Paroxysmal Nocturnal Hemoglobinuria

Slide 1 · Paroxysmal Nocturnal

Hemoglobinuria

Clonal stem-cell disease • complement-mediated hemolysis • thrombosis

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning outcomes

Trace an acquired PIGA mutation to loss of GPI-linked complement regulators.

Explain why PNH produces intravascular hemolysis, urinary iron loss, and thrombosis.

Interpret mixed normal and PNH cell populations in flow cytometry.

Relate PNH to aplastic anemia, myeloid neoplasms, and complement inhibition.

Slide 3 · Three interacting facets of PNH

Clonal hematopoiesis

Acquired PIGA mutation in a hematopoietic stem cell

Intravascular hemolysis

Complement attacks red cells lacking surface protection

Marrow / vascular disease

Aplasia, thrombosis, and occasional myeloid evolution

Slide 4 · PNH is an acquired genetic hemolytic anemia

The initiating defect is somatic, not a conventional inherited red-cell mutation.

PIGA is required to construct GPI anchors for selected cell-surface proteins.

Cells from the mutant stem-cell clone have reduced GPI-anchored proteins.

A genetic disorder can be acquired after conception.

Slide 5 · PNH is rare but potentially high risk

A US incidence of 2–5 per million; The excerpt does not specify the time denominator.

Its significance extends beyond anemia because thrombosis is a major cause of morbidity and death.

Clinical presentation varies: The stereotypical nocturnal attacks are not required.

Slide 6 · The initiating lesion occurs in a stem cell

Somatic PIGA mutation

Hematopoietic stem cell

Defective GPI anchoring

Mutant hematopoietic clone

Multilineage descendants

Red cells • white cells • platelets

Slide 7 · The abnormal phenotype spans blood lineages

Normal stem cell

HSC

Erythrocyte

Leukocyte

Platelet

GPI-linked proteins retained

PIGA-mutant stem cell

HSC

Erythrocyte

Leukocyte

Platelet

GPI-linked proteins deficient

The initiating PIGA lesion is carried across hematopoietic lineages.

Slide 8 · PIGA acts upstream of membrane-protein loss

Active PIGA

GPI-anchor biosynthesis

GPI anchor

Surface attachment of selected proteins

Functional protection

CD55 and CD59 on blood cells

The mutation disrupts the anchor, rather than independently mutating CD55 and CD59.

Slide 9 · Why a single PIGA mutation can suffice

PIGA is located on the X chromosome.

In cells with two X chromosomes, lyonization leaves one transcriptionally active copy.

An acquired pathogenic mutation in the active PIGA allele can remove functional GPI anchoring.

Slide 10 · Mutant and normal stem cells coexist

Normal stem cell

GPI anchors preserved

CD55+ / CD59+ progeny

PIGA-mutant stem cell

GPI anchors deficient

CD55− / CD59− progeny

Mixed populations are expected; This is clonal mosaicism, not a uniform change in every blood cell.

Slide 11 · A mutation alone may not explain clone expansion

Small PIGA-mutant populations have been described in otherwise healthy individuals.

Clinical disease generally requires expansion of the mutant population.

Immune-mediated selection as a hypothesis, not a proven universal mechanism.

Separate established PIGA causation from proposed selection pressure.

Slide 12 · Aplastic anemia provides an evolutionary context

Immune attack on marrow cells

Possible selective pressure

Relative survival of a PIGA-mutant clone

Clonal expansion

Coexisting PNH + marrow failure

Variable cytopenias

Hypothesized selection model: Not every PNH clone develops this way.

Slide 13 · The PNH–aplasia association is bidirectional

In a patient with PNH

Anemia may reflect red-cell destruction

Marrow failure can coexist and limit compensation

Other cytopenias call for marrow evaluation

In a patient with aplastic anemia

A PNH clone may coexist

Immune selection is one proposed explanation

Clinical significance depends on phenotype and clone behavior

Slide 14 · GPI anchors differ from transmembrane segments

Integral transmembrane protein

Extracellular

Cytoplasmic side

GPI-anchored protein

Extracellular

Cytoplasmic side

Protein spans membrane

Protein linked to outer-leaflet GPI

Slide 15 · GPI deficiency removes several proteins at once

The primary lesion is loss of a common anchoring mechanism.

