Autoimmune haemolytic anaemia is a condition in which autoantibodies directed against self red blood cell antigens cause premature erythrocyte destruction, leading to anaemia.

Autoimmune Haemolytic Anaemia (AIHA)

Autoimmune haemolytic anaemia (AIHA) is an acquired haemolytic anaemia caused by the production of autoantibodies directed against self-antigens on the surface of red blood cells (RBCs), leading to accelerated RBC destruction (haemolysis) and resultant anaemia ^[1]^[2].

Breaking down the name:

Auto = self → the immune system attacks its own cells
Immune = mediated by the adaptive immune system (antibodies)
Haemolytic = "haemo" (blood) + "lytic" (breaking/destroying) → destruction of red blood cells
Anaemia = "an" (without) + "haemia" (blood) → deficiency of haemoglobin/RBCs

The key concept is that this is a Type II hypersensitivity reaction (antibody-dependent cytotoxicity) — autoantibodies (IgG and/or IgM ± complement) bind to RBC surface antigens, marking them for destruction either by splenic macrophages (extravascular haemolysis) or by complement-mediated lysis (intravascular haemolysis) ^[3].

Core Concept – Why AIHA is Important

AIHA is the prototypical acquired haemolytic anaemia and can be primary (idiopathic) or secondary to a wide range of underlying conditions (SLE, CLL, drugs, infections). Recognising AIHA is critical because: (1) it is treatable, (2) it may be the presenting feature of a serious underlying disease, and (3) the thermal classification (warm vs cold) determines management.

Incidence: approximately 1–3 per 100,000 per year ^[4]
Warm AIHA accounts for ~70–80% of all AIHA cases; cold AIHA accounts for ~15–25%; mixed type is rare (~5%) ^[4]
Warm AIHA occurs at all ages but is more common in middle age and in females ^[4]
Cold agglutinin disease (CAD) has a median age of onset of ~65–70 years, with a slight female predominance
Paroxysmal cold haemoglobinuria (PCH) is very rare; classically seen in young children post-viral infection ^[1]
Secondary AIHA is common:
- Up to 11% of patients with CLL develop AIHA ^[4]
- SLE patients have a significant incidence of AIHA (~10%)
- AIHA may also occur after infections (e.g., Mycoplasma pneumoniae, EBV, CMV)
In Hong Kong, SLE is relatively prevalent (Orientals ~6 per 10,000), making lupus-associated AIHA an important consideration ^[5]

Category	Specific Risk Factors	Mechanism
Autoimmune diseases	SLE, rheumatoid arthritis, scleroderma, Sjögren syndrome, inflammatory bowel disease	Loss of self-tolerance → autoantibody production against RBC antigens
Lymphoproliferative disorders	CLL (most common), non-Hodgkin lymphoma, Hodgkin lymphoma	Dysregulated B-cell clones produce autoantibodies; immune dysregulation
Immunodeficiency	Common variable immunodeficiency (CVID), autoimmune lymphoproliferative syndrome (ALPS)	Impaired immune regulation → breakdown of tolerance ^[6]
Drugs	Penicillins, cephalosporins, methyldopa, fludarabine, quinidine	Drug-induced immune haemolysis via hapten, immune complex, or autoantibody mechanisms
Infections	Mycoplasma pneumoniae, EBV, CMV, HIV, hepatitis C	Molecular mimicry or polyclonal B-cell activation
Malignancy	Ovarian teratoma (dermoid cyst), thymoma	Ectopic immune tissue production or immune dysregulation
Prior transfusion/pregnancy	Alloimmunisation from prior exposure	Development of allo-antibodies that cross-react with self-antigens
Post-transplant	Haematopoietic stem cell transplant, solid organ transplant	Passenger lymphocyte syndrome; graft-vs-host immune dysregulation

Hong Kong Context

In Hong Kong, the most common secondary causes of AIHA to consider are SLE (high prevalence in Chinese women), CLL (in the elderly), infections (especially in immunocompromised), and drug-induced causes (given polypharmacy in the ageing population). Always check for underlying lymphoproliferative disease in elderly patients presenting with AIHA.

Anatomy and Function: The Red Blood Cell and Splenic Filtration

Understanding AIHA requires understanding the RBC surface:

RBC membrane: A lipid bilayer with embedded integral proteins (e.g., Band 3, glycophorin A) and an underlying cytoskeleton (spectrin, ankyrin, protein 4.1)
- The membrane expresses numerous blood group antigens (ABO, Rh, Kell, Duffy, etc.) — these are the targets of autoantibodies in AIHA
RBC lifespan: Normal ~120 days; in AIHA, this can be shortened to days
RBC deformability: Antibody-coated RBCs become less deformable → trapped in splenic sinusoids → phagocytosed

The spleen filters blood through its red pulp sinusoids (narrow ~2–3 μm slits between endothelial cells)
Normal flexible RBCs squeeze through; antibody-coated or spherocytic RBCs cannot → they are retained and phagocytosed by splenic macrophages
Macrophages have Fc receptors (FcγRI, FcγRIII) that recognise the Fc portion of IgG bound to RBCs → this triggers antibody-dependent cellular phagocytosis (ADCP)
Macrophages also have complement receptors (CR1, CR3) that recognise C3b/iC3b deposited on RBCs
The spleen can "nibble" portions of antibody-coated RBC membrane without destroying the whole cell → the RBC loses membrane but retains cytoplasm → spherocyte formation (the cell becomes spherical because it has less membrane relative to its volume)

Kupffer cells (hepatic macrophages) are important in clearing heavily opsonised RBCs (especially those coated with C3b or large amounts of IgG)
When there is massive complement activation (IgM-mediated cold AIHA), the liver becomes the dominant site of clearance

Classical pathway activation: IgM (pentameric, very efficient) or IgG (especially IgG1, IgG3) bound to RBC surface → C1q binding → cascade → C3b deposition → MAC formation (C5b-9)
Extravascular haemolysis: C3b-opsonised RBCs are cleared by macrophages (mainly hepatic Kupffer cells)
Intravascular haemolysis: Complete complement activation to MAC → pore formation → osmotic lysis of RBC within the vasculature

Aetiology and Pathophysiology

This is the most clinically important classification because it determines the mechanism of haemolysis, clinical presentation, associated conditions, and management strategy.

1. Warm AIHA

Warm AIHA: antibody is IgG class, reacts optimally at 37°C (body temperature), predominantly causes extravascular haemolysis in the spleen ^[1]^[4]

Loss of immune tolerance: In genetically predisposed individuals (HLA associations, complement deficiencies), environmental triggers (infections, drugs) cause breakdown of T-cell and B-cell tolerance to self-RBC antigens
Autoantibody production: Autoreactive B cells produce IgG autoantibodies (most commonly IgG1 and IgG3) directed against RBC surface antigens (often Rh system proteins, especially the e antigen)
Opsonisation: IgG binds to RBC surface → RBCs become "coated"
Extravascular haemolysis (primary mechanism):
- IgG-coated RBCs pass through the spleen
- Splenic macrophages recognise the Fc portion of IgG via FcγR receptors
- Macrophages either fully phagocytose the RBC or "nibble" off membrane → spherocyte formation
- Why spherocytes? Because the macrophage removes membrane but not cytoplasm → the cell volume stays the same but surface area decreases → the only geometric shape that maintains maximum volume with minimum surface area is a sphere
Complement involvement (partial):
- IgG1 and IgG3 can activate the classical complement pathway → C3b deposition on RBCs
- C3b is rapidly converted to iC3b by Factor I → iC3b is recognised by CR3 on hepatic Kupffer cells
- This leads to additional extravascular haemolysis in the liver
- Rarely, if complement activation goes all the way to MAC (C5b-9), intravascular haemolysis can occur, but this is uncommon in warm AIHA because regulatory proteins (CD55, CD59) on normal RBCs inhibit MAC

Category	Specific Conditions	Notes
Primary (idiopathic)	~50% of cases	No identifiable cause; diagnosis of exclusion
Autoimmune diseases	SLE (most common autoimmune association), RA, scleroderma, UC	Immune dysregulation → autoantibody production
Lymphoproliferative disorders	CLL (most common LPD association), NHL, Hodgkin lymphoma	Aberrant B-cell clones; immune dysregulation
Immunodeficiency	CVID, ALPS	Defective immune regulation ^[6]
Drugs	Methyldopa, fludarabine, penicillins, cephalosporins	Various mechanisms (see drug-induced section below)
Infections	HIV, hepatitis C	Immune dysregulation
Post-transplant	HSCT, solid organ transplant	Passenger lymphocyte syndrome

Evans syndrome: co-occurrence of ≥2 immune cytopaenias, most often AIHA + ITP ^[4]

Epidemiology: usually described in children but also in adults

Association: 50% idiopathic, otherwise may be associated with SLE, CVID, ALPS or other autoimmune diseases

Significance: important to identify underlying disorder as it is more difficult to treat ^[4]

2. Cold AIHA

Cold AIHA: antibody is IgM class, reacts optimally at 4°C (but can react up to 30–37°C depending on thermal amplitude), predominantly causes complement-mediated haemolysis ^[1]

IgM autoantibody production: Usually directed against the I antigen (or rarely i antigen) on RBC surface
- IgM is a pentameric immunoglobulin → extremely efficient at fixing complement (a single IgM molecule can activate C1q)
Cold-induced binding: When blood circulates through cooler peripheral areas (fingers, toes, ears, nose — skin temperature can drop to 28–31°C), IgM binds to RBCs
Complement activation: IgM-RBC complex activates the classical complement pathway → C1 → C4 → C2 → C3 convertase → C3b deposited on RBC surface
Two fates of C3b-coated RBCs:
- Extravascular haemolysis (predominant): C3b is degraded to iC3b by Factor I → iC3b is recognised by CR3 on hepatic Kupffer cells → phagocytosis in the liver
- Intravascular haemolysis (if complement cascade completes): C3b → C5 convertase → C5b-9 (MAC) → pore formation → osmotic lysis → free haemoglobin released into circulation → haemoglobinaemia → haemoglobinuria
Warming: As blood returns to central circulation (37°C), IgM detaches from RBCs (because the IgM only binds at lower temperatures), but complement components (C3b, C3d) remain on the RBC surface
- This is why the Direct Coombs test (DAT) in CAD is typically positive for C3d only (not IgG) — because the IgM has already dissociated before the blood sample reaches the lab

Type	Specific Conditions	Notes
Primary CAD	Clonal B-cell lymphoproliferative disorder (low-grade, often MYD88 mutation)	Chronic, insidious; now recognised as a distinct clonal lymphoproliferative entity
Secondary (infection-related)	Mycoplasma pneumoniae (anti-I), EBV/infectious mononucleosis (anti-i)	Usually self-limiting; polyclonal IgM; resolves with infection treatment
Secondary (lymphoma-related)	Waldenström macroglobulinaemia, marginal zone lymphoma, DLBCL	Monoclonal IgM; chronic course

Clinical Pearl – Thermal Amplitude Matters

The thermal amplitude of the cold agglutinin is more clinically relevant than the titre. A cold agglutinin that can react at temperatures close to 37°C (high thermal amplitude) causes more severe haemolysis than one that only reacts at 4°C, even if the latter has a higher titre. This is why some patients with high-titre cold agglutinins remain asymptomatic.

Very rare subtype of cold AIHA ^[1]
Caused by Donath-Landsteiner antibody: a biphasic IgG autoantibody (not IgM!) directed against the P antigen on RBCs
Biphasic mechanism: IgG binds to RBCs and fixes complement (up to C3) in the cold → when blood warms up centrally, complement cascade completes → MAC formation → intravascular haemolysis
Classically described in:
- Children post-viral infection (most common current presentation)
- Historically: tertiary syphilis (now extremely rare)
Clinical features: sudden onset of dark urine (haemoglobinuria), rigors, back pain, fever after cold exposure

Three main mechanisms:

Mechanism	Prototype Drug	Pathophysiology	DAT Pattern
Drug adsorption (hapten)	High-dose penicillins, cephalosporins	Drug binds to RBC membrane → acts as hapten → IgG antibodies form against drug-RBC complex → extravascular haemolysis	IgG positive
Immune complex (neoantigen)	Quinidine, quinine, NSAIDs	Drug-antibody immune complex forms in serum → loosely adsorbs to RBC → activates complement → intravascular haemolysis (can be severe and acute)	C3 positive
True autoantibody	Methyldopa, fludarabine	Drug induces production of true autoantibodies against RBC antigens (not against the drug itself) → indistinguishable from warm AIHA → extravascular haemolysis	IgG positive

Drug-Induced AIHA – A Tricky Diagnosis

Drug-induced AIHA can be difficult to distinguish from primary warm AIHA. The key is a thorough drug history and temporal correlation. If AIHA develops after starting a new drug (especially methyldopa, fludarabine, or cephalosporins), suspect drug-induced AIHA. The haemolysis should resolve after drug cessation (though methyldopa-induced autoantibodies may persist for months).

Both warm (IgG) and cold (IgM) autoantibodies coexist
Rare (~5% of AIHA)
Often secondary to SLE or lymphoproliferative disorders
Can be more refractory to treatment

Classification Summary

Feature	Warm AIHA	Cold Agglutinin Disease	Paroxysmal Cold Haemoglobinuria	Mixed
Frequency	70–80%	15–25%	Rare	~5%
Antibody class	IgG	IgM	IgG (biphasic)	IgG + IgM
Optimal temperature	37°C	4°C	Binds cold, lyses warm	Both
Target antigen	Rh system	I or i antigen	P antigen	Various
Complement fixation	Partial (C3b)	Yes (classical pathway)	Yes (biphasic, complete)	Yes
Primary haemolysis site	Spleen (extravascular)	Liver (extravascular) ± intravascular	Intravascular	Both
Spherocytes	Yes (characteristic)	Usually no	No	Variable
DAT pattern	IgG ± C3	C3 only	C3 (IgG if tested at 4°C)	IgG + C3

Category	Primary	Secondary
Definition	No identifiable underlying cause (idiopathic)	Associated with an underlying disease or drug
Proportion	~50% of warm AIHA	~50% of warm AIHA; most cold AIHA
Key associations	—	SLE, CLL, lymphoma, CVID, drugs, infections

