Hereditary Haemolytic Anaemias — A General Approach

A group of non-immune constitutional disorders in which red cell survival is shortened by defects of the membrane, energy and redox metabolism or erythropoiesis; pyruvate kinase deficiency serves as the prototype enzymopathy, PIEZO1/KCNN4 xerocytosis as the prototype stomatocytosis and the congenital dyserythropoietic anaemias as the differential diagnosis involving ineffective erythropoiesis. Hereditary spherocytosis, glucose-6-phosphate dehydrogenase deficiency and the haemoglobinopathies appear only in the differential diagnosis.

Literature and regulatory information updated: 10 October 2026.

0. Initial Reference Table

Table sources: GeneGuidance gene summaries, CDA I GeneReviews, 2015 International Council for Standardization in Haematology (ICSH) guideline and Orphanet [2, 4, 8, 9].

1. Introduction

1.1. Concept and classification

Hereditary haemolytic anaemias constitute a group of constitutional disorders in which red cells are removed from the circulation before the end of their normal lifespan because of an intrinsic defect. The European Hematology Association (EHA) Red Cell and Iron Working Group consensus groups them into red cell membrane defects, red cell enzymopathies, congenital dyserythropoietic anaemias, thalassaemic syndromes and haemoglobinopathies, and recalls that the spleen, through its red pulp and macrophages, is the main site of damaged red cell removal [3]. This functional architecture explains why splenectomy was historically the intervention common to almost the whole group and why its efficacy, well documented in spherocytosis, remains uncertain or is even harmful in other entities [3].

Classification by mechanism has immediate diagnostic consequences. Membrane defects alter red cell shape, deformability or cation and water content and therefore leave clues on the blood film, in red cell indices and in physical tests such as osmotic fragility and ektacytometry [4]. Enzymopathies reduce energy production or defence against oxidative stress without characteristically altering morphology, thus presenting as chronic nonspherocytic haemolytic anaemia (CNSHA) and requiring enzyme activity measurement or molecular analysis [5, 10]. CDAs are primarily disorders of erythropoiesis, with ineffective erythropoiesis and characteristic cytopathology of marrow erythroblasts, but include a haemolytic component and overlap clinically with the preceding two categories [3, 23]. Saudubray adds a metabolic perspective: glycolysis is the red cell's most important energy source, ten inborn errors of the glycolytic pathway are known, all autosomal recessive except phosphoglycerate kinase and glycerol kinase deficiencies (X-linked), and manifestations comprise haemolytic anaemia, metabolic myopathy or both [6].

Three functional groups are distinguished within enzymopathies. Glycolytic pathway defects (pyruvate kinase, glucose-6-phosphate isomerase, hexokinase, phosphofructokinase, aldolase A, triosephosphate isomerase and phosphoglycerate kinase) impair adenosine triphosphate (ATP) production; pentose phosphate pathway and glutathione metabolism defects (G6PD, γ-glutamylcysteine synthetase, glutathione synthetase and glutathione reductase) impair antioxidant defence; nucleotide metabolism defects (pyrimidine 5′-nucleotidase and adenylate kinase) impair nucleotide turnover and elimination [6, 16, 24]. Luzzatto's ASH Education Program review identifies at least 16 genetically determined red cell enzymopathies, ranging from ultrarare to rare, except G6PD deficiency, which is very common [5]. Among CNSHA causes, PK deficiency is the most frequent glycolytic defect and pyrimidine 5′-nucleotidase deficiency the third most frequent enzymopathy after G6PD and PK [15, 24].

1.2. Historical milestones and molecular discovery

The history of the group begins with the observation of congenital haemolytic anaemias without spherocytes. Selwyn and Dacie described nonspherocytic haemolytic anaemias whose in vitro haemolysis was corrected by ATP but not glucose; the 2019 international diagnostic consensus dates this work to 1956 and the PKD Burden of Disease group review to 1954, a divergence between secondary sources unresolved in the sources consulted [11, 17]. In 1961, Valentine, Tanaka and Miwa identified a specific red cell pyruvate kinase defect in three patients with congenital nonspherocytic haemolytic anaemia, making PK deficiency the first recognised glycolytic enzymopathy [17, 25]. The remaining glycolytic and redox enzymopathies were described in subsequent decades, and by 2019 more than 600 families with PK deficiency and more than 300 PKLR variants had been published [17].

The molecular era of CDA began with CDAN1 mapping to 15q15 from a cluster of 45 highly consanguineous Israeli Bedouins, published by Dgany and colleagues in 2002, who identified 12 variants in nine families in a 28-exon gene encoding the 1226-amino-acid protein codanin-1 [26]. In 2009, two independent groups associated CDA II with SEC23B, a component of the secretory pathway COPII coat: Schwarz and colleagues recapitulated the defect with interfering RNA and in zebrafish, while Bianchi and colleagues, starting from a proteomic and genomic approach to the CDAN2 locus on chromosome 20, found 12 variants in 13 patients from 10 families [27, 28]. In 2010, Arnaud and colleagues described a CDA caused by a dominant missense variant in the erythroid transcription factor KLF1, with abolition of aquaporin 1 and CD44 expression, and the 2016 consensus expanded the KLF1 variant spectrum to previously unrelated red cell disorders [29, 30].

In stomatocytoses, Zarychanski and colleagues identified PIEZO1 (then FAM38A) variants in 2012 in two multigenerational families with xerocytosis, the first genetic disease attributed to a mammalian mechanotransduction channel [31]. In 2013, Albuisson and colleagues confirmed PIEZO1 as the principal dehydrated stomatocytosis gene and demonstrated that variants, all in the C-terminal half, delay channel inactivation, namely exert a gain-of-function effect [32]. Gain-of-function variants in KCNN4, encoding the Gardos calcium-activated potassium channel, were described in 2015 and defined a less frequent and more anaemic subgroup [8, 13].

1.3. Nomenclature and reclassifications

Several designations coexist in the literature and require definition. Hereditary xerocytosis and dehydrated hereditary stomatocytosis (DHSt) are synonyms; the KCNN4-associated form is termed Gardos channelopathy, preferred by the French series because many of these patients show no laboratory signs of dehydration [13]. Overhydrated hereditary stomatocytosis (OHSt) corresponds to the dominant RHAG-associated phenotype, distinct from the recessive regulator-type Rh-null phenotype caused by the same gene [8]. CDA I is divided into Ia (CDAN1) and Ib (CDIN1, formerly C15orf41), with no phenotypic difference between them [9]. CDA II is known by the historical acronym HEMPAS, hereditary erythroblastic multinuclearity with positive acidified serum lysis test, referring to red cell lysis by acidified serum from 40–60% of healthy adults [9]. CDA IV is now subdivided into dominant IVa, associated with KLF1 p.(Glu325Lys), and recessive IVb [8].

Some historical designations may be misleading. The Orphanet entry “haemolytic anaemia due to DPGM deficiency” (ORPHA:714) corresponds to bisphosphoglycerate mutase deficiency, whose established phenotype is erythrocytosis rather than anaemia [8]. The most common TPI deficiency variant is cited as Glu104Asp in historical nomenclature, which does not count the initial methionine (historical numbering, = NM_000365.6:c.315G>C p.(Glu105Asp)), the designation used in recent reports [6, 8, 33]. The megaloblastic anaemia attributed to adenylate kinase deficiency in the introduction to Saudubray chapter 32 contradicts section 32.3.4 of the same book and the primary literature, which describe nonspherocytic haemolytic anaemia; the latter is the correct description [8, 24]. The HK1 gene is associated with five distinct entities that should not be merged: recessive haemolytic anaemia, dominant retinitis pigmentosa, Russe-type hereditary neuropathy, a neurodevelopmental disorder and congenital hyperinsulinism [8].

