Autoimmune hemolytic anemia (AIHA) is characterized by increased red blood cell (RBC) destruction and/or decreased RBC survival caused by autoantibodies directed against self-antigens on RBCs.1 The incidence of the disease has been reported to vary from 1 in 80,000 to 100,000 in a given population per year.2 Detection of RBC-bound immunoglobulins (Igs), like IgG, IgM, or IgA, and/or complement by direct antiglobulin test (DAT) remains the crucial serologic assay in the diagnosis of AIHA.3 A positive DAT is almost always seen in association with AIHA and forms the hallmark of the diagnosis.4 These autoantibodies react at various thermal amplitudes and, accordingly, AIHA is classified as warm, cold, or mixed AIHA.3,4
Previous studies have found that IgA class antibodies are present in approximately 14 percent of patients with warm AIHA (WAIHA) and are almost always associated with IgG.5,6 Nevertheless, WAIHA associated exclusively with IgA antibodies reacting optimally at 37°C remains rare.7,8 Reported cases of IgA-only WAIHA show increased disease severity, high-binding potency of IgA for RBCs, scant free antibody in serum, non-obvious blood group specificity, and re-fractoriness to first-line treatments like glucocorticosteroids.5 Approximately 90 percent of the IgA present in serum is monomeric, and IgA antibodies interact to a variable extent with the IgA Fc receptor FcαRI/CD89.9,10 IgA-mediated immune effector responses, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), NADPH oxidase activation, and cytokine release, are induced primarily by the activation of these FcαRI/CD89 receptors.11 Researchers have demonstrated that in IgA-only WAIHA, the IgA-coated RBCs fail to activate complement but trigger phagocytosis by monocytes
At our referral hospital–based immunohematology facility, we receive requests for complete characterization of autoantibodies in suspected cases of AIHA. In this study, we explored the patients with WAIHA, and we present our detailed work on characterizing patients with IgA WAIHA, both clinically and serologically, with regard to demographic profile, presenting symptoms, laboratory values, and immunohematologic and transfusion characteristics.
This study was conducted by the Apollo Multispeciality Hospital in Kolkata, India, from July 2012 to June 2021 and included 1249 patient samples with provisional diagnosis of “anemia under evaluation” or AIHA. Prior ethics clearance was obtained from the hospital ethics committee to conduct the study. A total of 348 patients had evidence of
Serum study by CAT included indirect antiglobulin tests (IATs) using commercial cell panels (ID-Dia Panel; Bio-Rad) and an autocontrol. Wherever indicated, adsorption studies were performed to investigate any underlying masked alloantibody using polyethylene glycol, as already described.14,15 In all agglutination studies, the agglutination reactions were graded as 4+, 3+, 2+, 1+, and negative and documented accordingly.
The current study included only those 214 AIHA patients who belonged to the WAIHA type; we further classified WAIHA into two groups: (1) IgA-associated WAIHA (IgA-only or IgA with IgG or complement or both) and (2) non–IgA-associated WAIHA. Demographic and clinical details of patients and their hematologic parameters such as hemoglobin (Hb), hematocrit (Hct), percentage of reticulocytes (Retic), total serum bilirubin (S.bil), and serum lactate dehydrogenase (LDH) were obtained from patient files and the Hospital Information System.
For patients requiring blood transfusion, blood samples and requisitions for packed RBCs (PRBCs) were obtained from the blood center for compatibility testing. For patients who developed underlying alloantibody(ies), corresponding antigen-negative blood was obtained from the inventory and subjected to crossmatching. In cases of incompatible crossmatch, “best match” or “least incompatible” PRBC units were selected for transfusion, as discussed by previous authors.16,17 All transfusions were performed slowly under close supervision and with regular monitoring of vital signs. Any adverse reaction to blood transfusion was managed accordingly and documented.