Clinical Features

Symptom	Pathophysiological Basis
Fatigue, malaise, decreased exercise tolerance	Decreased oxygen-carrying capacity due to anaemia → inadequate tissue oxygenation
Dyspnoea on exertion, palpitations	Compensatory ↑cardiac output to maintain tissue O₂ delivery → tachycardia and hyperdynamic circulation; reduced O₂ carrying capacity → exertional dyspnoea
Dizziness, syncope (if severe/acute)	Acute drop in Hb → cerebral hypoperfusion; may be postural
Jaundice (yellow discolouration of skin/sclera)	Increased unconjugated (indirect) bilirubin from breakdown of haemoglobin in phagocytosed RBCs → exceeds hepatic conjugation capacity → jaundice; No tea-coloured urine (unlike obstructive jaundice) because unconjugated bilirubin is water-insoluble and protein-bound, so it cannot be filtered by the kidneys ^[1]^[2]
Dark urine (coca-cola/red-brown urine)	Haemoglobinuria in intravascular haemolysis — free haemoglobin exceeds haptoglobin binding capacity → filtered by glomerulus → appears in urine; Also, excess urobilinogen from ↑bilirubin metabolism gives darker urine even in extravascular haemolysis
Back pain, abdominal pain	Intravascular haemolysis → free haemoglobin scavenges nitric oxide (NO) → smooth muscle spasm in GI tract and renal vasculature → pain. Also, acute splenic sequestration can cause LUQ pain
History of gallstones	Chronic haemolysis → ↑unconjugated bilirubin concentration in hepatic bile → promotes calcium bilirubinate (pigment) gallstone formation ^[1]^[2]
Cold-triggered symptoms (CAD)	IgM binds RBCs in cooler extremities → acrocyanosis (fingers/toes/ears turn blue-purple on cold exposure) ± Raynaud-like phenomenon; haemolysis worsened by cold
Paroxysms of dark urine after cold exposure (PCH)	Donath-Landsteiner IgG binds RBCs in cold → complement cascade completes on warming → massive intravascular haemolysis → haemoglobinuria
Symptoms of underlying disease	E.g., joint pain and rash (SLE), B-symptoms (lymphoma — fever, night sweats, weight loss), recurrent infections (CVID, CLL)

Important history to elicit from a patient with suspected haemolytic anaemia: Family history, Ethnicity, Infection, Drugs, Transfusion history, Co-existing illnesses such as autoimmune diseases / lymphoproliferative neoplasms ^[1]^[2]

Sign	Pathophysiological Basis
Pallor (conjunctival, palmar crease)	Reduced haemoglobin concentration → less oxyhaemoglobin in superficial vasculature → pale appearance ^[1]^[2]
Jaundice (scleral icterus, skin)	Pre-hepatic jaundice from ↑unconjugated bilirubin; scleral icterus detectable when total bilirubin > ~35 μmol/L; No tea-coloured urine distinguishes from obstructive jaundice ^[1]^[2]
Splenomegaly	The spleen is the primary site of extravascular haemolysis in warm AIHA → reticuloendothelial hyperplasia → splenic enlargement; also seen as work hypertrophy from chronic RBC clearance ^[1]^[2]
Hepatomegaly	Kupffer cell hyperplasia in the liver (especially in cold AIHA where C3b-coated RBCs are cleared hepatically)
Tachycardia, flow murmur	Compensatory ↑cardiac output and hyperdynamic circulation; ↓blood viscosity in severe anaemia → turbulent flow across valves
Postural hypotension	If acute severe haemolysis → effective volume depletion
Acrocyanosis (cold AIHA)	IgM-mediated RBC agglutination in peripheral cool vessels → vascular occlusion and cyanotic discolouration of fingers, toes, ears, nose
Leg ulcers (chronic severe cases)	Chronic tissue hypoxia in dependent areas; impaired microcirculation from RBC debris and free haemoglobin scavenging NO
Lymphadenopathy	If secondary to CLL, lymphoma, or other lymphoproliferative disorder
Signs of underlying disease	Malar rash, photosensitivity, oral ulcers (SLE); joint deformities (RA); generalised lymphadenopathy (CLL); skin changes (scleroderma)

High Yield – Clinical Features of Haemolytic Anaemia

The characteristic signs to look for in a patient with haemolytic anaemia are:

Pallor
Jaundice, without tea-coloured urine (distinguishes from obstructive/cholestatic jaundice)
Haemoglobinuria in intravascular haemolysis (e.g., PNH) — coca-cola coloured urine
Splenomegaly

History of gallstones should always be asked — chronic haemolysis → ↑unconjugated bilirubin → pigment gallstones ^[1]^[2]

Special Clinical Scenarios

Co-occurrence of AIHA + immune thrombocytopaenia (ITP) ± autoimmune neutropaenia ^[4]
Presents with features of both anaemia AND thrombocytopaenic bleeding (petechiae, purpura, mucosal bleeding)
May be idiopathic or secondary to SLE, CVID, ALPS
More difficult to treat than isolated AIHA

Most commonly in CLL (as much as 11% develop AIHA) ^[4]
Should alert to systemic symptoms (e.g., weight loss, loss of appetite, fever, lymphadenopathy) in elderly ^[4]
The AIHA may precede the diagnosis of CLL
Important to check lymphocyte count and peripheral blood film for smear cells

AIHA can be a presenting feature of SLE (part of the haematological criteria in the 2019 EULAR/ACR classification criteria)
Always check ANA, anti-dsDNA, complement levels (C3, C4) in a young woman presenting with AIHA
Lupus is associated with warm AIHA (IgG antibodies as part of the generalised autoantibody production)

A niche but important differential: Fulminant hepatic failure due to Wilson's disease presents with Coombs-negative haemolytic anaemia — the copper released from necrotic hepatocytes directly damages RBC membranes ^[7]
This is NOT true AIHA (Coombs-negative), but it enters the differential of haemolytic anaemia in a young patient with liver failure

This distinction is fundamental for understanding the different presentations and laboratory findings:

Feature	Extravascular Haemolysis	Intravascular Haemolysis
Primary site	Spleen (warm AIHA) or liver (cold AIHA)	Within blood vessels
Mechanism	Macrophage phagocytosis of opsonised RBCs	Complement-mediated MAC lysis
Main antibody type	IgG (warm)	IgM (cold) or drug immune complex
Spherocytes on PBS	Yes (membrane nibbling by macrophages)	No (cells lyse completely)
Haemoglobinaemia	Minimal	Yes (free Hb in plasma → pink plasma)
Haemoglobinuria	No	Yes (coca-cola urine)
Haptoglobin	↓ (consumed binding free Hb)	↓↓ (severely depleted)
Haemosiderinuria	No	Yes (chronic) — iron-laden tubular cells shed into urine
Methaemalbuminaemia	No	Yes (free haem binds albumin)
LDH	↑	↑↑
Unconjugated bilirubin	↑	↑
Jaundice	Yes	Yes
Splenomegaly	Yes (if chronic)	Usually no
Risk of acute renal injury	Low	Yes — free Hb is directly nephrotoxic (precipitates in tubules)

Understanding why each marker changes:

Marker	Change	Mechanism
↑ Reticulocyte count	Increased	Bone marrow compensatory response to anaemia → ↑erythropoietin → ↑reticulocyte production. Note: reticulocytes are larger than mature RBCs, which is why haemolytic anaemia can appear macrocytic (↑MCV)
↑ LDH (lactate dehydrogenase)	Increased	LDH is abundant in RBC cytoplasm; released upon RBC destruction
↓ Haptoglobin	Decreased	Haptoglobin binds free haemoglobin released from lysed RBCs → haptoglobin-Hb complex cleared by hepatocytes → haptoglobin consumed faster than it is produced. Most sensitive marker of haemolysis
↑ Unconjugated (indirect) bilirubin	Increased	Haem from destroyed RBCs → biliverdin → unconjugated bilirubin (by macrophage haem oxygenase). Exceeds hepatic conjugation rate
↑ Urobilinogen	Increased	More bilirubin conjugated by liver → more urobilinogen produced by gut bacteria → some reabsorbed (enterohepatic circulation) and excreted in urine
Polychromasia on PBS	Present	Reticulocytes stain bluish-grey with Wright stain due to residual RNA
Spherocytes on PBS	Present (warm AIHA)	Membrane loss from macrophage nibbling
RBC agglutination on PBS	Present (cold AIHA)	IgM cross-links RBCs at room temperature during slide preparation — can cause spuriously elevated MCV (machine counts clumps as single large cells)

Exam Trap – Spuriously Elevated MCV in Cold AIHA

In cold agglutinin disease, the automated CBC may show a falsely elevated MCV because RBC agglutinates are counted as single large cells by the machine. Warming the sample to 37°C before analysis corrects this artefact. Always check the peripheral blood film!

Feature	Warm AIHA	Cold Agglutinin Disease	PCH
Antibody	IgG	IgM	IgG (biphasic — Donath-Landsteiner)
Target	Rh antigens	I/i antigens	P antigen
Complement	Partial (C3)	Full cascade possible	Full cascade (biphasic)
Haemolysis type	Extravascular (spleen)	Extravascular (liver) ± intravascular	Intravascular
DAT	IgG ± C3	C3 only	C3 (IgG if tested cold)
Key associations	SLE, CLL, drugs, idiopathic	Mycoplasma, EBV, clonal B-cell disorder	Post-viral (children), syphilis (historical)
Spherocytes	Yes	No	No
Agglutination	No	Yes	No
Cold-triggered symptoms	No	Yes (acrocyanosis)	Yes (haemoglobinuria after cold)

High Yield Summary

Definition: AIHA = acquired haemolytic anaemia due to autoantibodies against self-RBC antigens → Type II hypersensitivity.

Classification by thermal reactivity:

Warm AIHA (70–80%): IgG, 37°C, extravascular haemolysis (spleen), spherocytes, DAT IgG ± C3
Cold agglutinin disease (15–25%): IgM, 4°C, complement-mediated (mainly extravascular via liver), DAT C3 only, acrocyanosis
PCH (rare): Biphasic IgG (Donath-Landsteiner Ab), P antigen, intravascular haemolysis after cold exposure
Mixed (~5%): Both IgG + IgM

Must-ask history: Family history, ethnicity, infections, drugs, transfusion history, co-existing autoimmune/lymphoproliferative disease, gallstones.

Cardinal clinical features: Pallor + jaundice (no tea-coloured urine) + splenomegaly ± haemoglobinuria (if intravascular).

Evans syndrome: AIHA + ITP — think SLE, CVID, ALPS.

CLL: Up to 11% develop AIHA — always consider in elderly with AIHA.

Key lab: ↑reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin, +ve DAT (IgG ± C3 in warm; C3 only in cold).

Drug-induced AIHA: Three mechanisms — hapten (penicillin), immune complex (quinidine), true autoantibody (methyldopa).

Active Recall - Autoimmune Haemolytic Anaemia

Differential Diagnosis of Autoimmune Haemolytic Anaemia

A patient presenting with features suggestive of AIHA — anaemia, jaundice, splenomegaly, reticulocytosis, elevated LDH, low haptoglobin — is really presenting with the syndrome of haemolytic anaemia. The Direct Antiglobulin Test (DAT / Direct Coombs test) is the pivotal investigation that separates immune from non-immune causes. However, before you even get to the DAT, you need to work through a structured differential of haemolytic anaemia, and even after confirming AIHA, you must systematically consider whether it is primary or secondary.

Think of it in two layers:

Layer 1: Is this really haemolytic anaemia? (vs other causes of anaemia with jaundice or reticulocytosis)
Layer 2: If haemolytic, is it immune-mediated (DAT-positive)? If so, what type and what is the underlying cause?

When a patient presents with features of haemolysis (anaemia + jaundice + ↑reticulocytes + ↑LDH + ↓haptoglobin ± splenomegaly), the differential includes all causes of haemolytic anaemia. The GC lecture slide provides a systematic classification ^[1]^[2]:

Acquired haemolytic anaemia — extrinsic factors causing red cell damage ^[1]:

Immune: (i) Autoimmune, (ii) Alloimmune (transfusion reactions, haemolytic disease of the newborn), (iii) Drug-induced immune

Heat, venoms and chemicals damaging the red cell membrane

Drugs and chemicals oxidizing haemoglobin and other cellular components

Red cell fragmentation syndromes: (i) Microangiopathic haemolytic anaemia (TTP), (ii) Mechanical haemolytic anaemia

Induced by micro-organisms: (i) Bacterial infection of red cells (e.g. C. perfringens), (ii) Parasitic infection of red cells (e.g. malaria)

The table below lists the key differentials for a patient presenting with haemolytic anaemia features, with the distinguishing features that help you narrow down to AIHA:

Differential Diagnosis	Key Distinguishing Features from AIHA	PBS Findings	DAT
Hereditary Spherocytosis (HS)	Family history (AD in 75%); lifelong history; ↓EMA binding on flow cytometry distinguishes from AIHA ^[4]^[8]	Spherocytes (same as warm AIHA!)	Negative (critical distinction — must do DAT to differentiate HS from warm AIHA)
G6PD Deficiency	X-linked; episodic haemolysis triggered by infection (most common), drugs, DKA; prevalence in HK: 4.5% males, 0.5% females ^[8]	Bite cells, Heinz bodies (not spherocytes); blister cells	Negative
Pyruvate Kinase Deficiency	AR; chronic haemolysis from birth; less common	Echinocytes, prickle cells	Negative
Thalassaemia (intermedia/HbH)	Family history, ethnicity (Southern Chinese in HK); microcytic; Hb electrophoresis diagnostic	Target cells, hypochromia, microcytosis	Negative
Sickle Cell Disease	Very rare in HK; characteristic crises	Sickle cells, target cells	Negative
Paroxysmal Nocturnal Haemoglobinuria (PNH)	Acquired intrinsic RBC defect; PIGA gene mutation → deficiency of GPI → deficiency of CD55, CD59 → complement-mediated lysis ^[1]; haemoglobinuria (classically dark morning urine), thrombosis in unusual sites (Budd-Chiari), pancytopenia	No specific morphology; may see polychromasia	Negative (Coombs-negative haemolysis!); diagnosed by flow cytometry showing ↓CD55/CD59 ^[9]
Microangiopathic Haemolytic Anaemia (MAHA) — TTP/HUS	Schistocytes (fragmented RBCs) on PBS; thrombocytopaenia; ADAMTS13 activity < 10% in TTP ^[10]; renal failure in HUS; clotting profile normal (unlike DIC)	Schistocytes, nucleated RBCs, polychromasia ^[10]	Negative ("test for immune-mediated hemolysis is typically negative") ^[10]
Mechanical Haemolytic Anaemia	Prosthetic heart valves, march haemoglobinuria	Schistocytes	Negative
Alloimmune Haemolytic Anaemia (transfusion reaction)	Temporal relationship with blood transfusion; ABO incompatibility causes acute reaction; delayed reactions from minor antigen mismatch ^[3]	Spherocytes possible	Positive (but antibody specificity is allo- not auto-)
Haemolytic Disease of the Newborn (HDN)	Neonatal presentation; maternal antibodies (anti-D, anti-Kell) crossing placenta	Spherocytes, polychromasia	Positive
Drug-Induced Immune Haemolytic Anaemia	Temporal relationship with drug initiation; resolves with drug cessation	Depends on mechanism	Positive (IgG and/or C3 depending on mechanism)
Infection-Related Haemolysis	Travel history (malaria — most important worldwide); Clostridium perfringens (gas gangrene / post-surgical); Mycoplasma and EBV can trigger cold AIHA	Malaria parasites in RBCs; spherocytes in Clostridium	Usually negative (unless triggering secondary AIHA)
Wilson's Disease (fulminant hepatic failure)	Young patient with no reason for fulminant liver failure; Coombs-negative haemolytic anaemia ^[7]; copper directly damages RBC membranes; low ceruloplasmin, high urinary copper	Spherocytes possible	Negative (critical to note)
Hypersplenism	Splenomegaly with pancytopaenia; dilutional effect from splenic pooling	No specific morphology	Negative
Anaemia of chronic disease	No haemolysis markers; ↓serum iron, ↓TIBC, ↑ferritin; ↑hepcidin trapping iron in RES ^[11]	No haemolysis features	Negative

High Yield — DAT-Negative Haemolytic Anaemias That Can Mimic AIHA

Not all haemolytic anaemia with spherocytes is AIHA! The two most important DAT-negative conditions with spherocytes on PBS are:

Hereditary Spherocytosis — distinguish with ↓EMA binding on flow cytometry and positive family history ^[4]^[8]
Wilson's disease with fulminant hepatic failure — Coombs-negative haemolytic anaemia is a very specific feature; the copper released from necrotic hepatocytes directly damages RBC membranes ^[7]

Also remember: PNH is Coombs-negative — diagnosed by flow cytometry for CD55/CD59 deficiency ^[1]^[9]

Layer 2 — Once AIHA Is Confirmed (DAT-Positive), What Is the Underlying Cause?

