Paroxysmal nocturnal haemoglobinuria is an acquired clonal disorder of haematopoietic stem cells caused by a mutation in the PIG-A gene, leading to deficiency of GPI-anchored complement-regulatory proteins (CD55 and CD59) and resulting in complement-mediated intravascular haemolysis, thrombosis, and bone marrow failure.

Paroxysmal Nocturnal Haemoglobinuria (PNH)

"Paroxysmal" = sudden, episodic attacks; "Nocturnal" = occurring at night; "Haemoglobinuria" = haemoglobin in the urine. The name describes the classic (but actually uncommon) presentation of dark-red/cola-coloured urine most noticeable upon waking — though we now understand the disease is far more than just nocturnal haemolysis.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired, clonal haematopoietic stem cell (HSC) disorder characterised by:

Complement-mediated intravascular haemolysis (the hallmark)
Bone marrow failure (overlap with aplastic anaemia)
Thrombophilia (tendency to form venous and arterial thrombi — the leading cause of death in untreated PNH)

It arises from a somatic mutation in the PIGA gene (phosphatidylinositol glycan class A) in one or more haematopoietic stem cells, leading to deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of blood cells ^[1]^[2]. Among the most clinically important GPI-anchored proteins lost are CD55 (decay-accelerating factor, DAF) and CD59 (membrane inhibitor of reactive lysis, MIRL), both of which normally protect cells from complement-mediated destruction ^[1]^[2].

Key Concept — Acquired Intrinsic Red Cell Defect

PNH is unique: it is an acquired disease but causes an intrinsic red cell defect. Most acquired haemolytic anaemias are extrinsic (e.g. autoimmune, drug-induced). PNH is the only acquired intrinsic red cell defect — the mutation lives within the stem cell, making every daughter cell inherently vulnerable to complement ^[1].

Parameter	Detail
Incidence	~1–1.5 per million per year (rare)
Prevalence	~10–20 per million
Age at diagnosis	Median ~30–40 years (can occur at any age; paediatric cases rare)
Sex	Slight M = F or M > F in some series
Ethnicity	Seen worldwide; slightly higher reported prevalence in Southeast Asia compared with Western populations; in Hong Kong, aplastic anaemia is more common than in the West (~4–6/million/year in Asia vs 2/million/year in the West), and ~20–30% of aplastic anaemia patients harbour PNH clones ^[3]
Overlap with AA	Up to 50% of aplastic anaemia (AA) patients have detectable PNH clones on high-sensitivity flow cytometry; conversely, ~20–30% of PNH patients have concurrent bone marrow hypoplasia ^[3]

In Hong Kong, given the higher incidence of aplastic anaemia in Asian populations, PNH clones are a relatively important clinical consideration when evaluating pancytopenia.

PNH arises from a somatic mutation, so there are no classic "lifestyle" risk factors. However, the following are associated with development or clinical unmasking of PNH:

Pre-existing aplastic anaemia (AA) — PNH clones emerge in the setting of immune-mediated marrow destruction. The PIGA-mutant HSC may have a survival advantage in an environment where normal HSCs are being destroyed by autoreactive T-cells ^[3].
Myelodysplastic syndromes (MDS) — small PNH clones can be detected in some MDS patients.
Age — young to middle-aged adults most commonly affected.
Prior immunosuppressive therapy for AA — PNH clones may expand after immunosuppression.

4. Relevant Anatomy & Physiology

To understand PNH, you need to understand how proteins are attached to the cell surface:

Many cell-surface proteins are anchored to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor.
The GPI anchor is a glycolipid structure synthesised in the endoplasmic reticulum. It acts like a "molecular hook" that tethers proteins to the lipid bilayer.
The PIGA gene (on the X chromosome, Xp22.1) encodes the enzyme catalysing the first step of GPI anchor biosynthesis (transfer of N-acetylglucosamine to phosphatidylinositol).
If PIGA is mutated → no GPI anchor is made → all GPI-anchored proteins are absent from the cell surface.

Protein	Normal Function	Consequence of Loss
CD59 (MIRL)	Prevents formation of the membrane attack complex (MAC, C5b-9) by blocking C9 insertion	Intravascular haemolysis — MAC punches holes in RBCs → lysis
CD55 (DAF)	Accelerates decay of C3 convertase (both classical and alternative pathway)	Enhanced C3b opsonisation → extravascular haemolysis (C3b-opsonised cells cleared by splenic macrophages)
CD16b (FcγRIIIb)	Neutrophil Fc receptor	Impaired immune function
CD14	LPS receptor on monocytes	Altered immune signalling
CD87 (uPAR)	Urokinase plasminogen activator receptor	Impaired fibrinolysis → contributes to thrombosis
Acetylcholinesterase	On RBC surface	No major clinical consequence (but historically used for testing)

Why CD59 Loss Is More Important Than CD55 Loss

CD59 is the last line of defence before MAC-mediated lysis. Loss of CD59 alone is sufficient to cause clinically significant intravascular haemolysis. Loss of CD55 alone causes only mild haemolysis. In PNH, both are absent → severe haemolysis.

The complement system is a cascade of ~30 serum proteins that form part of innate immunity. Three pathways converge on the same terminal pathway:

The alternative pathway is particularly relevant in PNH because it is constitutively active ("tick-over") — C3 spontaneously hydrolyses at a low rate, and this is normally kept in check by CD55 (DAF) on cell surfaces.
Without CD55 and CD59, PNH cells are sitting ducks for complement attack, especially via the alternative pathway.

The traditional explanation is:

During sleep, mild respiratory acidosis occurs (shallow breathing → CO₂ retention → slight drop in blood pH).
Lower pH enhances complement activation (alternative pathway is pH-sensitive).
Result: increased haemolysis overnight → morning haemoglobinuria with dark urine on waking.

Reality check: Only ~25% of PNH patients actually present with classic nocturnal haemoglobinuria. Most have chronic haemolysis throughout the day, and the "nocturnal" pattern is now considered more myth than rule. The name is historically rooted but clinically misleading.

5. Aetiology & Pathophysiology

The underlying defect is a somatic (acquired) loss-of-function mutation in the PIGA gene in a haematopoietic stem cell ^[1]^[2].

Key points about the PIGA gene:

Located on the X chromosome (Xp22.1).
Because males have only one X, a single mutation is sufficient (hemizygous). In females, because of X-inactivation (lyonisation), only one X is active in any given cell, so a single mutation on the active X also causes the phenotype.
This is why one somatic mutation is sufficient in either sex — it behaves functionally as a loss-of-function regardless of sex.
The mutation is somatic (not germline) → not inherited, arises during the patient's lifetime, and affects only the clone derived from the mutant stem cell.

A single PIGA mutation is necessary but not sufficient to cause clinical PNH. You need the PNH clone to expand to a significant proportion of haematopoiesis. Two models explain this:

Immune escape hypothesis (most accepted):
- In the setting of an autoimmune attack on HSCs (as in aplastic anaemia), normal HSCs expressing GPI-anchored proteins are preferentially targeted and destroyed by autoreactive cytotoxic T-cells.
- PIGA-mutant HSCs, lacking certain GPI-anchored surface proteins, are less visible to the immune system → selective survival advantage.
- Over time, the PNH clone expands to fill the marrow niche vacated by destroyed normal HSCs.
- This explains the strong clinical overlap between PNH and aplastic anaemia ^[3].
Additional second-hit mutations:
- Some PNH clones acquire additional mutations (e.g. in HMGA2, JAK2, BCL6) that confer a proliferative advantage.
- This may explain why some PNH clones persist and expand even after immune suppression.

5.3 Consequences of GPI-Anchor Deficiency

The loss of GPI-anchored proteins produces the three cardinal features of PNH:

Loss of CD59 → cannot prevent MAC (C5b-9) assembly → complement punches holes in RBC membranes → intravascular lysis → free haemoglobin released into plasma.
Loss of CD55 → cannot inactivate C3 convertase → more C3b deposited on RBC surface → enhanced opsonisation → extravascular haemolysis by splenic/hepatic macrophages (this becomes clinically relevant when patients are treated with C5 inhibitors like eculizumab — see later).
The haemolysis is chronic and ongoing, but can be accentuated by:
- Infections (complement activation via classical/lectin pathways)
- Surgery / trauma
- Pregnancy
- Vaccinations
- Exercise
- Sleep (as above, pH changes)

PNH is listed as a cause of hypercoagulability leading to venous thromboembolism ^[4].

Thrombosis is the leading cause of morbidity and mortality in PNH (~40–67% of deaths). Mechanisms include:

Mechanism	Explanation
Free haemoglobin scavenges nitric oxide (NO)	NO is a potent vasodilator and inhibitor of platelet aggregation. Free Hb binds NO avidly → NO depletion → vasoconstriction + platelet activation
Release of procoagulant microparticles	Lysed RBCs and activated platelets release phosphatidylserine-rich microparticles → promote thrombin generation
Loss of CD87 (uPAR) on monocytes/granulocytes	Impaired urokinase-mediated fibrinolysis
Complement activation on platelet surfaces	GPI-deficient platelets undergo complement-mediated activation → procoagulant state
Endothelial dysfunction from free Hb	Free Hb is toxic to endothelium → promotes thrombus formation
Inflammatory cytokines	Chronic haemolysis → inflammation → endothelial activation

Characteristic thrombotic sites in PNH (unusual/atypical sites are a red flag):

Hepatic veins → Budd-Chiari syndrome (most characteristic, almost pathognomonic for PNH when occurring in a young patient without other risk factors)
Cerebral venous sinuses → sagittal sinus thrombosis
Mesenteric veins → intestinal ischaemia, abdominal pain
Portal vein
Dermal veins
Deep veins → DVT/PE (conventional sites as well) ^[4]

High Yield — Thrombosis in PNH

Any young patient with thrombosis at an unusual site (especially Budd-Chiari syndrome, cerebral venous sinus thrombosis, or mesenteric vein thrombosis) should be screened for PNH. This is a classic exam question. The combination of haemolytic anaemia + thrombosis at an unusual site = think PNH ^[4].

PNH exists on a spectrum with aplastic anaemia.
Many patients have concurrent bone marrow hypoplasia → varying degrees of cytopenias beyond just anaemia.
Flow cytometry showing decreased CD55/CD59 should be performed in patients with aplastic anaemia to screen for PNH clones ^[3].
The marrow failure component can manifest as:
- Neutropenia → recurrent infections
- Thrombocytopenia → bleeding tendency (in addition to or despite the thrombotic tendency!)
- Reticulocytopenia (when marrow is failing, cannot compensate for haemolysis)

When RBCs lyse within blood vessels, the released contents cause a cascade of problems:

Why PNH Causes Iron Deficiency

This is counterintuitive — you would expect haemolysis to cause iron overload (as in thalassaemia). But in PNH, the haemolysis is intravascular → haemoglobin is filtered through the kidneys → iron is lost in the urine as haemoglobin and haemosiderin → chronic urinary iron loss → iron deficiency anaemia. This is unique to intravascular haemolytic conditions.

PNH is classified based on clinical presentation and the relationship with bone marrow failure. The International PNH Interest Group (I-PIG) classification is most widely used:

Category	Description	Clone Size	Key Features
Classical (Haemolytic) PNH	Predominantly haemolysis and thrombosis	Large (usually > 50%)	Clinical evidence of intravascular haemolysis; normal or near-normal bone marrow cellularity; no other marrow disorder
PNH in the setting of another bone marrow disorder	PNH coexisting with aplastic anaemia, MDS, or myelofibrosis	Variable (small to moderate)	Evidence of haemolysis AND concurrent bone marrow failure syndrome
Subclinical PNH	Small PNH clone detected by high-sensitivity flow cytometry without clinical/laboratory evidence of haemolysis	Very small (usually < 1%)	No haemolysis; detected during workup for AA or MDS; requires monitoring as clone may expand over time

Cell-Based Classification (Flow Cytometry)

Individual cells are classified based on the degree of GPI-anchor deficiency:

Type	GPI-Anchor Status	CD55/CD59 Expression	Sensitivity to Complement
Type I	Normal	Normal	Normal (these are the non-PNH cells)
Type II	Partial deficiency	Reduced	Intermediate sensitivity (~3–5× normal)
Type III	Complete deficiency	Absent	Very high sensitivity (~15–25× normal)

Most patients have a mixture of Type I, II, and III cells. The proportion of Type III cells correlates with clinical severity of haemolysis.