Multiple distinct membrane proteins can therefore be absent together.

The key hemolysis-relevant losses are CD55 and CD59; Robbins also names C8-binding protein.

Slide 16 · Membrane complement control: Two levels

CD55 / DAF

Destabilizes C3/C5 convertases

Limits complement amplification

CD59 / MIRL

Prevents terminal C5b–9 assembly

Blocks membrane pore formation

Verified correction to the supplied Robbins wording: CD59 does not principally inhibit C3 convertase.

Slide 17 · CD55: Decay-accelerating factor

CD55 is a GPI-anchored regulator at the convertase stage.

It accelerates dissociation of C3 convertases and also regulates C5 convertases.

Its loss permits increased complement activation on the cell surface.

Slide 18 · CD59: The terminal protective barrier

CD59 is a GPI-anchored membrane inhibitor of reactive lysis.

It prevents incorporation/polymerization of C9 in the developing membrane attack complex.

Loss of CD59 leaves a red cell susceptible to terminal complement-mediated lysis.

Slide 19 · CD55 and CD59 protect distinct checkpoints

C3 convertase activity

Normally restrained by CD55

C5 cleavage

C5a released; C5b generated

C5b–9 assembly

Normally restrained by CD59

Uncontrolled terminal attack

Intravascular RBC lysis

Slide 20 · Complement can operate without a new antibody

The alternative complement pathway has ongoing low-level activity.

Normal blood cells require surface regulatory proteins to avoid bystander injury.

Deficiency of the protective GPI-linked regulators makes PNH cells unusually vulnerable.

Slide 21 · Terminal complement creates membrane pores

C5 convertase

Cleaves C5 to C5a + C5b

C5b recruits C6–C9

Assembling membrane attack complex

Pore formation

Membrane integrity lost

Hemoglobin release

Intravascular hemolysis

Slide 22 · Normal and PNH erythrocytes face the same complement

Protected red cell

CD55

CD59

Complement attack is restrained

PNH red cell

C5b–9 MAC

Loss of CD55/CD59 permits lysis

Original RBC–complement schematic; Not a photomicrograph.

Slide 23 · Intravascular hemolysis is the central red-cell lesion

MAC injures red-cell membrane

Lytic pore formation

RBC ruptures within circulation

Free hemoglobin enters plasma

Hemoglobin-handling systems saturate

Hemoglobinuria / hemosiderinuria

Slide 24 · Biochemical effects of red-cell lysis

Premature red-cell destruction shortens circulating erythrocyte survival.

Anemia induces erythropoietin release and compensatory erythropoiesis.

The parent hemolytic-anemia section describes increased bilirubin products and reduced haptoglobin.

Slide 25 · Intravascular and extravascular hemolysis differ

Feature

Intravascular

Extravascular

Primary destruction

In bloodstream

Within phagocytes

Free plasma hemoglobin

Prominent

Usually less prominent

Hemoglobinuria

May occur

Not characteristic

Splenic sequestration

Not primary

Central mechanism

Slide 26 · The clinical label does not describe most cases

Only a minority have strictly paroxysmal, nocturnal hemoglobinuria.

That a chronic hemolytic pattern is more typical.

An absence of dramatic dark urine does not exclude PNH.

Do not require night-time attacks to suspect PNH.

Slide 27 · The nocturnal pattern has a proposed mechanism

A modest fall in blood pH during sleep may favor complement activity.

This can amplify hemolysis in susceptible red cells.

The finding explains the historical name without making it a diagnostic requirement.

Slide 28 · Hemoglobinuria follows overwhelming free hemoglobin

Intravascular RBC lysis

Hemoglobin released

Haptoglobin binding capacity depleted

Free circulating hemoglobin

Renal filtration

Hemoglobinuria

Slide 29 · Hemosiderinuria records chronic urinary iron loss

Renal tubular cells take up and catabolize filtered hemoglobin.