This is the critical next step. AIHA is not a final diagnosis — it is a manifestation of an underlying process. You must always search for secondary causes ^[1]^[2]^[4]^[5].

Important history to elicit from a patient with suspected haemolytic anaemia: Family history, Ethnicity, Infection, Drugs, Transfusion history, Co-existing illnesses such as autoimmune diseases and lymphoproliferative neoplasms ^[1]^[2]

DAT Pattern	Most Likely AIHA Type	Key Differentials to Consider
IgG positive, C3 negative	Warm AIHA	Primary (idiopathic); SLE; drugs (methyldopa); CLL; CVID
IgG positive, C3 positive	Warm AIHA with complement activation	SLE (often has both); drugs; aggressive warm AIHA
IgG negative, C3 positive	Cold agglutinin disease	Primary CAD (clonal B-cell LPD); secondary to Mycoplasma, EBV; lymphoma
IgG positive, C3 positive	Mixed AIHA	SLE; lymphoproliferative disorders
DAT negative ( < 3% of AIHA)	Cannot exclude AIHA if clinical picture is compelling	Low-affinity IgG; IgA-mediated AIHA; very low antibody titre below detection threshold ^[4]

Differential by AIHA Subtype

Category	Specific Conditions	Clinical Clues
Autoimmune diseases	SLE	Young female; malar rash; photosensitivity; joint pain; oral ulcers; positive ANA, anti-dsDNA; active SLE does NOT evoke a CRP response normally ^[12]
	RA, scleroderma, Sjögren, IBD	Joint symptoms; sicca symptoms; GI symptoms
Lymphoproliferative disorders	CLL (most common LPD cause — up to 11% develop AIHA) ^[4]	Elderly patient; lymphocytosis; smear/smudge cells on PBS; generalised lymphadenopathy
	NHL, Hodgkin lymphoma	B-symptoms (fever, night sweats, weight loss > 10%); lymphadenopathy; hepatosplenomegaly
Immunodeficiency	CVID	Recurrent sinopulmonary infections; hypogammaglobulinaemia; ITP, AIHA are well-recognised associations ^[6]
	ALPS	Childhood onset; massive lymphadenopathy + splenomegaly; elevated double-negative T cells
Drugs	Methyldopa (true autoantibody)	History of hypertension treatment; DAT positive for IgG; haemolysis may persist for months after stopping
	Fludarabine	CLL patient on treatment; profound immune suppression
	Penicillins, cephalosporins (hapten)	Recent antibiotic use; dose-dependent
	Quinidine, quinine (immune complex)	Acute, severe intravascular haemolysis; renal failure possible
Post-transplant	Passenger lymphocyte syndrome	After solid organ (esp. liver, kidney) or HSCT; donor lymphocytes produce anti-recipient RBC antibodies
Pregnancy	Pregnancy-associated AIHA	May overlap with pre-eclampsia/HELLP — must distinguish ^[13]
Ovarian teratoma	Dermoid cyst-associated AIHA	Rare; resolves after tumour removal

Evans syndrome (AIHA + ITP): 50% idiopathic; otherwise associated with SLE, CVID, ALPS ^[4]. When you see both AIHA and thrombocytopaenia, do NOT assume the low platelets are simply from the same autoimmune process without checking for TTP (which also has anaemia + low platelets but with schistocytes and a very different management) ^[10].

Category	Specific Conditions	Clinical Clues
Infections	Mycoplasma pneumoniae (anti-I) ^[1]^[5]	Recent atypical pneumonia; younger patient; self-limiting haemolysis
	EBV / Infectious mononucleosis (anti-i) ^[1]^[5]	Young adult; pharyngitis; atypical lymphocytes on PBS; heterophile antibody positive
	CMV	Fever; hepatitis; atypical lymphocytes
Lymphoproliferative disorders	Waldenström macroglobulinaemia	Monoclonal IgM (IgM paraprotein); hyperviscosity symptoms
	Marginal zone lymphoma, DLBCL	Lymphadenopathy; B-symptoms
Primary CAD	Clonal B-cell disorder (now classified as distinct entity, not "idiopathic")	Chronic; older patient; MYD88 mutation common; monoclonal IgMκ

Setting	Notes
Post-viral in children	Most common current presentation; self-limiting
Tertiary syphilis	Historical; now extremely rare

A few conditions can present with anaemia + jaundice and be confused with haemolytic anaemia. The key is the reticulocyte count and haemolysis markers:

Condition	Why It Mimics	Key Distinguishing Feature
Ineffective erythropoiesis (megaloblastic anaemia, MDS)	Anaemia + ↑LDH + ↑unconjugated bilirubin (from intramedullary destruction of defective RBC precursors)	Reticulocyte count is LOW (reticulocytopaenia — the marrow is producing defective cells that die before release); haptoglobin may be normal or low; MCV often > 110 fL in B12/folate deficiency
Gilbert syndrome	Unconjugated hyperbilirubinaemia + mild anaemia (co-incidental)	No haemolysis markers; bilirubin fluctuates with fasting/stress; UGT1A1 polymorphism
Acute hepatitis	Jaundice + anaemia (dilutional or from chronic disease)	Conjugated hyperbilirubinaemia predominates; ↑ALT/AST; no reticulocytosis
Bleeding with iron deficiency	Anaemia; may have mild ↑LDH	Iron studies diagnostic (↓ferritin, ↑TIBC); no jaundice; reticulocytes may be elevated but no haemolysis pattern

Exam Pearl — Reticulocyte Count Is Your Friend

The reticulocyte count is the single most useful initial test to distinguish between haemolytic anaemia (↑reticulocytes — the marrow is trying to compensate) and ineffective erythropoiesis / marrow failure (↓reticulocytes — the marrow cannot compensate). In AIHA, you expect reticulocytosis with polychromasia ^[1]^[5]. Exception: rarely, AIHA can have reticulocytopaenia if autoantibodies also target reticulocyte precursors (seen in some cases of Evans syndrome or parvovirus B19 co-infection causing pure red cell aplasia).

Distinguishing AIHA from Key Mimickers — Specific Comparisons

Feature	Warm AIHA	Hereditary Spherocytosis
Age of onset	Any age (middle-aged adults most common)	Usually childhood/neonatal
Family history	Negative	Positive (AD 75%, sporadic 25%) ^[8]
Spherocytes on PBS	Yes	Yes
DAT	Positive (IgG ± C3) ^[4]^[5]	Negative
EMA binding (flow cytometry)	Normal	Reduced — this is the diagnostic test for HS ^[4]^[8]
Osmotic fragility	May be increased (spherocytes are osmotically fragile regardless of cause)	Increased
Response to steroids	Yes	No (steroids target immune mechanism, not membrane defect)
Splenectomy	2nd-line treatment	Curative in many cases

Rule out alternative diagnosis with EMA binding for hereditary spherocytosis — this is specifically mentioned as part of the AIHA workup ^[4]

Feature	AIHA	TTP / MAHA
PBS morphology	Spherocytes	Schistocytes (fragmented RBCs) ^[10]
Platelets	Usually normal (unless Evans syndrome)	Thrombocytopaenia (consumed in microthrombi)
Coagulation	Normal	Normal (unlike DIC which has ↑PT, ↑APTT, ↑D-dimer, ↓fibrinogen)
DAT	Positive	Negative ^[10]
ADAMTS13	Normal	< 10% in acquired TTP ^[10]
Renal function	Usually normal	Often impaired (especially HUS)
Neurological features	Absent	Fluctuating neurological signs (TTP pentad)

Critical Distinction — Do Not Confuse Evans Syndrome with TTP

Both Evans syndrome (AIHA + ITP) and TTP present with anaemia + thrombocytopaenia. The critical differences: (1) PBS in Evans shows spherocytes while TTP shows schistocytes; (2) DAT is positive in Evans but negative in TTP; (3) TTP requires urgent plasma exchange while Evans requires immunosuppression. Getting this wrong can be fatal.

Feature	AIHA	PNH
Mechanism	Autoantibody-mediated	PIGA mutation → loss of GPI-anchored proteins (CD55, CD59) → unregulated complement activation → intravascular lysis ^[1]
Haemolysis type	Mainly extravascular (warm)	Mainly intravascular
DAT	Positive	Negative
Haemoglobinuria	Rare (unless severe cold AIHA)	Characteristic — classically dark morning urine
Thrombosis	Not a major feature	Thrombosis in unusual sites (hepatic/portal/cerebral veins) — leading cause of death ^[9]
Diagnostic test	DAT	Flow cytometry: ↓CD55, ↓CD59 ^[1]^[9]
Associated conditions	SLE, CLL, drugs	Aplastic anaemia, MDS ^[9]

Feature	AIHA (drug-induced immune)	G6PD Deficiency (drug-precipitated)
Mechanism	Immune — antibody-mediated	Oxidative — depleted glutathione cannot protect against oxidative damage
PBS	Spherocytes	Bite cells, Heinz bodies (not spherocytes) ^[8]
DAT	Positive	Negative
Timing	Days to weeks after drug initiation	1–3 days after oxidative trigger
Drug examples	Methyldopa, penicillin, cephalosporins	Anti-malarials, dapsone, nitrofurantoin, quinolones, sulphonamides (e.g., Septrin) ^[8]
Recovery	After drug cessation + immunosuppression	Self-limiting once drug stopped; G6PD assay after recovery confirms

The degree of splenomegaly offers information about the underlying aetiology and can help narrow the differential when evaluating a patient with haemolytic anaemia ^[14]:

Spleen Size	Differential
Minimal splenomegaly	Haemolytic anaemia (including AIHA, thalassaemia intermedia, HbH disease, autoimmune cytopaenia — ITP, AIHA) ^[14]
Moderate splenomegaly	Portal hypertension, haematological malignancies (lymphoma)
Massive splenomegaly	CML, myelofibrosis

In AIHA, splenomegaly is typically mild to moderate — if you find massive splenomegaly in a patient with AIHA, think harder about an underlying myeloproliferative or lymphoproliferative disorder as the primary pathology driving both the splenomegaly and the AIHA.

High Yield Summary — Differential Diagnosis of AIHA

Step 1 — Confirm haemolysis: ↑Reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin, polychromasia on PBS.

Step 2 — PBS morphology guides you:

Spherocytes → Warm AIHA vs Hereditary Spherocytosis (differentiate by DAT and EMA binding)
Schistocytes → MAHA (TTP/HUS) — DAT negative
Bite cells / Heinz bodies → G6PD deficiency — DAT negative
RBC agglutination → Cold agglutinin disease
No specific morphology → PNH (flow cytometry for CD55/CD59)

Step 3 — DAT is the key test:

DAT positive → Immune haemolysis (AIHA, alloimmune, drug-induced)
DAT negative → Non-immune (HS, G6PD, PNH, TTP, Wilson's)

Step 4 — Once AIHA confirmed, always search for secondary causes:

Warm: SLE, CLL, drugs (methyldopa), CVID
Cold: Mycoplasma, EBV, lymphoma, primary CAD

Key traps:

Evans syndrome (AIHA + ITP) vs TTP: spherocytes + DAT positive (Evans) vs schistocytes + DAT negative (TTP)
Wilson's disease: Coombs-negative haemolytic anaemia
PNH: Coombs-negative; diagnosed by flow cytometry (↓CD55, CD59)
DAT-negative AIHA exists in < 3% of cases

Active Recall - Differential Diagnosis of AIHA

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf (Haemolytic anaemia classification and clinical features slides) ^[2] Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf (Haemolytic anaemia section) ^[3] Senior notes: Block A - Fever after a blood transfusion_ transfusion and related problems.pdf (Acute transfusion reactions) ^[4] Senior notes: Ryan Ho Haemtology.pdf (Section 2.5.4.1 Warm AIHA) ^[5] Lecture slides: Haematology Introduction to Haematological investigations (CBP, Clotting).pdf (AIHA and haemolytic anaemia laboratory features) ^[6] Senior notes: Jerry's immunodeficiencies.pdf (CVID and associated AIHA) ^[7] Senior notes: Block A - Patients with non-viral chronic liver diseases.pdf (Wilson's disease — Coombs-negative haemolytic anaemia) ^[8] Senior notes: Maksim Medicine Notes.pdf (Haemolytic anaemia specific causes table) ^[9] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (Aplastic anaemia section — PNH) ^[10] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (TTP section — diagnosis) ^[11] Senior notes: Ryan Ho Chemical Path.pdf (Anaemia of chronic disease) ^[12] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (SLE diagnosis section) ^[13] Lecture slides: GC 115. I am pregnant medical problems complicating pregnancy.pdf ^[14] Senior notes: Block A - Splenomegaly_ common causes of splenomegaly; myeloproliferative diseases.pdf (Splenomegaly sizes and differentials)

Diagnostic Criteria, Algorithm & Investigations for AIHA

Unlike many autoimmune diseases (e.g., SLE with EULAR/ACR criteria), AIHA does not have a single universally agreed-upon set of formal diagnostic criteria with a numerical score. Instead, the diagnosis is established by demonstrating three things simultaneously:

Evidence of haemolysis (laboratory markers confirming accelerated RBC destruction)
Positive Direct Antiglobulin Test (DAT) confirming immune-mediated aetiology
Exclusion of other causes of DAT-positive haemolysis (alloimmune, drug-induced)

The working diagnostic framework can be summarised as follows:

Criterion	Requirement	Rationale
1. Anaemia	Hb below reference range (< 12.3 g/dL females, < 14.0 g/dL males)	Confirms inadequate red cell mass
2. Evidence of haemolysis	↑Reticulocyte count (usually > 2%), ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin, ↑methemalbumin ^[1]^[2]^[4]	Demonstrates accelerated RBC destruction with compensatory erythropoiesis
3. Positive DAT	Direct antiglobulin test positive for IgG and/or C3 ^[1]^[2]^[4]^[5]	Confirms that immunoglobulins and/or complement are bound to the RBC surface in vivo — the hallmark of immune-mediated haemolysis
4. Peripheral blood smear consistent with AIHA	Spherocytes (warm AIHA), RBC agglutination (cold AIHA), polychromasia ^[1]^[2]	Morphological evidence supporting the diagnosis and subtype
5. Exclusion of alternative diagnoses	Rule out hereditary spherocytosis (↓EMA binding), PNH (flow cytometry for CD55/CD59), alloimmune haemolysis (recent transfusion history), drug-induced ^[4]^[8]	AIHA is partly a diagnosis of exclusion among DAT-positive haemolytic anaemias

High Yield — The Diagnostic Triad of AIHA

The diagnosis of AIHA rests on demonstrating:

Anaemia with evidence of haemolysis (reticulocytosis + ↑LDH + ↓haptoglobin + ↑unconjugated bilirubin)
Positive DAT (IgG ± C3 for warm; C3 only for cold)
Exclusion of other DAT-positive conditions and non-immune haemolytic anaemias

There is no single scoring system — it is a clinical-laboratory synthesis.