7. Clinical Features

Symptom	Pathophysiological Basis
Dark/cola-coloured urine (haemoglobinuria), classically worse in the morning	Intravascular haemolysis → free Hb filtered by kidneys; "nocturnal" worsening due to complement activation from sleep-related respiratory acidosis (lower pH activates alternative pathway)
Fatigue and exercise intolerance	Anaemia (reduced O₂ carrying capacity) + NO depletion (impaired oxygen delivery) + chronic disease effects
Dyspnoea on exertion	Anaemia + possible pulmonary hypertension (NO scavenging → pulmonary vasoconstriction)
Dysphagia (difficulty swallowing)	Free Hb scavenges NO → oesophageal smooth muscle spasm/dystonia. This is a characteristic symptom of PNH and should trigger suspicion
Abdominal pain (episodic, can be severe)	NO depletion → mesenteric vasoconstriction + possible mesenteric vein thrombosis; also direct smooth muscle dystonia from NO scavenging
Erectile dysfunction	NO depletion → impaired smooth muscle relaxation in corpus cavernosum (same mechanism as why sildenafil works — it potentiates NO signalling)
Back pain	Free Hb causing renal tubular damage; also possibly related to micro-thrombosis in renal vessels
Headache	Cerebral vasoconstriction from NO depletion; also consider cerebral venous sinus thrombosis
Jaundice (mild to moderate)	Haemolysis → increased unconjugated bilirubin (pre-hepatic jaundice)
Leg swelling	DVT (thrombosis)
Sudden severe abdominal pain	Budd-Chiari syndrome (hepatic vein thrombosis) or mesenteric vein thrombosis
Recurrent infections	Neutropenia from bone marrow failure + impaired neutrophil function (loss of GPI-anchored immune receptors like CD16b)
Easy bruising/bleeding	Thrombocytopenia from bone marrow failure

The Triad of PNH Symptoms to Remember

Haemolysis → dark urine, anaemia, jaundice
Thrombosis → at unusual sites (Budd-Chiari, cerebral sinuses, mesenteric)
Cytopenias → from bone marrow failure (infections, bleeding)

Plus the NO-depletion symptoms: dysphagia, abdominal pain, erectile dysfunction, pulmonary hypertension

Sign	Pathophysiological Basis
Pallor	Anaemia from haemolysis ± marrow failure
Jaundice (usually mild, lemon-yellow tinge)	Unconjugated hyperbilirubinaemia from haemolysis (pre-hepatic jaundice). No tea-coloured urine (because unconjugated bilirubin is water-insoluble and does not enter urine) — the dark urine in PNH is from haemoglobin, not bilirubin ^[1]
Hepatomegaly	Budd-Chiari syndrome (hepatic vein thrombosis → hepatic congestion); also extramedullary haematopoiesis
Splenomegaly (usually mild or absent)	Unlike extravascular haemolytic anaemias, PNH is predominantly intravascular → spleen is NOT the main site of destruction. May have mild splenomegaly from extramedullary haematopoiesis
Skin pigmentation	Chronic haemolysis → haemosiderin deposition in skin
Signs of thrombosis	Depending on site: hepatomegaly + ascites (Budd-Chiari), focal neurological signs (cerebral sinus thrombosis), abdominal tenderness (mesenteric thrombosis), leg swelling (DVT)
Petechiae, purpura, mucosal bleeding	Thrombocytopenia from concurrent bone marrow failure
Signs of iron deficiency (if chronic)	Koilonychia (spoon nails), angular stomatitis, glossitis — from chronic urinary iron loss
Absent splenomegaly with haemolytic anaemia	This is a distinguishing feature — most haemolytic anaemias cause splenomegaly because the spleen is the site of RBC destruction. In PNH, destruction is intravascular → spleen is relatively spared

Clinical Pearl — Intravascular vs Extravascular Haemolysis

In intravascular haemolysis (including PNH), you expect: ^[5]

Haemoglobinaemia (pink/brown serum)
Haemoglobinuria (dark/cola urine)
Haemosiderinuria (Prussian blue stain of urine sediment)
↓↓ Haptoglobin (severely depleted)
Methaemalbumin (Schumm test positive)
Risk of AKI from free Hb toxicity to tubules
Risk of thrombosis from free Hb

In extravascular haemolysis, you expect: ^[5]

Splenomegaly (the spleen is doing the destroying)
↑ Unconjugated bilirubin (same in both, but relatively more prominent)
Gallstones (bilirubin stones)
↓ Haptoglobin (but not as severely depleted)
No haemoglobinuria or haemosiderinuria

PNH is predominantly intravascular, but when treated with C5 inhibitors (eculizumab), extravascular haemolysis becomes more prominent (because C3b opsonisation continues unchecked).

When you suspect PNH, ask about:

Colour of urine — especially first morning void (dark/cola-coloured?)
Timing — episodic worsening with infections, surgery, stress?
Thrombotic events — especially at unusual sites (Budd-Chiari, cerebral sinus, mesenteric)
Family history — PNH is NOT inherited (somatic mutation), so a positive family history of anaemia should redirect to inherited haemolytic anaemias ^[1]
Ethnicity ^[1]
Drug history — to exclude drug-induced haemolysis (e.g. oxidising drugs in G6PD deficiency)
Infection history — infections can trigger haemolytic crises and also be a complication of neutropenia
Transfusion history ^[1]
Co-existing illnesses — autoimmune diseases, lymphoproliferative neoplasms ^[1]
Prior diagnosis of aplastic anaemia or MDS
Symptoms of smooth muscle dystonia — dysphagia, abdominal pain, erectile dysfunction

This is a crucial concept for exams:

PNH and aplastic anaemia are closely related conditions that exist on a spectrum ^[3].
~20–30% of AA patients have detectable PNH clones (often subclinical).
~20–30% of PNH patients have concurrent marrow hypoplasia.
Flow cytometry for CD55/CD59 (or FLAER) should be performed in all patients with aplastic anaemia to screen for PNH clones ^[3].
The relationship is explained by the immune escape hypothesis: in AA, autoreactive T-cells destroy normal HSCs. PIGA-mutant HSCs escape this immune attack → selective expansion → PNH clone emerges.
Over time, an AA patient's PNH clone may expand → clinical PNH. Or a PNH patient may develop progressive marrow failure → AA/PNH overlap.

Aplastic anaemia occurring in association with PNH is listed as an acquired clonal abnormality under secondary causes of aplastic anaemia ^[3].

10. Why PNH Matters in Other Clinical Contexts

PNH is listed as a cause of intravascular haemolysis alongside fragmentation syndromes (DIC, TTP, HUS), mechanical haemolysis (heart valves), infections (malaria, C. perfringens), drug/toxin-related haemolysis, G6PD deficiency, and complement-induced immune haemolysis ^[5].

PNH is listed under acquired causes of hypercoagulability/thrombophilia contributing to venous thromboembolism ^[4]. It is specifically mentioned alongside:

Lupus anticoagulants
Antiphospholipid syndrome
Myeloproliferative neoplasms
Nephrotic syndrome
Homocysteinaemia ^[4]

In the nephrology context, PNH should be considered as a cause of a positive urine dipstick for blood (which detects haemoglobin) that is NOT due to glomerulonephritis — it is a false positive for haematuria because the dipstick detects free haemoglobin, not intact RBCs ^[6].

When evaluating pancytopenia, PNH should be in the differential alongside:

Aplastic anaemia
MDS
Leukaemia (especially hypoplastic)
Megaloblastic anaemia
Bone marrow infiltration

High Yield Summary

Definition: PNH = acquired, clonal HSC disorder from somatic PIGA mutation → loss of GPI-anchored proteins (CD55, CD59) → complement-mediated intravascular haemolysis + thrombosis + bone marrow failure.

The Three Pillars of PNH:

Intravascular haemolysis — loss of CD59 → MAC-mediated RBC lysis → haemoglobinaemia, haemoglobinuria, ↓↓haptoglobin, iron deficiency from urinary iron loss
Thrombosis — #1 killer; unusual sites (Budd-Chiari, cerebral sinuses, mesenteric veins); caused by NO depletion, procoagulant microparticles, impaired fibrinolysis
Bone marrow failure — overlap with aplastic anaemia; immune escape hypothesis explains clonal expansion

Key "Why" Points:

Why nocturnal? Sleep → mild respiratory acidosis → ↓pH → enhanced complement activation (mainly historical/oversimplified)
Why iron deficiency in haemolysis? Intravascular → Hb lost in urine → chronic urinary iron loss
Why thrombosis? Free Hb scavenges NO → platelet activation + vasoconstriction; loss of CD87 → impaired fibrinolysis
Why dysphagia/abdominal pain/ED? NO depletion → smooth muscle dystonia
Why overlap with AA? Immune escape: PIGA-mutant HSCs survive T-cell attack → selective clonal expansion

Screening: Flow cytometry for CD55/CD59 (or FLAER) in all AA patients, unexplained intravascular haemolysis, Coombs-negative haemolytic anaemia, thrombosis at unusual sites, unexplained cytopenias.

Classification: Classical PNH / PNH with another marrow disorder / Subclinical PNH.

Active Recall - Paroxysmal Nocturnal Haemoglobinuria (PNH)

^[1] Lecture slides: GC 047. Family history of anaemia.pdf; Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf ^[2] Lecture slides: GC 097. Many members of the family have anaemia (PATH).pdf; GC 097. Many members of the family have anaemia (File 2).pdf ^[3] Senior notes: Ryan Ho Haemtology.pdf (Section 2.4.2 — Aplastic Anaemia) ^[4] Lecture slides: Block A - Leg swelling and chest pain_ deep vein thrombosis; pulmonary embolism; Thrombophilia.pdf; Senior notes: Block A - Leg swelling and chest pain_ DVT; PE; Thrombophilia.pdf; MBBS Final MB (Surgery) (Felix PY Lai).pdf (p. 962) ^[5] Senior notes: Ryan Ho Haemtology.pdf (p. 36 — Classification by site of RBC destruction) ^[6] Senior notes: Block A - Nephrology Interactive Tutorial.pdf (Case 1 — false positive dipstick)

Differential Diagnosis of Paroxysmal Nocturnal Haemoglobinuria (PNH)

The challenge with PNH is that it is a clinical chameleon — patients rarely walk in saying "I have dark urine at night." Instead, PNH presents through one or more of its three pillars (haemolysis, thrombosis, cytopenias), and the differential diagnosis must be constructed around whichever clinical presentation is dominant. Think of it as: "What else could cause this picture?"

We will systematically cover the differential diagnosis organised by the presenting syndrome, because that is how you will encounter PNH in clinical practice and in exam questions.

2. DDx When PNH Presents as Haemolytic Anaemia

This is the most classic presentation. The key distinguishing features of PNH haemolysis are:

Intravascular (not extravascular)
Coombs-negative (not immune-mediated by autoantibodies)
Acquired (not inherited)
Only acquired intrinsic red cell defect — all other acquired haemolytic anaemias are extrinsic ^[1]

Causes of intravascular haemolysis include: ^[5]

Cause	Distinguishing Features from PNH	Mechanism
Fragmentation syndromes / MAHA (DIC, TTP, HUS)	Schistocytes on blood film; thrombocytopenia from platelet consumption; abnormal clotting in DIC (↑PT, ↑APTT, ↓fibrinogen, ↑D-dimer); ADAMTS13 < 10% in TTP	RBCs sheared by fibrin strands in damaged microvasculature
Mechanical haemolysis (prosthetic heart valve, ECMO, burns)	History of mechanical device or thermal injury; schistocytes on film	Physical trauma to RBCs
Bacterial infection (C. perfringens)	Fulminant sepsis picture; gas gangrene; very acute onset	Alpha-toxin (lecithinase) destroys RBC membrane
Parasitic infection (malaria)	Travel history; thick/thin blood film shows parasites; cyclic fevers	Plasmodium replicates inside and ruptures RBCs
G6PD deficiency	Episodic (triggered by oxidant drugs, fava beans, infections); Heinz bodies and bite cells on film; family history; more common in males (X-linked)	Oxidative stress → Heinz body formation → intravascular lysis
Drug/toxin-related haemolysis	Drug exposure history (e.g. dapsone, ribavirin, oxidant drugs)	Direct toxicity to RBC membrane or oxidative damage
Complement-induced lysis / immune-mediated intravascular haemolysis	Coombs test positive (usually IgM-mediated as IgM fixes complement well, e.g. ABO transfusion reaction, cold agglutinin disease); PNH is Coombs-negative	Antibody-mediated complement activation → MAC formation
Paroxysmal cold haemoglobinuria (PCH)	Rare; predominantly in children post-viral infection; Donath-Landsteiner antibody positive; cold-triggered	Biphasic IgG antibody binds RBCs in cold, fixes complement, causes lysis on rewarming
Severe immune haemolysis overwhelming RES	Coombs positive; underlying autoimmune disease or lymphoproliferative neoplasm	When extravascular mechanisms are saturated, spillover intravascular haemolysis occurs

High Yield — The Coombs Test Is Your Best Friend

The direct antiglobulin test (DAT / Coombs test) is the single most important test to separate immune from non-immune haemolysis.

Coombs-positive haemolysis = autoimmune haemolytic anaemia (AIHA) — warm (IgG) or cold (IgM/C3d)
Coombs-negative haemolysis = PNH, fragmentation syndromes, inherited RBC defects (G6PD, HS, haemoglobinopathies), drug/toxin-related, infections

PNH classically presents as Coombs-negative intravascular haemolytic anaemia. ^[7] If you see "Coombs-negative haemolytic anaemia" in a question with dark urine and no schistocytes on blood film → think PNH.