Iron can accumulate in tubular cells as hemosiderin.

Urinary shedding of iron-containing material can progressively deplete body iron.

Slide 30 · PNH can become iron deficient despite hemolysis

Persistent intravascular lysis

Heme iron released

Hemoglobin / renal iron loss

Urinary iron excretion

Progressive iron depletion

Iron-deficient erythropoiesis

Additional anemia

Beyond red-cell destruction alone

Slide 31 · The anemia reflects several simultaneous processes

Destruction

Complement-mediated lysis of circulating erythrocytes

Nutrient loss

Urinary iron loss can impair hemoglobin synthesis

Production failure

Coexisting aplasia may blunt marrow compensation

Slide 32 · The three major adverse trajectories

Hemolytic disease

Chronic complement injury and iron loss

Thrombotic disease

Venous clots, often in atypical sites

Stem-cell disease

Marrow aplasia or evolution to myeloid neoplasm

Slide 33 · Thrombosis dominates disease-related mortality

Thrombosis as the leading cause of PNH-related death.

Venous events are especially characteristic.

Thrombosis in approximately 40% of patients in its cited disease context.

A clot may be more immediately dangerous than the anemia.

Slide 34 · Recognize unusual venous thrombosis sites

Hepatic veins

Outflow obstruction should prompt consideration of PNH

Portal veins

Splanchnic thrombosis is an important clue

Cerebral veins

An unusual-site venous event can reveal PNH

Slide 35 · The thrombotic mechanism is incompletely settled

Complement activation and thrombotic risk are linked.

Robbins does not present a single established pathway explaining every event.

Candidate contributors include nitric oxide depletion and endothelial injury.

Mechanistic hypotheses are not equivalent to proven single-cause explanations.

Slide 36 · Free hemoglobin may reduce nitric oxide availability

Intravascular hemolysis

Extracellular hemoglobin released

Nitric oxide scavenging

Reduced NO bioavailability

Vascular dysfunction

Plausible contribution to thrombosis

Mechanistic hypothesis, not a complete explanation of thrombosis.

Slide 37 · Terminal complement may injure endothelium

Complement activation

Generation of C5b–9

Potential endothelial injury

Prothrombotic vascular state

Venous thrombosis

Clinical complication

Proposed contributory pathway; Association does not prove this sequence in every patient.

Slide 38 · PNH is a stem-cell disorder, not only an anemia

The clone arises in multipotent hematopoiesis.

Marrow failure and cytopenias may accompany complement-mediated hemolysis.

A fraction of patients later develop a myeloid neoplasm.

Slide 39 · Aplastic anemia impairs blood-cell production

Aplastic anemia is marrow hypocellularity with hematopoietic failure.

Anemia, neutropenia, and thrombocytopenia can occur together.

A patient with PNH may have both peripheral destruction and central production failure.

Slide 40 · PNH and immune marrow injury may intersect

Immune-mediated progenitor injury

Often implicated in aplastic anemia

Surviving mutant clone

Possible relative advantage

PNH phenotype alongside aplasia

Hemolysis ± pancytopenia

Model presented as a mechanistic hypothesis, not a proven inevitable sequence.

Slide 41 · Myeloid neoplasms represent clonal progression

Occasional evolution to acute myeloid leukemia or myelodysplastic neoplasm.

This in about 5% of affected patients.

This supports the importance of stem-cell genetic damage beyond the red-cell phenotype.

Slide 42 · Three patterns prompt diagnostic consideration

Unexplained hemolysis

Especially intravascular with urinary hemoglobin/iron loss

Atypical venous thrombosis

Hepatic, portal, or cerebral site

Marrow failure

Aplastic anemia with a possible PNH clone

Slide 43 · Flow cytometry demonstrates the cellular defect

A test for PNH looks for absent or decreased GPI-linked surface proteins.

Loss of erythrocyte CD59 and describes CD55/CD59 double-negative cells.

The method can demonstrate coexistence of affected and unaffected populations.

Slide 44 · Mixed populations are the hallmark of clonal mosaicism

Population A

CD55+/CD59+

Normal GPI-anchored phenotype

Population B

CD55−/CD59−

PNH-phenotype clone

Conceptual phenotype labels, not a reproduced clinical flow plot.

