PNH extravascular hemolysis: exploring clinical significance

TO THE EDITOR:

We read with great interest the article by Kulasekararaj et al¹ on extravascular hemolysis (EVH) in paroxysmal nocturnal hemoglobinuria (PNH).

C3-mediated EVH is a well-documented iatrogenic phenomenon inherent to C5 and all terminal-complement inhibitors (see figure panel A-B).2, 3, 4, 5 Although this phenomenon affects all patients with PNH on C5 inhibition to varying degrees, potentially limiting the therapeutic hematological benefits,^2,6,7 robust criteria defining clinically significant EVH (csEVH) are still missing.⁸ Thus, we appreciate the authors’ valuable and timely attempt to define prevalence and clinical impact of EVH in 2 C5-inhibitor trials.¹ However, their csEVH definition, based on the ALPHA trial inclusion criteria, remains largely arbitrary.

Complement activation and the fate of PNH erythrocytes across various therapeutic scenarios. (A) Steady state/untreated. (Left panel) PNH erythrocytes lack surface GPI-linked complement inhibitors, CD55 and CD59. Consequently, activation of the complement alternative pathway (AP) triggers uncontrolled formation of C3 and C5 convertases, leading to MAC assembly and subsequent intravascular hemolysis (IVH). The direct antiglobulin test (or flow cytometry) for C3 remains negative, as PNH erythrocyte undergoing membrane C3 activation are rapidly lysed before opsonization can occur. (Middle panel) Representative flow cytometry dot plot of erythrocytes stained for CD59 and C3d from a patient with PNH at diagnosis: no erythrocytes show C3 binding (normal, CD59⁺, erythrocytes in green; PNH, CD59^˗, erythocytes in red). (Right panel) The pie chart schematically depicts the fate of erythrocytes in patients with PNH, illustrating the relative proportions of nonhemolyzed (yellow background) and hemolyzed (pink background) erythrocytes. The full circle represents "normal" erythrocyte mass (and hemoglobin levels). (B) C5 inhibition. (Left panel) Blockade of the terminal-complement pathway at the level of C5 prevents MAC formation and IVH. However, upstream AP activity persists, and membrane C3 activation leads to C3 opsonization of PNH erythrocytes (positive direct antiglobulin test for C3). These erythrocytes undergo erythrophagocytosis in the spleen and liver,^2,9 resulting in extravascular hemolysis (EVH) of a fraction of PNH erythrocytes, thereby limiting the hematological efficacy of the treatment. (Middle panel) Flow cytometry analysis of the same patient during stable therapy with the C5 inhibitor eculizumab shows 1 normal population (CD59⁺C3d^˗) and 2 distinct PNH populations (CD59^˗): 1 with (C3⁺ PNH) and 1 without (C3^˗ PNH) C3 binding. (Right panel) The pie chart reflects the increased proportion of circulating (nonhemolyzed) PNH erythrocytes and the fraction of PNH erythrocytes spared from IVH that subsequently undergo EVH because C3 binding (pink background). (C) Proximal-complement inhibition (C3, factor B, or factor D). (Left panel) Blocking the proximal-complement pathway prevents both C3 convertase formation and C3 fragment generation. This mechanism inhibits initial membrane C3 activation, eventually preventing both IVH and EVH. Consequently, a significantly larger fraction of PNH erythrocytes is spared compared to C5 inhibition, leading to higher, often near-normal hemoglobin levels. (Middle panel) Flow cytometry analysis of the same patient after switching to stable therapy with the factor B inhibitor iptacopan shows no PNH erythrocytes with C3 binding. (Right panel) The pie chart reflects a significant increase in the proportion of circulating (nonhemolyzed) PNH erythrocytes which, unlike with C5 inhibition, are spared from both IVH and EVH because they are not opsonized by C3.MAC, membrane attack complex.

The article reports that csEVH affected 20.7% to 24.0% of patients, who exhibited only slightly reduced fatigue and near-normal quality-of-life (QoL) scores. This unexpected finding—that csEVH affects QoL minimally—is in apparent contrast with the meaningful improvements in patient-reported outcomes (PRO) associated with hemoglobin increase observed in patients treated with proximal-complement inhibitors,10, 11, 12 even those reaching near-normal hemoglobin levels.¹³

This discrepancy likely arises because the proposed criteria do not fully capture clinically-relevant anemia. Specifically, these criteria—based on a single-point determination (Hb < 9.5 g/dL and reticulocytes >120 × 10³/μL) regardless of transfusions—failed to identify over half (57.7%) of patients requiring blood transfusions, those who are most likely to experience reduced QoL. This suggests that csEVH may affect a much larger fraction of C5-inhibitor–treated patients. Moreover, novel PNH–specific PRO instruments—better suited to capture emotional, cognitive, and physical functioning from the patient’s perspective—may more sensitively track QoL variations and their possible relationship with hemoglobin levels in patients with C3-mediated EVH,^14,15 even variations associated with mild-to-moderate anemia.¹⁴

These observations underscore the necessity for a more refined definition of csEVH. To this end, the extensive datasets from the C5-inhibitor trials present valuable opportunities to establish robust csEVH definition that reliably identify all patients with PNH, likely much more than 20.7% to 24.0%, who would benefit most from proximal anticomplement therapies that, preventing C3-opsonization of PNH erythrocytes and subsequent EVH,^2,7,9 result in increased hemoglobin levels and significant clinical impact (see figure panel C).

Conflict-of-interest disclosure: R.N. reports lecture fees from Alexion Pharmaceuticals, AstraZeneca Rare Disease, and Roche; served as member of an investigator board for Alexion Pharmaceuticals, AstraZeneca Rare Disease, Novartis, and Roche; and reports travel fees from Roche. A.M.R. reports consulting fees and honoraria from Alexion Pharmaceuticals, AstraZeneca Rare Disease, Apellis, Novartis, Omeros, Roche, and Samsung; research funding from Alexion Pharmaceuticals, AstraZeneca Rare Disease, Novartis, and Roche; and served as a consultant for Amyndas.