When Coombs is negative, you need to think systematically:

Category	Conditions	How to Distinguish from PNH
Acquired, intrinsic	PNH	Flow cytometry for CD55/CD59 / FLAER
Acquired, extrinsic — non-immune	MAHA (TTP, HUS, DIC), mechanical, infections, drugs/toxins	Schistocytes, clotting profile, blood film for parasites, drug history
Inherited, intrinsic — membrane	Hereditary spherocytosis (HS), hereditary elliptocytosis (HE)	Family history; characteristic RBC morphology (spherocytes, elliptocytes); osmotic fragility test; predominantly extravascular (splenomegaly); onset in childhood ^[1]
Inherited, intrinsic — enzyme	G6PD deficiency, pyruvate kinase deficiency	Family history; male preponderance (X-linked for G6PD); episodic with oxidant triggers; Heinz bodies; enzyme assay ^[1]
Inherited, intrinsic — haemoglobin	Sickle cell disease, HbH disease, unstable haemoglobins (e.g. Hb Köln)	Family history; Hb electrophoresis; target cells, sickle cells on film; HbH inclusions ^[8]

Key differentiating principle: If inherited, think in terms of the components of the RBC that can go wrong — either the membrane, the metabolism (enzymes), or the haemoglobin ^[1]. PNH is the only acquired condition that causes an intrinsic RBC defect.

PNH is listed as an acquired cause of pancytopenia under "peripheral consumption" alongside DIC, TMA, and autoimmune-related causes (SLE) ^[9]^[10]. However, because of its overlap with aplastic anaemia, it can also be categorised under bone marrow failure.

The full differential diagnosis of pancytopenia is extensive ^[10]^[11]:

Category	Causes	How to Distinguish from PNH
BM failure — acquired	Aplastic anaemia (AA)	Hypocellular marrow without dysplasia; may coexist with PNH (up to 50% have PNH clones); flow cytometry for CD55/CD59 essential ^[3]^[7]
BM failure — congenital	Fanconi anaemia, dyskeratosis congenita, Schwachman-Diamond syndrome	Childhood onset; dysmorphic features (thumb abnormalities in Fanconi); chromosomal breakage test with diepoxybutane (DEB) ^[3]
BM infiltration — malignant	Acute leukaemia, chronic leukaemia, lymphoma, MDS, multiple myeloma, metastatic cancer	Abnormal cells/blasts on PBS/BM; dysplastic features in MDS; cytogenetics
BM infiltration — non-malignant	Primary myelofibrosis, TB	Tear-drop cells on PBS (myelofibrosis); dry tap on BM aspiration; AFB stain for TB
BM suppression	HIV, EBV, hepatitis-associated AA	Viral serology; hepatitis may precede AA by 2–3 months ^[3]
Nutritional	Megaloblastic anaemia (B12/folate deficiency)	MCV often > 110 fL; hypersegmented neutrophils; low serum B12 or RBC folate; responds to replacement ^[12]
Peripheral consumption	DIC, TTP/HUS	Schistocytes; abnormal clotting profile (DIC); ADAMTS13 < 10% (TTP)
Hypersplenism	Portal hypertension, haematological malignancies	Splenomegaly with dilutional cytopenias; liver disease features
Autoimmune	SLE (Evans syndrome — AIHA + ITP)	Coombs positive; ANA/anti-dsDNA positive; multi-system features
Drug-induced	Cytotoxic agents, chloramphenicol, carbimazole	Temporal relationship with drug exposure ^[3]

High Yield — PNH vs Aplastic Anaemia

Flow cytometry (↓CD55/CD59) for PNH should be performed in all patients with aplastic anaemia. ^[3]^[7] The two diseases sit on a spectrum:

AA → PNH: immune-mediated marrow destruction → PIGA-mutant HSCs escape → clonal expansion → PNH emerges
PNH → AA: progressive marrow failure in a PNH patient → pancytopenia worsens

Up to 50% of AA patients have detectable PNH clones ^[7]. This is a recurrent exam theme.

4. DDx When PNH Presents as Thrombosis at Unusual Sites

PNH is listed as an acquired cause of thrombophilia / hypercoagulability ^[4]^[13].

When a patient (especially young) presents with thrombosis at an unusual site (Budd-Chiari, cerebral venous sinuses, mesenteric veins, portal vein), the differential diagnosis for hypercoagulability must be systematically considered:

Condition	Distinguishing Features	Mechanism
PNH	Haemolysis markers (↑LDH, ↓haptoglobin, haemoglobinuria); Coombs-negative; flow cytometry CD55/CD59	NO scavenging by free Hb; procoagulant microparticles; impaired fibrinolysis (loss of CD87)
Antiphospholipid syndrome (APS)	Lupus anticoagulant, anticardiolipin Ab, anti-β2GP1 Ab; ↑APTT paradoxically; associated with SLE	Antibodies against phospholipid-binding proteins → endothelial activation, platelet activation
Myeloproliferative neoplasms (MPN) — especially JAK2-positive	High counts (RBCs in PV, platelets in ET); JAK2 V617F mutation; thrombosis in peculiar locations, most commonly mesenteric vein ^[14]	Hyperviscosity; JAK2-mutant cells activate endothelium and platelets; can clot even before overt cytosis develops ^[14]
Malignancy (Trousseau syndrome)	Known malignancy (especially adenocarcinoma — mucin secretion); migratory superficial thrombophlebitis	Tumour-derived procoagulant substances (tissue factor, mucin, cancer procoagulant)
Nephrotic syndrome	Heavy proteinuria ( > 3.5 g/day); hypoalbuminaemia; oedema	Loss of antithrombin III in urine → hypercoagulable state ^[4]
Pregnancy / oestrogen therapy	OCP use; pregnancy/puerperium	Oestrogen increases hepatic production of clotting factors
Heparin-induced thrombocytopenia (HIT)	Recent heparin exposure; paradoxical thrombocytopenia with thrombosis; anti-PF4/heparin antibodies	HIT antibodies activate platelets via FcγRIIa

Condition	Distinguishing Features
Antithrombin deficiency	Family history; VTE resistant to normal heparin doses; AD inheritance ^[13]
Protein C deficiency	Family history; warfarin-induced skin necrosis (in first few days — because protein C has a short half-life and is depleted before the procoagulant factors); AD ^[13]
Protein S deficiency	Family history; AD; cofactor of protein C ^[13]
Factor V Leiden	Most common inherited thrombophilia in Caucasians; NOT found in Chinese population ^[13]
Prothrombin G20210A mutation	Caucasian populations; gain-of-function mutation
Hyperhomocysteinaemia	Elevated homocysteine levels; associated with both arterial and venous thrombosis; no data in Chinese ^[13]

High Yield — Unusual Site Thrombosis DDx

When you see thrombosis at an unusual site in a young patient, your top differentials are:

PNH — especially Budd-Chiari syndrome; look for haemolysis markers
MPN (JAK2-positive) — screen for JAK2 mutation even if CBC doesn't show MPN features, as patients can clot before developing the cytosis ^[14]
Antiphospholipid syndrome — check lupus anticoagulant, aCL, anti-β2GP1
Inherited thrombophilia — protein C/S deficiency, antithrombin deficiency

Budd-Chiari syndrome in a young patient = screen for PNH AND JAK2 mutation.

The differential diagnosis of dark urine / positive urine dipstick for "blood" must distinguish haemoglobinuria from haematuria and myoglobinuria ^[6]:

Cause of Dark Urine	Mechanism	How to Distinguish
Haemoglobinuria (PNH, other intravascular haemolysis)	Free Hb filtered by kidneys after intravascular RBC lysis	Urine dipstick +ve for blood; urine microscopy shows NO intact RBCs; serum is pink/brown (haemoglobinaemia); ↓↓haptoglobin
Haematuria (glomerulonephritis, stones, tumour, infection)	Intact RBCs enter urine from urinary tract bleeding or glomerular leakage	Urine dipstick +ve for blood; urine microscopy shows intact RBCs ± dysmorphic RBCs/casts (if glomerular)
Myoglobinuria (rhabdomyolysis)	Myoglobin released from damaged skeletal muscle	Urine dipstick +ve for blood but NO RBCs on microscopy (like haemoglobinuria); massively elevated CK; serum is clear (myoglobin clears quickly from plasma)
Bilirubinuria (obstructive jaundice)	Conjugated bilirubin enters urine	Tea-coloured urine; +ve for bilirubin on dipstick; NOT positive for blood
Drugs/foods	Beetroot, rifampicin, nitrofurantoin	History of drug/food intake; urine dipstick negative for blood
Porphyria (acute intermittent porphyria)	Porphobilinogen in urine; darkens on standing	Abdominal pain, neuropsychiatric symptoms; urine porphobilinogen positive

In the nephrology context, PNH should be excluded as a cause of positive urine dipstick for blood (false positive for haematuria), because the dipstick detects free haemoglobin — not intact RBCs ^[6].

Exam Trap — Dark Urine Differential

A urine dipstick positive for "blood" does NOT necessarily mean haematuria. It detects the peroxidase activity of haem — which is present in both haemoglobin and myoglobin. Always confirm with urine microscopy:

RBCs present → true haematuria
No RBCs → haemoglobinuria (PNH, intravascular haemolysis) or myoglobinuria (rhabdomyolysis)

Then distinguish haemoglobinuria from myoglobinuria by checking serum colour (pink in haemoglobinuria, clear in myoglobinuria) and serum CK (massively elevated in rhabdomyolysis).

A special mention: Wilson's disease can present as fulminant hepatic failure with Coombs-negative haemolytic anaemia ^[15]. This is a rare but important differential:

Wilson's disease: Young patient with unexplained fulminant liver failure + Coombs-negative haemolytic anaemia + low haemoglobin. The haemolysis is caused by acute copper release from necrotic hepatocytes, which directly damages RBC membranes.
PNH: Would have chronic haemolysis rather than acute fulminant picture; no liver failure unless from Budd-Chiari; flow cytometry for CD55/CD59 distinguishes definitively.

Feature	PNH	AIHA (warm)	TTP/HUS	AA	MPN
Coombs test	Negative	Positive	Negative	N/A	N/A
Blood film	May be unremarkable or polychromasia	Spherocytes, polychromasia	Schistocytes	Normal morphology but low counts	High counts ± abnormal cells
Haemolysis type	Intravascular	Mostly extravascular	Intravascular	Not primary	Not primary
Haptoglobin	↓↓↓	↓	↓↓	Normal	Normal
Haemoglobinuria	Present	Usually absent	May be present	Absent	Absent
Thrombosis	Unusual sites	Not typical	Rare in TTP	Not typical (unless PNH clone)	Yes — unusual sites (esp JAK2+)
Bone marrow	Variable (normal to hypocellular)	Erythroid hyperplasia	Normal	Hypocellular	Hypercellular
Flow cytometry	↓CD55/CD59	Normal	Normal	Normal (unless PNH clone)	Normal
Key genetic test	PIGA mutation	N/A	ADAMTS13 activity	DEB test (Fanconi)	JAK2 mutation

High Yield Summary — Differential Diagnosis of PNH

When PNH presents as haemolytic anaemia:

Key DDx: AIHA (Coombs +ve), TTP/HUS (schistocytes), G6PD deficiency (oxidant triggers), hereditary spherocytosis (family Hx, spherocytes), infections (malaria, C. perfringens)
PNH is the only acquired intrinsic RBC defect — all others are extrinsic

When PNH presents as pancytopenia:

Key DDx: Aplastic anaemia (hypocellular marrow), MDS (dysplastic features), leukaemia (blasts), megaloblastic anaemia (B12/folate)
Screen all AA patients for PNH clones by flow cytometry

When PNH presents as thrombosis at unusual sites:

Key DDx: MPN (JAK2+), APS, inherited thrombophilia (AT/PC/PS deficiency), malignancy
Budd-Chiari in a young patient = screen for PNH and JAK2

When PNH presents as dark urine:

Distinguish haemoglobinuria from haematuria (no RBCs on microscopy) and myoglobinuria (clear serum, ↑CK)
PNH causes false positive urine dipstick for blood — not true haematuria

The definitive differentiating test for PNH is flow cytometry for GPI-anchored proteins (CD55, CD59) or FLAER assay.