1.4. Scope of this article

This article covers the general approach to the group. PK deficiency is developed in greater detail because it is the most frequent glycolytic enzymopathy, has a diagnostic consensus and management guidelines, and is the only entity in the group with approved targeted pharmacological treatment [12, 17, 18]. The remaining enzymopathies, the stomatocytoses, the elliptocytoses and the CDAs receive dedicated subsections whenever they differ. Hereditary spherocytosis, G6PD deficiency and the thalassaemias, sickle cell disease and unstable haemoglobins appear here only as differential diagnoses or comparators, for example in the cross-cutting splenectomy recommendations.

2. Molecular Genetics and Pathophysiology

2.1. The red cell as a system with limited reserves

The mature red cell has neither a nucleus nor mitochondria and depends entirely on anaerobic glycolysis to produce ATP, which powers cation pumps and membrane maintenance [8, 17]. Glycolysis is organised into ten steps, a preparatory ATP-consuming phase and a payoff phase producing ATP and NADH; the pentose phosphate pathway has an irreversible oxidative part generating NADPH and a reversible non-oxidative part [6]. Ionic balance rests on a high intracellular potassium and low sodium concentration, maintained by the ATP-dependent Na+/K+ pump; when passive leakage exceeds pump capacity, the red cell dehydrates or becomes overhydrated [4]. Approximately 65% of the membrane's cytoplasmic surface is covered by a spectrin and actin network with protein 4.1R, linked vertically to the bilayer by the band 3–ankyrin–protein 4.2 complex and the glycophorin C–p55–4.1R axis [4]. Each level has a family of disorders: vertical links fail mainly in spherocytosis, horizontal links in elliptocytosis and pyropoikilocytosis, cation transport in stomatocytoses and metabolism in enzymopathies [4, 8].

2.2. Pyruvate kinase deficiency

Pyruvate kinase (EC 2.7.1.40) catalyses the last glycolytic step, the irreversible conversion of phosphoenolpyruvate to pyruvate with production of one ATP molecule, and requires potassium and magnesium or manganese [17]. The reaction generates approximately 50% of red cell ATP [3]. There are four isoenzymes: L (liver, renal cortex, intestine) and R (red cell), produced by PKLR from two tissue-specific promoters, and M1 (muscle, heart, brain) and M2 (leucocytes, platelets, lung, spleen, kidney, adipose tissue), produced by alternative splicing of PKM; during erythroid differentiation, the M2 isoform is replaced by R [17]. The enzyme is a homotetramer with A, B, C (where the allosteric activator fructose-1,6-bisphosphate binds) and N-terminal domains [17].

Biallelic deficiency causes ATP depletion and accumulation of upstream intermediates, including 2,3-diphosphoglycerate (2,3-DPG), which may triple [17]. The energy-depleted red cell becomes rigid and is destroyed prematurely, mainly by extravascular haemolysis [8, 11]. Increased 2,3-DPG shifts the oxyhaemoglobin dissociation curve to the right, improving tissue oxygen release and explaining why many patients tolerate anaemia better than haemoglobin levels would predict [8, 17]. A second mechanism is distinctive: reticulocytes still depend on oxidative phosphorylation, but in the hypoxic splenic environment PK-deficient reticulocytes become dependent on glycolysis and are destroyed; after splenectomy they survive longer and the reticulocyte count typically rises to 50% or more [11, 17]. There is also an ineffective erythropoiesis component, with low hepcidin and elevated erythroferrone, intermediate in degree between spherocytosis and CDA, explaining iron overload in non-transfused patients [7, 34].

The PKLR gene lies at 1q22, has 12 exons and spans approximately 9.5 kb; exon 1 is exclusive to the erythroid isoform (transcript NM_000298, ENST00000342741) and exon 2 to the hepatic isoform (NM_181871), and the PK-R protein has 574 amino acids [17]. Inheritance is autosomal recessive and symptoms occur only in homozygotes or compound heterozygotes [17]. Most variants are missense; the most frequent are NM_000298.6:c.1529G>A p.(Arg510Gln), approximately 16% of alleles in northern Europe and the USA, and c.1456C>T p.(Arg486Trp), approximately 12% in southern Europe [17, 33]. A gain-of-function PKLR allele causes a dominant trait of elevated red cell ATP (OMIM 102900) [8].

The genotype–phenotype correlation is real but imperfect. Two non-missense alleles (nonsense, frameshift, splicing or large deletions) are associated with more severe disease, poorer quality of life and a poorer splenectomy response, and also predict lack of response to mitapivat [11, 15, 35]. However, severity varies between patients with the same genotype, and complications such as iron overload, gallstones and aplastic crises occur even with two missense alleles and mild anaemia [11]. Proposed modifiers include polymorphisms in other enzymes, epigenetic factors, the degree of ineffective erythropoiesis, splenic function and compensatory PK-M2 expression [11].

2.3. Other glycolytic enzymopathies

Glucose-6-phosphate isomerase (GPI) is a dimer interconverting glucose-6-phosphate and fructose-6-phosphate; the secreted form also acts as a cytokine (autocrine motility factor, neuroleukin), and the gene should not be confused with glycosylphosphatidylinositol anchor biosynthesis genes such as PIGA [8]. Mutant enzymes are unstable and residual activity is typically 10–40% [8, 36]. Hexokinase 1 has tissue-specific promoters for red cells, retina and Schwann cells, explaining the coexistence of haematological, retinal and neurological disorders without clinical overlap within the same gene [8].

Phosphofructokinase is a tetramer of M (muscle), L (liver) and P (platelet) subunits; red cells contain L and M, which tetramerise randomly, so M deficiency produces partial deficiency in red cells, with compensated haemolysis, and total deficiency in muscle [6]. Aldolase A is the only isoenzyme present in red cells and muscle, and the variants p.Cys338Tyr and p.Ala279Val, associated with a myopathic phenotype, are thermolabile, explaining fever as a rhabdomyolysis trigger [6]. Triosephosphate isomerase interconverts dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate, at an equilibrium of 20:1 favouring DHAP; in deficiency, red cell DHAP increases 20- to 60-fold and spontaneously converts to methylglyoxal, toxic through oxidative stress, DNA damage and apoptosis [6]. The most common variant destabilises the dimer and generates inactive monomers prone to aggregation, making TPI deficiency a conformational disorder; patients with null alleles have very severe haemolytic anaemia without neurological signs, and the deficiency also impairs synaptic vesicle cycling [6]. Phosphoglycerate kinase 1 is ubiquitous (PGK2 is exclusive to the testis); unstable variants with little effect on catalysis cause haemolysis with neurological dysfunction, whereas those markedly affecting catalysis and stability cause myopathy without haemolysis and cluster in the C-terminal domain [6]. Residual activity modulates phenotype: approximately 4–10% in pure myopathy and approximately 2.6% in multisystem forms [8, 37].

