AIHA is an acquired hemolysis condition where the immune system attacks red cell antigens.
Complement activation influences clinical presentation and is a therapeutic target.
A stepwise approach should be used when a patient presents with anemia:
Initial Investigations:
Normo-/macrocytic anemia
Raised reticulocyte count
Raised unconjugated bilirubin
Reduced haptoglobin
Blood smear with polychromasia or spherocytes/agglutination (Figure 1).
Note: None of these tests are fully sensitive or specific. Liver disease can increase LDH and reduce haptoglobin. Bilirubin can be normal in mild cases, and spherocytes might not always be visible. Reticulocytopenia can occur, possibly due to bone marrow infiltration or parvovirus B19 infection. Reticulocytopenia can be observed in a significant minority of patients (20% in one series), despite erythroid hyperplasia in the marrow, potentially due to immune attack on late-stage erythroid precursors or a lag in marrow responsiveness; this can persist and predict a more severe clinical course.
Significantly raised LDH, red cell fragments, or urinary hemosiderin suggests intravascular hemolysis.
Confirmation of Hemolysis
Further investigation is needed to confirm if hemolysis is immune-mediated, primarily using the direct antiglobulin test (DAT).
DAT Results:
Standard DAT shows IgG and/or complement (C3d) bound to the red cell membrane.
Autoantibodies can be of IgM and IgA classes (detected using extended DAT panel).
Exclude other causes of positive DAT, such as:
Delayed transfusion reaction
Alloimmune hemolysis post-transplant
Drug-induced immune hemolysis
Hemolytic disease of the newborn
Passive antibody deposition in liver disease, chronic infection, malignancy, SLE, renal disorders, IV Ig, or anti-thymocyte globulin.
DAT-Negative AIHA
AIHA can occur with a negative DAT due to low-affinity antibody or IgA-only antibody.
Diagnose by excluding other hemolysis causes, confirming with a sensitive technique, and noting response to steroid therapy.
Further Investigation
Investigate underlying conditions (~50% of patients) and perform serological tests to determine the AIHA type, as treatment varies.
A suggested diagnostic pathway for AIHA is shown in Figure 2.
A final diagnosis may integrate the clinical picture and specialist reference laboratory advice.
Types of AIHA
Warm AIHA:
~65% of patients; diagnosed with a consistent clinical picture and DAT positive for IgG only or C3d + IgG, excluding clinically significant cold antibody.
Primary Cold Agglutinin Disease (CAD):
Caused by underlying lymphoproliferative bone marrow disorder.
IgM antibodies active in vitro at low temperatures bind red cells in cooler peripheral circulation, causing agglutination and acrocyanosis or Raynaud’s disease.
IgM binds C1q, activating the classical complement pathway but dissociating centrally, leading to DAT positive for C3d only (sometimes weak IgG).
Extravascular removal of C3b-coated red cells occurs, but exacerbations can activate C5, causing intravascular hemolysis.
Diagnose with AIHA, DAT positive for C3d + IgG, consistent clinical picture, and high-titer cold reactive antibody (titer \ge 1:64 at 4°C).
Antibody thermal amplitude is usually \ge 30°C when red cells are suspended in 30% bovine albumin.
Serum electrophoresis usually detects a monoclonal paraprotein (typically IgMk); keep serum at 37-38°C from sampling.
Cold Agglutinin Syndrome (CAS):
Laboratory criteria consistent with clinically significant cold antibody associated with secondary disorders (infection, SLE, aggressive lymphoma).
Mixed AIHA:
Combination of warm IgG and cold IgM antibody.
DAT usually positive for IgG and C3d.
Cold-associated symptoms are rare; cold antibody may have a low titer (<1:64) but thermal amplitude up to 30-37°C.
Diagnose with DAT positive for IgG and C3d, cold antibody with thermal amplitude \ge 30°C , and appropriate clinical picture.
Paroxysmal Cold Hemoglobinuria (PCH):
Usually in children; severe and intravascular hemolysis but transient following infection.
Caused by a biphasic IgG antibody that binds to red cells at low temperatures and causes complement-mediated lysis as temperature increases.
Diagnose with AIHA and positive Donath-Landsteiner test.
Consider in patients with AIHA and DAT positive for C3d + IgG (sometimes negative) when CAD has been excluded, and there is hemoglobinuria, cold-associated symptoms, atypical serology, or age <18 years.
Autoantibody specificity is usually anti-P, unlike CAD (usually anti-I, sometimes anti-i or anti-PR).
Warm AIHA autoantibodies usually target a high-incidence antigen on the red cell surface (~3% have specificity, e.g., anti-e).
Treatment of AIHA
Secondary AIHA
Treat the underlying condition according to best practice.
If the associated condition does not require treatment, approach AIHA similarly to primary AIHA, but individualize treatment decisions.
In SLE, splenectomy benefits are less favorable compared to primary warm AIHA.
Evan’s syndrome (AIHA + immune thrombocytopenia [ITP]) has differences, including alternative causes of bilineage cytopenia, identifying secondary causes like autoimmune lymphoproliferative syndrome, and managing the relapsing/remitting clinical course.
B-cell malignancies are a common association (e.g., AIHA in ~5-10% of CLL patients).
In active CLL requiring treatment, combinations like RCD, R-CVP, and BR are effective.
New targeted therapies (obinutuzumab, ibrutinib, idelalisib, venetoclax) could potentially be beneficial; however, vigilance is needed for drug-induced immune cytopenias.
General Strategies for Primary AIHA
Treat any identified underlying cause (e.g., infection).
Provide folic acid supplementation to prevent deficiency.
Compatibility testing for transfusion:
Identify patient’s ABO CcDEe and K status and alloantibodies using techniques like auto- or alloadsorption or prewarming.