Active Recall - Differential Diagnosis of PNH

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf; Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf ^[3] Senior notes: Ryan Ho Haemtology.pdf (Section 2.4.2 — Aplastic Anaemia) ^[4] Lecture slides: Block A - Leg swelling and chest pain_ deep vein thrombosis; pulmonary embolism; Thrombophilia.pdf; Senior notes: MBBS Final MB (Surgery) (Felix PY Lai).pdf (p. 962) ^[5] Senior notes: Ryan Ho Haemtology.pdf (p. 36 — Classification by site of RBC destruction) ^[6] Senior notes: Block A - Nephrology Interactive Tutorial.pdf (Case 1 — false positive dipstick) ^[7] Senior notes: MBBS Final MB (Medicine) (Felix PY Lai).pdf (p. 1468–1470); MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (p. 624) ^[8] Senior notes: Block A - Many members of the family have anaemia.pdf ^[9] Senior notes: Ryan Ho Haemtology.pdf (p. 50 — Pancytopenia); Ryan Ho Fundamentals.pdf (p. 393) ^[10] Senior notes: MBBS Final MB (Medicine) (Felix PY Lai).pdf (p. 1460–1462) ^[11] Senior notes: MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (p. 772) ^[12] Senior notes: Block A - Pallor_ diagnosis of anaemia; nutritional anaemia; anaemia of systemic diseases.pdf ^[13] Senior notes: Ryan Ho Haemtology.pdf (p. 135 — Thrombophilia Screening) ^[14] Senior notes: Block A - Leg swelling and chest pain_ DVT; PE; Thrombophilia.pdf (p. 18 — MPN-associated thrombosis); Lecture slides: GC 086. Splenomegaly.pdf (Polycythaemia Vera) ^[15] Senior notes: Block A - Patients with non-viral chronic liver diseases.pdf (Wilson's disease)

Diagnostic Criteria, Diagnostic Algorithm & Investigations for PNH

Unlike many conditions, PNH does not have a single universally agreed-upon set of formal diagnostic criteria with numbered items you tick off (like the ACR/EULAR criteria for SLE or the WHO criteria for diabetes). Instead, the diagnosis rests on a two-step approach:

Clinical suspicion — recognising the presentation that should make you think "could this be PNH?"
Confirmatory laboratory testing — demonstrating the absence of GPI-anchored proteins on blood cell surfaces by flow cytometry

The reason there is no elaborate set of diagnostic criteria is that the flow cytometry finding is essentially pathognomonic: if you demonstrate absence of GPI-anchored proteins (CD55, CD59) on blood cells, and this is clonal (i.e. a discrete population of deficient cells coexisting with normal cells), the diagnosis is confirmed. No other condition causes this pattern.

This is arguably more important than the diagnostic test itself, because PNH is a rare disease that is easily missed if you don't think of it. The key clinical scenarios that should trigger PNH screening are:

High Yield — Indications for PNH Screening

Screen for PNH (flow cytometry for CD55/CD59) in the following clinical scenarios:

All patients with aplastic anaemia — PNH clones occur in up to 50% of patients with AA ^[3]^[7]
Coombs-negative intravascular haemolytic anaemia — especially with haemoglobinuria, ↓↓haptoglobin
Thrombosis at unusual sites — hepatic (Budd-Chiari), portal, mesenteric, cerebral venous sinuses — especially in young patients without other risk factors ^[7]
Unexplained cytopenias — especially when combined with haemolysis markers
Haemoglobinuria (dark/cola-coloured urine) ^[1]
Unexplained recurrent iron deficiency — with no GI blood loss identified
Positive urine dipstick for blood with no RBCs on microscopy — exclude PNH as a cause of false-positive urine dipstick ^[6]
MDS with unexplained haemolysis
Recurrent episodes of dysphagia, abdominal pain, or erectile dysfunction with anaemia (NO depletion symptoms)

3. The Confirmatory Test — Flow Cytometry for GPI-Anchored Proteins

Flow cytometry on peripheral blood is the MOST useful and accepted method to confirm diagnosis of PNH ^[7].

The principle is straightforward:

Flow cytometry uses fluorescently labelled monoclonal antibodies that bind to specific surface proteins.
In PNH, the GPI-anchored proteins are absent → the antibodies have nothing to bind to → the affected cells show reduced or absent fluorescence for these markers.
This creates a bimodal distribution: a population of normal cells (bright fluorescence) and a population of PNH cells (dim or absent fluorescence).

Marker	What It Detects	Cell Type Tested	Notes
CD55 (DAF)	GPI-anchored complement regulator	RBCs	Detects deficiency of GPI-anchored proteins including CD55 on RBCs ^[7]
CD59 (MIRL)	GPI-anchored complement regulator	RBCs	Detects deficiency of CD59 on RBCs ^[7] — the loss of CD59 is the primary driver of intravascular haemolysis
CD24	GPI-anchored protein on granulocytes	Granulocytes
CD16 (FcγRIIIb)	GPI-anchored Fc receptor	Neutrophils
CD14	GPI-anchored LPS receptor	Monocytes
FLAER (see below)	GPI anchor itself	WBCs (granulocytes, monocytes)	Most sensitive and specific modern method

FLAER = Fluorescent Labelled Aerolysin Reagent

Aerolysin is a bacterial toxin (from Aeromonas hydrophila) that specifically binds to the GPI anchor itself — not to any single GPI-anchored protein.
A fluorescently labelled, inactive variant of aerolysin (FLAER) is used in flow cytometry.
Normal cells with intact GPI anchors bind FLAER brightly. PNH cells lacking GPI anchors show absent or reduced FLAER binding.
Advantage over CD55/CD59 testing: FLAER detects the anchor directly, so it catches deficiency regardless of which specific protein is being assessed. It is also more sensitive for small clones.
Limitation: FLAER cannot be used on RBCs (because mature RBCs have low GPI anchor density making the signal unreliable). It is used on granulocytes and monocytes.

Why Test Granulocytes/Monocytes Rather Than Just RBCs?

Testing GPI-anchored proteins on granulocytes is actually more reliable than testing RBCs for several reasons:

RBC transfusions introduce normal donor RBCs that dilute the PNH clone → falsely reducing the apparent PNH RBC clone size
Complement-mediated destruction preferentially removes PNH Type III RBCs from circulation → underestimates the true clone size
Reticulocytes can be specifically gated on flow cytometry to assess the "true" RBC clone size
Granulocytes are not affected by transfusion or complement-mediated destruction, so they give a more accurate reflection of the underlying PNH clone size in the bone marrow

Testing both RBCs and granulocytes/monocytes is recommended by the International Clinical Cytometry Society (ICCS) guidelines.

Flow cytometry classifies individual cells into three populations:

Cell Type	GPI-Anchor Status	Fluorescence Pattern	Clinical Significance
Type I cells	Normal GPI-anchor expression	Normal fluorescence	These are the patient's remaining normal cells
Type II cells	Partial deficiency of GPI-anchored proteins	Intermediate/reduced fluorescence	Moderate sensitivity to complement (~3–5× normal). May represent cells with hypomorphic PIGA mutations
Type III cells	Complete absence of GPI-anchored proteins	Absent fluorescence	Very high sensitivity to complement (~15–25× normal). These are the cells that undergo clinically significant haemolysis

The PNH clone size = percentage of Type II + Type III cells. This is expressed separately for RBCs, granulocytes, and monocytes.

The granulocyte clone size is the most reliable measure of the true underlying PNH clone in the marrow.

Granulocyte PNH Clone Size	Clinical Significance
< 1%	Subclinical PNH — no clinical haemolysis; often detected during workup for AA/MDS; requires monitoring as clone may expand ^[7]
1–10%	May have mild haemolysis but often clinically compensated
10–50%	Moderate haemolysis; variable clinical severity
> 50%	Classical (haemolytic) PNH — significant intravascular haemolysis; high risk of thrombosis; likely symptomatic

Important: There is no absolute clone-size cutoff that defines "PNH" vs "no PNH." The diagnosis is a combination of clone size + clinical features + haemolysis markers.

The following algorithm represents the clinical decision-making pathway from suspicion to diagnosis:

5. Complete Investigation Panel — Systematic Approach

When PNH is suspected or confirmed, the following investigations should be performed. I will organise them by purpose:

Investigation	Expected Findings in PNH	Rationale / Interpretation
Flow cytometry for GPI-anchored proteins (CD55, CD59) on peripheral blood	Deficiency of GPI-anchored proteins including CD55 and CD59 on RBCs ^[7]	MOST useful and accepted method to confirm diagnosis of PNH ^[7]. Demonstrates absence of proteins that normally protect against complement. Test on both RBCs and granulocytes/monocytes
FLAER assay on granulocytes and monocytes	Absent/reduced FLAER binding on GPI-deficient populations	Binds GPI anchor directly; more sensitive than CD55/CD59 alone for small clones; cannot use on RBCs

Investigation	Expected Findings in PNH	Rationale / Interpretation
Serum LDH	↑ LDH ^[7]	Released from lysed RBCs (rich in LDH isoenzymes 1 and 2). LDH is often markedly elevated in PNH (can be 2–10× ULN) and is the most sensitive marker of ongoing haemolysis. Also used to monitor disease activity and treatment response
Serum haptoglobin	↓↓ Haptoglobin ^[7]	Free Hb released from intravascular haemolysis binds haptoglobin → Hb-haptoglobin complex rapidly cleared by liver → haptoglobin consumed and undetectable. Haptoglobin is the most sensitive marker of haemolysis — it drops before other markers rise
Serum bilirubin (indirect/unconjugated)	↑ Unconjugated bilirubin ^[7]	Haem breakdown → biliverdin → unconjugated bilirubin. Typically mildly elevated in PNH (usually < 85 µmol/L); higher levels suggest severe or acute haemolytic crisis
Reticulocyte count	↑ Reticulocytes (if marrow function preserved)	Compensatory marrow response to anaemia. May be inappropriately low or normal if concurrent bone marrow failure (AA/PNH overlap) — this is an important clue to coexistent marrow pathology
Direct antiglobulin test (Coombs test)	Negative	Excludes immune-mediated haemolysis (AIHA). PNH is complement-mediated but NOT antibody-mediated, so Coombs is negative. This is a defining feature ^[5]^[16]

High Yield — Why Is Coombs Negative in PNH?

The Coombs test detects antibodies (IgG) or complement fragments (C3d) bound to the RBC surface. In PNH, the haemolysis is caused by lack of complement regulatory proteins (CD55, CD59) on the RBC, NOT by antibodies targeting the RBC. The complement system is activated through the alternative pathway (spontaneous "tick-over"), not through antibody binding. Therefore, there are no anti-RBC antibodies to detect → Coombs negative.

One subtlety: C3d may occasionally be weakly positive in PNH (because complement IS activated on PNH cells), but anti-IgG is always negative. This can mimic cold AIHA, but the clinical context and flow cytometry resolve this.

These investigations distinguish intravascular from extravascular haemolysis:

Investigation	Expected Findings in PNH	Rationale / Interpretation
Urine for haemoglobin (haemoglobinuria)	Positive (dark/cola-coloured urine)	Free Hb spills into urine once haptoglobin is saturated; detected by urine dipstick as "blood positive" but no intact RBCs on microscopy ^[6]
Urine for haemosiderin (haemosiderinuria)	Positive — test urine for haemosiderin for haemosiderosis associated with intravascular haemolysis ^[7]	Renal tubular cells absorb filtered Hb → intracellular iron deposited as haemosiderin → when tubular cells are shed, haemosiderin appears in urine. Detected by Prussian blue stain of urine sediment. A reliable marker of chronic intravascular haemolysis (persists even when haemoglobinuria is intermittent)
Serum free haemoglobin	↑ (haemoglobinaemia)	Free Hb in plasma gives serum a pink (oxyHb) or brown (metHb) colour ^[5]
Methaemalbumin (Schumm test)	Positive	Once haptoglobin is saturated, free haem (as methaem) binds albumin → methaemalbumin. Detected by Schumm test. Indicates severe or chronic intravascular haemolysis ^[5]

The pathology of intravascular haemolysis in PNH: haemolysis occurs within the bloodstream → free Hb released into blood (haemoglobinaemia) → pink or brown serum. Free haem and Hb are toxic → risk of AKI, DIC, ↑ risk of thrombosis. Free Hb binds to haptoglobin → ↓ serum haptoglobin → once saturated, metHb binds albumin to form methaemalbumin (detectable on Schumm test). All mechanisms saturated → Hb excreted in urine → absorbed by tubular cells to form haemosiderin → haemosiderinuria when tubular cells shed → haemoglobinuria if overwhelmed ^[5].

Investigation	Expected Findings in PNH	Rationale / Interpretation
CBC	Anaemia (variable severity); may also show neutropenia and/or thrombocytopenia if marrow failure component	Normocytic or mildly macrocytic anaemia (macrocytosis from reticulocytosis and/or concurrent marrow stress). Pancytopenia suggests AA/PNH overlap
Peripheral blood smear	Polychromasia (reflecting reticulocytosis); no schistocytes (important to distinguish from MAHA); no spherocytes (distinguishes from AIHA/HS); no blasts or dysplastic cells	A relatively "unremarkable" film in the context of haemolysis should raise suspicion — in PNH, there are no characteristic morphological abnormalities on the film because the defect is a surface protein deficiency, not a structural RBC defect ^[16]

Exam Pearl — The 'Bland' Blood Film

Unlike most haemolytic anaemias that leave clues on the blood film (spherocytes in AIHA/HS, schistocytes in TTP/DIC, bite cells in G6PD, sickle cells in SCD), PNH has a remarkably unremarkable blood film apart from polychromasia. If you see haemolysis markers with a bland film and negative Coombs → think PNH.

Investigation	Expected Findings in PNH	Rationale / Interpretation
Serum ferritin	↓ or low-normal (in untreated, chronic PNH)	Chronic urinary iron loss → iron depletion. Counterintuitive in haemolysis (where you'd expect iron overload) — unique to intravascular haemolysis
Serum iron, TIBC	Low iron, high TIBC (iron deficiency pattern)	Same mechanism — urinary iron loss
Transferrin saturation	Low

Exception: Patients who have received multiple blood transfusions may develop iron overload despite PNH.