Bisphosphoglycerate mutase produces 2,3-bisphosphoglycerate; its deficiency reduces this metabolite, increases haemoglobin oxygen affinity (low P50) and causes compensatory erythrocytosis rather than anaemia. In the index homozygote, haemoglobin was 19.2 g/dL, haematocrit 58.9%, P50 19 mmHg (normal 25–32), 2,3-BPG 0.3 µmol/g Hb (normal 11.4–19.4) and enzyme activity 0.16 IU/g Hb (normal 4.13–5.43) [8].

2.4. Glutathione and nucleotide metabolism

Reduced glutathione protects red cells against oxidants. γ-Glutamylcysteine synthetase deficiency affects the catalytic subunit GCLC (6p), which should not be confused with the regulatory subunit GCLM; carriers have approximately 50% activity with normal glutathione [8, 16]. GSS (20q11.2) has 13 exons across 32 kb and more than 30 described variants, approximately 40% affecting splicing, prompting Saudubray to recommend supplementing genomic analysis with RNA transcript studies; splicing, frameshift and nonsense variants are associated with moderate and severe forms, and healthy carriers have approximately 55% activity [16]. Glutathione reductase has an enormous functional reserve: glutathione regeneration is impaired only with activity losses exceeding 80%, and functional equivalence to type A− G6PD deficiency requires losses exceeding 90%, so partial reductions rarely explain haemolysis [8, 38]. Activity depends on flavin adenine dinucleotide and thus riboflavin; the activation coefficient with and without this cofactor distinguishes genetic from nutritional deficiency [8, 16].

Red cell-restricted cytosolic pyrimidine 5′-nucleotidase 3A removes pyrimidine nucleotides during reticulocyte maturation; deficiency causes their accumulation and coarse basophilic stippling and chronic haemolytic anaemia through an as yet unknown mechanism [24]. Lead binds the active site of the same enzyme and produces an acquired phenocopy indistinguishable on the blood film and enzyme assay [8, 24]. Adenylate kinase 1 is cytosolic and expressed in muscle, brain and red cells; deficiency is associated with nonspherocytic haemolytic anaemia and, in a minority, psychomotor delay, but a brother with equally profound red cell deficiency without haemolysis has been reported, demonstrating that enzyme deficiency and disease are not equivalent [8, 24].

2.5. Membrane: skeleton and transport

In hereditary elliptocytosis (HE), defects affect horizontal skeletal links (α-spectrin, β-spectrin, protein 4.1R), reducing stability and deformability; the proportion by gene is approximately 65% for SPTA1, 30% for SPTB and 5% for EPB41 [8]. Elliptocytosis is usually monoallelic and hereditary pyropoikilocytosis (HPP) typically biallelic, at the severe end of the spectrum [4, 8]. The low-expression SPTA1 αLELY allele, present in 20–31% of chromosomes in Caucasian, African, Japanese and Chinese populations, is a modifier rather than a pathogenic variant: when in trans with an elliptocytosis allele, it worsens the phenotype and explains probands more severely affected than relatives [4, 8, 39]. The αLEPRA allele, with a population frequency of 3.6%, plays a similar role in spherocytosis [4].

Band 3, encoded by SLC4A1, is both a structural protein and an anion exchanger; the same gene causes dominant distal renal tubular acidosis (usually with normal red cell morphology), recessive distal tubular acidosis with haemolytic anaemia, frequent in Southeast Asia, dominant spherocytosis, Southeast Asian ovalocytosis and cryohydrocytosis [8]. Southeast Asian ovalocytosis (SAO) results from a heterozygous 27 bp deletion removing nine amino acids from band 3 and producing rigid ovalocytes [4, 8]. Complete band 3 null status causes severe transfusion-dependent haemolytic anaemia, dyserythropoiesis and complete tubular acidosis [8].

PIEZO1 (16q24.3, approximately 69.9 kb, 51 coding exons) encodes a non-selective mechanosensitive cation channel with a trimeric structure [40]. Gain-of-function variants delay channel inactivation; prolonged calcium influx activates the Gardos channel, with potassium and water efflux and red cell dehydration [13, 32]. Biallelic loss of function causes the opposite disorder, generalised lymphatic dysplasia with non-immune hydrops fetalis [8, 40]. KCNN4 gain-of-function variants act directly on the Gardos channel and include recurrent p.(Arg352His), sometimes de novo [8, 13]. In OHSt, the RHAG variants p.Ile61Arg and p.Phe65Ser open a cation pore in the Rh-associated glycoprotein, with leakage approximately six times greater than wild type in oocytes, elevated intracellular sodium, low potassium and water overload [8]. SLC2A1-associated cryohydrocytosis combines cold-induced cation leakage with neurological disease in the GLUT1 deficiency spectrum [4, 8]. In ABCB6-related familial pseudohyperkalaemia, passive potassium leakage manifests only in samples stored at or below room temperature [8]. ATP11C encodes a phosphatidylserine flippase (not a scramblase); phosphatidylserine internalisation was reduced approximately 10-fold in the index patient [8].

2.6. Congenital dyserythropoietic anaemias

CDAs result from defects in erythroid differentiation and proliferation and belong to conditions of ineffective erythropoiesis with predominantly single-lineage cytopenia [23]. In CDA I, CDAN1 loss of function accounts for approximately 80–85% of cases and CDIN1 for approximately 5–10%; the remaining 10% suggest regulatory variants or another locus [7, 9]. Codanin-1 binds ASF1, a negative regulator of chromatin assembly, and is stabilised by the nuclease CDIN1; the Cdan1-null mouse dies on embryonic day 6.5 [7, 9]. In CDA II, SEC23B loss of function alters the COPII secretory pathway and causes hypoglycosylation of proteins such as band 3 and band 4.5 (GLUT1), retention of endoplasmic reticulum beneath the membrane and failed cytokinesis, with binucleated erythroblasts with equal-sized nuclei [7, 27, 28]. Dominant CDA III results from KIF23 variants (MKLP1, a component of the cytokinetic centralspindlin complex), with the Swedish founder variant c.2747C>G p.(Pro916Arg); a recessive form is associated with RACGAP1 [7, 8]. In CDA IVa, KLF1 p.(Glu325Lys) (NM_006563.5:c.973G>A) binds poorly to the canonical motif and ectopically to other sites, dysregulating target genes [7, 33]. GATA1-related cytopenia combines macroplatelets with anaemia involving minimal to severe dyserythropoiesis [8].

2.7. Iron, erythroferrone and hepcidin

Iron overload without transfusions is a feature across the group. Erythroferrone, produced by expanding erythroblasts, inhibits hepatic hepcidin transcription and increases intestinal iron absorption; the mechanism is documented in non-transfusion-dependent thalassaemias, PK deficiency, sideroblastic anaemias and CDAs [23, 41, 42]. In CDA II, SEC23B loss of function also suppresses hepcidin through altered glycosylation in hepatic cells, supporting chelation in never-transfused patients [8, 43]. In xerocytosis, hepatic overload is independent of anaemia severity and transfusion regimen because PIEZO1 also acts in hepatocytes and macrophages: in cellular models and gain-of-function mice, variants suppress hepcidin through the RAS (RRAS) pathway and MAP kinases, and pharmacological inhibition of PIEZO1, RAS-GTPase or MEK1/2 restores HAMP expression in a preclinical model [44, 45]. In the French series, hypotheses also included chronic hypoxia, erythroblast proliferation and intestinal iron uptake through mutant channels [13].

