Journal of

Clinical Medicine

Article
Autoimmune Hemolytic Anemia as a Complication
of Congenital Anemias. A Case Series and Review
of the Literature
Irene Motta 1,2, * , Juri Giannotta 3 , Marta Ferraresi 1,4 , Kordelia Barbullushi 3,4 , Nicoletta Revelli 5 ,
Giovanna Graziadei 1 , Wilma Barcellini 3 and Bruno Fattizzo 3,6

1 General Medicine Unit, Rare Diseases Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,
20122 Milan, Italy; [email protected] (M.F.); [email protected] (G.G.)
2 Department of Clinical Sciences and Community Health, Università degli Studi di Milano, 20122 Milan, Italy
3 Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy;
[email protected] (J.G.); [email protected] (K.B.); [email protected] (W.B.);
[email protected] (B.F.)
4 University of Milan, 20122 Milan, Italy
5 Laboratorio di Immunoematologia di Riferimento, Dipartimento di Medicina Trasfusionale ed Ematologia,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy;
[email protected]
6 Department of Oncology and Oncohematology, University of Milan, 20122 Milan, Italy
* Correspondence: [email protected]; Tel.: +39-02-5503-3493



Abstract: Congenital anemias may be complicated by immune-mediated hemolytic crisis. Alloan-

 tibodies are usually seen in chronically transfused patients, and autoantibodies have also been
Citation: Motta, I.; Giannotta, J.; described, although they are rarely associated with overt autoimmune hemolytic anemia (AIHA),
Ferraresi, M.; Barbullushi, K.; Revelli, a serious and potentially life-threatening complication. Given the lack of data on the AIHA diag-
N.; Graziadei, G.; Barcellini, W.; nosis and management in congenital anemias, we retrospectively evaluated all clinically relevant
Fattizzo, B. Autoimmune Hemolytic AIHA cases occurring at a referral center for AIHA, hemoglobinopathies, and chronic hemolytic
Anemia as a Complication of anemias, focusing on clinical management and outcome. In our cohort, AIHA had a prevalence of 1%
Congenital Anemias. A Case Series
(14/1410 patients). The majority were warm AIHA. Possible triggers were recent transfusion, infec-
and Review of the Literature. J. Clin.
tion, pregnancy, and surgery. All the patients received steroid therapy as the first line, and about 25%
Med. 2021, 10, 3439. https://doi.org/
required further treatment, including rituximab, azathioprine, intravenous immunoglobulins, and
10.3390/jcm10153439
cyclophosphamide. Transfusion support was required in 57% of the patients with non-transfusion-
Academic Editor: Emmanuel Andrès
dependent anemia, and recombinant human erythropoietin was safely administered in one third of
the patients. AIHA in congenital anemias may be challenging both from a diagnostic and a thera-
Received: 16 May 2021 peutic point of view. A proper evaluation of hemolytic markers, bone marrow compensation, and
Accepted: 28 July 2021 assessment of the direct antiglobulin test is mandatory.
Published: 2 August 2021
Keywords: autoimmune hemolytic anemia; alloimmunization; thalassemia; sickle cell disease;
Publisher’s Note: MDPI stays neutral congenital hemolytic anemias
with regard to jurisdictional claims in
published maps and institutional affil-
iations.

1. Introduction
Congenital anemias include a broad spectrum of rare red blood cell (RBC) disorders
classified according to the affected RBC structure. They include hemoglobinopathies, namely
Copyright: © 2021 by the authors. sickle cell disease (SCD) and thalassemia syndromes, which are by far the most prevalent [1,2],
Licensee MDPI, Basel, Switzerland. and congenital hemolytic anemias (CHAs). In SCD, the abnormal hemoglobin (Hb), called
This article is an open access article hemoglobin S (HbS), tends to form polymers in erythrocytes that deform the structure
distributed under the terms and
of RBC [3]. Subsequent intravascular sickling results in hemolytic anemia and recurrent
conditions of the Creative Commons
occlusion of small vessels leading to vaso-occlusive crisis. In β-thalassemia, the precipi-
Attribution (CC BY) license (https://
tation of α-chains aggregates in erythroid precursors leads to ineffective erythropoiesis
creativecommons.org/licenses/by/
in the bone marrow and peripheral hemolysis in the intravascular and extravascular
4.0/).