Bone marrow aspiration and trephine biopsy are not required to diagnose PNH (flow cytometry on peripheral blood is sufficient), but are indicated in the following situations ^[7]^[3]:

Concurrent cytopenias → to assess marrow cellularity and exclude AA, MDS, or other marrow disorders
Suspected AA/PNH overlap → to determine the degree of marrow hypoplasia
Suspected MDS → to look for dysplasia and perform cytogenetics

Finding	Interpretation
Hypocellular marrow with decrease in all elements, replacement by fat/stromal cells	Concurrent aplastic anaemia ^[3]^[7]
Morphologically normal residual haematopoietic cells	Supports AA (vs MDS where cells are dysplastic) ^[3]^[7]
No BM infiltration by fibrosis/malignancy	Excludes myelofibrosis, leukaemia, metastatic infiltration ^[3]^[7]
Erythroid hyperplasia	Compensatory response to haemolysis (if marrow is not failing)
Dysplastic features	Suggests MDS/PNH overlap

Flow cytometry on bone marrow sample can also be used to detect PNH clones if peripheral blood is insufficient ^[7].

Cytogenetics (karyotyping / FISH) and molecular genetics should be performed on the marrow sample to exclude hypocellular myelodysplastic syndrome (MDS) ^[7].

Given that thrombosis is the leading cause of death in PNH, the following should be considered:

Investigation	Purpose
D-dimer	Screening for active thrombosis
Doppler ultrasound of hepatic veins	Screen for Budd-Chiari syndrome
CT venography / MR venography	Detect cerebral venous sinus thrombosis, mesenteric vein thrombosis, or hepatic vein thrombosis
Echocardiography	Assess for pulmonary hypertension (chronic NO depletion → pulmonary vasoconstriction)
Protein C, Protein S, Antithrombin levels	If concurrent thrombophilia screening needed (but note: PNH itself is the thrombophilic state)

Investigation	Expected Findings	Rationale
Serum creatinine / eGFR	May be elevated	Free Hb is directly toxic to renal tubules → acute and chronic kidney injury
Urinalysis	Haemoglobinuria, haemosiderinuria, proteinuria	Chronic tubular damage
Renal USS	Cortical haemosiderin deposition (seen as cortical signal changes on MRI)	Chronic intravascular haemolysis → iron deposition in kidneys

Investigation	Purpose
Serum B12, RBC folate	Exclude megaloblastic anaemia as cause of pancytopenia ^[7]
Viral serology (HIV, HAV/HBV/HCV, EBV/CMV)	Exclude viral causes of marrow failure ^[7]
Serum autoantibodies (ANA, anti-dsDNA)	Exclude SLE as cause of cytopenias/haemolysis ^[7]
LFT	Detects antecedent or ongoing hepatitis ^[7]; also baseline for monitoring hepatic complications (Budd-Chiari)
Serum EPO	Usually elevated (appropriate response to anaemia)
Chest X-ray	Baseline; assess for pulmonary hypertension
NT-proBNP	Screen for heart failure / pulmonary hypertension
Chromosome breakage analysis with diepoxybutane	Screen for Fanconi anaemia — especially in paediatric patients with aplastic anaemia ^[1]^[3]

These tests are no longer used clinically but may appear in exams or older textbooks:

Test	Principle	Why Obsolete
Ham test (acidified serum lysis test)	Patient's RBCs are incubated in acidified serum → pH drop activates complement → PNH cells lyse (lack CD55/CD59); normal cells do not	Replaced by flow cytometry — much more sensitive and specific; Ham test has poor sensitivity for small clones and cannot quantify clone size
Sucrose lysis test (sugar water test)	Low-ionic-strength sucrose solution promotes complement binding to RBCs → PNH cells lyse	Same limitations as Ham test — low sensitivity, cannot quantify, superseded by flow cytometry

These tests were the original methods for diagnosing PNH and explain the historical naming conventions. They exploit the same principle — PNH cells are hypersensitive to complement activation — but modern flow cytometry is vastly superior because it can detect small clones (< 1%), quantify clone size precisely, and classify cells into Type I/II/III.

Once PNH is confirmed, longitudinal monitoring is essential:

Parameter	Frequency	Purpose
CBC, reticulocyte count, LDH, haptoglobin, bilirubin	Every 3–6 months (or more frequently if unstable)	Monitor haemolysis activity and response to treatment
Flow cytometry (PNH clone size)	Every 6–12 months	Track clone expansion or contraction; especially important in subclinical PNH
Iron studies (ferritin, serum iron, TIBC)	Every 6–12 months	Monitor for iron deficiency (from urinary loss) or iron overload (from transfusions)
Renal function (creatinine, eGFR)	Every 3–6 months	Monitor for chronic kidney injury from haemoglobinuria
BM biopsy	As clinically indicated	Monitor for evolution to MDS, AML, or worsening marrow failure

AA patients with PNH clones require regular CBC to screen for development of clonal disorders — the condition may progress into MDS, AML, or clinical PNH over time ^[16].

High Yield Summary — Diagnosis of PNH

The diagnostic test: Flow cytometry on peripheral blood detecting deficiency of GPI-anchored proteins (CD55, CD59) — the MOST useful and accepted method ^[7]

The modern gold standard: FLAER assay on granulocytes/monocytes — binds the GPI anchor directly; most sensitive for small clones

Key supporting findings:

↑ LDH, ↓ haptoglobin, ↑ unconjugated bilirubin = haemolysis ^[7]
Negative Coombs test = not immune-mediated
Haemoglobinuria + haemosiderinuria = intravascular haemolysis ^[7]
No schistocytes, no spherocytes on PBS = not MAHA or AIHA
BM biopsy if cytopenias — assess for AA/MDS overlap

When to screen: AA (up to 50% have PNH clones), Coombs-negative haemolysis, thrombosis at unusual sites, unexplained cytopenias, false-positive urine dipstick for blood

Obsolete tests: Ham test (acidified serum lysis), sucrose lysis test — replaced by flow cytometry

Classification by flow cytometry: Type I (normal), Type II (partial deficiency), Type III (complete deficiency). Clone size = Type II + III percentage.

Active Recall - Diagnosis & Investigations of PNH

References

Management of Paroxysmal Nocturnal Haemoglobinuria (PNH)

Managing PNH is fundamentally about addressing the three pillars of the disease and tailoring treatment to the clinical subcategory:

Pillar	Therapeutic Goal	Key Interventions
Intravascular haemolysis	Block complement-mediated RBC destruction	Complement inhibitors (eculizumab, ravulizumab, etc.)
Thrombosis	Prevent and treat the #1 killer	Anticoagulation; complement inhibitors themselves reduce thrombotic risk
Bone marrow failure	Restore haematopoiesis	Immunosuppressive therapy (for AA component); allogeneic HSCT (curative)

Not every PNH patient needs the same treatment. A patient with a tiny subclinical clone detected during AA workup has very different needs from a patient with classical PNH having haemolytic crises and Budd-Chiari syndrome. The management must be risk-stratified.

3. Supportive Care — The Foundation for All PNH Patients

Regardless of whether a patient receives complement inhibitor therapy, supportive measures are important:

Dose: 1–5 mg/day
Rationale: Chronic haemolysis causes increased RBC turnover → increased folate consumption for DNA synthesis in erythroid precursors. Without supplementation, folate deficiency can develop, worsening the anaemia (megaloblastic component superimposed on haemolytic anaemia)
This is the same principle as in any cause of chronic haemolysis — folate supplementation is standard ^[17]

Rationale: PNH with intravascular haemolysis causes chronic urinary iron loss (haemoglobinuria + haemosiderinuria) → iron deficiency
Indication: Only when iron deficiency is documented (low ferritin, low transferrin saturation)
Caution: Iron supplementation provides substrate for new RBC production → these new reticulocytes are GPI-deficient → they are immediately susceptible to complement-mediated lysis → can paradoxically worsen haemolysis temporarily. Therefore, iron replacement should ideally be given under the cover of complement inhibitor therapy in patients with significant haemolysis

Indication: Symptomatic anaemia that is severe or refractory to other measures
Consideration: Transfused donor RBCs are normal (have intact CD55/CD59) → they are protected from complement → they survive normally in the patient's circulation. This is actually reassuring — transfusions work well in PNH
Avoid unnecessary transfusions due to risks of alloimmunisation, iron overload

May be used as adjunct in patients with suboptimal reticulocyte response, particularly those with concurrent marrow failure (AA/PNH overlap)
Not a standard first-line therapy

4. Complement Inhibitor Therapy — The Cornerstone of Modern PNH Treatment

This is the transformative advance in PNH management. Understanding these drugs requires understanding where they act in the complement cascade.

4.1 Terminal Complement Inhibitors (C5 Inhibitors)

"Eculizumab" → "ecu" from humanised; "-lizumab" = humanised monoclonal antibody. It is a humanised monoclonal IgG2/4 antibody that binds complement protein C5, preventing its cleavage into C5a and C5b.

Mechanism: By binding C5, eculizumab prevents:
- Formation of C5a (a potent anaphylatoxin and inflammatory mediator)
- Formation of C5b, which initiates assembly of the membrane attack complex (MAC, C5b-9)
- Without MAC → no pore formation in RBC membranes → intravascular haemolysis is dramatically reduced
Route & dosing: IV infusion every 2 weeks (after an induction phase of weekly dosing for 4 weeks)
Indications:
- Symptomatic intravascular haemolysis (LDH persistently elevated, transfusion-dependent anaemia, haemoglobinuria)
- PNH-related thrombosis
- End-organ damage from haemolysis (renal impairment, pulmonary hypertension)
- Debilitating NO-depletion symptoms (dysphagia, severe abdominal pain, erectile dysfunction)
Efficacy:
- Reduces intravascular haemolysis by ~85–90%
- Reduces transfusion requirement by ~50–70%
- Reduces thrombotic events by ~85%
- Improves quality of life, fatigue scores, and renal function
- Does NOT cure PNH — the PNH clone persists; treatment is lifelong (unless HSCT is performed)
Limitations — The Extravascular Haemolysis Problem:
- Eculizumab blocks C5 → no MAC formation → PNH RBCs survive in circulation but they still lack CD55 → C3b continues to be deposited on PNH RBC surfaces (because C3 convertase is NOT blocked by eculizumab)
- C3b-opsonised RBCs are recognised and phagocytosed by splenic and hepatic macrophages → extravascular haemolysis
- This is why many patients on eculizumab develop a positive Coombs test for C3d (C3b/C3d fragments on RBC surface) and may have persistent anaemia despite normalisation of LDH
- The Coombs test positivity in this context does NOT mean the patient has developed AIHA — it is a pharmacological consequence of blocking downstream complement while upstream opsonisation continues

Why Eculizumab Causes Extravascular Haemolysis — First Principles

Think of it this way: before eculizumab, PNH RBCs are destroyed quickly by MAC (intravascular lysis) — they don't survive long enough for C3b to accumulate. With eculizumab, MAC is blocked → PNH RBCs survive longer → but C3b keeps being deposited (CD55 is still absent) → C3b-coated RBCs are cleared by the reticuloendothelial system → extravascular haemolysis. The disease shifts from intravascular to extravascular destruction.

"Ravulizumab" = a next-generation, long-acting anti-C5 monoclonal antibody (also known as ALXN1210).

Mechanism: Same as eculizumab — binds C5 and prevents MAC formation
Key advantage: Engineered for extended half-life through pH-dependent C5 recycling
- After binding C5, the drug–C5 complex is internalised into endosomes
- At the low pH of the endosome, ravulizumab releases C5 (which is degraded) while the antibody itself is recycled back to the cell surface via the FcRn receptor
- Result: dosing interval extended to every 8 weeks (vs every 2 weeks for eculizumab) → much better patient convenience and adherence
Route & dosing: IV infusion every 8 weeks (after loading dose)
Efficacy: Non-inferior to eculizumab in clinical trials (ALXN1210-PNH-301/302)
Same limitation: Still causes C3b-mediated extravascular haemolysis (same issue as eculizumab)

These are newer agents that block the complement cascade upstream of C5, at the level of C3 or Factor B/D, preventing both C3b opsonisation and MAC formation:

Drug	Target	Route	Key Features
Pegcetacoplan	C3	Subcutaneous (self-administered); every 3 days or weekly	Binds C3 → prevents both C3b opsonisation AND MAC formation → reduces both intra- and extravascular haemolysis. Can be monotherapy or add-on to eculizumab. FDA/EMA approved
Iptacopan	Factor B	Oral (twice daily)	Factor B is essential for the alternative pathway C3 convertase (C3bBb). Blocking Factor B → no alternative pathway activation → no C3b deposition → no MAC. First oral complement inhibitor for PNH. FDA approved 2023
Danicopan	Factor D	Oral (three times daily)	Factor D cleaves Factor B → essential for alternative pathway activation. Blocking Factor D achieves similar effect as blocking Factor B. Approved as add-on to C5 inhibitors

Why "proximal" matters: By blocking at C3 or upstream (Factor B/D), these drugs prevent C3b from being deposited on RBC surfaces → the extravascular haemolysis problem seen with C5 inhibitors is eliminated. Patients may achieve Hb normalisation without transfusion.