2.8. Variants, nomenclature and founder effects

The following table brings together chapter variants standardised according to Human Genome Variation Society (HGVS) nomenclature in VariantValidator, on genome assembly GRCh38 and MANE Select (Matched Annotation from NCBI and EMBL-EBI) transcripts.

Nomenclature standardisation source: [33].

Other relevant variants are cited in source nomenclature because they were not standardised during data collection: the Bedouin CDA I founder variant, described in GeneReviews as NM_138477.4:c.3124C>T p.(Arg1042Trp) of CDAN1 [9]; the Swedish founder variant KIF23 c.2747C>G p.(Pro916Arg) [8]; the RHAG variants p.Ile61Arg and p.Phe65Ser [8]; the recurrent PGK1 variant c.491A>T p.(Asp164Val), associated with haemolysis, encephalopathy and parkinsonism [8]; GPI p.Arg347His, recurrent in Indian patients [8]; and TPI1 Glu104Asp in historical nomenclature (historical numbering, = NM_000365.6:c.315G>C p.(Glu105Asp)), present in approximately 80% of mutant alleles, with a severe phenotype and a common haplotype in all carrier families, indicating a single ancestor [6, 8, 48]. For the PIEZO1 variants of the French series, protein nomenclature was published without a transcript; the recurrent exon 51 variants were standardised in the table [13, 33].

Documented founder effects include: PKLR p.(Arg479His) in the Pennsylvania Amish, with a carrier frequency of 1/151 in the Mifflin (Pennsylvania) and Geauga (Ohio) Amish; a 1149 bp deletion removing PKLR exon 11 in Roma communities; CDAN1 c.3124C>T in Israeli Bedouins; multiple SEC23B founder alleles in different regions; KIF23 p.(Pro916Arg) in Västerbotten, Sweden; TPI1 p.(Glu105Asp) (Glu104Asp in historical numbering) with a common haplotype; an HK1 variant in an alternative untranslated exon in the Roma, causing neuropathy rather than haemolysis; and the three common SPTB variants in Thailand [8, 9, 17, 49, 50].

2.9. Allelic disorders and modifiers

Several allelic disorders span the group. PIEZO1 causes dominant gain-of-function xerocytosis and recessive loss-of-function lymphatic dysplasia; in the French series, one family with p.Gly782Ser and p.Arg808Gln in cis had severe perinatal oedema and one member with persistent lymphoedema, illustrating the overlap [13]. Missense variants in the PIEZO1 extracellular domain also define Er blood group system antigens, and anti-Er4 and anti-Er5 antibodies have been implicated in severe haemolytic disease of the fetus and newborn [8]. SEC23B is also associated with dominant Cowden syndrome type 7 [8]. In KLF1, heterozygous loss of function causes traits without disease, such as the In(Lu) phenotype and hereditary persistence of fetal haemoglobin, and acts as a modifier attenuating β-thalassaemia [8, 30]. KCNN4 is a cystic fibrosis modifier [8].

Among haemolysis modifiers, the UGT1A1 Gilbert genotype has particular clinical relevance. Gilbert syndrome affects 4–7% of the general population and results from homozygosity for the UGT1A1 promoter insertion A(TA)7TAA; approximately 50% of the European and North American population is heterozygous and 9% homozygous [51]. Prolonged neonatal jaundice occurs mainly in infants with the Gilbert genotype who also have G6PD deficiency, Coombs-negative ABO disease, spherocytosis or prolonged breastfeeding [51]. At the ARUP reference laboratory, 10% of 456 patients with unexplained haemolysis were homozygous for UGT1A1*28, eight with an additional pathogenic variant [52]. In PK deficiency, the UGT1A1 TA polymorphism contributes to gallstones [17]. HFE variants have been studied as iron modifiers in xerocytosis, without a ferritin difference between carriers and non-carriers in 45 patients [13]. Coinheritance of defects is another modifier: PKLR heterozygosity may coexist with CDA, spherocytosis or xerocytosis; coinheritance of PIEZO1 and SEC23B was associated with marked iron overload; and in the Neapolitan PIEZO1 cohort, five of nine patients in a microcytic group also had globin variants [17, 40, 53].

3. Clinical Spectrum and Manifestations by System

3.1. Manifestations common to the group

Chronic haemolysis produces a shared clinical picture whose intensity depends on the balance between destruction and marrow compensatory capacity. Markers are reticulocytosis, unconjugated hyperbilirubinaemia, elevated lactate dehydrogenase (LDH) and low or undetectable haptoglobin [8, 17]. Chronic jaundice has a psychosocial impact, and the PKD Burden of Disease group describes it as a stigma and a reason for bullying [11]. Splenomegaly, pigment gallstones, parvovirus B19 aplastic crises, extramedullary haematopoiesis, osteopenia, pulmonary hypertension and leg ulcers complete the picture, and iron overload, even without transfusions, is the complication that most distinguishes these disorders from spherocytosis [11, 13, 15]. Haemolytic crises occur with infections, pregnancy and surgery, and an aplastic crisis is recognised by a fall in haemoglobin with inappropriately low reticulocytosis [11].

3.2. Pyruvate kinase deficiency

The PK deficiency spectrum ranges from hydrops fetalis with intrauterine death to asymptomatic compensated haemolysis, including severe lifelong transfusion-dependent anaemia [11, 54]. The PKD Natural History Study (NCT02053480) included 254 molecularly confirmed patients and is the reference cohort [15]. Perinatal complications were frequent, with anaemia requiring transfusion, hyperbilirubinaemia, hydrops and prematurity; 93% of newborns received phototherapy and 46% exchange transfusion [15]. Children up to five years of age were often transfusion-dependent until splenectomy, performed in 150 of the 254 patients (59%) [15]. The most frequent complications were iron overload (48%) and gallstones (45%), followed by aplastic crises, osteopenia or bone fragility, extramedullary haematopoiesis, post-splenectomy sepsis, pulmonary hypertension and leg ulcers; these complications occurred at all baseline haemoglobin levels, supporting iron and gallstone surveillance in all patients and not only those with more severe anaemia [15]. These figures describe patients referred to specialist centres, many already having undergone splenectomy and transfusions, rather than the natural history of untreated patients.

The diagnostic consensus adds clinical details. Anaemia tends to improve with age, early-onset cases have a more severe course and splenomegaly is present in approximately 80% [17]. Gallstones become more frequent after the first decade and also after splenectomy [17]. Rare complications include kernicterus, leg ulcers, gallstone pancreatitis, splenic abscess, spinal cord compression from extramedullary haematopoiesis, pulmonary hypertension, thromboembolism (particularly after splenectomy) and rare cases of fulminant liver failure [17]. Extramedullary haematopoiesis is predominantly paravertebral, may compress nerve roots or the spinal cord and may be mistaken for malignant tumours [11]. Patients requiring transfusions or with severe disease are diagnosed neonatally or in early childhood, whereas those rarely needing transfusions are often diagnosed only in adulthood, after being labelled as having thalassaemia trait, iron deficiency, spherocytosis or inflammatory anaemia, with complications attributed to Gilbert syndrome or hereditary haemochromatosis [11]. Anaemia tolerance decreases with age and transfusion independence may come and go throughout life [11].

3.3. Rare enzymopathies

Rare enzymopathies differ mainly in their extrahaematological involvement, summarised in the table.