Statistical analysis was performed using software (SPSS, version 13; IBM, Armonk, NY). Results were calculated as
A total of 214 patients with WAIHA were studied, of whom 17 (7.9%) belonged to the IgA-associated WAIHA group. Two IgA-only WAIHA cases were found during our investigation (Fig. 1). Table 1 describes the demographic, laboratory, and immunohematologic characteristics of the patients with IgA-associated WAIHA. A total of 10 patients were female (male:female [M:F] = 1:1.4). The median age of these patients was 50 years (range 27–68 years), and 70.6 percent were aged 40 years or older. The mean Hb in this group of patients was 5.58 g/dL. Mean Hb in patients younger than 40 years was 5.74 ± 0.54 g/dL; the group ≥40 years of age showed a mean Hb of 5.47 ± 1.23 g/dL. All IgA-associated WAIHA patients revealed high DAT reactivity (median DAT 4+). Monospecific DAT revealed 12 patients with IgG+IgA WAIHA, three with IgG+IgA+C3, and two with IgA-only WAIHA.
Patient no. | Demographics | Laboratory values | Immunohematologic details | Transfusion (PRBC units) | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Age (years) | Sex | Hb (g/dL) | Retic (%) | S.Bil (mg/dL) | LDH (IU/L) | DAT (poly) | DAT (mono) | IAT | ||
1 | 68 | M | 6.7 | 5.5 | 3.4 | 798 | 4+ | IgG+IgA | 3+ | 1 |
2 | 53 | M | 7.1 | 5 | 2.8 | 654 | 3+ | IgG+IgA | 2+ | None |
3 | 55 | F | 5.9 | 6.1 | 3.9 | 880 | 4+ | IgG+IgA | 3+ | 2 |
4 | 43 | F | 4.8 | 9.5 | 5.1 | 1102 | 4+ | IgA | 0 | 2 |
5 | 52 | F | 3.5 | 12.6 | 7.2 | 1349 | 4+ | IgG+IgA+C3 | 4+ | 3 |
6 | 50 | F | 5.9 | 8.2 | 4.1 | 901 | 4+ | IgG+IgA | 4+ | 2 |
7 | 39 | F | 5.5 | 7.9 | 4 | 873 | 4+ | IgG+IgA | 4+ | 2 |
8 | 37 | M | 6.4 | 6.6 | 3.3 | 995 | 3+ | IgG+IgA | 3+ | 2 |
9 | 60 | M | 7.3 | 5.1 | 3.1 | 709 | 3+ | IgG+IgA | 2+ | None |
10 | 51 | F | 4.6 | 10.2 | 7.3 | 1512 | 4+ | IgA | 0 | 3 |
11 | 29 | F | 5.1 | 7.8 | 5.1 | 893 | 4+ | IgG+IgA | 3+ | 2 |
12 | 59 | M | 3.9 | 13.4 | 6.9 | 1334 | 4+ | IgG+IgA+C3 | 4+ | 3 |
13 | 41 | F | 5.7 | 7.7 | 4.5 | 921 | 4+ | IgG+IgA | 4+ | 2 |
14 | 35 | M | 6.2 | 6.8 | 4 | 605 | 4+ | IgG+IgA | 3+ | 1 |
15 | 27 | F | 5.5 | 8.1 | 3.7 | 819 | 4+ | IgG+IgA | 4+ | 2 |
16 | 57 | M | 5.8 | 8 | 3.4 | 843 | 4+ | IgG+IgA | 3+ | 2 |
17 | 41 | F | 4.4 | 9.8 | 7 | 1260 | 4+ | IgG+IgA+C3 | 4+ | 3 |
Ig = immunoglobulin; WAIHA = warm autoimmune hemolytic anemia; Hb = hemoglobin (normal range 13–17 g/dL); Retic = reticulocytes (normal range 0.5-2.5%); S.Bil = serum bilirubin (normal up to 1 mg/dL); LDH = lactate dehydrogenase (normal range 266–500 IU/L); DAT = direct antiglobulin test; poly = polyspecific; mono = monospecific; IAT = indirect antiglobulin test; PRBC = packed red blood cell; C = complement.