High Yield — Meningococcal Vaccination Before Complement Inhibitor Therapy

Eculizumab is a monoclonal antibody that inhibits the complement pathway by binding to C5 and preventing formation of membrane attack complex. Infections by Neisseria meningitidis is expected to be the MOST COMMON in patients on eculizumab ^[18].

Why? The MAC (C5b-9) is the body's primary defence against Neisseria species (meningococcus and gonococcus). These encapsulated gram-negative diplococci are uniquely susceptible to complement-mediated killing by MAC. When you block MAC formation with C5 inhibitors:

The patient loses their ability to kill Neisseria
Risk of fulminant meningococcal septicaemia/meningitis increases by 1000–2000-fold

Mandatory pre-treatment measures:

Meningococcal vaccination (MenACWY + MenB) at least 2 weeks before starting complement inhibitor therapy
If treatment is urgent and cannot wait 2 weeks → start prophylactic antibiotics (penicillin V or ciprofloxacin) as a bridge until vaccination takes effect
Some centres recommend continuing prophylactic antibiotics throughout treatment regardless of vaccination status (penicillin V 500 mg BD or ciprofloxacin 500 mg OD)
Patient education: carry an alert card; seek immediate medical attention for fever, headache, neck stiffness, rash

This is the same principle as overwhelming post-splenectomy infection (OPSI) by encapsulated organisms ^[19] — loss of complement function mirrors functional asplenia for the purpose of Neisseria defence.

Other infection considerations:

Gonococcal infections: also increased risk (same mechanism — Neisseria gonorrhoeae)
Pneumococcus, Haemophilus: These are primarily opsonophagocytosis-dependent (C3b pathway), so risk is not as dramatically increased with C5 inhibitors, but may be increased with proximal complement inhibitors (C3/Factor B/D inhibitors)

Contraindication / Caution	Rationale
Unresolved Neisseria meningitidis infection	C5 blockade removes the primary defence; would worsen active infection
Not vaccinated against meningococcus without antibiotic cover	Unacceptable infection risk
Known hypersensitivity to the drug	Anaphylaxis risk
Hepatic impairment (for oral agents like iptacopan)	Altered drug metabolism; dose adjustment may be needed
Pregnancy (relative caution)	Limited safety data; must weigh risk vs benefit (PNH itself carries high pregnancy-related morbidity/mortality from thrombosis, so complement inhibitors are often continued)
Breakthrough haemolysis	Can occur with missed doses or dose delays → must ensure strict adherence to dosing schedule; also with complement-amplifying conditions (infections, surgery, pregnancy)

5. Anticoagulation — Preventing the #1 Killer

Thrombosis is the leading cause of morbidity and mortality in PNH (accounting for ~40–67% of deaths in untreated patients) ^[4]^[7].

Historically: Before complement inhibitors, guidelines recommended primary prophylactic anticoagulation with warfarin for all PNH patients with large clones (granulocyte clone > 50%) even without prior thrombotic events, given the extremely high thrombotic risk
In the complement inhibitor era: Eculizumab/ravulizumab dramatically reduces thrombotic risk (~85% reduction) → many expert guidelines no longer mandate primary anticoagulation in patients on complement inhibitors who have never had thrombosis
Decision: Individualised risk-benefit assessment

All PNH patients who have had a thrombotic event should receive long-term anticoagulation
Warfarin (target INR 2.0–3.0) has the most evidence in PNH-specific thrombosis
DOACs (direct oral anticoagulants): increasingly used; no PNH-specific trial data but reasonable based on extrapolation from general VTE guidelines
Duration: Usually lifelong if the PNH clone persists — because the thrombophilic state is ongoing
Complement inhibitor therapy should be initiated/continued simultaneously (treats the underlying cause of thrombosis, not just preventing clot formation)

Standard anticoagulation protocols (heparin → transition to oral anticoagulation)
Thrombolysis: May be considered for life-threatening thrombosis (massive PE with haemodynamic instability, extensive cerebral sinus thrombosis) — same principles as for any VTE ^[4]
Urgent initiation of complement inhibitor therapy: If not already on treatment, eculizumab should be started urgently alongside anticoagulation

6. Treatment of the Bone Marrow Failure Component

When PNH coexists with aplastic anaemia (AA/PNH overlap), the marrow failure component must be addressed:

Regimen: Horse antithymocyte globulin (ATG) + ciclosporin — the standard first-line treatment for severe aplastic anaemia
Rationale: AA is caused by T-cell-mediated destruction of HSCs → IST suppresses the autoreactive T-cells → allows residual HSCs (including PNH clones) to recover
Caveat: IST may allow the PNH clone to expand (since it was the PNH clone that had the immune escape advantage in the first place). Monitor PNH clone size after IST

EPO (erythropoietin-stimulating agents): For patients with suboptimal reticulocyte response
G-CSF: Occasionally used for severe neutropenia, but with caution (theoretical concern about promoting clonal evolution)

7. Allogeneic Haematopoietic Stem Cell Transplantation (HSCT) — The Only Cure

Allogeneic HSCT is the only curative treatment for PNH. It replaces the patient's PIGA-mutant haematopoietic stem cells with normal donor stem cells.

HSCT is not first-line for most PNH patients because complement inhibitors are highly effective and HSCT carries significant transplant-related mortality (5–10% in matched sibling donor transplants). HSCT is reserved for:

Indication	Rationale
Severe aplastic anaemia / PNH overlap (especially in young patients with a matched sibling donor)	Standard indication for severe AA; cures both the AA and PNH simultaneously
Life-threatening thrombotic complications refractory to anticoagulation + complement inhibitors	When medical therapy cannot control the thrombotic diathesis
Failure of or intolerance to complement inhibitor therapy	Some patients have breakthrough haemolysis or cannot access complement inhibitors
Transformation to MDS or AML	Clonal evolution of the PNH/AA stem cell population
Young patients in resource-limited settings without access to lifelong complement inhibitor therapy	Complement inhibitors are extremely expensive (~US$500,000/year); HSCT offers a one-time cure

Donor: Matched sibling donor preferred; matched unrelated donor or haploidentical transplant if no sibling donor
Conditioning: Reduced-intensity conditioning (RIC) increasingly used to reduce transplant-related toxicity
Complications: Graft-versus-host disease (GVHD), graft failure, infection, transplant-related mortality
Outcomes: Overall survival 70–90% at 5 years with matched sibling donor (variable with other donor types)

Pregnancy in PNH is high-risk due to:

Increased complement activation during pregnancy (physiological)
Hypercoagulable state of pregnancy compounded by PNH thrombophilia
Risk of haemolytic crises, thrombosis (especially postpartum), and foetal loss

Management principles:

Continue or initiate complement inhibitor therapy (eculizumab has been used in pregnancy; safety data are reassuring but limited)
Thromboprophylaxis with LMWH throughout pregnancy and for 6 weeks postpartum
Close monitoring by a multidisciplinary team (haematology, obstetrics, maternal-foetal medicine)
Folic acid supplementation (also serves as pre-conception folate for neural tube defect prevention)

Complication	Management
Haemolytic crisis (triggered by infection, surgery, etc.)	Supportive (transfusion, fluids, monitoring for AKI); complement inhibitor dose escalation or additional dose; treat the trigger
Budd-Chiari syndrome	Anticoagulation (heparin → warfarin/DOAC); consider thrombolysis if acute/severe; TIPS or liver transplant if refractory; start complement inhibitor urgently
Pulmonary hypertension	Complement inhibitor therapy (addresses NO depletion); standard PHT management if severe
Iron deficiency	Iron supplementation, ideally under complement inhibitor cover (to prevent worsening haemolysis from new reticulocyte production)
Chronic kidney disease	Complement inhibitor therapy slows progression; standard CKD management
Smooth muscle dystonia symptoms (dysphagia, abdominal pain, erectile dysfunction)	Complement inhibitor therapy (restores NO bioavailability); symptomatic relief (PDE5 inhibitors for erectile dysfunction)

Parameter	Frequency	Purpose
LDH	Every visit (monthly initially, then every 2–3 months)	Most sensitive marker of ongoing intravascular haemolysis; should normalise on C5 inhibitors
CBC, reticulocyte count	Every visit	Monitor anaemia response; persistent anaemia despite normal LDH → suspect extravascular haemolysis
Haptoglobin, bilirubin	Every visit	Haemolysis markers
PNH clone size (flow cytometry)	Every 6–12 months	Track clone stability; sudden expansion may herald disease progression
Iron studies	Every 6–12 months	Monitor for iron deficiency (ongoing urinary loss) or iron overload (from transfusions)
Renal function	Every 3–6 months	Monitor for haemoglobin-related nephropathy
Direct Coombs test	If persistent anaemia on C5 inhibitor	C3d positivity suggests C3b-mediated extravascular haemolysis (expected pharmacological consequence, not AIHA)
Meningococcal vaccination status	Annual review	Ensure boosters are given; maintain antibiotic prophylaxis awareness

Clinical Scenario	Treatment Approach
Subclinical PNH (clone < 1%, no haemolysis)	Observation; serial flow cytometry + CBC every 6–12 months
Mild/compensated haemolysis	Supportive care (folic acid, iron if deficient); monitor; anticoagulation if high thrombotic risk
Symptomatic haemolysis / transfusion-dependent	Complement inhibitor (eculizumab or ravulizumab); consider proximal inhibitor if suboptimal response
PNH with thrombosis	Anticoagulation + complement inhibitor therapy; lifelong anticoagulation
PNH/AA overlap	IST (ATG + ciclosporin) for marrow failure; complement inhibitor for haemolysis; consider HSCT if young + matched donor
Refractory / life-threatening disease	Allogeneic HSCT (only curative option)
Pregnancy	Complement inhibitor + LMWH thromboprophylaxis; multidisciplinary care

High Yield Summary — Management of PNH

Complement inhibitors are the cornerstone of modern PNH treatment:

C5 inhibitors (eculizumab, ravulizumab): Block MAC formation → dramatically reduce intravascular haemolysis and thrombosis. Limitation: do not prevent C3b opsonisation → residual extravascular haemolysis
Proximal complement inhibitors (pegcetacoplan [anti-C3], iptacopan [anti-Factor B], danicopan [anti-Factor D]): Block upstream → prevent both C3b opsonisation and MAC → address extravascular haemolysis problem

Critical safety measure: Meningococcal vaccination is mandatory before complement inhibitor therapy. Neisseria meningitidis is the MOST COMMON infection risk because MAC is the primary defence against Neisseria ^[18]

Anticoagulation: Thrombosis is the #1 killer; lifelong anticoagulation after thrombotic event; primary prophylaxis debated in complement inhibitor era

HSCT: Only cure; reserved for severe AA/PNH overlap, refractory disease, transformation to MDS/AML, or resource-limited settings without access to complement inhibitors

Supportive care: Folic acid (all patients); iron supplementation under complement inhibitor cover; transfusion as needed

Not curative except HSCT: Complement inhibitors suppress but do not eliminate the PNH clone; treatment is lifelong

Active Recall - Management of PNH

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf; Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf ^[4] Lecture slides: Block A - Leg swelling and chest pain_ deep vein thrombosis; pulmonary embolism; Thrombophilia.pdf ^[7] Senior notes: MBBS Final MB (Medicine) (Felix PY Lai).pdf (p. 1464–1470); MBBS Final MB (Pediatrics) (Felix PY Lai).pdf (p. 620–624) ^[17] Senior notes: Ryan Ho Haemtology.pdf (p. 39 — management of haemolytic anaemia: folic acid supplementation); Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf (G6PD management) ^[18] AOS material: AOS - Microbio.pdf (Question 4 — eculizumab and Neisseria meningitidis) ^[19] Senior notes: Gen Clerk Anaes + Microbiology Summary.pdf (post-splenectomy infection — encapsulated organisms)

Complications of Paroxysmal Nocturnal Haemoglobinuria (PNH)

PNH is not simply "anaemia from haemolysis." It is a systemic disease whose complications touch nearly every organ system. The complications arise from the three pathophysiological pillars — intravascular haemolysis, thrombophilia, and bone marrow failure — and from the downstream consequences of chronic free haemoglobin release (particularly NO depletion). Understanding the "why" behind each complication is what makes them predictable, not just a list to memorise.

