# Autoimmune Hemolytic Anemia

## Introduction

Autoimmune hemolytic anemia (AIHA) is characterized by the production of autoantibodies directed against self-red blood cell antigens, leading to accelerated RBC destruction. With an incidence of 1 to 3 per 100,000 per year, it is an uncommon but clinically important condition that may present as a primary (idiopathic) disorder or as a manifestation of an underlying disease. Classification is based on the thermal reactivity of the pathogenic antibody: warm antibodies operate optimally at 37 degrees Celsius, cold antibodies at 4 degrees Celsius, and mixed AIHA involves both types simultaneously.

## Classification

### Warm AIHA (wAIHA) - 70-80% of AIHA

Warm AIHA accounts for the majority of cases and is mediated by IgG autoantibodies, typically directed against antigens of the Rh blood group system. These antibodies exhibit maximal activity at 37 degrees Celsius, which is body temperature. The predominant mechanism of red cell destruction is extravascular hemolysis through splenic Fc-receptor-mediated phagocytosis: splenic macrophages recognize the Fc portion of IgG bound to red cells and either engulf them completely or partially strip the membrane, producing the characteristic spherocytes seen on the peripheral smear. The DAT pattern shows IgG positivity, with or without concurrent C3d positivity.

### Cold Agglutinin Disease (CAD) - 15-25% of AIHA

Cold agglutinin disease is mediated by IgM autoantibodies, most commonly directed against the I or i antigens on the red cell surface. These antibodies exhibit maximal activity at 4 degrees Celsius and agglutinate red cells at temperatures encountered in the peripheral circulation (fingers, toes, ears, nose). The IgM antibodies activate the classical complement pathway, depositing C3b on the red cell surface. The fate of these C3b-coated cells depends on the degree of complement activation: partial activation leads to C3b opsonization and extravascular hemolysis in the liver (by hepatic Kupffer cells, which have complement receptors), while full activation to the membrane attack complex causes intravascular hemolysis. The DAT is characteristically positive for C3d only, with IgG negative. The cold agglutinin titer is typically greater than 1:64 and often exceeds 1:1000, but the thermal amplitude (the highest temperature at which agglutination occurs) correlates more closely with clinical severity than the titer itself.

### Paroxysmal Cold Hemoglobinuria (PCH) - <5%

Paroxysmal cold hemoglobinuria is the least common form but has a distinctive and elegant pathophysiology. The Donath-Landsteiner antibody is a biphasic IgG autoantibody directed against the P antigen on red cells. It binds to red cells and fixes complement in cold temperatures encountered in the peripheral circulation, and when these complement-coated cells return to the warm central circulation, complement activation proceeds to completion, causing intravascular lysis. Historically associated with tertiary syphilis, PCH is now seen most frequently as a post-viral syndrome in children. The DAT shows C3d positivity, and the diagnosis is confirmed by the Donath-Landsteiner test.

### Mixed AIHA - ~5%

Mixed AIHA involves the simultaneous presence of both warm IgG and cold IgM autoantibodies. The DAT is positive for both IgG and C3d, and a significant cold agglutinin titer is present. This form typically has a more severe clinical course and is often secondary to systemic lupus erythematosus.

| Feature | Warm AIHA | Cold Agglutinin Disease | Paroxysmal Cold Hemoglobinuria | Mixed AIHA |
|---|---|---|---|---|
| Frequency | 70-80% | 15-25% | <5% | ~5% |
| Antibody class | IgG | IgM | IgG (biphasic) | IgG + IgM |
| Target antigen | Rh system | I or i antigen | P antigen | Multiple |
| Optimal temperature | 37°C | 4°C | Binds at cold, lyses at 37°C | 37°C + cold |
| DAT pattern | IgG ± C3d | C3d only | C3d only | IgG + C3d |
| Hemolysis type | Extravascular (spleen) | Extravascular (liver) ± intravascular | Intravascular | Both |
| Steroids effective | Yes (70-85%) | No (usually) | Supportive only | Partially |
| Splenectomy effective | Yes (60-70%) | No | No | Variable |
| Common associations | CLL, SLE, idiopathic | Lymphoproliferative, Mycoplasma, idiopathic | Post-viral (children), syphilis | SLE |

## Etiology

### Primary (Idiopathic)

Approximately 50% of warm AIHA cases and the majority of cold agglutinin disease cases in elderly patients are primary, meaning no underlying cause is identified.