On analyzing the 214 patients with WAIHA, we observed that
IgA-associated WAIHA (N = 17) | Non-IgA-associated WAIHA (N = 197) | Statistics ( |
||
---|---|---|---|---|
Two-tailed |
95% Confidence interval | |||
Hb (g/dL) | 5.58 ± 1.07 | 6.71 ± 1.16 | 0.0001 | –1.701 to –0.553 |
Hct (%) | 16.9 ± 2.73 | 19.5 ± 3.32 | 0.002 | –4.232 to –0.971 |
Retic (%) | 9.13 ± 2.39 | 7.38 ± 1.93 | 0.0005 | 0.769 to 2.732 |
S.Bil (mg/dL) | 4.63 ± 1.54 | 3.52 ± 1.93 | 0.022 | 0.165 to 2.051 |
LDH (IU/L) | 967.5 ± 258.9 | 793.2 ± 269.3 | 0.010 | 40.692 to 208.317 |
Values are presented as mean ± standard deviation, unless otherwise marked.
Ig = immunoglobulin; WAIHA = warm autoimmune hemolytic anemia; Hb = hemoglobulin; Hct = hematocrit; Retic = reticulocytes; S.Bil = serum bilirubin; LDH = lactate dehydrogenase.
Table 3 depicts the clinical, immunohematologic, and transfusion characteristics of the patients with WAIHA. The median age of the patients with non–IgA-associated WAIHA was 43 years, with a female preponderance (M:F = 1:1.7). Secondary WAIHA was found in 11 (64.7%) patients with IgA-associated WAIHA, and 116 (58.9%) patients had underlying etiology in the non-IgA WAIHA group. The major underlying diseases included lymphoproliferative diseases (57.5%), systemic lupus erythematosus (19.7%), and rheumatoid arthritis (11.9%). Over 90 percent of patients in both categories presented with weakness and pallor. Clinical manifestations, such as fever (29.4%), jaundice (23.5%), and organomegaly (41.2%) were observed more in the IgA-associated WAIHA patients. No statistical significance was observed when comparing the clinical characteristics of patients between the two groups. While strong DAT reactivity (≥2+) was observed in 153 (71.5%) patients with WAIHA, a median 4+ DAT and 4+ IAT were found in the IgA WAIHA group. Fifteen patients with IgA-associated WAIHA (this group also included the two IgA-only cases) received 32 units of PRBC transfusions, of which 27 (84.4%) were “best match” or “least incompatible.” A total of 86 patients with non-IgA WAIHA received 123 units of blood, of which 99 (80.5%) were compatible. Statistical analysis of the immunohematologic and transfusion parameters between the two groups showed that blood transfusion was significantly higher in the IgA-associated WAIHA patients (88.2% vs. 43.7%,
Characteristics | IgA-associated WAIHA ( |
Non-IgA-associated WAIHA ( |
|
---|---|---|---|
Age in years (range) | 50 (27–68) | 43 (20–71) | — |
Sex (male:female) | 1:1.4 | 1:1.7 | 0.736 |
Primary WAIHA | 6 (35.3) | 81 (41.1) | 0.639 |
Secondary WAIHA | 11 (64.7) | 116 (58.9) | |
Lymphoproliferative disease | 6 (35.3) | 67 (34.1) | — |
Systemic lupus erythematosus | 3 (17.6) | 22 (11.2) | — |
Rheumatoid arthritis | 1 (5.9) | 14 (7.1) | — |
Autoimmune hepatitis | 1 (5.9) | 4 (2) | — |
Glomerulonephritis | 0 | 5 (2.5) | — |
Thyrotoxicosis | 0 | 2 (1) | — |
Polyarthritis nodosa | 0 | 1 (0.5) | — |
Sarcoidosis | 0 | 1 (0.5) | — |
Fever | 5 (29.4) | 34 (17.2) | 0.213 |
Jaundice | 4 (23.5) | 33 (16.7) | 0.478 |
Organomegaly | 7(41.2) | 71 (36) | 0.672 |
Palpitation | 10 (58.8) | 102 (51.8) | 0.576 |
Dyspnea | 7(41.2) | 65 (33) | 0.493 |
DAT positive | 17 (100) | 197 (100) | — |
DAT <2 + | 0 (0) | 61 (31) | 0.0067 |
DAT ≥2+ | 17 (100) | 136 (69) | |
DAT strength | 4+ (1+ to 4+) | 3+ (1+ to 4+) | — |
IAT positive | 15 (88.2%) | 128 (65%) | — |
IAT <2+ | 0 (0) | 48 (37.5) | 0.0036 |
IAT ≥2+ | 15 (100) | 80 (62.5) | |
IAT strength | 4+ (1+ to 4+) | 2+ (1+ to 4+) | — |
Autocontrol strength | 3+ (1+ to 4+) | 2+ (1+ to 4+) | — |
Blood group discrepancy | 1 (5.9) | 9 (4.6) | 0.805 |
Patients with new alloantibody | 1 (5.9) | 14 (7.1) | 0.849 |
Patients transfused ( |
15 (88.2) | 86 (43.7) | 0.0004 |
PRBC units crossmatched ( |
118 | 309 | — |
PRBC units transfused ( |
32 | 123 | — |
Compatible PRBC units transfused | 5 (15.6) | 99 (80.5) | 0.983 |
“Least incompatible” PRBC units transfused | 27 (84.4) | 24 (19.5) | |
PRBC units transfused per patient (mean ± SD) | 2.13 ± 0.92 | 1.43 ± 0.68 | — |
