 months, resulting in remission of AIHA and restoration of
ESA-responsiveness which persisted during tapering off gluco-
corticoids. IgG4 decreased to 15 mg/dL.
3. Discussion
Due to drastic progress in the management of CKD, anemia
is generally recognized as a controllable disorder. Hemolytic
anemia, however, is a relatively uncommon complication and
may go unnoticed, especially when the result of DAT turns out
negative. Although DAT-negative AIHA per se manifests milder
anemia,[3] the underlying CKD may act in concert to exacerbate
anemia with refractoriness to ESA. The definitive diagnosis of
this disease entity requires expert knowledge and evaluation,
and the successful treatment is followed by the improved qual-
ity of disease management, including the conversion from ESA-
resistance to responsiveness.
Although there have been reported only a few cases with
DAT-positive AIHA and IgG4-RD,[4] no other report has
illustrated a DAT-negative AIHA patient with IgG4-RD.
Furthermore, the association between AIHA and IgG4 or
whether IgG4 levels directly affect positive/negative DAT
remains unclarified. Given the involvement of IgG4 in Th1/
Th2 T-cell subset modulation[5] and the association between
Th1/Th2 and AIHA,[6] however, it can reasonably be inferred
 Kitamura et al. • Med Case Rep Study Protoc (2023) 4:5www.md-cases.com
that elevated IgG4 plays a substantial role in the development
of AIHA.
Author contributions
Conceptualization: Koichi Kitamura.
Data curation: Koichi Kitamura.
Investigation: Koichi Kitamura.
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Methodology: Koichi Kitamura.
Project administration: Koichi Kitamura, Toshihiko Suzuki.
Supervision: Koichi Hayashi, Keiichi Iwanami, Toyomi
X1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 03/07/2024
Kamesaki, Toshihiko Suzuki.
Writing – original draft: Koichi Kitamura.
Writing – review & editing: Koichi Kitamura, Koichi Hayashi,
Keiichi Iwanami, Toyomi Kamesaki, Toshihiko Suzuki.
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