2. Thrombotic Complications — The #1 Killer

PNH presents with hypercoagulability (thrombosis) in atypical locations — hepatic, portal, mesenteric, cerebral — which is the leading cause of death ^[7]. The mechanisms are multifactorial:

Free Hb scavenges NO → loss of NO's anti-platelet and vasodilatory effects → platelet activation + vasoconstriction
Procoagulant microparticles from lysed RBCs and complement-activated platelets → promote thrombin generation
Loss of CD87 (uPAR) on GPI-deficient monocytes/granulocytes → impaired urokinase-mediated fibrinolysis
Complement-mediated activation of GPI-deficient platelets → procoagulant state
Endothelial dysfunction from chronic free Hb exposure and complement deposition

Complication	Site	Why This Site?	Clinical Presentation	Prognosis
Budd-Chiari syndrome	Hepatic veins	Most characteristic thrombotic complication of PNH; hepatic veins are low-flow vessels susceptible to thrombosis in hypercoagulable states; NO depletion may preferentially affect hepatic sinusoidal endothelium	Acute: sudden RUQ pain, hepatomegaly, ascites, jaundice. Chronic: progressive portal hypertension, hepatic fibrosis, liver failure	Life-threatening; may require TIPS, liver transplant; or can be managed with anticoagulation + complement inhibitors if caught early
Cerebral venous sinus thrombosis	Dural venous sinuses (sagittal, transverse)	Cerebral sinuses are low-pressure, low-flow systems; hypercoagulable states disproportionately affect venous sinuses	Severe headache (often progressive, worse lying down), papilloedema, seizures, focal neurological deficits, altered consciousness	Can be fatal or cause permanent neurological disability; anticoagulation is mainstay
Mesenteric vein thrombosis	Superior/inferior mesenteric veins	Low-flow splanchnic circulation; NO depletion causes mesenteric vasoconstriction compounding the thrombotic tendency	Severe abdominal pain (often out of proportion to examination findings), nausea, vomiting, bloody stools (late), peritonitis if bowel infarction	Bowel ischaemia/infarction can be fatal; may require surgical resection
Portal vein thrombosis	Portal vein	Similar to mesenteric — low-flow splanchnic system	Abdominal pain, splenomegaly, ascites; can lead to portal hypertension	Contributes to chronic liver disease
DVT and PE	Lower limb deep veins, pulmonary arteries	Conventional VTE sites also affected due to systemic hypercoagulability ^[4]	Leg swelling, dyspnoea, pleuritic chest pain, haemodynamic instability	Standard DVT/PE management + complement inhibitors
Dermal vein thrombosis	Cutaneous vessels	Dermal microvasculature affected by NO depletion and procoagulant state	Painful, purpuric skin lesions that may mimic vasculitis	Usually self-limiting but indicates high systemic thrombotic risk

High Yield — Thrombosis in Unusual Sites

Any young patient presenting with thrombosis at an unusual site — especially Budd-Chiari, cerebral sinus, or mesenteric vein thrombosis — must be screened for PNH (flow cytometry) and MPN (JAK2 mutation). ^[4] This is repeatedly examined. The combination of Coombs-negative haemolytic anaemia + unusual-site thrombosis = PNH until proven otherwise.

3. Renal Complications

The kidneys are a major target organ in PNH because they are the front line for filtering the toxic free haemoglobin released by intravascular haemolysis.

Free haem and Hb are toxic → risk of AKI ^[5]
Mechanism: Free Hb is filtered at the glomerulus → in the acidic environment of the renal tubular lumen, Hb precipitates → direct cytotoxicity to tubular epithelial cells → acute tubular necrosis (ATN)
Can be precipitated by haemolytic crises (infections, surgery, complement-amplifying events) when massive Hb is released suddenly
Additionally, NO scavenging causes renal vasoconstriction → reduced renal blood flow → ischaemic injury

Repeated subclinical episodes of Hb-induced tubular injury → progressive tubular scarring
Haemosiderin deposition in renal cortex (visible on MRI as cortical signal changes) → chronic tubulointerstitial inflammation and fibrosis
Approximately 21% of PNH patients develop CKD stage 3 or worse over time
Complement inhibitor therapy (eculizumab/ravulizumab) significantly reduces free Hb exposure and slows CKD progression

Chronic haemoglobinuria causes tubular iron overload → oxidative stress → further tubular damage
A vicious cycle: haemolysis → Hb → kidney damage → reduced EPO production → worsened anaemia

This complication is counterintuitive and frequently tested.

In most haemolytic anaemias, iron is retained (recycled by macrophages from destroyed RBCs) → risk of iron overload (e.g. thalassaemia major)
In PNH, the haemolysis is intravascular → free Hb is filtered by kidneys → iron is lost in urine as haemoglobin and haemosiderin → chronic urinary iron loss → iron deficiency anaemia ^[5]
The iron deficiency superimposes on the haemolytic anaemia, worsening the overall anaemia
Paradoxically, treating the iron deficiency (giving iron supplements) can worsen haemolysis by providing substrate for new RBC production — these new reticulocytes are GPI-deficient and immediately vulnerable to complement

Why Iron Deficiency in PNH Is a Unique Exam Point

If an examiner gives you a patient with haemolytic anaemia AND iron deficiency — this should immediately make you think of intravascular haemolysis, specifically PNH. In extravascular haemolysis (e.g. AIHA, hereditary spherocytosis), iron is retained; you would NOT expect iron deficiency.

Mechanism: Free Hb scavenges NO → loss of NO-mediated pulmonary vasodilation → chronic pulmonary vasoconstriction → pulmonary arterial hypertension (PAH)
Additionally, in situ microthrombosis in pulmonary vasculature (chronic thromboembolic component) contributes
Clinical presentation: Progressive dyspnoea on exertion, reduced exercise capacity, right heart failure (elevated JVP, peripheral oedema, hepatomegaly)
Incidence: Up to 40% of untreated PNH patients have elevated pulmonary artery pressures on echocardiography
Management: Complement inhibitor therapy (restores NO bioavailability by reducing free Hb); standard PAH therapies if needed (phosphodiesterase-5 inhibitors, endothelin receptor antagonists)

This is the same mechanism as in sickle cell disease — both have chronic intravascular haemolysis → NO depletion → pulmonary hypertension. The common underlying pathophysiology is chronic free Hb in circulation.

NO is the master regulator of smooth muscle relaxation (endothelium releases NO → activates guanylyl cyclase → cGMP → smooth muscle relaxation). When free Hb scavenges NO, smooth muscle throughout the body goes into inappropriate spasm:

Complication	Anatomical Site	Clinical Presentation	Pathophysiology
Oesophageal spasm / dysphagia	Oesophageal smooth muscle	Difficulty swallowing, chest pain with swallowing	NO depletion → oesophageal smooth muscle spasm
Abdominal pain crises	Intestinal smooth muscle + mesenteric vasculature	Episodic severe abdominal pain, may mimic acute abdomen	NO depletion → intestinal smooth muscle spasm + mesenteric vasoconstriction; must also exclude mesenteric vein thrombosis
Erectile dysfunction	Corpus cavernosum smooth muscle	Inability to achieve/maintain erection	NO depletion → impaired cavernosal smooth muscle relaxation (same pathway targeted by PDE5 inhibitors like sildenafil)
Back pain	Renal vasculature, paraspinal vasculature	Episodic lower back pain, sometimes severe	Renal vasoconstriction + haemoglobin-induced renal tubular injury

These symptoms are often episodic and correlate with haemolytic episodes (because more haemolysis → more free Hb → more NO scavenging). They respond dramatically to complement inhibitor therapy, often within days of starting treatment.

7. Bone Marrow Failure and Haematological Complications

PNH exists on a spectrum with aplastic anaemia → many patients have concurrent bone marrow hypoplasia
This manifests as:
- Anaemia (from both haemolysis AND reduced marrow production → "double hit")
- Neutropenia → recurrent bacterial/fungal infections
- Thrombocytopenia → bleeding tendency (paradoxically coexisting with thrombotic tendency!)

There is an appreciable risk of developing clonal mutations → PNH may progress into a clonal disorder, e.g. MDS, AML ^[20]
The PIGA-mutant HSC, by virtue of being a long-lived clonal stem cell, can acquire additional somatic mutations over time
The risk is estimated at ~1–5% over the lifetime
Monitoring: require regular CBC to screen for development of these disorders ^[20]
Clinical clues to transformation:
- New-onset worsening cytopenias not explained by haemolysis
- Appearance of blasts or dysplastic cells on peripheral blood smear
- New cytogenetic abnormalities on bone marrow examination

Superimposed aplastic crisis (e.g. from parvovirus B19 infection → red cell aplasia → sudden severe anaemia with reticulocytopenia)
The marrow, already compromised, cannot compensate → life-threatening drop in Hb

Mechanism: Chronic haemolysis → increased unconjugated bilirubin production → hepatic excretion of excess bilirubin into bile → supersaturation → pigment (bilirubin) gallstones
This is the same complication seen in any chronic haemolytic anaemia (hereditary spherocytosis, thalassaemia, sickle cell disease) ^[5]
Type: Black pigment stones (calcium bilirubinate)
Clinical consequence: Biliary colic, cholecystitis, cholangitis
Important to distinguish from obstructive jaundice secondary to gallstones (which produces conjugated hyperbilirubinaemia and tea-coloured urine) versus the pre-hepatic jaundice of haemolysis (unconjugated hyperbilirubinaemia, no tea-coloured urine) ^[1]

9. Infections

Infections are typically bacterial (sepsis, pneumonia, UTI) but invasive fungal infection is an important cause of death ^[3]
The neutropenia from marrow failure makes patients susceptible to the same spectrum of infections seen in aplastic anaemia

Eculizumab blocks C5 and prevents formation of membrane attack complex. Neisseria meningitidis infection is the MOST COMMON infection risk in patients on eculizumab ^[18].

Why Neisseria? The MAC (C5b-9) is the primary host defence against Neisseria species. Unlike most bacteria that are killed by opsonophagocytosis (C3b pathway), Neisseria are uniquely dependent on MAC-mediated killing. Block MAC → Neisseria thrives
Risk of fulminant meningococcal septicaemia with DIC, purpura fulminans, and death within hours
Prevention: Mandatory meningococcal vaccination (MenACWY + MenB) + prophylactic antibiotics
With proximal complement inhibitors (blocking C3, Factor B/D), the infection risk spectrum broadens because C3b-mediated opsonisation is also impaired → increased susceptibility to all encapsulated organisms (pneumococcus, Haemophilus influenzae), similar to functional asplenia ^[19]

Exam Pearl — Encapsulated Organism Risk

The infection risks of complement inhibitor therapy mirror those of splenectomy / functional asplenia: encapsulated organisms that rely on complement and splenic clearance. The mnemonic "Some Nasty Killers Have Some Capsule Protection" — Streptococcus pneumoniae, Neisseria meningitidis, Klebsiella, Haemophilus influenzae, Salmonella, Cryptococcus, Pseudomonas — applies to both post-splenectomy patients and those on complement inhibitors ^[19].

PNH in pregnancy is a high-risk situation:

Complication	Mechanism
Haemolytic crises	Pregnancy physiologically increases complement activation → worsened haemolysis
Venous thromboembolism	Pregnancy is itself a hypercoagulable state (↑clotting factors, venous stasis) → superimposed on PNH's thrombophilic state → dramatically elevated VTE risk, especially postpartum
Pre-eclampsia/eclampsia	Possibly related to endothelial dysfunction from NO depletion and complement dysregulation
Foetal loss / IUGR	Placental thrombosis and endothelial dysfunction
Postpartum haemorrhage	If concurrent thrombocytopenia from marrow failure

Historical maternal mortality in PNH was ~10–20%; with complement inhibitor therapy and LMWH thromboprophylaxis, outcomes have improved dramatically, though pregnancy remains high-risk.

11. Complications of Treatment

Can occur with:
- Missed or delayed doses (complement blockade wears off → sudden MAC formation → massive haemolysis)
- Complement-amplifying conditions: infections, surgery, pregnancy, vaccinations
- Pharmacokinetic breakthrough: some patients metabolise the drug faster (especially with concurrent infections that increase complement production)
Presents as sudden dark urine, falling Hb, rising LDH
Management: Supplemental dose of complement inhibitor; treat the trigger

As discussed in the management section: C5 inhibitors block MAC but allow C3b opsonisation → extravascular haemolysis by splenic/hepatic macrophages
Manifests as persistent anaemia despite normalised LDH; new positive DAT for C3d
Not a "complication" in the traditional sense but a pharmacological limitation

If the patient undergoes allogeneic HSCT, the standard transplant complications apply ^[21]:

Timing	Complications
Early ( < 1 year)	Graft rejection; acute GvHD (skin rash, hepatitis, diarrhoea); veno-occlusive disease of liver; neutropenic infections (bacterial, fungal); mucositis; haemorrhage
Late ( > 1 year)	Chronic GvHD; secondary malignancy; cardiovascular disease; endocrine dysfunction (hypothyroidism, hypogonadism, T2DM); infertility; cataracts (from TBI); osteoporosis (from steroids); relapse

Organ System	Complication	Pathophysiology
Vascular	Thrombosis (Budd-Chiari, cerebral sinus, mesenteric, DVT/PE)	NO depletion, procoagulant microparticles, impaired fibrinolysis, platelet activation
Renal	AKI, CKD, haemosiderinuria	Free Hb nephrotoxicity, haemosiderin deposition, NO-mediated vasoconstriction
Haematological	Iron deficiency anaemia	Urinary iron loss from intravascular haemolysis
Haematological	Pancytopenia	Concurrent bone marrow failure (AA/PNH overlap)
Haematological	MDS/AML transformation	Clonal evolution of PIGA-mutant HSCs
Pulmonary	Pulmonary hypertension	NO depletion → pulmonary vasoconstriction; microthrombosis
GI	Dysphagia, abdominal pain crises	NO depletion → smooth muscle dystonia
GI	Mesenteric ischaemia/infarction	Mesenteric vein thrombosis
Hepatobiliary	Budd-Chiari syndrome; pigment gallstones	Hepatic vein thrombosis; chronic haemolysis → bilirubin stones
Reproductive	Erectile dysfunction; pregnancy complications	NO depletion; hypercoagulability in pregnancy
Infectious	Meningococcal infection (on eculizumab); bacterial/fungal infections (neutropenia)	MAC blockade; bone marrow failure
Neurological	Cerebral sinus thrombosis; headache	Thrombosis; NO depletion → cerebral vasoconstriction

High Yield Summary — Complications of PNH

The #1 killer is thrombosis — at unusual sites (Budd-Chiari, cerebral sinuses, mesenteric veins). Mechanism: NO depletion from free Hb scavenging + procoagulant microparticles + impaired fibrinolysis.