Slide 45 · What a CD55/CD59 pattern means

Cell phenotype

Interpretation

CD55+ / CD59+

GPI-linked regulators retained

CD55− / CD59−

Typical loss of both regulators in PNH clone

Mixed positive and negative groups

Residual normal hematopoiesis coexists with mutant clone

This table is an educational reconstruction of the Robbins Fig. 14.13 description.

Slide 46 · Contemporary diagnostic practice uses multiple lineages

High-sensitivity testing can assess erythrocytes, granulocytes, and monocytes.

Leukocyte clone fractions are less distorted by prior red-cell transfusion and selective hemolysis.

The selected lineage and gating strategy must be documented.

Slide 47 · FLAER interrogates the GPI anchor itself

Fluorescent aerolysin derivative (FLAER) binds GPI anchors.

Granulocyte/monocyte assays combine FLAER with lineage and GPI-linked markers.

Interpret results with laboratory-specific controls and analytical sensitivity.

Slide 48 · Why red-cell clone size may underestimate burden

Erythrocytes

PNH red cells may be destroyed by complement

Transfused cells contribute normal phenotypes

Measured fraction may underrepresent marrow clone

Granulocytes / monocytes

Represent another progeny compartment

Less affected by RBC transfusion and lysis

Useful for clonal assessment

Slide 49 · Flow-cytometry interpretation requires controls

One absent signal in one lineage is not sufficient for comprehensive characterization.

Confirm GPI deficiency with an appropriate multiparameter panel and multiple cellular populations.

Consider technical gating, specimen quality, and recent transfusion before interpretation.

Slide 50 · PNH vs immunohemolytic anemia

Feature

PNH

Immunohemolytic anemia

Primary lesion

Acquired GPI-anchor defect

Red-cell reactive antibody

Complement vulnerability

Lost CD55/CD59 protection

Antibody-associated mechanisms vary

Key diagnostic approach

GPI-protein flow cytometry

Direct antiglobulin testing

RBC destruction

Characteristically intravascular

Often extravascular in warm type

Slide 51 · PNH among causes of intravascular hemolysis

Category

Example mechanism

Distinguishing principle

GPI-anchor defect

PNH / complement MAC

Deficient GPI-linked proteins

Mechanical trauma

Valve or microvascular shear

Physical fragmentation

Immune complement

Some antibody-associated hemolysis

Evidence of immunoglobulin/complement binding

Infection / toxin

Parasite or toxin-mediated injury

Trigger-specific context

Slide 52 · Clinical case: Chronic hemolysis without dark urine

A patient has persistent laboratory features of hemolysis but reports no nocturnal attacks of visibly red urine. Does this exclude PNH?

Assess whether PNH could still produce chronic hemolysis.

Name the most direct phenotypic test.

Predict whether all circulating red cells must be abnormal.

Slide 53 · Case interpretation: The name can mislead

Chronic hemolysis is compatible with PNH.

Flow cytometry for deficient GPI-linked proteins is the defining diagnostic approach.

Mixed normal and deficient populations are expected when normal stem-cell progeny persist.

Slide 54 · Clinical case: Hepatic-vein thrombosis

A patient presents with unexplained hepatic venous thrombosis and anemia. What unified hematologic process should be considered?

Identify the thrombotic complication pattern.

Connect red-cell complement sensitivity to the initiating clone.

Select a test that could support the hypothesis.

Slide 55 · Case interpretation: Recognize the vascular clue

PNH can present with thrombosis in atypical venous territories.

The PIGA-mutant hematopoietic clone generates complement-sensitive cells.

Peripheral-blood flow cytometry can identify GPI-deficient populations.

Slide 56 · Complement inhibition intervenes at C5

Complement activation upstream

C3 convertases may still operate

C5 inhibitor

Prevents generation of C5a and C5b

MAC cannot form

Reduced intravascular hemolysis

Slide 57 · Why C5 inhibition changes the clinical course

Blocking terminal complement prevents formation of the lytic C5b–9 complex.