Several particular features merit emphasis. In Tarui disease, compensated haemolysis with elevated bilirubin and reticulocytosis distinguishes the condition from McArdle disease, and two exercise findings are discriminating: absence of the “second wind” (none of 5 patients with PFK deficiency experienced it, compared with all 29 McArdle patients) and worsening performance with carbohydrates, which in a crossover experiment in four patients reduced maximal work capacity by 60–70% and maximal oxygen consumption by 30–40% [6, 8]. In GPI deficiency, osmotic fragility and the eosin-5′-maleimide (EMA) binding test are normal and morphology is little altered, and some patients have been misdiagnosed with spherocytosis [8]. In PGK1 deficiency, the French national series described Charcot-Marie-Tooth-like axonal sensorimotor polyneuropathy, and heterozygous women may have anaemia or, rarely, neurological disease: a heterozygous mother with normal red cell activity developed parkinsonism at 36 years [6, 8]. In GSS deficiency, a first uneventful pregnancy was described in a woman with the moderate form [16]. In GCLC deficiency, a sulphonamide precipitated psychosis and marked haemolysis in one patient [8].

3.4. Xerocytosis (PIEZO1) and Gardos channelopathy (KCNN4)

The retrospective series from Bicêtre Hospital (France, 1993–2017) included 126 patients from 64 families and describes the natural history of xerocytosis [13]. Probands' mean age at diagnosis was 32±18 years, ranging from the perinatal period to over 88 years [13]. Chronic nonspherocytic haemolysis was present in 84%, hyperferritinaemia in 36%, perinatal oedema in 17% and post-splenectomy thrombosis in 11% of the total [13]. Gallstones affected 52% of the 67 assessed patients, increasing with age (22% before 20 years, 62% between 20 and 40 and 80% after 40), and splenomegaly affected 56% [13]. Mean haemoglobin was 131±20 g/L and 68% had no anaemia; no patient depended on regular transfusions and eight had occasional transfusions; mean corpuscular volume (MCV) was 97±8 fL, reticulocytes 294±102×10⁹/L and mean corpuscular haemoglobin concentration (MCHC) 351±13 g/L, exceeding 360 g/L in 38% [13]. Seven adults with PIEZO1 variants had haemoglobin above 160 g/L, two referred for suspected polycythaemia [13]. Of the 19 patients diagnosed before one year of age, seven of 15 had neonatal jaundice (six receiving phototherapy, one exchange transfusion), 48% had haemoglobin below 120 g/L in the first month and five received transfusions, with progressive improvement with age [13].

Gardos channelopathy causes more anaemia. In the same series, patients with KCNN4 had haemoglobin of 102±13 g/L versus 134±19 g/L in those with PIEZO1 (P<0.001), reticulocytes of 178±63 versus 308±106×10⁹/L and MCHC of 332±13 versus 354±24 g/L; stomatocytes were present in 65% of patients with PIEZO1, generally rare, and absent in 10 of 12 with KCNN4 [13]. Haemoglobin also varied with the PIEZO1 variant: 121±11 g/L with p.(Leu2495_Glu2496dup), 131±15 g/L with p.(Arg2456His) and 144±17 g/L with the others [13]. The GeneGuidance entry reports 13 families with Gardos channelopathy described worldwide by 2022 and some KCNN4 variants causing anaemia with stomatocytosis without dehydration [8].

Perinatal oedema was documented in 22 patients from 13 families, all genotyped patients having PIEZO1: hydrops in six, pleural effusion in five, ascites in eleven, hydramnios in two and pericardial effusion in two, with one in utero death at 27 weeks, one medical termination of pregnancy at 28 weeks and one death at 15 days; only three had anaemia, suggesting a lymphatic mechanism, and resolution was complete in 14 [13]. In 13 of these patients there was no known family history, and recurrence occurred in all informative families [13]. In Gardos channelopathy, perinatal oedema had not been observed until a 2022 report of fetal loss from non-immune hydrops with a KCNN4 deletion [8, 58].

Iron overload increases with age: ferritin at diagnosis was 273±141 ng/mL before 20 years, 717±441 ng/mL between 20 and 40 and 1409±653 ng/mL after 40, and in adults with KCNN4 reached 1702±1048 ng/mL versus 656±428 ng/mL with PIEZO1 [13]. There were three cases of diabetes, one hepatocellular carcinoma with liver transplantation, hypogonadism, hypothyroidism and three cases of osteoporosis [13]. Pseudohyperkalaemia, caused by potassium leakage from red cells stored at room temperature, was present in 18 of 35 patients (51%) and can lead to unnecessary treatment of non-existent hyperkalaemia [8, 13]. Thrombosis without splenectomy occurred in four patients, and post-splenectomy thrombosis is addressed in section 7.2 [13].

The Neapolitan cohort of 176 patients from 141 families with rare PIEZO1 variants (2020–2024) defined three subtypes [40]. The first corresponds to the typical form, with haemoglobin of 11.65±2.16 g/dL, MCV of 93.4±11.8 fL, reticulocytes of 6.22±4.78%, ferritin of 886±715 ng/mL and diagnosis at 42±6.5 years. The second has microcytic anaemia (haemoglobin 9.78±1.31 g/dL, MCV 66.9±10.1 fL) with near-normal reticulocytes and ferritin of 115±96 ng/mL, and five of the nine patients also had globin variants. The third is mild or subclinical (haemoglobin 14.56±1.5 g/dL), with elevated ferritin in approximately 30% [40]. C-terminal domain variants were associated with low indices, mild haemolysis and a tendency towards iron overload, and inner helix variants with marked hyperferritinaemia [40]. Half the patients were referred with a general suspicion of red cell disease and the second category with suspected iron disorders, such as primary hyperferritinaemia, haemochromatosis or refractory iron deficiency [40]. Xerocytosis may also present with polycythaemia, marrow failure or myelodysplastic syndrome, and a predisposition to colorectal adenomatous polyposis has been described [40].

3.5. Other stomatocytoses and related entities

OHSt is very rare (approximately 20 cases or families worldwide) and more severe than xerocytosis: macrocytic haemolytic anaemia of fluctuating severity, with haemoglobin of 80–100 g/L, MCV of 120–140 fL and MCHC of 240–280 g/L, splenomegaly or hepatosplenomegaly, gallstones and a strong tendency towards iron overload independent of transfusions [3, 4, 8]. In the regulator-type Rh-null phenotype, chronic haemolytic anaemia is compensated, mild to moderate, with stomatocytes, spherocytes and increased osmotic fragility, but the dominant problem concerns transfusion: anti-Rh29 alloimmunisation develops after transfusion or pregnancy, allowing only Rh-null blood to be transfused [8]. Cryohydrocytosis causes mild to moderate haemolysis with pseudohyperkalaemia; the SLC2A1-associated form adds the paediatric neurological disorder of GLUT1 deficiency [4, 8]. ABCB6-related familial pseudohyperkalaemia is a laboratory artefact without disease, with the R276W allele (NM_005689.4:c.826C>T p.(Arg276Trp)) present in approximately 1 in 327 donors [8, 33]. Recessive sitosterolaemia causes mild haemolysis with marked stomatocytosis without cation leakage, and macrothrombocytopenia [4]. X-linked ATP11C deficiency causes mild chronic haemolysis, often compensated, with normal morphology and hepatic iron overload; in a 2026 series of 10 individuals from 7 families, a 53-year-old man was initially suspected of having haemochromatosis and a 68-year-old heterozygous woman had anaemia due to skewed X-inactivation [8, 59].