Genotyping can be useful, especially for complex cases.
Full compatibility testing can take 4-6 hours, and units will not be labeled “compatible.”
Without a history of transfusion or pregnancy, an alloantibody is unlikely.
If anemia is life-threatening, transfuse ABO Rh and K matched red cells rather than waiting for full compatibility testing.
Venous thromboembolism prophylaxis:
Thrombosis risk is underestimated but remains important.
VTE events occur in up to 20-25% of patients, usually when hemolysis is active.
Larger studies show higher rates of venous and arterial thrombosis.
Risk increases with higher LDH levels and previous splenectomy.
Thromboprophylaxis with low-molecular-weight heparin is recommended for in-patients with acute exacerbation and considered in ambulatory patients during severe exacerbations (hemoglobin < 85 g/L ).
Strategies Specific to CAD Patients
Treat bacterial or febrile illnesses promptly.
Avoid cold weather and protect distal extremities.
Keep the patient warm in the hospital, especially perioperatively.
Do not administer cold fluids IV; use an in-line blood warmer.
Strategies for Patients Receiving Steroids
Provide antacid therapy (e.g., proton pump inhibitor) if there is an additional risk factor.
Advise on lifestyle modification (smoking cessation, limiting alcohol intake to \le 2 U/d , and engaging in regular weight-bearing exercise).
Maintain adequate daily vitamin D (600-800 IU) and calcium (1000-1200 mg) intake, if possible, or supplements, if needed.
For adults \ge 40 years, estimate initial absolute fracture risk using FRAX and bone mineral density testing to determine whether an oral bisphosphonate should also be prescribed.
Primary Warm AIHA
Watchful waiting may be appropriate in mild asymptomatic anemia cases.
First-line therapy: Glucocorticoids (typically prednis(ol)one at a starting dose of 1 mg/kg).
Response rate is ~80%, but ~60% lose their response upon weaning or discontinuing steroids.
Two phase 3 prospective randomized trials have compared predniso(lo)ne with predniso(lo)ne-rituximab as first-line treatment for warm AIHA:
A Danish study randomized 64 patients: response rate to prednisolone-rituximab was significantly higher (75% vs 36%; P = 0.003) at 12 months, with no difference in adverse reactions.
A French study randomized 32 patients: the primary end point of overall response at 12 months was significantly higher (75% vs 31%; P = 0.032) in patients receiving rituximab, without excess adverse events.
First-line rituximab with a relatively short course of steroids has been suggested for elderly patients or those with comorbidities.
Second-line treatment: Rituximab is favored over splenectomy in recent UK and French guidelines.
In a meta-analysis of 154 patients, the overall response rate of warm AIHA to rituximab was 79%.
Adverse events occurred in 38 of 364 (14%) AIHA patients receiving rituximab, including 18 severe infections.
Approximately 25% to 50% of responding patients will relapse within 2 to 3 years.
Splenectomy:
Approximately 70% of patients with primary warm AIHA respond to splenectomy.
Alternative, relatively nontoxic immunosuppressive therapies: mycophenolate mofetil, azathioprine, and cyclosporin can also be used as third-line treatment.
Patients who are steroid sensitive but refractory or relapsing after third-line therapies may tolerate longer-term treatment with low-dose predniso(lo)ne ( \le 10 mg ), which can be used in conjunction with a steroid-sparing agent.
Alternatives with greater potential for toxicity include cyclophosphamide, alemtuzumab, and hematopoietic stem cell transplantation.
Rescue therapies:
For severe transfusion-dependent hemolysis, steroids (if the patient is known to be sensitive) or IV Ig (approximately one third of patients will respond) can be used.
Recombinant erythropoietin can be considered when hemolysis is severe with a poor reticulocyte response; however, parvovirus, hematinic deficiency, and marrow infiltration should also be considered.
Emergency splenectomy or partial splenic embolization in unfit patients has been used acutely to treat severe hemolysis.
C1 esterase inhibitor (C1-INH) may have a role in stabilizing severe hemolysis if the DAT is positive for C3d.
Future prospects for primary warm AIHA include inhibition of the following:
B cells with alternative anti-CD20 mAbs (e.g., obinutuzumab)
Mammalian target of rapamycin (e.g., sirolimus)
Proteasomes (e.g., bortezomib)
Spleen tyrosine kinase (Syk) (e.g., fostamatinib)
Neonatal Fc receptor (FcRn)
Bruton tyrosine kinase (BTK)
Complement pathways
Primary CAD
Patients with mild asymptomatic anemia can be monitored without specific treatment, which should be reserved for symptomatic anemia, severe circulatory symptoms, or transfusion dependence.
First-line treatment is usually rituximab (375 mg/m2 weekly for 4 weeks).
Approximately 50% responded in 2 prospective case series, but complete responses were rare.
The median response duration was only 11 months.
Rituximab has been combined with bendamustine (BR) or fludarabine in 2 prospective uncontrolled studies.
Combination therapy provides more opportunity for a durable response but with a greater risk for toxicity.
Emergency treatment:
In addition to blood transfusion, daily or alternative-day plasma exchange of 1 to 1.5 times the plasma volume with albumin has been used as a bridging therapy.
Patients with acute severe intravascular hemolysis may also respond to the C5 inhibitor eculizumab.
Future prospects:
Inhibition of the classical complement pathway is a rational and promising therapeutic approach for CAD.
The C3 inhibitor APL2 is being evaluated in warm AIHA and CAD.
Given the clonal nature of CAD, newer targeted therapies for B-cell malignancies, such as the BTK inhibitor ibrutinib and B-cell receptor pathway–modulating drugs (e.g., idelalisib and venetoclax), might have activity in CAD, although the hypothesis awaits investigation.