### Secondary Causes

Lymphoproliferative disorders represent the most important secondary associations. Chronic lymphocytic leukemia (CLL) is the single most common association, and AIHA may be the presenting manifestation of CLL. Lymphomas and Waldenstrom macroglobulinemia are additional associations. Autoimmune diseases, particularly systemic lupus erythematosus, are frequently associated, and the combination of AIHA with immune thrombocytopenia is termed Evans syndrome. Infections including Mycoplasma pneumoniae (which causes an acute cold agglutinin syndrome with anti-I antibodies), Epstein-Barr virus (anti-i antibodies), CMV, HIV, and hepatitis may trigger AIHA. Drug-induced causes are discussed separately below. Post-transplant AIHA occurs in 5 to 15% of allogeneic HSCT recipients. Immune checkpoint inhibitors (PD-1/PD-L1 and CTLA-4 inhibitors) are an increasingly recognized cause. Primary immunodeficiency states, including autoimmune lymphoproliferative syndrome (ALPS) and common variable immunodeficiency (CVID), predispose to AIHA.

<image>A clinical classification diagram of autoimmune hemolytic anemia showing the three main types (warm AIHA, cold agglutinin disease, and paroxysmal cold hemoglobinuria) arranged in three columns. For each type, display: the antibody class (IgG vs IgM), thermal reactivity range (shown as a thermometer graphic), DAT pattern, predominant mechanism of hemolysis (extravascular vs intravascular), site of RBC destruction (spleen vs liver), typical secondary associations, and characteristic peripheral smear findings (spherocytes for warm AIHA, RBC agglutination for CAD). Include a fourth smaller column for mixed AIHA. Use a consistent color scheme with warm tones for warm AIHA and cool tones for cold AIHA. Medical education poster format.</image>

## Diagnosis

### Laboratory Findings

The anemia in AIHA can be profound, with hemoglobin falling below 5 g/dL in severe cases. Reticulocytosis is expected but may be absent in 15 to 20% of patients due to marrow suppression, anti-reticulocyte antibodies, or concurrent nutritional deficiency. Hemolytic markers are elevated (LDH, indirect bilirubin) and haptoglobin is decreased or absent. The peripheral smear demonstrates spherocytes in warm AIHA and agglutination (clumps of red cells) in cold agglutinin disease, along with polychromasia reflecting reticulocytosis.

### Direct Antiglobulin Test (DAT)

The DAT is performed first with polyspecific reagent, and if positive, monospecific reagents (anti-IgG and anti-C3d) are applied. IgG alone indicates warm AIHA, C3d alone indicates cold agglutinin disease or PCH, and IgG plus C3d indicates warm AIHA with complement fixation or mixed AIHA. The strength of the DAT (graded 1+ to 4+) does not correlate with the severity of hemolysis. DAT-negative AIHA occurs in approximately 5 to 10% of cases and may be due to IgA-mediated hemolysis (not detected by standard reagents), low-affinity IgG that washes off during testing, or subthreshold antibody levels. More sensitive techniques such as gel/column methodology or mitogen-stimulated DAT can detect these cases.

### Additional Workup

For suspected CAD, cold agglutinin titer and thermal amplitude should be measured. For suspected PCH, the Donath-Landsteiner test (incubating patient serum with normal RBCs at 4 degrees then 37 degrees, with hemolysis confirming the diagnosis) is performed. Flow cytometry should be obtained in all cases to screen for an underlying CLL or other lymphoproliferative disorder. CT imaging is indicated if lymphoma is suspected. ANA and anti-dsDNA are checked if SLE is suspected. Immunoglobulin levels and serum protein electrophoresis complete the secondary cause evaluation.

## Drug-Induced Immune Hemolytic Anemia (DIIHA)

### Mechanisms

Drug-induced immune hemolytic anemia can occur through several mechanisms. Drug-dependent antibodies (hapten or immune complex mechanism) react only in the presence of the offending drug; cephalosporins are now the most common cause, followed by piperacillin, NSAIDs, and fludarabine. Drug-independent autoantibody induction occurs when a drug triggers the formation of true autoantibodies that persist after drug discontinuation; methyldopa is the classic example, with 15% of treated patients developing a positive DAT though fewer than 1% develop clinical hemolysis. Non-immunologic protein adsorption (NIPA) occurs with high-dose beta-lactam antibiotics, which modify the RBC membrane to cause nonspecific protein binding, producing a positive DAT without hemolysis.