Transfusion reaction | 2 (11.8) | 15 (7.6) | 0.543 |
History of previous transfusion | 11 (64.7) | 107 (54.3) | 0.408 |
Values are presented as median (range) or
Ig = immunoglobulin; WAIHA = warm autoimmune hemolytic anemia; DAT = direct antiglobulin test; IAT = indirect antiglobulin test; PRBC = packed red blood cell; SD = standard deviation.
More than half (55.1%) of the patients with WAIHA had history of blood transfusion, and 81.3% (109 of 134) of the female patients had history of pregnancy. RBC allo-immunization was noted more in the non-IgA patients with WAIHA (14 of 197, 7.1%). The newly detected alloantibodies in this group were anti-E (
AIHA is one of the oldest recognized autoimmune disorders; women are twice as likely to have AIHA as are men. Diagnostic pitfalls, treatment failure, life-threatening complications, and absence of evidence-based guidelines often make the disease medically challenging.18,19 Case studies on IgA WAIHA are ample in the literature, but original studies on clinical and immunohematologic characteristics of IgA-associated WAIHA are scarce. In this study, we explored WAIHA with special consideration for IgA-associated and non–IgA-associated WAIHA.
Sokol et al.5 observed that 124 of 5235 patients with AIHA had IgA autoantibodies, and, of this group, only six had IgA-only AIHA, for an incidence of 0.14 percent. The authors commented that autoimmune hemolysis due to IgA antibodies alone is rare and that, most commonly, IgA acts synergistically with other immunoglobulins (usually IgG) with or without complement.20 The rarity of IgA-only AIHA has been discussed in the literature, and the reported incidence in some series ranges from 0.2 to 2.7 percent.21,22 We investigated 17 patients with IgA autoantibodies, accounting for 7.9 and 4.9 percent of the total patients with WAIHA (
Female individuals show a stronger immune response than male individuals because of variations in hormones and genetic and environmental factors. As a result, the female preponderance for AIHA has been regularly observed.23,24 The present study revealed a female preponderance for AIHA, with a M:F ratio of 1:1.4 and 1:1.7, respectively, in IgA-associated and non–IgA-associated WAIHA.
The median age of patients in the present study was 50 years (IgA-associated WAIHA group) and 43 years (non–IgA-associated WAIHA group). Most authors have described a high incidence of AIHA in individuals above 40 years of age, with peak incidence being between 60 and 70 years of age.1,2,19
While the thought that IgA autoantibodies alone can activate complement remains controversial, increasing evidence suggests that they possibly can activate complement and cause intravascular hemolysis via the alternative pathway.20 A minimal role of complement activation in IgA-associated WAIHA was also reported by previous authors.12,13 We observed that only 3 of 17 (17.6%) patients with IgA-associated WAIHA had complement coating their RBCs. All of these patients presented with severe anemia (Hb ≤4.4 g/dL) and jaundice (S.bil ≥6.9 mg/dL). Two women (>40 years of age) with IgA-only WAIHA arrived in the emergency department of the hospital in altered sensorium with severe pallor and organomegaly (Table 1). In their study, Sokol et al.5 found that all six patients with IgA-only AIHA had IgA and C3d coating their RBCs and two of them had severe intravascular hemolysis, which is in concordance with the evidence that IgA antibodies are capable of activating complement by an alternative or lectin pathway.5,20,25
A secondary cause of AIHA has been determined in 20–80 percent of reported studies; these causes include lymphoproliferative disorders, autoimmune disorders, infections, immunodeficiency disorders, and tumors.1,2,28 Lymphoproliferative disorders account for approximately half of the cases of secondary warm AIHA.1,2 We observed secondary WAIHA in 11 (64.7%) and 116 (58.9%) patients of IgA-associated and non–IgA-associated WAIHA, respectively. The most common underlying ailment in our study was lymphoproliferative diseases (57.5%).