DAT-Negative AIHA — The 3% Exception

The antiglobulin test is diagnostic for AIHA and is negative in fewer than 3% of cases ^[4]. DAT-negative AIHA exists and should be suspected when haemolysis markers are strongly positive, spherocytes are present, the patient responds to steroids, and hereditary spherocytosis has been excluded by EMA binding. Causes of DAT-negative AIHA include: (1) low-affinity IgG that washes off during the test, (2) IgA-mediated AIHA (standard DAT uses anti-IgG and anti-C3, not anti-IgA), (3) antibody titre below the detection threshold of the assay. Specialised DAT techniques (micro-column, flow cytometric DAT, mitogen-stimulated DAT) may pick up these cases.

The DAT uses polyspecific anti-human globulin (AHG) reagent initially. If positive, monospecific reagents are used to determine exactly what is coating the RBC:

DAT Pattern	AIHA Classification	Antibody	Mechanism	Key Associations
IgG positive, C3 negative	Warm AIHA	IgG (usually IgG1/IgG3)	Extravascular haemolysis via FcγR on splenic macrophages	Primary; SLE; drugs (methyldopa); CLL
IgG positive, C3 positive	Warm AIHA with complement activation or Mixed type	IgG + complement	More severe — both splenic (IgG) and hepatic (C3b) clearance	SLE; aggressive warm AIHA; mixed type
IgG negative, C3 positive only	Cold agglutinin disease	IgM (already dissociated by the time of testing) + C3d remains	Complement-mediated extravascular (liver) ± intravascular	Mycoplasma; EBV; primary CAD; lymphoma ^[1]^[5]^[8]
Both negative ( < 3%)	DAT-negative AIHA	Low-affinity IgG, IgA, or subthreshold titre	As per warm AIHA	Requires specialised testing

Case 6 from Haematology Introduction lecture ^[5]: A 35-year-old woman presents with symptoms of anaemia. Investigations show: Haemoglobin 7.5 g/dL, WCC 9.5 × 10⁹/L, Platelets 200 × 10⁹/L, peripheral blood spherocytes ++, reticulocyte count 10%, total bilirubin 55 μmol/L, LDH 950 IU/L, Direct antiglobulin test positive for immunoglobulin G and complement. This is the classic warm AIHA presentation — all the pieces fit: anaemia with reticulocytosis, spherocytes, haemolysis markers elevated, and DAT positive for IgG + C3.

The algorithm follows the logical sequence taught in the GC lecture and senior notes ^[2]^[4]:

"After confirming a patient has haemolysis, order a direct antiglobulin test to determine whether the patient is undergoing autoimmune-mediated destruction" ^[2]

"Some portion of the healthy population will have positive DAT but no haemolysis, so you can't blindly do this test for everyone or you'll get a bunch of false positives — the pre-test probability must be good, must ensure that patient has a form of haemolysis first" ^[2]

Investigation Modalities — Comprehensive Breakdown

Investigation	Expected Finding in AIHA	Interpretation / Why
CBC — Haemoglobin	Reduced	Confirms anaemia; severity guides urgency of management
MCV	Normal or mildly macrocytic ^[1]^[2]^[4]	Why macrocytic? Because reticulocytes are larger than mature RBCs, so they drag up the average MCV ^[2]. In cold AIHA, RBC agglutinates can cause spuriously elevated MCV (machine counts clumps as single large cells — warm the sample to 37°C to correct)
MCHC	May be elevated (> 36 g/dL)	↑MCHC suggests spherocytosis — spherocytes have concentrated haemoglobin because membrane is lost but cytoplasm is retained ^[15]
RDW	Increased	Anisocytosis from mixed population of normal RBCs, spherocytes, and large reticulocytes
Reticulocyte count	Elevated (usually > 2%, often 5–20%) ^[1]^[2]^[4]	Compensatory bone marrow response — erythropoietin drives increased red cell production. This is the key distinguishing feature from marrow failure (aplastic anaemia, MDS) where reticulocytes are LOW
WCC and Platelets	Usually normal	If thrombocytopaenia also present → consider Evans syndrome (AIHA + ITP) ^[4]. If pancytopaenia → consider underlying LPD, aplastic anaemia, or PNH

High Yield — MCV in Haemolytic Anaemia

"Anaemia (mildly macrocytic usually) with reticulocytosis" ^[1]. The MCV in AIHA is typically normal-to-mildly elevated (100–110 fL) due to reticulocytosis. If MCV is > 110 fL, think of megaloblastic anaemia mimicking haemolysis (ineffective erythropoiesis also causes ↑LDH and ↑unconjugated bilirubin, but reticulocytes will be LOW). If MCV is < 80 fL, consider iron deficiency from chronic intravascular haemolysis (haemosiderinuria → iron loss) or concurrent thalassaemia trait.

These are the biochemical footprints of RBC destruction. Understanding why each changes is essential:

Marker	Expected in AIHA	Mechanism from First Principles
↑ LDH (Lactate Dehydrogenase)	Elevated (often 2–5× ULN)	LDH is an enzyme abundant in RBC cytoplasm. When RBCs are destroyed, LDH is released into the plasma. According to Prof Anskar Leung in data interpretation, LDH is the one marker to immediately differentiate haemolysis — if high, indicates high turnover ^[2]. Note: LDH is not specific — also elevated in liver disease, malignancy, tissue ischaemia
↓ Haptoglobin	Reduced or undetectable	Haptoglobin ("hapto" = to fasten/bind) is a plasma protein that binds free haemoglobin released from lysed RBCs. The haptoglobin-Hb complex is rapidly cleared by hepatocytes. In haemolysis, haptoglobin is consumed faster than the liver can produce it → serum levels fall. Most sensitive marker of haemolysis ^[15]. Caveats: haptoglobin is a positive acute phase reactant (may be falsely "normal" in concurrent inflammation); congenital ahaptoglobinaemia exists (rare)
↑ Unconjugated (indirect) bilirubin	Elevated	Haem from destroyed RBCs → biliverdin → unconjugated bilirubin (via haem oxygenase in macrophages). Rate of production exceeds hepatic conjugation capacity → accumulates in blood → jaundice. Unconjugated bilirubin is water-insoluble → cannot be excreted in urine → no tea-coloured urine ^[1]^[2]
↑ Methemalbumin	Elevated (especially intravascular haemolysis)	"Once haptoglobin is fully saturated, haem binds to albumin, forming methemalbumin" ^[15]. This indicates severe/intravascular haemolysis where haptoglobin has been completely consumed
Haemoglobinaemia	Present in intravascular haemolysis	Free haemoglobin in plasma (pink/red plasma). Occurs when free Hb exceeds haptoglobin binding capacity
Haemoglobinuria	Present in intravascular haemolysis	Free Hb filtered by glomerulus → dark (coca-cola) urine. Haemoglobinuria in intravascular haemolysis e.g. PNH ^[1]
Haemosiderinuria	Present in chronic intravascular haemolysis	Renal tubular cells absorb filtered haemoglobin → iron stored as haemosiderin → tubular cells sloughed into urine. Detected by Prussian blue staining of urine sediment. Indicates chronic intravascular haemolysis (PNH, chronic cold AIHA)
Urine urobilinogen	Increased	More bilirubin produced → more conjugated → more urobilinogen in gut → some reabsorbed and excreted in urine

Laboratory features of haemolytic anaemia ^[1]^[5]:

Anaemia (mildly macrocytic usually) with reticulocytosis

Increase in unconjugated bilirubin, LDH

Reduced serum haptoglobin

Increased in methemalbumin

The PBS is your "eyes" on the disease. Different haemolytic conditions produce characteristic morphological changes:

PBS Finding	Seen In	Mechanism
Polychromasia	All haemolytic anaemias with compensatory reticulocytosis ^[1]^[2]	Reticulocytes retain residual RNA → stain bluish-grey with Wright/Giemsa stain. Polychromasia is the visual correlate of reticulocytosis
Spherocytes	Warm AIHA, hereditary spherocytosis ^[1]^[2]^[5]	In warm AIHA: splenic macrophages "nibble" membrane from IgG-coated RBCs → cell loses surface area but retains volume → spherical shape. Why spherocytes also form in HS: underlying membrane protein defect (spectrin, ankyrin) causes membrane instability → progressive membrane loss
RBC agglutination	Cold agglutinin disease ^[1]^[2]^[5]	IgM pentamers cross-link multiple RBCs at room temperature (the temperature at which the smear is prepared) → visible clumps on the smear. This also causes spuriously elevated MCV on automated analysers
Schistocytes (fragmented RBCs)	MAHA — TTP, HUS, DIC, mechanical haemolysis ^[1]^[2]	RBCs are sheared as they pass through fibrin strands or damaged microvasculature → fragmentation. NOT seen in AIHA — if you see schistocytes, reconsider the diagnosis
Nucleated RBCs (normoblasts)	Severe haemolysis	Extreme erythropoietic stress → premature release of nucleated RBC precursors from bone marrow

Blood film findings in haemolytic anaemia ^[1]^[5]:

Polychromasia

Spherocytes (hereditary spherocytosis, immune haemolytic anaemia)

RBC fragmentation (microangiopathic haemolysis)

RBC agglutination (cold agglutinin disease)

Step 4: Direct Antiglobulin Test (DAT / Direct Coombs Test) — The Pivotal Investigation

This is the single most important test for diagnosing AIHA. Understanding exactly how it works is essential.

Take the patient's washed RBCs (washing removes unbound serum proteins)
Add anti-human globulin (AHG) reagent — this reagent contains antibodies against human IgG and human C3d
If the patient's RBCs are coated with IgG or C3 in vivo, the AHG bridges adjacent coated RBCs → visible agglutination = POSITIVE DAT
If the patient's RBCs are NOT coated, no bridging → no agglutination = NEGATIVE DAT

The DAT tells you: "Are there immunoglobulins and/or complement components physically stuck to the surface of this patient's red blood cells?"

The initial polyspecific AHG detects any coating. If positive, monospecific anti-IgG and anti-C3d reagents determine what is on the RBC surface — this is critical for classifying the AIHA subtype:

Monospecific Result	Interpretation
Anti-IgG positive	IgG autoantibody bound to RBC → Warm AIHA
Anti-C3d positive	Complement fragments on RBC → Cold AIHA (IgM already dissociated) or complement activation in warm AIHA
Both positive	IgG + complement → severe warm AIHA, mixed type, or SLE-associated
Both negative (with positive polyspecific)	Rare; may be due to IgA, IgM warm antibody, or low-density IgG

Direct antiglobulin test — positive in immune haemolytic anaemia ^[1]^[5]

Direct Coombs test: useful in detecting prior antibody binding to RBC using anti-IgG, anti-C3 ^[4]

Feature	Direct Antiglobulin Test (DAT)	Indirect Antiglobulin Test (IAT)
What it tests	Antibodies already bound to patient's RBCs in vivo ^[4]	Free autoantibodies circulating in patient's serum ^[4]
Sample	Patient's washed RBCs	Patient's serum + donor/reagent RBCs
Purpose in AIHA	Diagnostic — confirms immune haemolysis	Supportive — identifies free autoantibodies; important for cross-matching and predicting transfusion compatibility
How it works	AHG added directly to patient's RBCs	Patient's serum incubated with test RBCs → then AHG added → if serum contains auto-antibodies that bound to test RBCs, agglutination occurs

Indirect Coombs test: useful in detecting autoreactive antibodies in serum ^[4]

Exam Pearl — DAT Positive ≠ AIHA Automatically

A positive DAT is found in ~0.1% of healthy blood donors and ~1–15% of hospitalised patients (drugs, infections, passive transfer of antibodies). A positive DAT is only meaningful in the context of demonstrated haemolysis. You must first confirm haemolysis markers before interpreting a positive DAT as AIHA. Conversely, a negative DAT does not completely exclude AIHA (negative in < 3%).

Step 5: Identify the Underlying Cause — "What Is Driving the AIHA?"

This is where the diagnostic algorithm branches into searching for secondary causes. AIHA is not a final diagnosis — always search for the underlying aetiology ^[1]^[2]^[4].

Investigation	Expected Finding	Rationale
ANA (antinuclear antibody)	Positive in SLE	Sensitive screening test for SLE
Anti-dsDNA antibodies	Positive in SLE (specific)	Highly specific for SLE; correlates with disease activity and lupus nephritis
Complement levels (C3, C4)	↓ in active SLE	Complement consumed by immune complex deposition. ↓C3, C4 correlates with disease activity ^[12]
ESR, CRP	ESR ↑ in active SLE; Active SLE does NOT normally evoke a CRP response ^[12]	If CRP is elevated in SLE → suspect intercurrent infection
Anti-phospholipid antibodies	Positive in APS (may co-exist with SLE)	Lupus anticoagulant, anti-cardiolipin, anti-β2-glycoprotein I
Urinalysis	Haematuria, proteinuria	Screen for lupus nephritis

Investigation	Expected Finding	Rationale
CBC differential count	Lymphocytosis (≥ 5 × 10⁹/L in CLL)	CLL is the most common LPD to cause AIHA — up to 11% develop AIHA ^[4]
PBS	Smudge / smear cells (CLL)	Fragile CLL lymphocytes lyse during smear preparation → characteristic artefact
Flow cytometry (peripheral blood)	CD5+, CD19+, CD20+, CD23+ (CLL immunophenotype)	Confirms clonal B-cell population
Serum immunoglobulins	↓ IgG, IgA, IgM (hypogammaglobulinaemia in CLL and CVID)	CLL: progressive immune dysfunction; CVID: defective B-cell maturation ^[6]^[16]
CT neck/chest/abdomen/pelvis	Lymphadenopathy, splenomegaly, hepatomegaly	Staging for lymphoma / CLL
Bone marrow biopsy	May show lymphomatous infiltration	Not always required if peripheral blood flow cytometry is diagnostic
Serum protein electrophoresis + immunofixation	Monoclonal band (M-protein) — particularly IgM in Waldenström	Cold AIHA may be driven by monoclonal IgM from Waldenström or marginal zone lymphoma

Investigation	Expected Finding	Rationale
Mycoplasma pneumoniae serology (cold agglutinins, IgM)	Positive in Mycoplasma-associated cold AIHA	Anti-I cold agglutinins develop during Mycoplasma infection → typically self-limiting
EBV serology (VCA IgM, EBNA)	Positive in infectious mononucleosis	Anti-i cold agglutinins may develop; atypical lymphocytes on PBS
CMV, HIV, Hepatitis B/C serology	Positive if underlying infection	HIV and HCV can cause secondary AIHA; HBV screening essential before rituximab

Investigation	Approach	Key Drugs
Thorough drug history	Temporal correlation between drug initiation and onset of haemolysis	Methyldopa (true autoantibody), fludarabine, penicillins, cephalosporins (hapten), quinidine (immune complex) ^[1]^[5]^[8]
Drug-dependent antibody testing	Specialised test: patient's serum + drug + donor RBCs → if haemolysis, confirms drug-dependent antibody	Available in reference labs