Renal complications: AKI (free Hb nephrotoxicity) and CKD (chronic haemosiderin deposition + tubular injury). Free haem and Hb are toxic → risk of AKI ^[5].

Iron deficiency in haemolysis: Paradox unique to intravascular haemolysis — iron lost in urine, not retained as in extravascular haemolysis.

NO depletion syndrome: Pulmonary hypertension, dysphagia, abdominal pain crises, erectile dysfunction — all from smooth muscle dystonia due to NO scavenging.

Clonal evolution: Risk of MDS/AML transformation (~1–5% lifetime); requires regular CBC monitoring ^[20].

Infection risk: Neisseria meningitidis is the MOST COMMON infection on eculizumab (MAC is the primary defence against Neisseria) ^[18]. Neutropenia from marrow failure → bacterial/fungal infections.

Gallstones: Pigment stones from chronic unconjugated hyperbilirubinaemia.

Pregnancy: High-risk — haemolytic crises + dramatically elevated thrombotic risk; requires complement inhibitors + LMWH.

Active Recall - Complications of PNH

References

^[1] Lecture slides: GC 047. Family history of anaemia.pdf; Senior notes: Block A - Family history of anaemia_ inherited causes of anaemia; haemolytic anaemia; aplastic anaemia.pdf ^[3] Senior notes: Ryan Ho Haemtology.pdf (Section 2.4.2 — Aplastic Anaemia, p. 32) ^[4] Lecture slides: Block A - Leg swelling and chest pain_ deep vein thrombosis; pulmonary embolism; Thrombophilia.pdf ^[5] Senior notes: Ryan Ho Haemtology.pdf (p. 36 — intravascular haemolysis pathology: free Hb toxic → risk of AKI, DIC, thrombosis) ^[7] Senior notes: MBBS Final MB (Medicine) (Felix PY Lai).pdf (p. 1464 — PNH presents with thrombosis at atypical locations as leading cause of death) ^[18] AOS material: AOS - Microbio.pdf (Question 4 — eculizumab and Neisseria meningitidis) ^[19] Senior notes: Block A - Splenomegaly_ common causes of splenomegaly; myeloproliferative diseases.pdf (p. 20 — OPSI, encapsulated organisms); Maksim Surgery Notes.pdf (p. 153 — splenectomy complications) ^[20] Senior notes: Adrian Lui Pediatrics Notes.pdf (p. 369 — appreciable risk of clonal disorders, monitoring with regular CBC) ^[21] Senior notes: Ryan Ho Haemtology.pdf (p. 156 — Complications and Prognosis of HSCT)

High Yield Summary

The Three Pillars of PNH:

Intravascular haemolysis — loss of CD59 → MAC-mediated RBC lysis → haemoglobinaemia, haemoglobinuria, ↓↓haptoglobin, iron deficiency from urinary iron loss
Thrombosis — #1 killer; unusual sites (Budd-Chiari, cerebral sinuses, mesenteric veins); caused by NO depletion, procoagulant microparticles, impaired fibrinolysis
Bone marrow failure — overlap with aplastic anaemia; immune escape hypothesis explains clonal expansion

Key "Why" Points:

Why nocturnal? Sleep → mild respiratory acidosis → ↓pH → enhanced complement activation (mainly historical/oversimplified)
Why iron deficiency in haemolysis? Intravascular → Hb lost in urine → chronic urinary iron loss
Why thrombosis? Free Hb scavenges NO → platelet activation + vasoconstriction; loss of CD87 → impaired fibrinolysis
Why dysphagia/abdominal pain/ED? NO depletion → smooth muscle dystonia
Why overlap with AA? Immune escape: PIGA-mutant HSCs survive T-cell attack → selective clonal expansion

Classification: Classical PNH / PNH with another marrow disorder / Subclinical PNH.

High Yield Summary — Differential Diagnosis of PNH

When PNH presents as haemolytic anaemia:

Key DDx: AIHA (Coombs +ve), TTP/HUS (schistocytes), G6PD deficiency (oxidant triggers), hereditary spherocytosis (family Hx, spherocytes), infections (malaria, C. perfringens)
PNH is the only acquired intrinsic RBC defect — all others are extrinsic

When PNH presents as pancytopenia:

Key DDx: Aplastic anaemia (hypocellular marrow), MDS (dysplastic features), leukaemia (blasts), megaloblastic anaemia (B12/folate)
Screen all AA patients for PNH clones by flow cytometry

When PNH presents as thrombosis at unusual sites:

Key DDx: MPN (JAK2+), APS, inherited thrombophilia (AT/PC/PS deficiency), malignancy
Budd-Chiari in a young patient = screen for PNH and JAK2

When PNH presents as dark urine:

Distinguish haemoglobinuria from haematuria (no RBCs on microscopy) and myoglobinuria (clear serum, ↑CK)
PNH causes false positive urine dipstick for blood — not true haematuria

The definitive differentiating test for PNH is flow cytometry for GPI-anchored proteins (CD55, CD59) or FLAER assay.

High Yield Summary — Diagnosis of PNH

The diagnostic test: Flow cytometry on peripheral blood detecting deficiency of GPI-anchored proteins (CD55, CD59) — the MOST useful and accepted method ^[7]

The modern gold standard: FLAER assay on granulocytes/monocytes — binds the GPI anchor directly; most sensitive for small clones

Key supporting findings:

↑ LDH, ↓ haptoglobin, ↑ unconjugated bilirubin = haemolysis ^[7]
Negative Coombs test = not immune-mediated
Haemoglobinuria + haemosiderinuria = intravascular haemolysis ^[7]
No schistocytes, no spherocytes on PBS = not MAHA or AIHA
BM biopsy if cytopenias — assess for AA/MDS overlap

When to screen: AA (up to 50% have PNH clones), Coombs-negative haemolysis, thrombosis at unusual sites, unexplained cytopenias, false-positive urine dipstick for blood

Obsolete tests: Ham test (acidified serum lysis), sucrose lysis test — replaced by flow cytometry

Classification by flow cytometry: Type I (normal), Type II (partial deficiency), Type III (complete deficiency). Clone size = Type II + III percentage.

High Yield Summary — Management of PNH

Complement inhibitors are the cornerstone of modern PNH treatment:

C5 inhibitors (eculizumab, ravulizumab): Block MAC formation → dramatically reduce intravascular haemolysis and thrombosis. Limitation: do not prevent C3b opsonisation → residual extravascular haemolysis
Proximal complement inhibitors (pegcetacoplan [anti-C3], iptacopan [anti-Factor B], danicopan [anti-Factor D]): Block upstream → prevent both C3b opsonisation and MAC → address extravascular haemolysis problem

Anticoagulation: Thrombosis is the #1 killer; lifelong anticoagulation after thrombotic event; primary prophylaxis debated in complement inhibitor era

HSCT: Only cure; reserved for severe AA/PNH overlap, refractory disease, transformation to MDS/AML, or resource-limited settings without access to complement inhibitors

Supportive care: Folic acid (all patients); iron supplementation under complement inhibitor cover; transfusion as needed

Not curative except HSCT: Complement inhibitors suppress but do not eliminate the PNH clone; treatment is lifelong

High Yield Summary — Complications of PNH

Renal complications: AKI (free Hb nephrotoxicity) and CKD (chronic haemosiderin deposition + tubular injury). Free haem and Hb are toxic → risk of AKI ^[5].

Iron deficiency in haemolysis: Paradox unique to intravascular haemolysis — iron lost in urine, not retained as in extravascular haemolysis.

NO depletion syndrome: Pulmonary hypertension, dysphagia, abdominal pain crises, erectile dysfunction — all from smooth muscle dystonia due to NO scavenging.

Clonal evolution: Risk of MDS/AML transformation (~1–5% lifetime); requires regular CBC monitoring ^[20].

Gallstones: Pigment stones from chronic unconjugated hyperbilirubinaemia.

Pregnancy: High-risk — haemolytic crises + dramatically elevated thrombotic risk; requires complement inhibitors + LMWH.

Paroxysmal Nocturnal Haemoglobinuria

1. Definition

2. Epidemiology

3. Risk Factors

4. Relevant Anatomy & Physiology

4.1 The GPI Anchor System

4.2 Key GPI-Anchored Proteins Lost in PNH

4.3 The Complement System — Quick Refresher

4.4 Why "Nocturnal"?

5. Aetiology & Pathophysiology

5.1 The PIGA Mutation — The Initiating Event

5.2 Clonal Expansion — Why Does the Mutant Clone Take Over?

5.3 Consequences of GPI-Anchor Deficiency

5.3.1 Intravascular Haemolysis

5.3.2 Thrombophilia

5.3.3 Bone Marrow Failure

5.4 Downstream Consequences of Chronic Intravascular Haemolysis

6. Classification

7. Clinical Features

7.1 Symptoms (with pathophysiological basis)

7.2 Signs (with pathophysiological basis)

7.3 Important History Points to Elicit [1]

8. Pathophysiology Summary Diagram

9. Relationship Between PNH and Aplastic Anaemia

10. Why PNH Matters in Other Clinical Contexts

10.1 As a Cause of Haemolytic Anaemia

10.2 As a Cause of Thrombophilia

10.3 As a Cause of Positive Urine Dipstick

10.4 As a Cause of Pancytopenia

Active Recall - Paroxysmal Nocturnal Haemoglobinuria (PNH)

References

1. Differential Diagnosis Framework — By Presenting Syndrome

2. DDx When PNH Presents as Haemolytic Anaemia

2.1 Intravascular Haemolysis — Differential Diagnosis

2.2 Coombs-Negative Haemolytic Anaemia — Systematic DDx

3. DDx When PNH Presents as Pancytopenia

4. DDx When PNH Presents as Thrombosis at Unusual Sites

4.1 Acquired Thrombophilia

4.2 Inherited Thrombophilia

5. DDx When PNH Presents as Dark Urine (Haemoglobinuria)

6. DDx When PNH Presents as Coombs-Negative Haemolytic Anaemia with Liver Failure

7. Diagnostic Differentiation Summary Table

8. Clinical Approach to Differentiating PNH — Decision Algorithm

Active Recall - Differential Diagnosis of PNH

1. Can We "Diagnose" PNH the Way We Diagnose Diabetes or SLE? — Understanding the Diagnostic Framework

2. When to Suspect PNH — Clinical Indications for Testing

3. The Confirmatory Test — Flow Cytometry for GPI-Anchored Proteins

3.1 Why Flow Cytometry?

3.2 What Markers Are Tested?

3.3 FLAER Assay — The Gold Standard

3.4 Interpretation of Flow Cytometry Results — PNH Cell Types

3.5 Clone Size and Clinical Correlation

4. Diagnostic Algorithm

5. Complete Investigation Panel — Systematic Approach

5.1 Confirming the Diagnosis

5.2 Demonstrating Haemolysis

5.3 Evidence of Intravascular Haemolysis (Specific)

5.4 Complete Blood Count & Peripheral Blood Smear

5.5 Iron Studies

5.6 Bone Marrow Examination

5.7 Thrombophilia and Thrombosis Workup

5.8 Renal Function Assessment

5.9 Additional / Supportive Investigations

6. Historical / Obsolete Tests (For Completeness)

7. Monitoring After Diagnosis

8. Summary of Diagnostic Approach — Key Decision Points

Active Recall - Diagnosis & Investigations of PNH

1. Principles of Management — The Big Picture

2. Management Algorithm

3. Supportive Care — The Foundation for All PNH Patients

3.1 Folic Acid Supplementation

3.2 Iron Supplementation (When Indicated)

3.3 Blood Transfusion

3.4 Erythropoietin (EPO)

4. Complement Inhibitor Therapy — The Cornerstone of Modern PNH Treatment

4.1 Terminal Complement Inhibitors (C5 Inhibitors)

Eculizumab

Ravulizumab

4.2 Proximal Complement Inhibitors — Addressing the Extravascular Haemolysis Problem

4.3 Critical Safety Concern — Meningococcal Infection Risk