### Approach

Drug-induced hemolysis typically manifests 7 to 10 days after initiation (or immediately upon re-exposure). The offending drug should be discontinued, and supportive care with steroids or transfusion is provided if the hemolysis is severe. Hemolysis usually resolves within 1 to 2 weeks of drug cessation.

## Treatment of Warm AIHA

### First-Line

Corticosteroids remain the cornerstone of first-line therapy for warm AIHA. Prednisone at 1 to 1.5 mg/kg/day produces response rates of 70 to 85%, with improvement typically expected within 1 to 3 weeks. The steroid taper must be gradual, extending over 4 to 6 months, as rapid dose reduction frequently leads to relapse. Pulse methylprednisolone at 1 g IV for 3 days is reserved for severe or life-threatening anemia. Folic acid supplementation at 1 to 5 mg daily is recommended to support the increased erythropoietic demand.

### Second-Line

Rituximab, an anti-CD20 monoclonal antibody given at 375 mg/m2 weekly for 4 doses, achieves response rates of 80 to 90% with a median response duration exceeding 2 years. It is now considered early second-line therapy and is increasingly used in combination with steroids as initial treatment based on the ACTR randomized trial, which demonstrated superior relapse-free survival at 36 months with the combination (70%) compared to prednisone alone (45%). Splenectomy, historically a standard second-line option with response rates of 60 to 70%, is falling out of favor given the availability of rituximab, though it remains an option for refractory cases. The current consensus increasingly favors rituximab before splenectomy.

### Third-Line and Beyond

For refractory disease, options include mycophenolate mofetil (1 g BID, response rate approximately 60%), azathioprine (1 to 2 mg/kg/day, slower onset over 3 to 6 months, response rate approximately 50%), cyclosporine, and danazol (400 to 800 mg/day, approximately 40% response rate with virilizing side effects). Sutimlimab, an anti-C1s complement inhibitor FDA-approved for CAD, is under investigation for warm AIHA. Fostamatinib, a SYK inhibitor approved for ITP, has demonstrated some activity in warm AIHA in off-label use.

### Refractory/Life-Threatening wAIHA

For life-threatening hemolysis unresponsive to steroids and rituximab, IVIG provides temporary benefit with a response rate of approximately 40% (less reliable than in ITP). Plasma exchange has limited efficacy for IgG-mediated disease given the large volume of distribution of IgG. Cyclophosphamide at 500 to 750 mg/m2 pulsed or 1 to 2 mg/kg orally may be effective. Alemtuzumab is reserved as a last resort given the profound immunosuppression it produces.

## Treatment of Cold Agglutinin Disease

### General Measures

The first-line intervention in cold agglutinin disease is avoidance of cold exposure, which prevents peripheral complement activation. Intravenous fluids and blood products should be warmed before administration. Critically, corticosteroids are ineffective in most cases of CAD because IgM production by B cells is not steroid-sensitive, unlike the IgG-producing plasma cells in warm AIHA. Splenectomy is also ineffective in CAD because the site of red cell destruction is predominantly hepatic (complement-mediated), not splenic.

### Pharmacologic Therapy

Rituximab monotherapy achieves response rates of 50 to 60%, with a median response duration of approximately 11 months. The combination of rituximab with bendamustine improves response rates to approximately 70% with more durable responses and is now considered the standard first-line pharmacologic treatment for CAD requiring therapy. Sutimlimab (Enjaymo), a humanized anti-C1s monoclonal antibody that blocks the classical complement pathway, received FDA approval in 2022 for CAD. In the CARDINAL trial, 54% of patients achieved a hemoglobin increase of 2 g/dL or more by week 5, with rapid onset of response. Importantly, sutimlimab does not reduce the cold agglutinin titer itself but treats the downstream complement-mediated destruction. Patients on sutimlimab require vaccination against encapsulated organisms and ongoing infection monitoring.