J. Clin. Med. 2021, 10, 3439. https://doi.org/10.3390/jcm10153439 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2021, 10, 3439 2 of 11

compartments. Consequent anemia and hypoxia stimulate erythroid precursor prolifer-

ation in the medullary and extramedullary compartments [2]. CHAs are heterogeneous
conditions, with either dominant, recessive, or X-linked inheritance, exhibiting a clinical
course ranging from mild fully compensated anemia to chronic severe hemolysis. They
include defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to
defective erythropoiesis.
In the most severe forms of all these disorders, transfusions and iron chelation are
currently the main treatment strategy [4–8]. The chronic course of both hemoglobinopathies
and CHAs may be complicated by the abrupt drop of Hb values due to several causes, in-
cluding increased destruction/sequestration (i.e., hemolytic crisis) and reduced/inhibited
erythropoiesis (i.e., aplastic crisis). The latter recognizes various triggers, particularly
parvovirus B19 infection [9], while the former is mainly immune-mediated. In particular,
alloantibodies (alloAbs) are usually seen in chronically transfused patients and may cause
severe transfusion reactions. Autoantibodies (autoAbs) have also been described, although
they are rarely associated with overt autoimmune hemolytic anemia (AIHA). Autoim-
munity may occur through several mechanisms, including modification of RBC mem-
brane antigens, molecular mimicry, hidden epitopes spreading, and innocent bystander
destruction [10]. Finally, both the increased destruction and impaired erythropoiesis may
coexist when the autoimmune attack is directed against erythrocyte precursors [11,12].
Given the lack of data on the AIHA diagnosis and management in congenital anemias,
we retrospectively evaluated all the clinically relevant AIHA cases that occurred at our
hospital, a referral center for AIHA, hemoglobinopathies, and CHAs, focusing on clinical
management and outcome. A review of the available literature is also provided.

2. Materials and Methods

We retrospectively collected clinical, laboratory, and treatment data from electronic
medical records of the patients with congenital anemias followed at Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico who developed AIHA over a period of 20 years, be-
tween January 1991 and December 2020. The patients belonged to a cohort of 1410 followed
at the Hematology Unit and the Rare Diseases Center of Fondazione IRCCS Ca’ Granda
Ospedale Maggiore Policlinico, Milan, Italy, including 410 non-transfusion-dependent
thalassemia (NTDT), 260 transfusion-dependent thalassemia (TDT), 190 SCD and sickle
thalassemia, and 550 CHA cases, namely hereditary spherocytosis (HS), stomatocytosis
(HSt), elliptocytosis (HE), and enzymopathies (mainly including pyruvate kinase defi-
ciency). Direct and indirect antiglobulin test (DAT and IAT) results were revised by
an experienced immune hematologist, and the diagnosis of AIHA was made according
to international guidelines [13]. Reticulocyte count was collected when available, and the
bone marrow responsiveness index (BMRI) was calculated according to the formula “abso-
lute reticulocyte count × patient’s Hb/normal Hb” using a cutoff of 121 to discriminate
well-compensated hemolytic anemia from an ineffective response [14].
Response to AIHA therapy was defined as complete response (CR) (Hb > 12 g/dL and
normalization of all hemolytic markers), partial response (PR) (Hb > 10 g/dL or at least
2 g/dL increase in Hb, and no transfusion requirement) [15,16], and no response (NR).
The study was approved by the ethical review committee of the coordinating center
“Comitato Etico Milano Area 2” and was carried out according to the principles established
by the Declaration of Helsinki. A literature review by searching for the terms “congenital
anemia”, “thalassemia”, “sickle cell disease”, “hereditary spherocytosis”, “hereditary
elliptocytosis”, “autoimmune hemolytic anemia”, “auto-antibodies” in indexed articles
in MEDLINE via PubMed and the National Library of Medicine in the last 50 years
was performed.
J. Clin. Med. 2021, 10, 3439 3 of 11