3.6. Elliptocytosis, pyropoikilocytosis and ovalocytosis

Hereditary elliptocytosis is the most common primary membrane protein disorder worldwide, but most heterozygotes are asymptomatic, with elliptocytes without fragmentation, and diagnosis is often incidental [8]. The International Council for Standardization in Haematology (ICSH) guideline distinguishes asymptomatic carriers, elliptocytosis with variable haemolysis, elliptocytosis with transient infantile poikilocytosis (which may require transfusions in the first year and evolves towards the parental phenotype) and HPP [4]. HPP causes severe transfusion-dependent anaemia from the neonatal period (haemoglobin 60–100 g/L), with marked poikilocytosis, fragmentation, microspherocytosis, very low MCV and characteristic thermal instability [4, 8]. Fatal hydrops has been described in one case of β-spectrin homozygosity [4]. Patients with near-complete α-spectrin deficiency remained transfusion-dependent after splenectomy, and severity correlates with residual α-spectrin [8, 60]. Complications include pigment gallstones, splenomegaly, parvovirus B19 aplastic crisis and neonatal jaundice [8].

Screening of 1505 Thai newborns found heterozygosity for one of three common SPTB variants in 13 infants (0.86%); this heterozygosity was an independent risk factor for jaundice requiring phototherapy (relative risk 5.01; 95% CI 2.56–9.79), most infants had neither anaemia nor a diagnosis at birth and coinheritance with G6PD deficiency worsened anaemia and jaundice [8]. Southeast Asian ovalocytosis is usually asymptomatic in adults, may be haemolytic in childhood and protects against malaria; in isolation it does not cause renal tubular acidosis, but coinheritance with a recessive SLC4A1 variant is associated with distal tubular acidosis with haemolytic anaemia [4, 8].

3.7. Congenital dyserythropoietic anaemias

CDA I causes moderate to severe macrocytic anaemia. It rarely presents in utero with severe anaemia and hydrops, potentially requiring intrauterine transfusion; more frequently it presents neonatally with hepatomegaly, early jaundice and growth restriction, and some patients are diagnosed only in childhood or adulthood [9]. Among 70 Bedouin newborns with CDA I, 45 (64%) were symptomatic; of these, 65% had hepatomegaly, 53% early jaundice and 27% were small for gestational age, and most required at least one neonatal transfusion [9]. After the neonatal period, anaemia is moderate and lifelong (mean haemoglobin 85±6 g/L in a series of 21 patients), with splenomegaly in 80%, gallstones in 4 of 21 before 30 years and only 2 of 21 transfusion-dependent during follow-up to 37 years; 5 of the 21 (23%) died as adults, mainly from iron overload [9]. In follow-up of the Israeli group, 3 of 32 adults (9%) died between 46 and 59 years from sepsis or severe pulmonary arterial hypertension, all previously splenectomised, and osteoporosis was the rule [9]. Distal limb anomalies (syndactyly, hypoplastic nails, fourth metatarsal duplication) occur in 4–14%, and scoliosis, short stature, flattened vertebrae and angioid streaks with visual loss in adults aged 45–57 years have been described [9, 21]. Age at diagnosis in 21 patients ranged from 0.1 to 47 years (median 17.3) [21].

CDA II is the most common CDA, with more than 200 patients described in 2017; anaemia ranges from mild to severe, with jaundice, splenomegaly in 50–60%, transfusion dependence in up to 15% and iron overload in most after 20 years [3, 9]. A meta-analysis of 205 cases (172 unrelated) identified 84 causal SEC23B variants, defined three genotypic groups with phenotypic correlation and proposed a clinical index distinguishing well-compensated haemolysis from ineffective erythropoiesis; normal or near-normal haemoglobin does not exclude diagnosis [8, 50]. CDA III is the rarest: in the large Swedish family from Västerbotten anaemia was mild, many considered themselves healthy, 45% reported anaemia symptoms and there was an excess of monoclonal gammopathy and multiple myeloma (approximately 20%) and macular degeneration with angioid streaks [8, 9]. CDA IVa combines ineffective erythropoiesis and haemolysis with circulating erythroblasts, splenomegaly, fetal haemoglobin (HbF) elevated up to 40%, normal MCV, red cells lacking CD44 and aquaporin 1 and severity ranging from moderate to fetal anaemia with hydrops; some patients with p.(Glu325Lys) had 46,XY differences of sex development [8, 9]. Recessive CDA IVb causes severe haemolytic anaemia from the first day, with numerous erythroblasts, hypochromic microcytosis and HbF of approximately 18–47% [8]. GATA1-related cytopenia ranges from anaemia with minimal dyserythropoiesis to hydrops requiring intrauterine transfusion, with haematocrit of 16–35% and macroplatelets, and may progress to myelodysplastic syndrome or aplasia [8].

3.8. Fetal and neonatal period

Non-immune hydrops fetalis is the most severe presentation. In a systematic review of 5437 cases, haematological causes (haemoglobinopathies, red cell aplasias, dyserythropoiesis) accounted for 564 cases (10.4%), behind cardiovascular (21.7%) and chromosomal causes (13.4%), with 17.8% of unknown cause and 5.7% due to lymphatic dysplasia [61]. Within this group, hydrops has been described in PK deficiency, GPI deficiency, CDA I, CDA IV, HPP from β-spectrin homozygosity, GATA1 deficiency and, through a lymphatic rather than anaemic mechanism, in xerocytosis and biallelic PIEZO1 loss of function [4, 8, 9, 13, 15]. Among metabolic disorders, transaldolase deficiency presents neonatally or prenatally with hepatosplenomegaly, liver dysfunction, anaemia and thrombocytopenia, and Pearson syndrome and some congenital disorders of glycosylation may present as hydrops [6, 62].

Severe neonatal jaundice is the most frequent presentation in early-onset forms. Jaundice persisting beyond 2–4 weeks requires investigation, and unconjugated bilirubin is neurotoxic, with a risk of kernicterus and deafness [51]. In spherocytosis, haemoglobin may be normal at birth and fall rapidly in the first month [4]. In HPP and elliptocytosis with infantile poikilocytosis, neonatal anaemia is severe and may require transfusions in the first year [4]. Haptoglobin has diagnostic value only after six months of age [17].

4. Clinical Recognition and Warning Signs

4.1. When to suspect, by age

In the fetus, suspicion arises with non-immune hydrops, fetal anaemia detected by Doppler or isolated effusions. Assessment includes a three-generation pedigree covering fetal losses, family history of anaemia and consanguinity, ethnic origin and exclusion of maternal–fetal alloimmunisation and parvovirus B19 infection; if fetal death occurs, tissue and DNA should be retained [61]. In a parent with xerocytosis, fetal oedema may occur without anaemia [13].

In newborns, warning signs are severe unexplained indirect hyperbilirubinaemia, particularly if requiring exchange transfusion, anaemia with reticulocytosis and prolonged neonatal jaundice [17, 51]. History should include consanguinity, ethnic origin, prolonged neonatal jaundice in a sibling or parent, reactions to drugs or foods and previous phototherapy or exchange transfusion [51]. Haematological investigation begins with the Coombs test, Rh and ABO incompatibility and examination for abnormal red cells [51]. The combination of unexplained haemolytic anaemia and high-anion-gap metabolic acidosis in a newborn should prompt testing for 5-oxoproline in urinary organic acids to exclude GSS deficiency [8].