Investigation	When to Order	Expected Finding
Cold agglutinin titre + thermal amplitude	All suspected cold AIHA	Titre > 1:64 at 4°C is significant; thermal amplitude (highest temperature at which agglutination occurs) is more clinically relevant than titre — high thermal amplitude (close to 37°C) = more severe haemolysis
Donath-Landsteiner test	Suspected PCH (children with acute post-viral intravascular haemolysis)	Biphasic IgG haemolysin detected: patient's serum + P-antigen-positive RBCs incubated at 4°C then warmed to 37°C → haemolysis confirms PCH
Flow cytometry for CD55/CD59	Suspected PNH (Coombs-negative haemolysis, thrombosis, pancytopaenia)	↓CD55 and ↓CD59 on RBCs/granulocytes ^[1]^[9]
EMA binding (flow cytometry)	Suspected hereditary spherocytosis (Coombs-negative with spherocytes)	↓EMA binding diagnostic of HS ^[4]^[8]
Bone marrow examination	Not routinely needed in AIHA; indicated if suspecting underlying LPD, MDS, aplastic anaemia, or unexplained cytopaenias	May show erythroid hyperplasia (compensatory); excludes marrow infiltration

Step	Investigation	Key Finding in AIHA	Notes
1	CBC + Reticulocyte count	Anaemia (normo/macrocytic), reticulocytosis	Reticulocytes drag up MCV; if reticulocytes low, reconsider diagnosis
2	LDH	Elevated	Most immediately useful marker of haemolysis ^[2]
2	Serum haptoglobin	Reduced or undetectable	Most sensitive marker; caution if concurrent inflammation
2	Unconjugated bilirubin	Elevated	Pre-hepatic jaundice; no tea-coloured urine
2	Methemalbumin	Elevated (if haptoglobin fully consumed)	Indicates severe/intravascular haemolysis ^[1]^[2]
3	Peripheral blood smear	Spherocytes (warm), RBC agglutination (cold), polychromasia	Essential for morphological classification
4	Direct Antiglobulin Test (DAT)	Positive for IgG ± C3 (warm) or C3 only (cold)	Diagnostic for AIHA; negative in < 3% ^[4]
4	Indirect Antiglobulin Test (IAT)	Positive for free autoantibodies in serum	Helps with cross-matching for transfusion
5	ANA, anti-dsDNA, C3/C4	Positive ANA/anti-dsDNA, ↓C3/C4 in SLE	Always check in young females with warm AIHA
5	CBC differential, PBS, flow cytometry	Lymphocytosis, smudge cells, CD5+CD19+CD23+	Screen for CLL
5	Serum immunoglobulins	↓Ig levels	CVID or CLL
5	Mycoplasma / EBV / CMV serology	Positive serology	Infection-triggered cold AIHA
5	HIV, HBV, HCV serology	Positive serology	HIV-associated AIHA; HBV screen before rituximab
5	Drug history	Temporal correlation with offending drug	Drug-induced AIHA
5	Cold agglutinin titre + thermal amplitude	Titre > 1:64; high thermal amplitude	Cold AIHA subclassification
5	Serum protein electrophoresis	Monoclonal IgM band	Waldenström / clonal CAD
5	CT scan	Lymphadenopathy, organomegaly	Staging for LPD
Special	EMA binding	↓EMA = HS	Rule out hereditary spherocytosis when DAT negative ^[4]^[8]
Special	Flow cytometry CD55/CD59	↓CD55/CD59 = PNH	Rule out PNH when DAT negative ^[1]^[9]
Special	Donath-Landsteiner test	Biphasic haemolysis = PCH	Children with acute post-viral intravascular haemolysis
Special	Bone marrow examination	Erythroid hyperplasia; excludes infiltration	Not routine; for diagnostic uncertainty or suspected underlying marrow disease

Example Case Interpretation (GC Lecture Case 6)

A 35-year-old woman presents with symptoms of anaemia. Haemoglobin: 7.5 g/dL; WCC: 9.5 × 10⁹/L; Platelet count: 200 × 10⁹/L; Peripheral blood: spherocytes ++; Reticulocyte count: 10%; Total bilirubin: 55 μmol/L; LDH: 950 IU/L; Direct antiglobulin test: positive for immunoglobulin G and complement ^[5]

Step-by-step interpretation:

Anaemia confirmed: Hb 7.5 g/dL (severe)
Reticulocytosis: 10% (markedly elevated → compensatory erythropoiesis)
Haemolysis markers: ↑Total bilirubin 55 μmol/L, ↑LDH 950 IU/L → confirmed haemolysis
PBS: Spherocytes ++ → immune haemolysis or HS
DAT: Positive for IgG AND complement (C3) → Warm AIHA with complement activation
Normal WCC and platelets → isolated AIHA (not Evans syndrome)
Next step: Search for underlying cause — autoimmune screen (SLE), LPD screen (CLL), drug history

Sequence of investigations to arrive at a diagnosis of AIHA ^[2]:

Order CBC → confirm anaemia, with compensatory increase in reticulocytes

Order blood film → observe microspherocytes

More blood parameters → observe increased LDH, increased unconjugated bilirubin, decrease in haptoglobin, increase in methemalbumin

With a diagnosis of haemolysis, confirm whether it is immune mediated by ordering a direct antiglobulin / Coombs' test → observe IgG in warm AIHA, C3d in cold AIHA

Transfusion in AIHA is notoriously difficult because autoantibodies interfere with cross-matching:

The IAT (indirect Coombs) is used for cross-matching — in AIHA, the patient's serum autoantibodies react with virtually ALL donor RBCs → all units appear "incompatible"
Test for allo-antibodies (type and screen) which may have developed from previous pregnancies or transfusion → risk of transfusion reaction ^[4]
The blood bank must use adsorption techniques (auto-adsorption or allo-adsorption) to remove autoantibodies and unmask any underlying allo-antibodies before issuing compatible blood
Transfusion should not be withheld in life-threatening anaemia simply because of a "positive crossmatch" — use "least incompatible" units if allo-antibodies have been excluded
In cold AIHA, use a blood warmer to prevent cold agglutination during transfusion

High Yield Summary — Diagnosis of AIHA

Diagnosis rests on 3 pillars:

Evidence of haemolysis (↑reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin)
Positive DAT (IgG ± C3 = warm; C3 only = cold)
Exclusion of other causes (HS → EMA binding; PNH → CD55/CD59; MAHA → schistocytes)

DAT pattern classifies AIHA: IgG ± C3 = warm; C3 only = cold (IgM already dissociated); Both negative in < 3% (DAT-negative AIHA).

Always search for secondary causes: SLE (ANA, anti-dsDNA, C3/C4), CLL (differential count, smudge cells, flow cytometry), drugs (methyldopa, fludarabine), infections (Mycoplasma, EBV), immunodeficiency (CVID — Ig levels).

GC Case 6: Classic warm AIHA — Hb 7.5, spherocytes, retic 10%, ↑LDH, ↑bilirubin, DAT positive for IgG + complement.

Remember: LDH is the immediate differentiator for haemolysis; haptoglobin is the most sensitive marker; DAT is the diagnostic test — but only order it AFTER confirming haemolysis to avoid false positive interpretation.

Active Recall - Diagnosis of AIHA

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf (Haemolytic anaemia classification, clinical features, laboratory features, AIHA slide) ^[2] Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf (Haemolytic anaemia section, AIHA diagnosis sequence) ^[4] Senior notes: Ryan Ho Haemtology.pdf (Section 2.5.4.1 Warm AIHA — laboratory findings, DAT, EMA binding, Evans syndrome) ^[5] Lecture slides: Haematology Introduction to Haematological investigations (CBP, Clotting).pdf (AIHA slide, Case 6, haemolytic anaemia laboratory features) ^[6] Senior notes: Jerry's immunodeficiencies.pdf (CVID and associated AIHA) ^[7] Senior notes: Block A - Patients with non-viral chronic liver diseases.pdf (Wilson's disease — Coombs-negative haemolytic anaemia) ^[8] Senior notes: Maksim Medicine Notes.pdf (Haemolytic anaemia specific causes — HS, G6PD, AIHA investigations) ^[9] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (Aplastic anaemia section — PNH flow cytometry) ^[12] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (SLE diagnosis — CRP, complement levels) ^[15] Senior notes: Block A - Fever after a blood transfusion_ transfusion and related problems.pdf (Haemolysis investigation — haptoglobin, methemalbumin mechanism) ^[16] Senior notes: MBBS Final MB (Medicine) (Felix PY Lai).pdf (CLL diagnosis — CBC, flow cytometry, immunoglobulins)

Management of AIHA

Before diving into specific agents, understand the four pillars of AIHA management as laid out in the GC lecture slides ^[1]^[2]:

AIHA Treatment ^[1]:

Transfusion if necessary

Folate supplement

Decrease further haemolysis, e.g.:

Steroid or other immunosuppressants in autoimmune haemolytic anaemia

Avoidance of certain drugs in drug-induced haemolytic anaemia

Keep warm in cold haemagglutinin disease

Splenectomy

Treat the underlying cause (e.g. AIHA due to underlying lymphoproliferative disorder) if necessary

These four pillars map directly onto the pathophysiology:

Pillar 1 (Transfusion): Replace what has been lost — restore oxygen-carrying capacity
Pillar 2 (Folate): Supply the raw materials the marrow needs for compensatory erythropoiesis
Pillar 3 (Decrease haemolysis): Interrupt the immune destruction pathway — either suppress the immune system (steroids, rituximab), remove the site of destruction (splenectomy), remove the trigger (drugs, cold), or block complement
Pillar 4 (Treat underlying cause): AIHA is often secondary — treat SLE, CLL, stop the offending drug, treat the infection

The management differs fundamentally between warm and cold AIHA:

Primary chronic cold agglutinin disease (CAD) has been considered more difficult to treat than warm-antibody AIHA. This is because of the lack of efficacy of therapy with corticosteroids or other non-specific immunosuppressive agents in cold AIHA ^[2]

Aspect	Details
Indication	Transfusion if necessary ^[1] — life-threatening anaemia (Hb < 5–6 g/dL or symptomatic cardiac ischaemia, haemodynamic compromise, heart failure)
Challenge	Autoantibodies in the patient's serum react with virtually ALL donor RBCs → cross-match appears "incompatible" → this is a serological challenge, not a clinical contraindication
Key principle	Test for allo-antibodies (type and screen) which may have developed from previous pregnancies or transfusion → risk of transfusion reaction ^[4]. The blood bank must adsorb out autoantibodies to identify any underlying allo-antibodies (which cause true haemolytic transfusion reactions)
Decision	Transfusion should NOT be withheld in life-threatening anaemia just because cross-match is "incompatible" — use "least incompatible" units once allo-antibodies have been excluded. Transfuse slowly with close monitoring
Cold AIHA specifics	Use a blood warmer and pre-warmed IV fluids ^[8] to prevent cold agglutination during transfusion. Keep the patient warm
Rate	Transfuse slowly (1 unit over 3–4 hours) with careful monitoring for transfusion reactions

Exam Pearl — Never Withhold Blood in Life-Threatening AIHA

A common exam mistake is stating "do not transfuse because cross-match is incompatible." In AIHA, the autoantibody reacts with almost all donor cells — this does NOT mean transfusion is contraindicated. The real danger is missing an underlying allo-antibody. Once allo-antibodies are excluded by adsorption studies, transfuse the "least incompatible" unit. The transfused cells will be destroyed at the same rate as the patient's own cells (by the autoantibody), but they buy time for immunosuppressive therapy to take effect.

Aspect	Details
Indication	Folate supplement — generally recommended for any form of haemolysis, to replenish the raw material ^[1]^[2]
Rationale	Compensatory erythropoiesis ↑ bone marrow RBC production by 6–8× → this markedly increases folate consumption (folate is essential for DNA synthesis in rapidly dividing erythroid precursors). Without supplementation, the marrow may become folate-depleted → megaloblastic crisis superimposed on haemolytic anaemia → worsening anaemia with reticulocytopaenia
Dose	Folic acid 1–5 mg/day orally
Duration	Throughout the period of active haemolysis

Pillar 3: Decrease Further Haemolysis

This is the core of AIHA management and differs fundamentally between warm and cold types.

WARM AIHA — Treatment Ladder

Systemic steroids: oral prednisolone 1 mg/kg/day as 1st line → 50–90% effective ^[4]

Aspect	Details
Drug	Oral prednisolone 1 mg/kg/day (typically 60–80 mg/day for an average adult) ^[4]^[8]
Mechanism	↓Autoantibody production (suppresses autoreactive B and T cells) + ↓macrophage destruction (downregulates FcγR expression on splenic macrophages, reducing phagocytosis of IgG-coated RBCs) ^[4] — a dual mechanism that attacks both arms of the pathophysiology
Why steroids work quickly	The macrophage-suppressing effect begins within days (reduced Fc receptor avidity), whereas the antibody-reducing effect takes weeks (B cells have a longer lifespan). This is why you see initial clinical improvement before the DAT becomes negative
Expected response	Expect response (↑Hb, ↓haemolysis markers) in up to 3 weeks ^[4]. Most patients show some improvement within 1–2 weeks
Tapering	Taper slowly from 1 week after response to up to 10 weeks afterwards, as insufficient dose will only be apparent in 3–4 weeks ^[4]. Typical approach: once Hb stable > 10 g/dL and haemolysis markers improving, begin reducing by 10 mg every 1–2 weeks to ~20 mg, then by 5 mg every 2–4 weeks, then by 2.5 mg increments. Target: lowest effective dose, ideally off steroids entirely
Response rate	50–90% effective ^[4] (initial response); however, only ~20–30% achieve sustained remission off steroids
When to consider steroid failure	No response after 3 weeks of adequate dosing, or relapse during taper, or inability to taper below ≥15 mg/day (steroid-dependent)
Side effects	Cushingoid features (moon face, buffalo hump, truncal obesity), hyperglycaemia, osteoporosis, gastritis/PUD, immunosuppression, adrenal suppression, myopathy, cataracts, AVN of femoral head
Prophylaxis during steroids	PPI for gastric protection; calcium + vitamin D ± bisphosphonate for bone protection if prolonged course; monitor blood glucose

Why Taper Slowly?

Steroids suppress the autoimmune process at a dose-dependent level. If you taper too quickly, the immune attack resumes before adequate immunological tolerance has been restored. The 3–4 week lag ^[4] is because antibody-producing B cells and plasma cells have a turnover time of weeks — so the full effect of dose reduction on antibody production is only apparent after this delay. If you cut the dose and the patient relapses 3 weeks later, it's too late — you have to go back up and start again.