3. Results
3.1. AIHA in the Congenital Anemias Cohort
AIHA complicated the clinical course of 14 (1%) patients regularly followed for
congenital anemias, including nine β-thalassemia (mainly non-transfusion-dependent,
n = 7), two SCD/β-thalassemia, one SCD, one HS, and one HE patients. Table 1 shows
the main clinical laboratory findings collected during the AIHA episode. The median
Hb values dropped from 9.5 g/dL to 5.9 g/dL during the AIHA crisis, with a significant
increase in LDH. Reticulocyte response (i.e., BMRI > 121) was adequate to Hb levels in 7/11
(64%) of the evaluated subjects. AIHA cases were classified as warm (n = 11, of whom four
were fixing complement), cold (n = 2), and DAT-negative (n = 1, patient #4, diagnosed after
the exclusion of other causes of hemolysis and steroid response). Of note, eight patients
exhibited anti-RBC alloAbs along with autoAbs. We were not able to identify a trigger in
all the events; however, possible triggers were recent transfusion (n = 5), infection (n = 3),
pregnancy (n = 2), and surgery (n = 1). All the patients received steroid therapy as the
first-line, but the second- and third-line treatments were necessary in four (28.5%) and
two (14%) patients, respectively, and included rituximab (n = 2), azathioprine (n = 2),
intravenous immunoglobulins (n = 1), and cyclophosphamide (n = 1). Transfusion support
was required in eight patients (57%) with non-transfusion-dependent congenital anemia,
and recombinant human erythropoietin (rhEPO) was safely administered in five patients
(36%). Hydroxycarbamide (HU) for the underlying condition was given to four patients
during the acute event to obtain a reduction in transfusion requirements, while one was
already on chronic treatment. On the whole, four CR, seven PR, and three NR were
registered, with the median time to response of 2.5 months (range, 0.5–11). Notably, one HE
(#2) and two TDT (patients ##3 and 5) subjects experienced more than one AIHA episode,
and one patient (#14) died during acute AIHA because of underlying liver failure.

3.2. Literature Review

The data available in the literature on RBC autoAb/AIHA occurrence in congenital
anemias are summarized in Tables 2 and 3 and mainly derive from retrospective studies,
case series, or case reports on thalassemia and SCD. Most studies are focused on alloAbs
in transfused patients and report the presence of autoAbs in this context. Notably, all the
reports except for one in β-thalassemia [17] evaluated the frequency of autoAbs rather than
that of AIHA. Finally, only one study from our group evaluated the presence of autoAbs in
HS, by using the more sensitive mitogen-stimulated DAT [11,18].
J. Clin. Med. 2021, 10, 3439 4 of 11

Table 1. Characteristics and clinical management of the patients with congenital hemolytic anemias (CHAs) experiencing autoimmune hemolytic anemia (AIHA) referred to our institution.

Age (Years)/ Underlying CHA, Usual Hb Year of AIHA Hb Nadir during LDH (U/L) (xULN), ARC (×109 /L), sEPO Response at Last Crisis, Time
ID Trigger DAT Positivity Treatments
Sex Category Spleen status Values (g/dL) Occurrence AIHA (g/dL) UB (mg/dL) BMRI (U/L) to Restore Usual Hb Values

430 (2), 230; PR,

1 26/F HS 9 2018 - 6.3 50 C IV mPDN 1 mg/kg/day, folic acid, vitamin B12, oral iron
4.3 104 15 days

530 (2), 310; PR,

2 81/M HE 11 2008, 2009, 2010 - 7.3 27.3 IgG IV mPDN 500 mg for 2 days, then 2 mg/kg/day, rhEPO, folic acid
1.74 141 1.5 months

TDT,
3 50/F 8 1991, 2003, 2008 - NA NA NA NA IgG Oral PDN, azathioprine, cyclophosphamide PR, NA
splenectomy

PR,
4 30/F NTDT 10 2007 Pregnancy 4 NA NA NA NA, alloAb+ IV mPDN, rhEPO, transfusions (HU for NTDT)
>3 months

5 50/M TDT 10 2009, 2012 - NA NA NA NA IgG Oral PDN, azathioprine CR, NA

NTDT 559 (2.5), 396; CR,

6 49/M 9.5 2013 - 7.6 NA IgG+C, AlloAb+ Oral PDN 5 mg/day, folic acid (HU for NTDT)
splenectomy 2.46 188 11 months

