Autoimmune Haemolytic Anaemia

Autoimmune haemolytic anaemia


Cell-mediated immune red cell destruction

§        Monocytes and macrophages have cell surface receptors for Fc portion of IgG and for antigenic determinants on activated C3


Fc receptor mechanism - IgG

§        Macrophages have Fc receptors for IgG1 and IgG3

§        IgG coated red cells are destroyed

§        warm AIHA ~75% of antibodies are IgG

§        Spleen

§        Splenic vasculature allows very close contact between rbcs and splenic macrophages – main site for removal of IgG coated cells.

§        Partial Phagocytosis leads to the formation of spherocytes


C3 receptor mechanism – IgM

§        IgM binds to the cell at 20oC leading to C3 deposition on the cell surface.  The IgM elutes from the cell surface at 37oC leaving C3

§        C3 receptors - CR1 and CR3 on macrophages (particularly hepatic Kupfer cells)

§        CR1 is specific for an antigenic site in the C3c region of activated C3b which is not exposed on native C3 (circulating native C3 is not bound)

§        The largest concentration of C3b binding macrophages is in the liver – main site for destruction of rbcs in CHAD.



IgG antibodies

IgM antibodies



(except for Donath Lansteiner Abs - PCH)



Macrophages in spleen

(except PCH – intravascular haemolysis)

Complement activation

-         intravascular haemolysis

-         macrophages in liver


IgG positive

C3 positive

(False positives are often C3)

Ab specificity

10-15% against Rh antigens

Often polyclonal/ pan reacting

Monoclonal / polyclonal

-         anti-i – EBV

-         anti-I - mycoplasma








Causes of a positive DAT

Antibodies made by the patient

Abs to autologous epitopes (AIHA)

§   Triggered by infections (Mycoplasma / EBV)

Abs to allogenic red cells

Abs to drugs adsorbed onto the surface of red cells

Normal Abs reacting to new antigenic epitopes exposed by rbc damage (eg some cephalosporins)

Antiphospholipid antibodies

Non specific binding (e.g. hypergammaglobulinaemia)

C3 found in 10% of hospital inpatients due to adsorption of immune complexes onto the surface of red cells

Allogenic antibodies



FFP / Platelets (e.g. with high titre anti-A)

Passenger lymphocytes (solid organ malignancy)

False positives

EDTA – in vitro

Normal people – 1:10,000 – no cause





DAT negative AIHA

§        IgA mediated (0.2 – 2.5%)

§        Seen on diamed card which has a well for IgA

§        Low affinity IgG (need 300-4000 Igs per cell to detect with normal DAT) – sensitivity improved by

§        Column agglutination techniques

§        ELISA

§        Flow cytometry

§        Elution of antibody from red cells

§        Polybrene – reduces the electrostatic forces between red cells allowing agglutination at lower antibody levels


False negative DATs

1.      Failure to wash cells

2.      Excessive agitation

3.      Ab that is readily dissociated and washed away

4.      Impotent antisera (or antisera lacking the subclass specificity)


Other factors influencing red cell destruction

§        Bone marrow

§        Autoantibodies may be directed against reticulocytes/ erythroblasts (30% have transient reticulocytopenia)

§        Folate deficiency

§        Lymphoid infiltration

§        Reticuloendothelial function

§        Function reduced in SLE

§        Methyldopa reduces RE clearance of Ig coated cells

§        Hypocomplementaemia

§        In CHAD, the continuous complement activation will deplete levels and offer partial protection against complement-mediated lysis



§        Idiopathic

§        30%

§        Secondary

§        Auto-immune

§        Evans syndrome

§        AIHA and ITP

§        The antibodies are distinct and do not cross-react

§        Higher incidence of underlying illness

§        Eg. SLE, RA, sjogrens

§        Lymphoproliferative disease / other malignancies

§        CLL, low-grade NHL, HD

§        Polyclonal antibodies secondary to immune dysregulation

§        Drugs

§        Drug may be the target antigen by binding to rbc membrane eg. Penicillin

§        Interaction with membrane results in neoantigen eg. Quinidine

§        Drug forms the auto-antibody eg. Methyldopa

§        Often DAT positive but without haemolysis

§        Purine analogues

§        Association with blood transfusion

§        Close temporal relationship with allo-immunisation

§        Association with HSCT - ALLOIMUNE

§        Patient is group A and donor O (minor)

§        antibody produced by ‘passenger lymphocytes’

§        Resolves when patient transfused group O/ engrafts

§        Intravascular haemolysis

§        Rx = reduced plasma volume of stem cell product/ transfuse with compatible or group O cells/ corticosteroids/ exchange transfusion

§        If patient group O and donor A (major)