As described by Pirofsky,29 WAIHA has a highly variable clinical presentation. In cases of secondary disease, the symptoms of AIHA may precede the recognition of the underlying illness by months to years, but ultimately the symptoms of the underlying disorder dominate.29 More than 90 percent of patients in our study presented with weakness, fatigue, and pallor; in secondary AIHA, however, the symptoms of underlying disorders were predominant. Clinical manifestations like fever, jaundice, and organomegaly were observed more often in the patients with IgA-associated WAIHA.
A strongly reactive DAT signifies heavily coated RBCs that are easily recognized by the mononuclear phagocyte system causing hemolysis.1,2,30 We found the polyspecific DAT to be negative for both patients with IgA-only WAIHA in the initial investigation. Considering the clinical condition of the patients—severe ongoing hemolysis and anemic crisis—we performed monospecific DATs and revealed the underlying IgA bound to their RBCs. Such experience of DAT– AIHA was also shared by previous authors.31 We observed that all patients of IgA-associated WAIHA and 69 percent of patients with non-IgA WAIHA had a DAT strength of ≥2+. While IgG alone was detected in 129 (65.4%) patients with non-IgA WAIHA, multiple autoantibodies (IgG+C3) were found in the remaining patients.
Free autoantibodies alone or with underlying masked alloantibodies in serum was observed in 127 (59.3%) patients with WAIHA. Most patients with IgA-associated WAIHA (88.2%) and 62.9 percent of patients with non-IgA WAIHA reported an IAT strength of ≥2+, which signifies increased disease severity in the former group. A literature search showed that frequency of IAT positivity in individuals with AIHA is variable. Young et al.32 investigated a very low frequency of 4.6 percent; on the other hand, Petz and Garratty33 and Wheeler et al.34 reported IAT positivity of 70 percent and 57 percent, respectively. Determination of ABO blood group in AIHA is a frequent problem, caused by the autoantibodies bound to the RBCs or in the patient’s serum. Whereas Zhu et al.35 found blood group discrepancy in 31.6 percent of patients with AIHA, Das et al.28 observed a discrepancy of 6.8 percent in their 59 patients studied. The present study found ABO group discrepancy in 4.7 percent of samples, all of which were resolved by applying recommended methods.14
Petz36 once emphasized that free autoantibodies in serum do not always show incompatible crossmatch, although underlying masked alloantibody(ies) make the work more complicated. Other researchers have revealed that excess serum autoantibodies in WAIHA are more prone to cause crossmatch incompatibility.37 We observed alloimmunization (anti-E>anti-c>anti-C) in 11 (5.1%) patients with WAIHA, with the majority (93.3%) in the non-IgA WAIHA group. The majority of our patients (55.1%) reported previous transfusion of whole blood or PRBCs in one or more hospitals.
Previous studies on AIHA concluded that the decision to transfuse in AIHA should be based on the clinical condition of the patient and that no critical patient should be denied blood transfusion due to serologic incompatibility.17,36 In addition, Shirey et al.38 described that the major causes of blood transfusion in AIHA include the critical condition of the patient, the complex serology, severe anemia, and profound ongoing hemolysis. Interestingly, in the present study, we observed that 88.2 percent of patients with IgA-associated WAIHA received blood transfusions compared with only 43.7 percent of patients with non-IgA WAIHA (
We conclude that characterization of WAIHA with particular emphasis on IgA-associated and non–IgA-associated WAIHA is essential to evaluate the disease characteristics, access the degree of hemolysis, understand the immunohematologic behaviors of the antibodies, and manage blood transfusions.