33/F Pregnancy and 599 (2), 439; PR,

7 NTDT 9.5 2018 5.1 96.8 IgG+C, alloAb+ IV mPDN 1 mg/kg/day, folic acid, transfusions
transfusion 1.73 160 2 months

NTDT, Transfusion, 510 (2), 392; IgG+C, IV mPDN 1.5 mg/kg/day, rituximab 375 mg/s.m., folic acid, PR,
8 43/F 12 2018 6.3 NA
splenectomy pielonephritis 0.83 174 alloAb+ vitamin B12 (HU for NTDT) >3 months

7; Oral PDN 0.6 mg/kg/day, rhEPO, folic acid, vitamin B12,

9 67/M β-thal trait 9.5 2019 - 6.9 227 (1.06), 1.28 173 IgG, alloAb+ NR
3 transfusions

601 (2.8), 246;

10 24/F NTDT 8.5 2019 - 5.6 NA IgG, alloAb+ Oral PDN 0.5 mg/kg/day, folic acid, transfusions NR
4.31 98

IV mPDN 1.5 mg/kg/day + high-dose boli 1 g/day for 3 days,

1378 (6.4), 30; IVIG, rituximab 375 mg/s.m., rhEPO, folic acid, vitamin B12, CR,
11 54/M NTDT 9.5 2019 - 2.3 NA C, alloAb+
4.06 4 transfusions (HU for NTDT, imiglucerase for 4 months
β-glucocerebrosidase deficiency)

Surgery, infection, 4696 (21.9), 436; IgG+C, CR,

12 34/F SCD 10.5 2020 4.5 3365 IV mPDN 1 mg/kg/day, folic acid, vitamin B12, transfusions
transfusion 3.33 140 alloAb+ 2.5 months

Start of chronic
SCD/β-thal, 1235 (5.7), 668; IV mPDN 1.5 mg/kg/day, rhEPO, folic acid, transfusions (HU for PR,
13 58/F 8 2020 transfusion 6.3 NA IgG
splenectomy 1.67 301 SCD/thal) 20 days
support

Infection, 791 (3.7), 557;

14 55/M SCD/ β-thal 8.5 2020 5 130 IgG IV mPDN 1 mg/kg/day, folic acid, transfusions NR, dead
transfusion 33 174

599,
Summary
49.5 9.5 5.9 227–4696 392 113.4
median, - - - - - -
24–81 8–12 2.3–7.6 2.46 7–668 27.3–3365
range
0.83–33

HS: hereditary spherocytosis, HE: hereditary elliptocytosis, TDT: transfusion-dependent thalassemia, NTDT: non-transfusion-dependent thalassemia, SCD: sickle cell disease, Hb: hemoglobin, LDH: lactate
dehydrogenase, ULN: upper limit of normality, UB: unconjugated bilirubin, ARC: absolute reticulocyte count, sEPO: serum erythropoietin, DAT: direct antiglobulin test, C: complement, AlloAb: alloantibodies,
IV: intravenous, mPDN: methylprednisolone, PR: partial response, rhEPO: recombinant human erythropoietin, NR: non-response, HU: hydroxycarbamide, PDN: prednisone, CR: complete response, IVIG:
intravenous immunoglobulins.
J. Clin. Med. 2021, 10, 3439 5 of 11

Overall, autoAb frequency ranges from 1% to 28.2% in β-thalassemia [17,19–26], from