§        Haemolysis reduced by removing rbcs from the donor product


§        Abs produced by donor lymphocytes against donor red cells

§        More common in transplants for metabolic disorders

§        High mortality

§        Association with solid organ transplants

§        Proportional to the mass of lymphocytes transplanted – ALLOIMMUNE

§        Usually transient as lymphocytes don’t engraft



§        Thromboembolism

§        Especially in patients with a coexistent lupus anticoagulant             

§        LPD

§        RFs – age, underlying auto-immune disorder, coexistent serum gammopathy



Treatment of warm AIHA

§        Corticosteroids

§        2/3 respond, 20% achieve CR

§        Danazol

§        Immunosupressive

§        Azathioprine

§        MMF

§        Cyclophosphamide

§        Cyclosporin

§        IVIG

§        Esp if IgG is the main component on the red cell surface

§        Response in up to 40%

§        Children >adults

§        Splenectomy

§        IgG coated cells respond best

§        33% CR, 33% PR, 33% no response

§        Monoclonal antibodies

§        Rituximab/ alemtuzimab



§        Idiopathic

§        Mostly benign

§        Acrocyanosis

§        Stasis in peripheral circulation due to red cell agglutination

§        Haemolysis depends on the ability of the Ab to activate complement on the red cell surface

§        Monoclonal IgM kappa – usually against I (mycoplasma)/ i (EBV) (occ. Pr)

§        DAT – C3 positive, because IgM elutes in-vitro

§        Mostly extravascular in the liver

§        Lymphoproliferative disorders

§        Monoclonal

§        Infections

§        Mycoplasma pneumoniae/ infectious mononucleosis

§        Polyclonal

§        Haemolysis 2-3 weeks after the infection, usually mild and self-limiting

§        Treatment

§        Avoid cold

§        Blood warmers

§        Folic acid

§        danazol

§        Chlorambucil esp if underlying LPD

§        Monoclonal antibodies

§        Rituximab (may be used in combination with cyclophosphamide)

§        Steroids / IVIG

§        NOT normally helpful

§        Splenectomy

§        NOT normally helpful, as cells coated with C3d and therefore mostly removed in liver


Paroxysmal cold haemoglobinuria

§        Usually in children following viral infections/ vaccination

§        Self-limiting but can persist for up to  months

§        Acute attacks of intravascular haemolysis

§        Caused by an IgG anti-P specificity autoantibody  = biphasic (Donath-Landsteiner) (Causes haemolysis at cold temperatures despite being IgG).

§        Donath – Landstenier test

§        Direct – 2 samples from patient:

§        Control kept at 37oC – should have no haemolysis

§        Test sample cooled to 4oC for 1h followed by warming to 37oC for 20 mins – centrifuge – examine supernatant for haemolysis = positive test

§        Can give false negatives (weak antibody, insufficient complement either in serum or on red cells to cause haemolysis) – reaction can be improved by indirect 

§        Indirect Donath-Landsteiner:

§        Small quantity (1:9) O PP+ red cells put into the patients serum

§        Papainised (exposes more P-antigens) pooled O-cells

§        Add FFP at the end to provide complement

§        Can use pp i.e. P- cells as a control

§        Anitbodies bind in the peripheral circulation at <20C and cause lysis by complement activation in the central vessels at 37C

§        polyclonal

§        Specificity for P antigen

§        pp cells rarely available

§        Steroids may be useful

§        Splenectomy has no role



§        Immunoglobulins with reversible precipitation at low temperatures

§        Form immune complexes

§        Not haemolysis

§        Type1

§        Assoc with LPD

§        Monoclonal Ig, usually IgM

§        Type II

§        Assoc with Hep C, SLE, sjogrens

§        Type III



Drug induced haemolysis

Two immunological mechanisms

1.      Drug dependent immune

§   Antibodies can react with red cells when the drug is bound to the red cell surface OR

§   Antibodies react with the drug and are then adsorbed onto the red cell surface

§   Antibodies bind to a combination of the drug and the cell membrane but not both separately

§   Haemolysis ceases when the drug is withdrawn

§   Two main mechanisms

§   Complement lysis

§   Severe intravascular haemolysis which subsides when the drug is withdrawn

§   DAT strongly positive for C3 and sometimes IgG

§   E.g quinine, rifampicin, hydrochlorothiazide

§   Extravascular haemolysis

§   DAT +ve for IgG (sometimes C3 as well)

§   E.g. penicillin / cephalosporins / tetracycline

§        Can test for the antibody by mixing the patient’s serum with enzyme treated O red cells mixed with the drug (esp. penicillin)


2.      Drug-induced autoimmune

§   The larger part of the epitope is thought to be on the red cell membrane and the antibodies can therefore bind even when the drug isn’t present

§   Antibodies serologically identical to warm AIHA

§   E.g. alpha-methyldopa (the most common - antihypertensive), L-dopa, chlordiazepoxide, indomethicin