2nd Line: Rituximab or Splenectomy

2nd line: rituximab (to ↓Ab production) or splenectomy (to ↓RBC destruction, 50–60% response) if poorly responsive, relapsed or steroid-dependent ^[4]

Indications for 2nd line therapy:

No response to steroids after 3 weeks
Relapse during steroid taper
Steroid-dependent (cannot taper below ~15 mg/day without relapse)
Intolerable steroid side effects

Aspect	Details
Drug	Rituximab — a chimeric monoclonal anti-CD20 antibody ("Ritux-i-mab" → "rituxi" targets, "mab" = monoclonal antibody)
Mechanism	↓Autoantibody production ^[4] — CD20 is expressed on mature B lymphocytes (but NOT plasma cells). Rituximab depletes CD20+ B cells → removes the precursor pool that differentiates into autoantibody-producing plasma cells → over weeks, autoantibody titre falls → haemolysis decreases
Regimen	Standard: 375 mg/m² IV weekly × 4 doses. Low-dose alternative: 100 mg fixed dose weekly × 4 (similar efficacy, less cost, fewer infusion reactions — increasingly used)
Response rate	~70–80% overall response; ~50% complete response. Onset of response: 2–8 weeks (takes time for B-cell depletion to translate into reduced antibody levels)
Duration of response	Variable; median 1–2 years. Some patients achieve long-lasting remission; others relapse and may respond to retreatment
Advantages over splenectomy	Non-invasive; preserves splenic immune function; can be repeated
Contraindications / Cautions	Hepatitis B reactivation — MUST screen HBsAg and anti-HBc before starting rituximab. If HBsAg+ or anti-HBc+ (even if HBsAg−), give antiviral prophylaxis (entecavir) to prevent fatal HBV reactivation. Also: progressive multifocal leukoencephalopathy (PML) — very rare; hypogammaglobulinaemia with prolonged use; infusion reactions
Pre-treatment screening	HBV serology (HBsAg, anti-HBs, anti-HBc), HCV, HIV; baseline immunoglobulins

Rituximab and HBV — Critical Safety Point

In Hong Kong, ~8% of the population are chronic HBV carriers. Rituximab-induced B-cell depletion removes immunological control over HBV → viral reactivation can cause fulminant hepatitis and death. Always screen for HBV before rituximab and give antiviral prophylaxis if any marker of past or current HBV exposure is positive.

Aspect	Details
Rationale	The spleen is the primary site of extravascular haemolysis in warm AIHA (splenic macrophages phagocytose IgG-coated RBCs). Removing the spleen removes the main "destruction factory" → ↓RBC destruction, 50–60% response ^[4]
Indication	Refractory ITP or AIHA ^[14]; poorly responsive, relapsed, or steroid-dependent warm AIHA; when rituximab has failed or is contraindicated
Response rate	~50–60% achieve sustained remission ^[4]; some patients achieve partial response (reduced steroid requirement)
Approach	Laparoscopic preferred (fewer complications, shorter recovery)
Pre-splenectomy requirements	(1) Vaccinations: Pneumococcal (PCV13 + PPSV23), Meningococcal (MenACWY + MenB), Haemophilus influenzae type b (Hib) — ideally ≥2 weeks before elective splenectomy. (2) Consider perioperative VTE prophylaxis (post-splenectomy thrombocytosis increases VTE risk)
Post-splenectomy complications	Risk of infection — life-threatening infection from encapsulated bacteria → overwhelming post-splenectomy infection (OPSI) ^[14]^[17]: Pneumococcus, Neisseria meningitidis, Haemophilus influenzae, Capnocytophaga ^[17]
Post-splenectomy prophylaxis	Long-term (often lifelong) oral penicillin V prophylaxis (250 mg BD) or amoxicillin; patient education to seek urgent medical attention for any febrile illness; MedicAlert bracelet
Post-splenectomy blood findings	Spurious leukocytosis, increased platelet counts, Howell-Jolly bodies on PBS ^[14] — these are expected and NOT pathological

Splenectomy for therapeutic purposes: refractory ITP or AIHA (less common) — for patients who are on massive doses of treatment, don't want to give patients so much treatment ^[14]

3rd line: alternative immunosuppressants, e.g. azathioprine, cyclophosphamide ^[4] Caution in young due to risk of secondary neoplasm with prolonged immunosuppression ^[4]

Drug	Mechanism	Dose	Notes
Azathioprine	Purine analogue → inhibits DNA synthesis in rapidly dividing lymphocytes → ↓autoantibody production	1–2.5 mg/kg/day	Slow onset (2–3 months); used as steroid-sparing agent; check TPMT/NUDT15 genotype before starting (HK population has higher NUDT15 variant frequency → increased myelosuppression risk)
Mycophenolate mofetil (MMF)	Inhibits inosine monophosphate dehydrogenase → selectively inhibits lymphocyte proliferation	500 mg–1 g BD	Well-tolerated; increasingly used as steroid-sparing; teratogenic (contraindicated in pregnancy)
Cyclophosphamide	Alkylating agent → cross-links DNA → cytotoxic to lymphocytes	IV pulse or oral	Reserved for refractory cases; risk of infertility, haemorrhagic cystitis, secondary malignancy
Ciclosporin	Calcineurin inhibitor → blocks IL-2 transcription → suppresses T-cell activation	3–5 mg/kg/day	Used in some refractory cases; nephrotoxic; requires drug level monitoring

Drug	Mechanism	Status
Sutimlimab	Monoclonal antibody against complement C1s → blocks classical complement pathway activation → prevents complement-mediated haemolysis	FDA-approved for cold agglutinin disease (2022); under investigation for warm AIHA
Fostamatinib	Spleen tyrosine kinase (SYK) inhibitor → blocks FcγR-mediated phagocytosis in spleen	Approved for refractory ITP; used off-label/investigated in warm AIHA
Complement inhibitors (eculizumab, pegcetacoplan)	Anti-C5 (eculizumab) or C3 inhibitor (pegcetacoplan) → blocks complement cascade	Primarily for PNH; case reports/trials in complement-driven AIHA

Immunoglobulin therapy — as an immunomodulatory: probable benefit in autoimmune haemolytic anaemia ^[18]

Aspect	Details
Indication	Probable benefit — autoimmune haemolytic anaemia ^[18]; used as a temporising measure in acute, severe warm AIHA when rapid response needed (e.g., preoperatively, severe symptomatic anaemia unresponsive to initial steroids)
Mechanism	Blocks FcγR on macrophages (saturates the receptors with exogenous IgG → reduces phagocytosis of antibody-coated RBCs); also modulates complement activation and anti-idiotype effects
Dose	0.4 g/kg/day for 5 days or 1 g/kg/day for 2 days ^[18]
Limitations	Response is transient (days to weeks); expensive; not a long-term solution; supplies limited
Contraindications	Previous history of allergy to IVIG. Caution but not absolute contraindication in IgA deficiency ^[18] (risk of anaphylaxis from anti-IgA antibodies in IgA-deficient patients)

Cold AIHA management is fundamentally different from warm AIHA because:

The haemolysis is complement-mediated (not FcγR-mediated) → steroids have minimal effect (steroids suppress macrophage Fc receptor function and antibody production, but the main destruction in cold AIHA is complement-driven)
The site of destruction is mainly the liver (Kupffer cells clearing C3b-coated RBCs) → splenectomy has minimal benefit (the spleen is not the primary destruction site)
IgM autoantibodies are produced by clonal B cells → rituximab targeting CD20+ B cells is effective

The most important difference in pharmacological treatment between warm and cold AIHA: Primary chronic cold agglutinin disease has been considered more difficult to treat than warm-antibody AIHA because of the lack of efficacy of therapy with corticosteroids or other non-specific immunosuppressive agents ^[2]

Line	Treatment	Details
General measures	Cold avoidance — the cornerstone ^[1]^[2]^[8]	Keep extremities warm (gloves, warm clothing); pre-warmed IV fluids ^[8]; warm environment; avoid cold drinks/foods. This prevents IgM binding to RBCs in peripheral circulation
Mild / Stable	Cold avoidance alone	Many patients with chronic primary CAD have mild, compensated haemolysis that does not require pharmacological therapy
1st line (symptomatic)	Rituximab ^[8]	CD20+ B-cell depletion reduces the clonal B-cell population producing pathogenic IgM. Response rate ~50–60%. Rituximab monotherapy or rituximab + bendamustine for primary CAD
NOT effective	Steroids — NOT effective in cold AIHA ^[2]	Steroids do not significantly suppress complement-mediated haemolysis or reduce IgM production from clonal B cells
NOT effective	Splenectomy — NOT effective in cold AIHA	The liver (not spleen) is the main site of C3b-mediated clearance
Newer 1st line	Sutimlimab (anti-C1s)	FDA-approved (2022) specifically for cold agglutinin disease. Blocks classical complement pathway at C1s → prevents C3b deposition on RBCs → stops complement-mediated haemolysis. Rapid onset (within days). Does NOT reduce IgM titre (it blocks downstream effect). Given as IV infusion
Acute / Severe	Plasmapheresis ^[8]	Physically removes circulating IgM (which is a large pentameric molecule confined mostly to intravascular space → efficiently removed by plasma exchange). Temporising measure — effect is transient as IgM is rapidly re-synthesised
Refractory	Bortezomib (proteasome inhibitor), ibrutinib (BTK inhibitor)	Target plasma cells or B-cell signalling; used in refractory CAD
Infection-associated (secondary)	Treat the underlying infection	Mycoplasma: antibiotics (macrolides/doxycycline); EBV: supportive. The cold AIHA usually resolves spontaneously once the infection clears

Why Steroids Don't Work in Cold AIHA — From First Principles

In warm AIHA, steroids work by: (1) suppressing autoantibody production and (2) downregulating splenic macrophage FcγR. In cold AIHA, the destruction is mediated by complement (C3b opsonisation → hepatic Kupffer cell clearance ± MAC-mediated intravascular lysis). Steroids do NOT significantly inhibit the complement cascade. Furthermore, the IgM in primary CAD is produced by a clonal B-cell population that is relatively resistant to steroid-induced apoptosis (unlike the polyclonal autoreactive B cells in warm AIHA). This is why rituximab (which depletes the clonal CD20+ B cells) and complement inhibitors (which directly block the effector pathway) are effective, while steroids are not.

Principle	Details
Avoidance of certain drugs in drug-induced haemolytic anaemia ^[1]	Stop the offending drug immediately — this is the most important intervention
Hapten mechanism (penicillin)	Haemolysis resolves within days to weeks of drug cessation as drug-coated RBCs are cleared
Immune complex mechanism (quinidine)	Haemolysis can be acute and severe (intravascular) → may need supportive care including transfusion, fluid resuscitation, monitoring for AKI
True autoantibody mechanism (methyldopa)	Haemolysis may persist for weeks to months after drug cessation because the autoantibodies have already been induced and have an independent lifespan. May require a short course of steroids
General	Supportive care (folate, transfusion if needed); steroids if haemolysis does not resolve after drug withdrawal

Aspect	Details
Children (post-viral)	Self-limiting — supportive care (warming, transfusion if needed, folate). Resolves spontaneously as the viral infection clears
Acute management	Keep warm; transfuse through a blood warmer if needed; steroids may be tried but evidence is limited
Prognosis	Excellent in children — usually a single episode with complete recovery

Treat the underlying cause (e.g. AIHA due to underlying lymphoproliferative disorder) if necessary ^[1]

Underlying Cause	Treatment Approach
SLE	Hydroxychloroquine (all SLE patients); immunosuppression with steroids + steroid-sparing agents (azathioprine, MMF); treat lupus nephritis if present ^[19]
CLL	Disease-specific chemotherapy/immunotherapy (e.g., ibrutinib, venetoclax, FCR regimen). Treating the CLL often resolves the AIHA. Note: fludarabine (purine analogue used in CLL) can itself cause AIHA — avoid in CLL patients with active AIHA
Other lymphomas	Disease-specific chemoimmunotherapy
CVID	IVIG replacement therapy (to correct hypogammaglobulinaemia); treat AIHA with steroids/rituximab as for primary warm AIHA
Mycoplasma / EBV infection	Treat the infection; cold AIHA usually self-limited
Drug-induced	Stop the drug (see above)

Parameter	Expected Change	Timeline
Haemoglobin	Rising (target > 10 g/dL)	Within 1–3 weeks on steroids
Reticulocyte count	Initially rises further (bone marrow response), then normalises as haemolysis slows	1–2 weeks
LDH	Falls	Within days to weeks
Haptoglobin	Rises (no longer consumed)	May take weeks to normalise
Unconjugated bilirubin	Falls	Days to weeks
DAT	May remain positive for weeks to months even after clinical remission	Do not rely on DAT alone to guide treatment — use clinical response and haemolysis markers

DAT Persistence

The DAT can remain positive long after clinical remission because low levels of autoantibody may still coat RBCs without causing significant haemolysis. Treat the patient, not the lab test. If Hb is stable and haemolysis markers are normal, the patient is in remission regardless of DAT status.

Special Situations

More difficult to treat than isolated AIHA ^[4]
Same treatment ladder as warm AIHA (steroids → rituximab → splenectomy → immunosuppressants)
Must address both the anaemia AND the thrombocytopaenia
If platelet count is dangerously low (< 20 × 10⁹/L) with active bleeding: IVIG can be used for rapid but transient platelet increment ^[18]
Thrombopoietin receptor agonists (eltrombopag, romiplostim) may be considered for the ITP component ^[17]
Always search for underlying SLE, CVID, ALPS

Steroids are the mainstay (prednisolone is preferred; avoid dexamethasone and betamethasone as they cross the placenta more readily)
Rituximab: avoid if possible (crosses placenta in 2nd/3rd trimester → neonatal B-cell depletion)
IVIG: safe in pregnancy and can be used for acute episodes ^[18]
Splenectomy: safest in 2nd trimester if absolutely necessary
MMF and cyclophosphamide are teratogenic — absolutely contraindicated
Azathioprine: considered relatively safe in pregnancy (6-MP is metabolised by the placenta)

Step	Action
1	Assess clinical urgency — is Hb life-threateningly low?
2	Send extended type and screen — blood bank performs adsorption to remove autoantibodies and detect underlying allo-antibodies
3	If allo-antibodies excluded → use "least incompatible" units
4	Warm AIHA: standard transfusion with monitoring
5	Cold AIHA: use blood warmer and pre-warmed IV fluids ^[8]; keep patient warm
6	Transfuse slowly (1 unit over 3–4 hours); monitor for transfusion reactions

Treatment	Key Contraindications
Steroids	Uncontrolled diabetes (relative); active TB (relative); systemic fungal infection; known steroid psychosis
Rituximab	Active HBV without prophylaxis (risk of fatal reactivation); severe active infection; known hypersensitivity
Splenectomy	Inadequate vaccination (relative — vaccinate and proceed); unfit for surgery; cold AIHA (ineffective)
Azathioprine	TPMT/NUDT15 deficiency (increased myelosuppression); pregnancy (relative — lower doses may be used)
MMF	Pregnancy (teratogenic); breastfeeding
Cyclophosphamide	Pregnancy; active cystitis; young patients (long-term malignancy risk) ^[4]
IVIG	Previous allergy to IVIG; caution in IgA deficiency ^[18]
Sutimlimab	Risk of encapsulated organism infections (blocks classical complement); must vaccinate
Steroids in cold AIHA	Not a contraindication per se, but not effective — do not waste time ^[2]

Feature	Warm AIHA	Cold AIHA
1st line	Prednisolone 1 mg/kg/day ^[4]	Cold avoidance ^[1]^[2]^[8]
Pharmacological 1st line	Prednisolone	Rituximab (if treatment needed) or Sutimlimab ^[8]
Steroids effective?	Yes (50–90%) ^[4]	No ^[2]
Splenectomy effective?	Yes (50–60%) ^[4]	No (liver is main destruction site)
Rituximab	2nd line ^[4]	1st line pharmacological ^[8]
IVIG	Probable benefit ^[18]	Less useful (IgM not well removed by IVIG mechanism)
Plasmapheresis	Minimal role (IgG is distributed in extravascular space, poorly removed)	Effective acutely ^[8] (IgM is mainly intravascular, efficiently removed)
Complement inhibitors	Emerging role	Sutimlimab approved for CAD

High Yield Summary — Management of AIHA

Four Pillars (GC lecture):

Transfusion if necessary (do NOT withhold — use "least incompatible" after excluding allo-antibodies)
Folate supplementation (replenish raw material for compensatory erythropoiesis)
Decrease haemolysis:
- Warm: Steroids (1st line, 1 mg/kg/day prednisolone) → Rituximab or Splenectomy (2nd line) → Azathioprine/Cyclophosphamide (3rd line)
- Cold: Cold avoidance → Rituximab or Sutimlimab → Plasmapheresis for acute episodes → Steroids NOT effective
- Drug-induced: Stop the drug
Treat underlying cause (SLE, CLL, infection, CVID)

Key differences warm vs cold:

Steroids work in warm (↓FcγR + ↓Ab production) but NOT cold (complement-mediated, clonal IgM)
Splenectomy works in warm (spleen = site of destruction) but NOT cold (liver = main site)
Rituximab is 2nd line in warm but 1st line pharmacological in cold

Rituximab caution: Screen for HBV before starting — risk of fatal reactivation.