0.8% to 42% in SCD [27–30], and reaches 61% in HS (likely due to more sensitive tech-
nique) [11]. Few data are available about the Ab type, with warm antibodies (IgG+) being
reported in half of the DAT-positive cases, IgG+ complement (C)—in approximately one
third of cases, and C+—in the remaining cases. Studies are consistent in identifying alloAbs,
transfusion exposure, and splenectomy as risk factors for the development of autoAbs.
Interestingly, only two observational studies and a few case reports evaluated clinically
relevant AIHA, with the prevalence ranging from 1.8% to 6.4% [17,28]. The majority of
autoAbs were warm, with or without complement fixation. A recent longitudinal study
by Khaled et al. showed that 25 subjects developed AIHA among 385 β-thalassemia pe-
diatric patients [17]. All the patients were transfusion-dependent, and the frequency of
AIHA was inversely proportional to the number of blood transfusions received. AIHA
was triggered by vaccination in two patients and by Mycoplasma pneumoniae infection in
another. This study also showed that splenectomy in DAT-positive subjects was associated
with an increased risk of AIHA in thalassemic patients. Finally, considering the autoAb
type, thalassemic patients with IgG+ and C+ DAT were at higher risk for clinically overt
AIHA. Overall, most AIHA patients presented with severe anemia and all required ther-
apy. First-line steroids were the most frequent strategy, associated with IVIG in some
cases. The use of various cytotoxic/immunosuppressive drugs is described for refractory
cases, including azathioprine, mycophenolate mofetil, cyclophosphamide, methotrexate,
vincristine, anti-thymocyte globulin, and actinomycin D. Splenectomy was performed
for AIHA in selected refractory cases [17,31,32]. The use of rhEPO has been reported
only in one retrospective study [28], and only one case of thalassemia with AIHA was
successfully treated with rituximab [33]. Responses to various therapies are difficult to
establish given the heterogeneity of treatments and the small number of patients in each
report. Finally, across the various reports, two pediatric patients had a fatal outcome. These
were one SCD case with both autoAbs and alloAbs who developed an over-hemolytic
transfusion reaction [29] and one β-thalassemia case who had received multiple lines of
immunosuppressive therapies for AIHA (prednisone, cyclophosphamide, methotrexate,
vincristine, anti-human lymphocyte globulin, and actinomycin D; splenectomy) and died
due to infection [32].

Table 2. Studies and case series/reports about RBC autoAbs/AIHA in thalassemia.

Type of Study; Number of pts; Pediatric/Adult;

Main Findings
Objective of the Study Sex

- Frequency of RBC autoAbs: 22.6%

- AIHA: 6.4% of the entire population, 28.7% of
the subjects with RBC autoAbs
- DAT: IgG+ 65.5%, IgG+ C+ 32.1%, IgM+ 0.2%
- AIHA risk factor: prior alloimmunization, β-TI,
splenectomy, the first 72 transfusions, family
Longitudinal study; 385; history of AIHA, presence of polyspecific
to identify predictive factors of AIHA pediatric; autoAbs, AB blood type
in patients with RBC autoAbs F 57.5%, M 42.5% - AIHA protective factor: transfusion with
[17]
phenotypic and leukoreduced blood
- First-line treatment: prednisone 2 mg/kg/d
- Second-line treatments: IV methylprednisolone
1000 mg/m2 /day for three days (n = 5),
azathioprine (n = 7), mycophenolate mofetil
(n = 4), splenectomy (n = 2)
J. Clin. Med. 2021, 10, 3439 6 of 11

Table 2. Cont.

Type of Study; Number of pts; Pediatric/Adult;

Main Findings
Objective of the Study Sex

500; - Frequency of RBC autoAbs: 1%

Prospective observational study;
pediatric and adult; - No association with alloimmunization
incidence of RBC autoAbs [19]
F 57%, M 43%
407; - Frequency of RBC autoAbs: 6.5%
Cross-sectional study
pediatric and adult; - Risk factor: alloAbs
[20]
F 55%, M 45%

301; - Frequency of RBC autoAbs: 15.9%

Frequency of RBC autoAbs - DAT: IgG+ 56.3%, IgG+ C+ 10.4%, C+ 33.3%
NA;
[21] - Risk factor: alloAbs
NA

319; - Frequency of RBC autoAbs: 28.2%

Frequency of RBC autoAbs
pediatric and adult; - Risk factor: age, splenectomy, number of
[22]
F 26.3%, M 73.7% transfusions

- Frequency of RBC autoAbs: 1.8 %

Case-control study; 280;
- DAT: IgG+ 100%
prevalance of autoAbs pediatric and adult;
- All (n = 5) developed clinically significant AIHA
[23] F 33.3%, M 66.7%
- Treatment: steroids