<image>A treatment algorithm for autoimmune hemolytic anemia divided into two parallel pathways: warm AIHA on the left and cold agglutinin disease on the right. For warm AIHA: First-line: prednisone 1-1.5 mg/kg/day (±rituximab based on ACTR trial) → assess response at 3 weeks. If responsive, taper steroids over 4-6 months. If relapse or refractory: second-line options shown as a decision tree including rituximab (if not given upfront), splenectomy, mycophenolate, or azathioprine. Third-line: cyclophosphamide, danazol, or clinical trials. For cold agglutinin disease: First step: cold avoidance, warm blood products. If treatment required: rituximab ± bendamustine OR sutimlimab (complement inhibition). Show sutimlimab mechanism as an inset: blocking C1s in the classical complement pathway to prevent C3b opsonization and MAC formation. Include response rates for each therapy. Clean clinical algorithm format.</image>

## Transfusion in AIHA

### Challenges

Transfusion in AIHA presents unique challenges. The autoantibody typically reacts with all red cells in the crossmatch panel, creating a pan-reactive pattern that makes compatibility testing difficult. The blood bank must perform additional testing to rule out clinically significant alloantibodies that may be masked by the autoantibody. Extended phenotype or genotype matching, particularly for Rh and Kell antigens, is essential to prevent alloimmunization.

### Practical Approach

The most critical principle is that transfusion should never be withheld in life-threatening anemia. The "least incompatible" blood is appropriate and will provide temporary oxygen-carrying capacity, even though the transfused cells will hemolyze at approximately the same rate as the patient's own cells. Transfusion should proceed slowly with close monitoring. When clinically stable, allowing time for the blood bank to complete a thorough workup is appropriate. For patients with CAD, a blood warmer should be used to prevent exacerbation of cold-mediated hemolysis.

## Special Situations

### Evans Syndrome

Evans syndrome is defined by the co-occurrence of AIHA with immune thrombocytopenia (ITP), with or without autoimmune neutropenia. It may be primary or secondary to SLE, ALPS, CVID, or CLL. Treatment follows the principles of warm AIHA management, with IVIG providing acute benefit for the thrombocytopenia component. Rituximab is particularly effective in this setting. Children with Evans syndrome should be screened for ALPS.

### AIHA Post-Transplant

AIHA occurs in 5 to 15% of allogeneic HSCT recipients. The mechanism often involves donor-derived antibodies targeting recipient residual red blood cells in the setting of major ABO-incompatible transplantation. Passenger lymphocyte syndrome in solid organ transplantation presents similarly.

## Key Clinical Pearls

- Cold agglutinin disease is a clonal B-cell lymphoproliferative disorder (MYD88 wild-type, typically), NOT simply "autoimmune" - this is why it responds to B-cell directed therapy and not steroids
- In CAD, MCV may be spuriously elevated due to RBC agglutination in the analyzer; warming the sample corrects this
- Thermal amplitude of the cold agglutinin is more clinically relevant than the titer; an antibody active at 30C is more dangerous than one active only at 4C
- Never withhold transfusion in life-threatening AIHA; the transfused cells will hemolyze at the same rate as patient's own cells but will provide temporary oxygen-carrying capacity
- Consider underlying CLL in any older adult with new-onset AIHA; flow cytometry is essential
- Rituximab combined with short-course steroids as initial therapy for warm AIHA is supported by the ACTR trial and should be discussed early

## References
1. Jager U, et al. Diagnosis and treatment of autoimmune hemolytic anemia in adults: Recommendations from the First International Consensus Meeting. Blood Rev. 2020;41:100648.
2. Birgens H, et al. A phase III randomized trial comparing glucocorticoid monotherapy versus glucocorticoid and rituximab in patients with autoimmune haemolytic anaemia (ACTR trial). Br J Haematol. 2021;193(3):e118-e121.
3. Berentsen S, et al. Rituximab plus bendamustine in chronic cold agglutinin disease: results of a prospective multicenter trial. Blood. 2017;130(4):537-541.
4. Roth A, et al. Sutimlimab in patients with cold agglutinin disease (CARDINAL study). Blood. 2022;139(17):2553-2564.
5. Hill QA, et al. Guidelines on the management of drug-induced immune and secondary autoimmune, haemolytic anaemia. Br J Haematol. 2017;177(2):208-220.