Clinical perspectives, immunohematologic insights, and transfusion management in IgA-associated autoimmune hemolytic anemia

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 in vivo hemolysis with a positive DAT; others were diagnosed with hemolytic anemia due to various causes. These latter patients were DAT– for any Ig and/or complement. The remaining DAT+ patients with in vivo hemolysis were further subjected to immunohematologic evaluation per the departmental protocol. Patients diagnosed with cold AIHA and mixed AIHA were excluded from the study. Detailed immunohematologic characterization was performed using the sensitive gel-based column agglutination technology (CAT) (Bio-Rad, Cressier s/Morat, Switzerland). Blood group discrepancy, if any, was resolved using recommended methods.¹⁴ Samples positive for polyspecific DAT were subjected to monospecific DATs (anti-IgG, anti-IgM, anti-IgA, anti-C3c, and anti-C3d) using dedicated gel cards (DC-Screening I; Bio-Rad). Cold acid elution was performed on DAT+ RBCs per the standard method.¹⁴ An eluate was tested against reagent group O RBCs at three different temperatures (4°C, 22°C, and 37°C) to determine the thermal amplitude of the autoantibody and its specificity.

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 N (%), median, and mean ± standard deviation. Demographic, clinical, and immunohematologic variables were compared between IgA-associated WAIHA and non–IgA-associated WAIHA. Qualitative variables and quantitative variables were analyzed using the χ² test and t test, respectively, and a p value <0.05 was considered statistically significant.

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.

On analyzing the 214 patients with WAIHA, we observed that in vivo hemolytic markers were significantly abnormal in the IgA-associated WAIHA group when compared with the non-IgA group (Table 2). Parameters such as Hb (5.58 g/dL) and Hct (16.9%) were significantly lower (p ≤ 0.002), and other hemolytic parameters like Retic (9.13%), S.bil (4.63 mg/dL), and LDH (967.5 IU/L) were significantly higher in the IgA-associated group (p ≤ 0.02).

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%, p = 0.0004). Blood group discrepancy was encountered in 10 (5.1%) patients with WAIHA, which was resolved before initiating the compatibility testing. Except for the IgA-only cases, free autoantibody in serum (IAT ≥2+) was found in all patients with IgA-associated WAIHA. Sixty-nine (35%) patients in the non-IgA WAIHA group did not reveal any free serum autoantibody.

Fig. 1

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 (n = 8, 53.3%), anti-c (n = 3, 20%), anti-C (n = 2, 13.3%), and anti-s (n = 1, 6.7%). New alloantibodies developed at a lower rate in patients with IgA WAIHA, with only anti-Fy^a identified in one of the patients. All patients with alloimmunization had history of blood transfusion elsewhere before visiting our hospital. A total of 17 (7.9%) patients experienced adverse events to blood transfusion, with a majority suffering from febrile non-hemolytic transfusion reaction (n = 11, 64.7%) and allergic reactions like urticaria and itching (n = 4, 23.5%).

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 (N = 214) and AIHA (N = 348), respectively. Among the total cases of AIHA, only two patients in our study had IgA-only autoantibody, for an incidence of 0.57 percent.

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 In vivo hemolytic markers such as Hb, Hct, Retic, S.bil, and LDH were significantly abnormal in patients with IgA-associated WAIHA when compared with patients with non–IgA-associated WAIHA (p ≤ 0.022). These significant differences in laboratory values may be due to multiple Ig coating of the RBCs in IgA-associated WAIHA, the high-binding potency of IgA for RBCs, and the potentiality of IgA antibodies to induce severe hemolysis through FcαRI-mediated erythrophagocytosis, ADCC, NADPH oxidase activation, and cytokine release.^1,2,5,12,26 In 2005, Wikman et al.²⁷ studied the correlation of low Hb, high Retic, high LDH, high S.bil, and low haptoglobin with in vivo autoimmune hemolysis. They classified hemolysis into severe when three parameters were abnormal, moderate when one or two parameters were abnormal, and no hemolysis when the laboratory parameters were within normal range.²⁷

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 (p = 0.004). Whereas 32 units of PRBCs were transfused to the patients with IgA-associated WAIHA (2.13 units/patient), with 84.4 percent (27 of 32) being incompatible, only 123 units (1.43 units/patient) were needed for the transfusion of patients with non-IgA WAIHA, with only 19.5 percent (24 of 123) incompatibility. Only 17 (7.9%) patients complained of minor adverse events to blood transfusion; the remaining patients tolerated the transfusion well.

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.