- Frequency of RBC autoAbs: 16.5%

200;
Frequency of RBC autoAbs - Risk factor: age, duration of transfusion support
pediatric and adult;
[24] and the total number of transfusions,
F 53%, M 47%
splenectomy

- Frequency of RBC autoAbs: 22.8%

Retrospective and prospective 118; - 37% transient autoAbs without any treatment
observational study; prevalence of pediatric and adult; - DAT: IgG+ 48%, IgG+ C+ 52%
RBC autoAbs; risk factor analysis [25] F 50%, M 50% - Risk factor: alloAbs, splenectomy

49;
Frequency of RBC autoAbs
pediatric and adult; - Frequency of RBC autoAbs: 2.14 %
[26]
F 49%, M 51%

- DAT: IgG+
- Treatment:
- Case 1: High-dose IVIG (CR)
- Case 2: prednisone therapy (ineffective); then
azathioprine (PR); then High-dose IVIG (CR).
Splenectomy for significant splenomegaly
4; - Case 3: prednisone therapy (ineffective); then
Case series of AIHA
pediatric; High-dose IVIG (PR).
[31]
F 25%; M 75% - Case 4: prednisone therapy (PR); then
High-dose IVIG (CR). Recurrence after therapy
interruption: treatment with IVIG repeated,
with CR
- The standard dose of 2 g/kg was used for IVIG
treatment
J. Clin. Med. 2021, 10, 3439 7 of 11

Table 2. Cont.

Type of Study; Number of pts; Pediatric/Adult;

Main Findings
Objective of the Study Sex

- Both cases of AIHA presented with severe

anemia
- DAT: C+ (case 1); negative at diagnosis, then
2; strongly positive (case 2)
Case report of AIHA
pediatric; - Treatment: transfusions, IVIG
[34]
F 50%, M 50% 2 g/kg + methylprednisolone 30 mg/kg/d for
3 days, followed by prednisolone 2 mg/kg/d in
both cases
- Response: PR weeks later

- AIHA presenting with severe anemia

- DAT: initially negative, IgG+ after splenectomy
- Treatment: transfusion, oral prednisone
30 mg/day, cyclophosphamide 50 mg/day,
and methotrexate 2.5 mg/day: no hemolysis
reduction. Treatment with vincristine
1; 1 mg/week, anti-human lymphocyte globulin
Case report of AIHA 125 mg/day IV, and actinomycin D
pediatric;
[32] 15 mcg/kg/day for 5 days led to transient
F
decrease in transfusion requirement.
Splenectomy (for splenomegaly): no
improvement in hemolysis
- The patient died at the age of 45 months of
bronchopneumonia, pericardial effusion, and
congestive heart failure

- AIHA presenting with severe anemia

- DAT: IgG+ C+. IAT positive as well
- First-line treatment: prednisolone 45 mg/day,
1; then IVIG 20 mg in association (CR)
Case report of AIHA pediatric;
[33] - Second-line treatment at reactivation one year
M
later: rituximab 375 mg/m2 (four doses) and
regular transfusion of red cell antigen (major
and minor)-matched blood (CR)

RBC: red blood cells, autoAbs: autoantibodies; alloAbs: alloantibodies; AIHA: autoimmune hemolytic anemia; IV: intravenous; IVIG:
intravenous immunoglobulin; F: female; M: male, CR: complete remission; PR: partial remission; DAT: direct antiglobulin test (with
monospecific antisera unless otherwise specified).

Table 3. Studies and case series/reports about RBC autoAbs/AIHA in SCD and CHAs.

Type of Study Number of pts;

Objective of the Study Pediatric/Adult; Main Findings
Disease Sex

- Freque;ncy of RBC autoAbs: 12%

Prospective study 158 - DAT: C+ (n = 12)
SCD, TDT Pediatric and adult - The antibody did not appear to be clinically important
[27] NA since it became thermally reactive at 4 ◦ C and not at 37 ◦ C
- Risk factors: alloAbs
J. Clin. Med. 2021, 10, 3439 8 of 11

Table 3. Cont.