In children and adults, the PKD diagnostic consensus recommends suspecting an enzymopathy in chronic anaemia of variable severity, splenomegaly or jaundice with normal or near-normal morphology, in transfusion-dependent patients without an aetiological diagnosis, and in splenectomised patients without a diagnosis whose reticulocyte count rises after surgery [17]. Elevated MCHC with decreased osmotic fragility and pseudohyperkalaemia suggests xerocytosis [4, 8]. In adults, hyperferritinaemia or hepatic iron overload with negative HFE testing and subtle haemolysis signs should suggest xerocytosis, PK deficiency, CDA or ATP11C deficiency [8, 40, 47]. Erythrocytosis with compensated haemolysis may correspond to xerocytosis with high-affinity haemoglobin [13, 40]. Pigment gallstones at a young age, unexplained splenomegaly and an incomplete response to splenectomy performed for presumed spherocytosis should also prompt diagnostic reassessment [4, 15].

4.2. Extrahaematological signs guiding diagnosis

The association of haemolysis with other system signs narrows the differential. Progressive neurological disease from the first months, recurrent respiratory infections and respiratory failure suggest TPI deficiency [6, 8]. Exertional myopathy with rhabdomyolysis and myoglobinuria, with or without early parkinsonism, seizures or stroke-like episodes, suggests PGK1 in a boy, and cramps with exercise intolerance and hyperuricaemia suggest Tarui disease [6, 8]. Fever-triggered rhabdomyolysis suggests aldolase A deficiency [8]. Cataracts with haemolytic crises after broad beans or oxidant drugs suggest glutathione reductase deficiency [16]. Coarse basophilic stippling requires blood lead measurement before accepting pyrimidine 5′-nucleotidase deficiency [8]. Distal limb anomalies with macrocytic anaemia suggest CDA I, and macroplatelets with anaemia in a boy suggest GATA1-related cytopenia [8, 9]. Distal renal tubular acidosis with haemolytic anaemia suggests SLC4A1 [8].

4.3. Emergencies

Situations requiring immediate action are aplastic crisis (a rapid haemoglobin fall with low reticulocytes, usually from parvovirus B19), haemolytic crisis with infection, acute haemolysis after abrupt mitapivat discontinuation and, in splenectomised patients, fever, which should be treated as possible fulminant sepsis [3, 11, 18]. Rhabdomyolysis in PGK1 and ALDOA deficiencies may cause acute renal failure and hyperkalaemia [8]. Metabolic decompensation in GSS deficiency with infections may be fatal [16]. After splenectomy, abdominal pain or fever in the first week should raise suspicion of portal or splenic vein thrombosis [3]. Hyperkalaemia in a patient with xerocytosis should be confirmed in an immediately processed sample before any treatment [8].

4.4. Newborn screening

The consulted sources do not describe population newborn screening programmes for these disorders. Saudubray reports a symptomatic patient with GSS deficiency identified through newborn screening by tandem mass spectrometry [16]. Thai SPTB screening in 1505 newborns was a research study rather than a public health programme [8].

5. Diagnosis: Criteria, Tests and Algorithms

5.1. General algorithm

There is no single formal diagnostic criterion for the group. Sources converge on a stepwise algorithm: Luzzatto's review proposes four steps (recognise chronic haemolysis, exclude acquired causes, exclude haemoglobinopathies and membrane disorders, identify the deficient enzyme), acknowledging that a DNA panel can combine steps 3 and 4 [5]. The stepwise approach of the PKD Burden of Disease group starts with haemolysis tests, then excludes haemoglobinopathies and paroxysmal nocturnal haemoglobinuria (PNH, particularly in adults), next investigates PK deficiency and membrane disorders, and only then rare hereditary and acquired causes, with the option of bringing forward PKLR and KLF1 molecular testing [11]. Paroxysmal nocturnal haemoglobinuria is part of the differential diagnosis. The table integrates these proposals with the sources for each step.

The ordering of steps 4 to 7 is disputed. The 2015 ICSH guideline places molecular analysis as a case-by-case decision, often the last option, reserved for recessive inheritance, suspected de novo variants and compound heterozygosity [4]. The PKD diagnostic consensus allows two strategies, enzyme activity followed by PKLR sequencing or an NGS panel followed by functional confirmation of novel variants [17]. The 2022 Korean guideline proposes NGS before membrane protein or enzyme analysis because of genetic testing availability [66], and the ARUP national reference laboratory recommends NGS in all unexplained haemolysis and in newborns with hyperbilirubinaemia [52]. In practice, the panel tends to be brought forward in newborns without family studies, transfused patients and when shipping samples for functional assays is time-consuming [17].

5.2. Request, sample and morphology

The request should state the provisional diagnosis, clinical data, direct antiglobulin test result, reticulocytes, haemolysis markers, family history and date of last transfusion [4, 17]. The sample should be pre-transfusion; otherwise, ICSH notes that eight weeks are required for complete clearance of transfused cells [4]. Morphology effectively guides diagnosis when assessed by an experienced observer.

5.3. Membrane tests

The ICSH guideline describes tests and their limitations [4]. Classical osmotic fragility (OF) uses 14 sodium chloride concentrations and sensitivity increases after 24 h incubation at 37 °C, but specificity is low: it is abnormal in autoimmune anaemia, after recent transfusion, in G6PD and PK deficiencies and with unstable haemoglobins. The acidified glycerol lysis test (AGLT) has normal lysis above 900 s and false positives in autoimmune anaemia, approximately one-third of pregnant women, chronic kidney disease and myelodysplastic syndrome. The flow cytometric EMA binding test detects spherocytosis, SAO, cryohydrocytosis and some CDA II cases; in a Bristol audit, 4 of 403 patients (approximately 0.9%) with suspected spherocytosis and normal EMA had protein deficiency on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), so EMA combined with a second test probably detects all cases; there are no universal reference intervals because fluorescence scales differ between cytometers [4].

Osmotic gradient ektacytometry measures the deformability index across an osmolality gradient and produces characteristic profiles: in spherocytosis, the maximum index falls and the minimum point (Omin) rises; in elliptocytosis the curve is trapezoidal; in HPP the maximum index is very low; in SAO rigid cells do not deform; in xerocytosis the maximum index is normal and the curve is shifted left [4]. ICSH considers it the only simple reliable screening test for DHSt, with three limitations: the sickle cell disease profile is similar, thalassaemia and iron deficiency produce flattened profiles and autoimmune anaemia overlaps, particularly in jaundiced newborns, requiring a negative antiglobulin test [4]. Samples should be tested the same day or after overnight storage at 4 °C; after 24 h the profile tends towards overhydration [4]. In the French series, 87% of probands were diagnosed through the typical profile, but the 6 families with atypical or normal profiles all had KCNN4 variants, meaning ektacytometry does not identify Gardos channelopathy and genetic testing should be performed for unexplained haemolysis even with normal ektacytometry [13]. Ektacytometry also does not predict course or severity and is unreliable after recent transfusion [8]. ADVIA 2120 analyser reticulocyte indices distinguished 15 patients with Gardos channelopathy from 79 with PIEZO1 with 100% sensitivity and specificity, using ΔMCV thresholds of 8.6 fL and ΔMCHC of −5.5 g/L; the series is small and used a single analyser [8].