Splenectomy caution: Vaccinate against encapsulated organisms (Pneumococcus, Meningococcus, Hib); lifelong penicillin prophylaxis.

IVIG: Probable benefit in AIHA; dose 0.4 g/kg/day × 5 days; contraindicated if previous IVIG allergy; caution in IgA deficiency.

Active Recall - Management of AIHA

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf (AIHA treatment slide) ^[2] Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf (AIHA treatment principles, warm vs cold difference) ^[4] Senior notes: Ryan Ho Haemtology.pdf (Section 2.5.4.1 Warm AIHA — management: steroids, rituximab, splenectomy, 3rd line) ^[8] Senior notes: Maksim Medicine Notes.pdf (AIHA management table — warm and cold) ^[14] Senior notes: Block A - Splenomegaly_ common causes of splenomegaly; myeloproliferative diseases.pdf (Splenectomy indications, complications, post-splenectomy findings) ^[17] Senior notes: Gen Clerk Anaes + Microbiology Summary.pdf (Post-splenectomy encapsulated organism infections); Block A - Abnormal bleeding after tooth extraction.pdf (ITP treatment including IVIG, TPO-RA) ^[18] Lecture slides: Handbook of Internal Medicine 2024.pdf (IVIG indications, dosing, contraindications — AIHA as probable benefit) ^[19] Senior notes: Ryan Ho Rheumatology.pdf (SLE management — HCQ, immunosuppression regimens)

Complications of AIHA

Complications of AIHA arise from three sources: (1) the haemolysis itself and its downstream consequences, (2) the underlying disease driving the AIHA, and (3) the treatment used to manage it. Understanding the pathophysiology of each complication from first principles is essential.

A. Complications of the Haemolytic Process

Aspect	Details
Mechanism	Chronic haemolysis → ↑unconjugated bilirubin concentration in hepatic bile → promotion of stone formation within the gallbladder ^[1]^[2]. Unconjugated bilirubin is conjugated by the liver and excreted into bile. When bilirubin load chronically exceeds normal, the bile becomes supersaturated with calcium bilirubinate → nucleation → pigment (black) gallstones
Type of stone	Calcium bilirubinate pigment stones — characteristically black, radio-opaque (unlike cholesterol stones which are radiolucent)
Clinical significance	May cause biliary colic, acute cholecystitis, choledocholithiasis (CBD obstruction → obstructive jaundice), cholangitis, gallstone pancreatitis
Why important in AIHA specifically	Any chronic haemolytic anaemia predisposes to pigment gallstones. History of gallstones is one of the key histories to elicit in a patient with suspected haemolytic anaemia ^[1]^[2]
Management	Cholecystectomy if symptomatic; in patients undergoing splenectomy for AIHA, concomitant cholecystectomy may be considered if gallstones are present

Aspect	Details
Cause	Parvovirus B19 infection — the virus has a specific tropism for erythroid progenitor cells (binds to globoside / P antigen on erythroid precursors → directly infects and lyses them)
Mechanism	Parvovirus B19 causes a temporary cessation of erythropoiesis lasting ~7–10 days. In a normal person, this is insignificant (RBC lifespan ~120 days — a 10-day production halt barely dents the Hb). But in AIHA, the RBC lifespan is already drastically shortened (days instead of months), so the patient is entirely dependent on compensatory reticulocytosis to maintain Hb. When erythropoiesis stops → reticulocyte count drops to near zero → rapid and precipitous fall in Hb → potentially life-threatening anaemia
Clinical features	Sudden worsening of anaemia, absent reticulocytosis (paradoxically low reticulocytes in a patient with known haemolysis), fatigue, dyspnoea, pallor. May be preceded by a febrile illness
Diagnosis	Reticulocytopaenia in the setting of known haemolytic anaemia + Parvovirus B19 serology (IgM) or PCR
Management	Transfusion support; self-limiting once parvovirus infection clears (7–10 days); IVIG for immunocompromised patients who cannot clear the virus

Aplastic Crisis — Why Is It So Dangerous in Haemolytic Anaemia?

Think of it this way: in chronic AIHA, the patient's haemoglobin is at a steady state only because the bone marrow is working at 6–8× normal output to replace the RBCs being destroyed. If you suddenly switch off that compensatory erythropoiesis (parvovirus B19), the patient has no reserve — Hb plummets rapidly. It is like removing the pump from a sinking boat.

Aspect	Details
Mechanism	Chronic compensatory erythropoiesis consumes large amounts of folate (required for DNA synthesis in rapidly dividing erythroid precursors). If folate stores are not replenished, the marrow develops megaloblastic changes → megaloblastic crisis with worsening macrocytic anaemia and paradoxical reticulocytopaenia
Prevention	Folate supplementation — generally recommended for any form of haemolysis, to replenish the raw material ^[1]^[2]. Folic acid 1–5 mg/day
Why it matters	A preventable complication — always prescribe folic acid in any patient with chronic haemolysis

Aspect	Details
Mechanism	Primarily seen in intravascular haemolysis (severe cold AIHA, PCH, or fulminant warm AIHA with complement activation). Free haemoglobin released into the plasma is filtered by the glomerulus → haemoglobin precipitates in the renal tubules → acute tubular necrosis. Additionally, free haemoglobin scavenges nitric oxide (NO) → renal vasoconstriction → ↓renal blood flow → ischaemic AKI ^[15]. Haem also has direct oxidative toxicity to tubular epithelial cells
Risk factors	Massive acute intravascular haemolysis, dehydration, pre-existing CKD
Prevention	Aggressive IV hydration to maintain urine output; alkalinisation of urine (to keep haemoglobin more soluble in alkaline pH); prompt treatment of the underlying haemolysis
Same mechanism as ABO incompatible transfusion reaction AKI	Free haemoglobin causes platelet activation + binding of NO → smooth muscle dystonia → renal vasoconstriction → AKI ^[15]

Aspect	Details
Mechanism	AIHA is a recognised risk factor for venous thromboembolism (VTE). Multiple mechanisms: (1) Free haemoglobin scavenges NO → endothelial dysfunction and vasoconstriction; (2) Phosphatidylserine exposure on damaged RBC membranes is pro-coagulant; (3) Release of RBC-derived microparticles activates the coagulation cascade; (4) Inflammatory cytokines from the autoimmune process promote a hypercoagulable state; (5) Associated antiphospholipid antibodies in SLE-related AIHA
Incidence	VTE risk is elevated ~2–5× compared to age-matched controls
Common sites	DVT, PE; in cold AIHA/PNH, thrombosis may occur in unusual sites (splanchnic veins, cerebral sinuses)
Prevention	VTE prophylaxis should be considered in hospitalised patients with active AIHA, especially during acute haemolytic crises; treat underlying AIHA promptly

Aspect	Details
High-output cardiac failure	Chronic severe anaemia → compensatory ↑cardiac output (↑heart rate, ↑stroke volume) → if sustained, leads to high-output heart failure. Complications of anaemia: cardiac ischaemia, ↑thrombocytopenic bleeding, ↑mortality ^[15]
Cardiac ischaemia	Acute/severe anaemia → symptoms associated with cardiac ischaemia ^[20]. Reduced oxygen-carrying capacity → myocardial oxygen demand exceeds supply → angina or NSTEMI, especially in patients with pre-existing coronary artery disease
Mechanism	Every gram of Hb carries 1.34 mL of O₂. A drop from 14 to 7 g/dL halves the blood's O₂-carrying capacity. The heart compensates by increasing output, but if the demand-supply mismatch is severe enough, ischaemia occurs

Aspect	Details
Mechanism	Two sources: (1) Chronic extravascular haemolysis → macrophages phagocytose RBCs and release iron from haem → iron is recycled but in excess → stored as ferritin/haemosiderin → progressive tissue iron deposition; (2) Repeated transfusions — each unit of packed red cells contains ~200–250 mg of iron, and the body has no active mechanism to excrete excess iron
Organs affected	Liver (hepatic fibrosis/cirrhosis), heart (cardiomyopathy, arrhythmias), pancreas (diabetes mellitus), endocrine organs (hypogonadism, hypothyroidism)
Monitoring	Serial serum ferritin levels; cardiac MRI T2* for cardiac iron loading; liver MRI (FerriScan)
Management	Iron chelation therapy (desferrioxamine, deferasirox, deferiprone) if ferritin persistently elevated (typically > 1000 μg/L) or after ≥20 units of packed cells

Aspect	Details
Mechanism	Chronic extravascular haemolysis in warm AIHA causes reticuloendothelial hyperplasia in the spleen → splenomegaly → pooling of blood cells in the enlarged spleen → hypersplenism (cytopaenia from sequestration/destruction of all blood cell lines) ^[14]
Consequence	Worsening anaemia (beyond what autoimmune destruction alone would cause), leucopaenia (↑infection risk), thrombocytopaenia (↑bleeding risk)
Management	Treatment of underlying AIHA; splenectomy if hypersplenism is contributing significantly to cytopaenias

Since ~50% of AIHA is secondary, complications of the driving disease are major contributors to morbidity:

Underlying Disease	Key Complications
SLE	Lupus nephritis (proteinuria, haematuria → CKD); CNS lupus (seizures, psychosis); serositis (pleurisy, pericarditis); antiphospholipid syndrome → recurrent thrombosis, recurrent fetal loss ^[21]
CLL	Infections (hypogammaglobulinaemia); Richter transformation to aggressive DLBCL; autoimmune cytopaenias (AIHA, ITP — Evans syndrome); disease progression ^[4]
Lymphoma	Disease-related organ damage; B-symptoms; transformation
CVID	Recurrent sinopulmonary infections; bronchiectasis; autoimmune disease (ITP, AIHA); granulomatous disease; increased risk for GI and lymphoid malignancies ^[6]

C. Complications of Treatment

Treatment-related complications are a frequent source of exam questions.

Complication	Mechanism
Cushing syndrome	Exogenous glucocorticoid excess → moon face, buffalo hump, truncal obesity, striae, easy bruising
Hyperglycaemia / steroid-induced diabetes	Steroids ↑hepatic gluconeogenesis + ↓peripheral glucose uptake + ↑insulin resistance
Osteoporosis ± pathological fractures	Steroids ↓osteoblast activity + ↑osteoclast activity + ↓intestinal calcium absorption + ↑renal calcium excretion
Immunosuppression / infections	Steroids suppress T-cell function, ↓cytokine production → ↑risk of opportunistic infections (TB reactivation, Pneumocystis, fungal infections)
Adrenal suppression	Prolonged exogenous steroids suppress the HPA axis → if steroids are abruptly stopped, acute adrenal insufficiency (Addisonian crisis) → hence the need for slow taper
Peptic ulcer disease	Steroids ↓prostaglandin synthesis → ↓gastric mucosal protection; especially if combined with NSAIDs
AVN of femoral head	Disruption of microvascular blood supply to femoral head — a feared long-term complication
Myopathy	Proximal muscle weakness from catabolic effects of steroids
Psychiatric effects	Insomnia, mood disturbance, psychosis (steroid psychosis)

Risk of infection — life-threatening infection from encapsulated bacteria → overwhelming post-splenectomy infection (OPSI) ^[14]

Complication	Mechanism
Overwhelming post-splenectomy infection (OPSI)	The spleen is critical for opsonisation and clearance of encapsulated bacteria. Without it, these organisms can proliferate unchecked → fulminant sepsis with high mortality. Pathogens: Pneumococcus, Neisseria meningitidis, Haemophilus influenzae, Capnocytophaga ^[17]
Post-splenectomy thrombocytosis	Loss of splenic platelet pooling → increased platelet counts ^[14]; if platelets > 1000 × 10⁹/L, risk of thrombosis → prophylactic aspirin considered ^[22]
Spurious leukocytosis	Loss of splenic margination pool → increased cell counts on CBC ^[14] — this is expected and NOT pathological
Howell-Jolly bodies on PBS	Nuclear remnants normally removed by splenic pitting function → persist in asplenic patients. An expected finding, NOT pathological ^[14]
Surgical complications	Bleeding (splenic bed is vascular), injury to surrounding structures (pancreatic tail → fistula; stomach; diaphragm → left basal atelectasis, pleural effusion) ^[22]

Prevention of OPSI	Details
Vaccination	PCV13 + PPSV23 (repeat Q5 years), Hib vaccine, Meningococcal ACWY vaccine, influenza vaccine ^[22] — ideally ≥2 weeks pre-splenectomy if elective
Antibiotic prophylaxis	Long-term (often lifelong) penicillin V prophylaxis; alternative: amoxicillin
Patient education	MedicAlert bracelet; seek urgent medical attention for any febrile illness; empirical antibiotics for any fever before culture results

Complication	Mechanism
HBV reactivation	B-cell depletion removes immune control over latent HBV → viral reactivation → fulminant hepatitis. Critical in Hong Kong (~8% HBsAg carrier rate). Prevention: pre-treatment HBV screening + antiviral prophylaxis
Hypogammaglobulinaemia	Prolonged B-cell depletion → ↓immunoglobulin production → ↑infection risk
Infusion reactions	Cytokine release during initial infusions → fever, chills, rigors, hypotension. Managed with slowing infusion rate + premedication (paracetamol, antihistamine, hydrocortisone)
Progressive multifocal leukoencephalopathy (PML)	JC virus reactivation in immunosuppressed state → fatal demyelinating brain disease. Extremely rare but devastating
Late-onset neutropaenia	Can occur weeks to months after rituximab; usually self-limiting

Drug	Key Complications
Azathioprine	Myelosuppression (especially in TPMT/NUDT15-deficient patients — important in HK Chinese population); hepatotoxicity; ↑infection risk; long-term ↑lymphoma risk
Cyclophosphamide	Myelosuppression; haemorrhagic cystitis (acrolein metabolite → bladder epithelial damage — prevent with mesna); infertility (gonadal toxicity); secondary neoplasm risk with prolonged immunosuppression ^[4]; alopecia
MMF	GI side effects (nausea, diarrhoea); myelosuppression; teratogenicity (absolutely contraindicated in pregnancy)

Caution in young due to risk of secondary neoplasm with prolonged immunosuppression ^[4]

Complication	Mechanism
Alloimmunisation	Each transfusion exposes the patient to foreign RBC antigens → development of allo-antibodies that complicate future cross-matching and increase risk of delayed haemolytic transfusion reactions. This is particularly problematic in AIHA patients who may require multiple transfusions ^[4]
Iron overload	Repeated transfusions (see above)
Febrile non-haemolytic transfusion reaction	Recipient antibodies react with donor leukocyte antigens or cytokines in stored blood → fever, chills
Transfusion-associated circulatory overload (TACO)	Volume overload especially in elderly or patients with cardiac compromise
Transfusion-related acute lung injury (TRALI)	Donor antibodies against recipient HLA/neutrophil antigens → non-cardiogenic pulmonary oedema

D. Disease-Course Complications

Warm AIHA has a high relapse rate: only ~20–30% achieve sustained remission after initial steroids
Many patients have a relapsing-remitting course requiring multiple treatment lines
Each relapse necessitates re-evaluation for secondary causes (new underlying LPD? Drug exposure? Infection?)