Reductions in hemolysis and transfusion requirements.

It also reports a large reduction in thrombotic risk with C5 inhibition.

Slide 58 · C5 blockade does not repair the stem-cell mutation

Effect achieved

Suppresses terminal complement attack

Reduces complement-mediated RBC lysis

Can reduce downstream clinical complications

What remains

PIGA-mutant clone persists

GPI-anchor deficiency remains

Marrow disease needs independent assessment

Slide 59 · Residual anemia under C5 blockade: A further mechanism

C3 deposition can continue when proximal complement activation is not blocked.

C3-opsonized red cells may undergo extravascular clearance.

This is one reason control of intravascular lysis does not guarantee normal hemoglobin.

Slide 60 · Complement-targeted drugs require infection precautions

Terminal complement has an important role in defense against Neisseria meningitidis.

Serious or fatal meningococcal infection as a treatment hazard.

Inhibitor choice must be paired with infection-prevention planning.

The infection risk is clinically actionable, not a theoretical footnote.

Slide 61 · Risk reduction does not eliminate meningococcal risk

CDC recommends meningococcal vaccination for people receiving complement inhibitors.

Vaccination and antimicrobial approaches may offer incomplete protection.

Suspected meningococcal disease requires urgent evaluation despite prevention measures.

Slide 62 · Marrow-directed therapy addresses a different component

Immunosuppressive therapy can help some patients with accompanying marrow aplasia.

It targets immune marrow suppression rather than directly replacing GPI anchors.

Distinguish treatment of hemolysis from treatment of marrow failure.

Slide 63 · Hematopoietic transplantation is potentially curative

Replacing the abnormal hematopoietic stem-cell compartment can remove the source of the clone.

Transplantation as the curative strategy.

It is usually reserved for serious concurrent aplastic anemia or myeloid malignancy because of treatment risk.

Slide 64 · Management logic depends on the dominant phenotype

Hemolysis / thrombosis

Complement-directed treatment and complication assessment

Marrow aplasia

Evaluate hematopoietic failure; Consider marrow-directed therapy

Myeloid malignancy

Specialist neoplasm-directed evaluation; Transplantation may be relevant

Slide 65 · High-yield synthesis: Mutation to clinical harm

PIGA-mutant HSC

Mixed mutant and normal progeny

GPI-linked CD55 / CD59 deficiency

Complement dysregulation

C5b–9-mediated erythrocyte lysis

Hemolysis + urinary iron loss

Systemic complications

Thrombosis; Marrow disease

Slide 66 · Check your understanding

Which statement identifies the mechanistic distinction most important for understanding PNH?

A. CD55 blocks terminal C9 insertion; CD59 degrades C3 convertase.

B. CD55 accelerates convertase decay; CD59 blocks MAC formation.

C. PIGA directly encodes the CD59 protein.

D. PNH affects only mature erythrocytes.

Slide 67 · Mechanistic answer: B

CD55 acts mainly at the C3/C5 convertase stage.

CD59 protects at terminal membrane attack complex assembly.

A PIGA mutation disrupts the shared GPI anchoring of multiple proteins.

Slide 68 · Clinical integration: Diagnostic priorities

Think of PNH when hemolysis coexists with unusual venous thrombosis or marrow aplasia.

Demonstrate GPI-linked protein deficiency in blood-cell populations by flow cytometry.

Interpret clonal mosaicism and the difference between red-cell and leukocyte fractions.

Assess hemolytic, thrombotic, and marrow components separately.

Slide 69 · Selected source references

Robbins, Cotran & Kumar: Pathologic Basis of Disease. 11th ed. 2026. Chapter 14, pp. 579–580, 589–590, 599–601.

Brodsky RA. Paroxysmal nocturnal hemoglobinuria. Blood. 2014;124:2804–2811.

Risitano AM et al. The complement alternative pathway in PNH. Immunological Reviews. 2023.

Singapore expert consensus on PNH diagnosis and treatment. Ann Acad Med Singap. 2024.

US CDC. Meningococcal disease and complement inhibitors. Updated March 2026.