Type of Study Number of pts;

Objective of the Study Pediatric/Adult; Main Findings
Disease Sex

- Frequency of RBC autoAbs: 3.6% of the total, 8–9.7% of

the transfused
- DAT: panagglutinin with no apparent specificity (83%)
Retrospective study 167 - Only 2/12 patients developed AIHA
SCD Pediatric and adult - Risk factors: alloAbs
[28] NA - Treatment and response of AIHA:
- case 1 (IgG): steroids, IVIG, EPO, and RBC transfusions.
CR after 2 months
- case 2: unknown

- Frequency of RBC autoAbs: 8%

Retrospective study.
- DAT: IgG+ (n = 9), IgG+ C+ (n = 5)
Frequency, serological
184 - AIHA 4/14 patients (all IgG+ C+)
characteristics, and clinical
Pediatric - Risk factors: alloAbs
significance of autoimmunization
NA - Treatment: corticosteroids, RBC transfusion
in pediatric patients
- Response: one fatal hemolytic reaction following
SCD [29]
transfusion

- Frequency of RBC autoAbs: 42%

SCD 12
- DAT: IgG+ (n = 1), C+ (n = 4)
[30] Pediatric and adult
- Risk factors: alloAbs, chronic transfusion

- DAT: IgG+ C+
Case series 5 - Risk factors: alloAbs
SCD Pediatric and adult - Treatment: steroids; mercaptopurine in one patient
[35] F 2, M 3 - Response: CR/PR; autoAbs reverted to negative in all the
patients after hospital discharge

- Case 1: severe anemia (Hb 2.8 g/dL). Treated with

Case series and review of the 2
prednisone, IVIG, and RBC transfusion
literature Adult
- Case 2: severe anemia (Hb 4 g/dL), LDH was 3175 IU,
SCD [36] 1 M, 1 F
reticulocytes 28%. IgG+. Treated with prednisone with CR

- Frequency of RBC autoAbs by mitogen-stimulated DAT:

61%—IgG fraction bound to α- and β-spectrin, Band 3,
91
HS and Band 4.9
adult
[11] - The positive cases displayed increased reticulocytosis and
40 F, 51 M
slightly reduced hemoglobin (Hb) values compared to the
negative ones

TDT: transfusion-dependent thalassemia, SCD: sickle cell disease; RBC: red blood cells, autoAbs: autoantibodies; alloAbs: alloantibodies;
AIHA: autoimmune hemolytic anemia; IV: intravenous; IVIG: intravenous immunoglobulin; F: female; M: male, DAT: direct antiglobulin
test (with monospecific antisera unless otherwise specified); EPO: erythropoietin; CR: complete remission; NA: not available.

4. Discussion
Although rare, AIHA is a serious and potentially life-threatening complication of
congenital anemias. Our study represents the largest cohort of congenital anemias in
which the prevalence and treatment of AIHA are evaluated. In our analysis, AIHA had
a prevalence of 1%, which is lower than that reported in the paper by Khaled et al. [17],
which, however, included pediatric thalassemic patients only. Regarding triggers, we also
observed that previous splenectomy, recent transfusions, infections, and pregnancy may be
associated with the development of anti-RBC autoimmunity. However, in more than half
of the patients, the trigger was not identified. Different prevalence between studies can
J. Clin. Med. 2021, 10, 3439 9 of 11

be related either to heterogeneous populations or frequency of potential triggers. Indeed,