Evans syndrome (AIHA + ITP) may develop during the course of initially isolated AIHA ^[4]
The appearance of thrombocytopaenia in a patient with known AIHA should prompt:
- Check for concurrent ITP (megakaryocytes normal/increased on BM)
- Exclude TTP (schistocytes on PBS, ADAMTS13 activity)
- Screen for SLE, CVID, ALPS

AIHA may be the first manifestation of a broader autoimmune syndrome
Patients may subsequently develop:
- Autoimmune neutropaenia
- Pure red cell aplasia (anti-erythropoietin antibodies or anti-erythroblast antibodies)
- Autoimmune thrombocytopaenia (completing Evans syndrome)

Category	Complication	Key Mechanism
Haemolysis	Pigment gallstones	↑Unconjugated bilirubin → calcium bilirubinate stones
	Aplastic crisis	Parvovirus B19 → cessation of erythropoiesis
	Folate deficiency	↑Folate consumption by compensatory erythropoiesis
	AKI	Free Hb → tubular necrosis + NO scavenging → vasoconstriction
	VTE	Free Hb/NO scavenging, phosphatidylserine exposure, inflammation
	Cardiac ischaemia / HF	↓O₂ carrying capacity → demand-supply mismatch
	Iron overload	Chronic haemolysis + repeated transfusions
	Hypersplenism	Reticuloendothelial hyperplasia → splenic sequestration
Underlying disease	Lupus nephritis, Richter transformation, CVID complications	Disease-specific
Steroids	Cushing syndrome, DM, osteoporosis, infections, AVN	Exogenous glucocorticoid excess
Splenectomy	OPSI, thrombocytosis, surgical complications	Loss of splenic immune/filtration function
Rituximab	HBV reactivation, hypogammaglobulinaemia, PML	B-cell depletion → loss of immune control
Immunosuppressants	Myelosuppression, secondary neoplasm, infertility	Cytotoxic/immunosuppressive effects
Transfusion	Alloimmunisation, iron overload, TACO, TRALI	Repeated foreign antigen exposure; volume/iron load
Disease course	Relapse, Evans syndrome evolution	Ongoing autoimmune dysregulation

High Yield Summary — Complications of AIHA

Complications of haemolysis itself:

Pigment gallstones — chronic ↑unconjugated bilirubin; always ask about gallstone history
Aplastic crisis — Parvovirus B19 knocks out compensatory erythropoiesis → life-threatening Hb drop
Folate deficiency — prevent with folic acid supplementation in all chronic haemolysis
AKI — intravascular haemolysis → free Hb nephrotoxicity + NO scavenging → renal vasoconstriction
VTE — AIHA is a hypercoagulable state
Cardiac ischaemia / high-output HF — severe anaemia

Treatment complications (high yield for exams):

Steroids: Cushing, DM, osteoporosis, infections, AVN, adrenal suppression
Splenectomy: OPSI from encapsulated organisms (Pneumococcus, Meningococcus, Hib, Capnocytophaga); post-splenectomy thrombocytosis; Howell-Jolly bodies
Rituximab: HBV reactivation (screen before starting!); hypogammaglobulinaemia; PML
Immunosuppressants: Secondary neoplasm risk with prolonged use (especially in young patients)
Transfusion: Alloimmunisation (complicates future cross-matching); iron overload

Active Recall - Complications of AIHA

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf (AIHA treatment slide, haemolytic anaemia clinical features — gallstones) ^[2] Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf (AIHA treatment principles, folate supplementation, gallstone mechanism) ^[4] Senior notes: Ryan Ho Haemtology.pdf (Section 2.5.4.1 Warm AIHA — Evans syndrome, CLL association, management, 3rd line caution in young) ^[6] Senior notes: Jerry's immunodeficiencies.pdf (CVID complications — AIHA, malignancy risk) ^[14] Senior notes: Block A - Splenomegaly_ common causes of splenomegaly; myeloproliferative diseases.pdf (Splenectomy indications, complications, post-splenectomy CBC findings) ^[15] Senior notes: Block A - Fever after a blood transfusion_ transfusion and related problems.pdf (AKI mechanism in intravascular haemolysis — free Hb, NO scavenging, DIC) ^[17] Senior notes: Gen Clerk Anaes + Microbiology Summary.pdf (Post-splenectomy encapsulated organism infections — Pneumococcus, Neisseria, Hib, Capnocytophaga) ^[20] Senior notes: Block A - Pallor_ diagnosis of anaemia; nutritional anaemia; anaemia of systemic diseases.pdf (Acute anaemia → cardiac ischaemia) ^[21] Senior notes: Ryan Ho Rheumatology.pdf (SLE complications — lupus nephritis, APS) ^[22] Senior notes: Maksim Surgery Notes.pdf (Splenectomy indications, specific complications, OPSI prevention — vaccination list)

High Yield Summary

Definition: AIHA = acquired haemolytic anaemia due to autoantibodies against self-RBC antigens → Type II hypersensitivity.

Classification by thermal reactivity:

Warm AIHA (70–80%): IgG, 37°C, extravascular haemolysis (spleen), spherocytes, DAT IgG ± C3
Cold agglutinin disease (15–25%): IgM, 4°C, complement-mediated (mainly extravascular via liver), DAT C3 only, acrocyanosis
PCH (rare): Biphasic IgG (Donath-Landsteiner Ab), P antigen, intravascular haemolysis after cold exposure
Mixed (~5%): Both IgG + IgM

Must-ask history: Family history, ethnicity, infections, drugs, transfusion history, co-existing autoimmune/lymphoproliferative disease, gallstones.

Cardinal clinical features: Pallor + jaundice (no tea-coloured urine) + splenomegaly ± haemoglobinuria (if intravascular).

Evans syndrome: AIHA + ITP — think SLE, CVID, ALPS.

CLL: Up to 11% develop AIHA — always consider in elderly with AIHA.

Key lab: ↑reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin, +ve DAT (IgG ± C3 in warm; C3 only in cold).

Drug-induced AIHA: Three mechanisms — hapten (penicillin), immune complex (quinidine), true autoantibody (methyldopa).

High Yield Summary — Differential Diagnosis of AIHA

Step 1 — Confirm haemolysis: ↑Reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin, polychromasia on PBS.

Step 2 — PBS morphology guides you:

Spherocytes → Warm AIHA vs Hereditary Spherocytosis (differentiate by DAT and EMA binding)
Schistocytes → MAHA (TTP/HUS) — DAT negative
Bite cells / Heinz bodies → G6PD deficiency — DAT negative
RBC agglutination → Cold agglutinin disease
No specific morphology → PNH (flow cytometry for CD55/CD59)

Step 3 — DAT is the key test:

DAT positive → Immune haemolysis (AIHA, alloimmune, drug-induced)
DAT negative → Non-immune (HS, G6PD, PNH, TTP, Wilson's)

Step 4 — Once AIHA confirmed, always search for secondary causes:

Warm: SLE, CLL, drugs (methyldopa), CVID
Cold: Mycoplasma, EBV, lymphoma, primary CAD

Key traps:

Evans syndrome (AIHA + ITP) vs TTP: spherocytes + DAT positive (Evans) vs schistocytes + DAT negative (TTP)
Wilson's disease: Coombs-negative haemolytic anaemia
PNH: Coombs-negative; diagnosed by flow cytometry (↓CD55, CD59)
DAT-negative AIHA exists in < 3% of cases

High Yield Summary — Diagnosis of AIHA

Diagnosis rests on 3 pillars:

Evidence of haemolysis (↑reticulocytes, ↑LDH, ↓haptoglobin, ↑unconjugated bilirubin)
Positive DAT (IgG ± C3 = warm; C3 only = cold)
Exclusion of other causes (HS → EMA binding; PNH → CD55/CD59; MAHA → schistocytes)

DAT pattern classifies AIHA: IgG ± C3 = warm; C3 only = cold (IgM already dissociated); Both negative in < 3% (DAT-negative AIHA).

GC Case 6: Classic warm AIHA — Hb 7.5, spherocytes, retic 10%, ↑LDH, ↑bilirubin, DAT positive for IgG + complement.

High Yield Summary — Management of AIHA

Four Pillars (GC lecture):

Transfusion if necessary (do NOT withhold — use "least incompatible" after excluding allo-antibodies)
Folate supplementation (replenish raw material for compensatory erythropoiesis)
Decrease haemolysis:
- Warm: Steroids (1st line, 1 mg/kg/day prednisolone) → Rituximab or Splenectomy (2nd line) → Azathioprine/Cyclophosphamide (3rd line)
- Cold: Cold avoidance → Rituximab or Sutimlimab → Plasmapheresis for acute episodes → Steroids NOT effective
- Drug-induced: Stop the drug
Treat underlying cause (SLE, CLL, infection, CVID)

Key differences warm vs cold:

Steroids work in warm (↓FcγR + ↓Ab production) but NOT cold (complement-mediated, clonal IgM)
Splenectomy works in warm (spleen = site of destruction) but NOT cold (liver = main site)
Rituximab is 2nd line in warm but 1st line pharmacological in cold

Rituximab caution: Screen for HBV before starting — risk of fatal reactivation.

Splenectomy caution: Vaccinate against encapsulated organisms (Pneumococcus, Meningococcus, Hib); lifelong penicillin prophylaxis.

IVIG: Probable benefit in AIHA; dose 0.4 g/kg/day × 5 days; contraindicated if previous IVIG allergy; caution in IgA deficiency.

High Yield Summary — Complications of AIHA

Complications of haemolysis itself:

Pigment gallstones — chronic ↑unconjugated bilirubin; always ask about gallstone history
Aplastic crisis — Parvovirus B19 knocks out compensatory erythropoiesis → life-threatening Hb drop
Folate deficiency — prevent with folic acid supplementation in all chronic haemolysis
AKI — intravascular haemolysis → free Hb nephrotoxicity + NO scavenging → renal vasoconstriction
VTE — AIHA is a hypercoagulable state
Cardiac ischaemia / high-output HF — severe anaemia

Treatment complications (high yield for exams):

Steroids: Cushing, DM, osteoporosis, infections, AVN, adrenal suppression
Splenectomy: OPSI from encapsulated organisms (Pneumococcus, Meningococcus, Hib, Capnocytophaga); post-splenectomy thrombocytosis; Howell-Jolly bodies
Rituximab: HBV reactivation (screen before starting!); hypogammaglobulinaemia; PML
Immunosuppressants: Secondary neoplasm risk with prolonged use (especially in young patients)
Transfusion: Alloimmunisation (complicates future cross-matching); iron overload

Autoimmune Haemolytic Anaemia

Definition

Epidemiology

Risk Factors

Anatomy and Function: The Red Blood Cell and Splenic Filtration

The Red Blood Cell (RBC)

The Spleen — The "Quality Control" Organ

The Liver — Secondary Site of Clearance

The Complement System

Aetiology and Pathophysiology

Classification by Thermal Reactivity of the Autoantibody

1. Warm AIHA

Pathophysiology — Step by Step

Aetiology of Warm AIHA

2. Cold AIHA

2a. Cold Agglutinin Disease (CAD)

Pathophysiology — Step by Step

Aetiology of Cold Agglutinin Disease

2b. Paroxysmal Cold Haemoglobinuria (PCH)

3. Drug-Induced Immune Haemolytic Anaemia (DIIHA)

4. Mixed-Type AIHA

Classification Summary

By Thermal Reactivity (Primary Classification)

By Primary vs Secondary Aetiology

Clinical Features

Symptoms

Signs

Special Clinical Scenarios

Evans Syndrome

AIHA with Underlying CLL

AIHA in SLE

Fulminant Wilson's Disease

Pathophysiology of Haemolysis — Extravascular vs Intravascular

Haemolysis Markers — Laboratory Correlates

Summary of Key Pathophysiological Distinctions

Active Recall - Autoimmune Haemolytic Anaemia

References

Framing the Differential — Why Is This Tricky?

Layer 1 — Differential Diagnosis of the Haemolytic Anaemia Syndrome

Layer 1 — Systematic Differential Diagnosis Table

Layer 2 — Once AIHA Is Confirmed (DAT-Positive), What Is the Underlying Cause?

DAT Pattern Guides the Differential

Differential by AIHA Subtype

Warm AIHA — Secondary Causes

Cold AIHA — Secondary Causes

Paroxysmal Cold Haemoglobinuria (PCH) — Differential

Layer 1 Expanded — Non-Haemolytic Conditions That Can Mimic AIHA

Distinguishing AIHA from Key Mimickers — Specific Comparisons

AIHA vs Hereditary Spherocytosis

AIHA vs TTP/MAHA

AIHA vs PNH

AIHA vs Drug-Induced Non-Immune Haemolysis (e.g., G6PD Precipitated by Drugs)

Differential Diagnosis Algorithm for AIHA

Splenomegaly Size — Differential Clue

Active Recall - Differential Diagnosis of AIHA

Diagnostic Criteria — Does AIHA Have Formal Diagnostic Criteria?

DAT Subtype Interpretation — Classifying AIHA

Diagnostic Algorithm

Investigation Modalities — Comprehensive Breakdown

Step 1: Confirm Anaemia and Reticulocyte Response

Step 2: Haemolysis Markers

Step 3: Peripheral Blood Smear (PBS)

Step 4: Direct Antiglobulin Test (DAT / Direct Coombs Test) — The Pivotal Investigation

How the DAT Works — First Principles

Why Use Monospecific Reagents?

Indirect Antiglobulin Test (IAT / Indirect Coombs Test)

Step 5: Identify the Underlying Cause — "What Is Driving the AIHA?"

A. Autoimmune Disease Screen

B. Lymphoproliferative Disease Screen

C. Infection Screen

D. Drug History Review

E. Additional Investigations for Specific Scenarios

Comprehensive Investigation Summary Table

Interpreting the Investigations — Putting It All Together

Transfusion Considerations — An Important Investigation-Adjacent Topic

Active Recall - Diagnosis of AIHA

Overarching Principles of Treatment

Management Algorithm — Overview

Pillar 1: Transfusion — Acute Stabilisation

Pillar 2: Folate Supplementation