infections are more frequent in children and in certain regions, and transfusion strategies
and blood product may differ. Additionally, more severe CHAs with early transfusion
requirement during childhood may be at higher risk of allo- and autoimmunization. Finally,
it is largely accepted that immune system maturation is a dynamic concept evolving along
with age and that the type and severity of several autoimmune conditions consistently vary
from infancy to adulthood and elderly age. More importantly, in our study, we evaluated
the prevalence of clinically overt AIHA, whilst most reports deal with the presence of
anti-RBC autoAbs only (DAT positivity). As a matter of fact, the diagnosis of AIHA may
be challenging in chronic hemolytic patients and, even more, in those on transfusions. In
the former, hemolytic features are already present, and AIHA should be suspected in case
of sudden drop of Hb levels or significant worsening of hemolytic markers. In the latter,
a further flag may be the decrease of pretransfusion hemoglobin or the increase in the
transfusion need. In addition, most patients in our series also exhibited anti-RBC alloAbs,
which is a known finding in congenital anemias, particularly in transfusion-dependent
ones. One of the key points is the distinction of autoAbs from alloAbs either in transfusion-
dependent or non-transfusion-dependent conditions. In fact, besides the importance of
assigning the best-matched RBC units to the patient, recognition of the “true” AIHA is
pivotal for proper therapy. In this study, all the patients received steroids, and mostly
responded, whilst about 25% required further treatment. Of note, about 1/3 of the patients
received rhEPO, which has been shown to be effective in primary AIHA [37] avoiding
possible myelotoxicity of immunosuppressants. Generally, therapy of AIHA in the context
of congenital anemias is not codified, and therefore the guidelines available for primary
forms are applied. If steroids are given cautiously in patients with congenital anemias
and several comorbidities, second-line therapies raise even more concerns. Hydroxyurea,
which is extensively used in SCD and some NTDT patients to increase the total amount of
hemoglobin and fetal hemoglobin, has been recently demonstrated to alleviate complement
activation in sickle cell patients, thus acquiring a potential role in AIHA management [38].
Use of plasma exchange (PEX) has also been reported in very severe AIHA [39] in case
of steroids/IVIG refractoriness. No cases of CHA-related AIHA receiving PEX have been
reported; however, its use in CHA should be carefully evaluated, especially in SCD with
the risk related to increased viscosity.
In this regard, a close collaboration with a transfusion medicine specialist is funda-
mental to perform DAT with more sensitive methods (microcolumn and solid phase tests,
washings with low ionic strength solutions, or experimental methods) [10], as well as to
characterize the alloAbs by studying the eluate and performing extended phenotyping and
genotyping when required.
The pathophysiology underlying the development of anti-RBC autoAbs in congenital
anemias is object of several hypotheses. Some mechanisms include the exposure to foreign
antigens, as occurs in transfusion-dependent patients developing alloAbs, or during preg-
nancy, the molecular mimicry after infections, the spread of hidden epitopes during the
hemolytic process through the deformation of erythrocyte membranes due to exposure to
neoantigens, and the release of the free heme. The latter is involved in post-translational
diversification of circulating Abs, may induce complement activation on SCD RBCs, thus
increasing the risk of autoimmune reactions [40]. Additionally, the abnormal structure of
SCD and thalassemic RBCs (prematurely expressing senescence antigens) may be more
easily recognized by the immune system, as also shown by the increased amounts of
RBC-bound IgG detected in these patients. Finally, the ineffective erythropoiesis typical of
hemoglobinopathies, along with a proinflammatory bone marrow milieu, may also favor
the development of anti-erythroblast autoAbs.

5. Conclusions
In conclusion, AIHA in the context of congenital anemias may be challenging both
from a diagnostic and a therapeutic point of view. Clinical suspicion should be high
J. Clin. Med. 2021, 10, 3439 10 of 11

and prompt a proper evaluation of hemolytic markers, bone marrow compensation, and
assessment of the DAT positivity for alloAbs and autoAbs.

Author Contributions: Conceptualization, I.M., B.F. and W.B.; data curation, M.F., K.B., J.G., I.M.,
B.F. and N.R.; writing—original draft preparation, I.M., J.G., B.F. and W.B.; writing—review and
editing, G.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was approved by the ethical review committee of
the coordinating center Comitato Etico Milano Area 2 and was carried out according to the principles
established by the Declaration of Helsinki.
Informed Consent Statement: Informed consent was obtained from all the subjects involved in
the study.
Data Availability Statement: The data presented in this study are available in the article.
Conflicts of Interest: J.G., M.F., K.B., N.R., G.G. declare no conflict of interest. I.M. reports receiving
consultancy honoraria from Sanofi-Genzyme and Amicus Therapeutics; W.B. provided consultancy
services to Agios, Alexion, Apellis, Biocryst, Bioverativ, Incyte, Momenta, and Novartis, and received
lecture fees/congress support from Alexion, Incyte, Novartis, and Sanofi; B.F. reports receiving
consultancy honoraria from Novartis, Amgen, and Momenta.

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