SDS-PAGE quantifies spectrin, ankyrin, band 3, protein 4.2 and protein 4.1R, detects hypoglycosylated band 3 in CDA II and does not detect GLUT1, RhAG or PIEZO1; 8–11% of spherocytosis cases show no detectable reduction [4]. Flame photometry measures plasma potassium and intracellular sodium and potassium in fresh samples and samples stored at room temperature and refrigerated, with a paired control, and confirms the cation defect [4]. The table summarises stomatocytosis parameters in the ICSH guideline.

Source: [4].

5.4. Enzyme assays and the PK deficiency model

The ICSH reference assay for PK, following Beutler, is spectrophotometric at 340 nm, coupled to NADH oxidation by LDH, and expressed in IU/g Hb [17]. The 2019 international consensus, involving 24 experts from 20 centres and endorsed by ERN-EuroBloodNet, established preanalytical and analytical conditions [17].

Source: [17].

False-normal results arise from marked reticulocytosis (reticulocytes contain more PK), recent transfusion, incomplete removal of leucocytes and platelets (the leucocyte isoenzyme is more active), compensatory M2 expression and kinetically abnormal mutants with near-normal in vitro activity [17]. An example illustrates the problem: a patient with 55% reticulocytes had PK within the reference interval, but a PK/HK ratio of 3.9 versus 18.5 in controls [17]. The consensus calculated that one packed red cell unit in a 60 kg adult with haemoglobin of 8 g/dL produces approximately 12% contamination on the day of transfusion, falling to 6% at 50 days [17]. False positives arise from poor storage and secondary PK deficiencies in KLF1 variants and in acute myeloid leukaemias and myelodysplastic syndromes [17]. There are no systematic sensitivity and specificity studies; at one centre, receiver operating characteristic (ROC) curve analysis found mean activity of 38.5% of controls in 34 patients, 56.6% in 41 heterozygotes and 155% of the lower limit in 30 patients with other haemolytic disorders, with cut-offs of 81.5% and 96.9% [17]. The degree of activity reduction does not predict severity [11, 17]. Additional tests include upstream glycolytic intermediates, thermostability after one hour at 53 °C, which revealed thermolabile variants in patients with repeatedly normal activity, and activity at low substrate concentration [17]. There is no external quality control programme for the PK assay [17].

Similar limitations apply to other enzymopathies. In GPI deficiency, high reticulocytosis increased measured activity to 30–40% in a patient with a variant described in another context at 18% [8]. In adenylate kinase deficiency, activity in affected individuals ranges from undetectable to 30%, heterozygotes have intermediate values and transfusions, bleeding, deficiencies and coexisting haemolysis confound results [8]. In glutathione reductase deficiency, riboflavin deficiency produces secondary deficiency [8, 16]. In PGK1 deficiency, heterozygous women may have normal red cell activity [8]. In ALDOA deficiency, near-normal red cell activity does not exclude a muscular phenotype from thermolabile alleles [8]. In HK1 deficiency, normal red cell activity does not exclude other disorders of the gene, and intronic hyperinsulinism variants escape coding-only sequencing [8]. In pyrimidine 5′-nucleotidase deficiency, classical screening is ultraviolet spectrophotometry of the red cell nucleotide extract, with an abnormal purine/pyrimidine ratio, and heterozygotes are difficult to detect [8]. In ADA deficiency with severe combined immunodeficiency, diagnosis by activity below 1% in red cells or dried blood spots is valid only without prior transfusion [8].

5.5. Molecular tests

Sequencing has practical advantages: DNA is easy to handle and ship, requires little volume, is unaffected by transfusion and facilitates family and prenatal studies [17]. In the European Union and Japan, PKLR sequencing is mandatory to confirm PK deficiency, and six of seven European centres always perform it; in the USA, only two of five centres performed it routinely because of insurance coverage limitations [17]. To be recognised as a PKD centre of expertise within ENERCA, a laboratory must provide PK activity and PKLR analysis [17].

Panel yield varies with population and study design and should be interpreted as the proportion of patients with an identified pathogenic or likely pathogenic variant, rather than clinical sensitivity.

Technical limitations are known. Capture NGS misses large deletions, deep intronic variants and regulatory regions, particularly affecting recessive disorders [4, 17]. Up to 10% of PK-deficient patients have only one variant on PKLR exonic sequencing; in 13 families with one or no variants, genome sequencing identified deep intronic variants in five, and some European centres use MLPA for deletions [11, 17]. Patients with only one identified allele may be excluded from trials requiring two alleles, as happened to a 47-year-old woman transfused every three weeks despite splenectomy, with PK activity of 2.5 U/g Hb and a single variant, c.460G>A p.(Ala154Thr), with a negative 46-gene panel [11]. In CDA I, in a series with CDAN1 sequencing without splicing or large deletion testing, 60% of patients had two identified variants, 28% only one and 11% none; these are proportions of detected variants rather than clinical test sensitivity [9]. In GSS deficiency, approximately 40% of variants affect splicing and Saudubray recommends supplementing investigations with RNA analysis [16]. PGK1 pseudogenes on chromosomes 19, 21 and X complicate analysis [8]. Low-expression αLELY and αLEPRA variants should be included in panels and reports because they explain recessive phenotypes and greater severity than expected [52, 70].

Variant interpretation requires caution. In PIEZO1, many polymorphic variants are listed in databases and novel variants require phenotypic or functional methods, such as ektacytometry or electrophysiology [13]. The Neapolitan reclassification curated 2565 non-null PIEZO1 variants on transcript NM_001142864.4 using a Bayesian classifier according to American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP) rules and ClinGen weights: 1005 variants of uncertain significance (VUS) were reclassified (487 as pathogenic or likely pathogenic and 518 as benign or likely benign) and 1345 remained uncertain [40]. In PKLR, novel variants are VUS until functional confirmation by enzyme activity, Western blot, reverse transcription followed by PCR (RT-PCR) or reporter assays, and four European centres had homozygotes or compound heterozygotes with normal activity [17]. In patients with deficiency and no PKLR variants, KLF1 should be considered [17]. Reports should state the gene, versioned transcript, HGVS nomenclature, zygosity, ACMG/AMP class and, where relevant, the presence of low-expression alleles and modifiers such as UGT1A1; segregation in parents confirms the trans phase and helps classify novel variants [17, 52]. Segregation also reveals parental mosaicism, as in the case described in section 8.3 [11].

5.6. Bone marrow and electron microscopy

Bone marrow aspiration is indicated when reticulocytosis is inadequate for the degree of anaemia. In CDA I, marrow shows erythroid hyperplasia, few binucleated erythroblasts and interchromatin bridges in 0.6–2.8% of erythroblasts; electron microscopy shows spongy (“Swiss cheese”) heterochromatin in up to 60% of erythroblasts and nuclear membrane invaginations, distinguishing type I from other CDAs without separating Ia from Ib [8, 9]. In CDA II, more than 10% (up to 50%) of mature erythroblasts are bi- or multinucleated with equal-sized nuclei, electron microscopy shows a double membrane from residual endoplasmic reticulum and SDS-PAGE shows thinner, faster-migrating band 3 and band 4.5 [7–9]. CDA III has giant multinucleated erythroblasts with up to 12 nuclei [9]. None of these findings identifies the gene, and molecular confirmation is the standard [8]. Marrow morphology can mislead: in a series of 36 patients with suspected CDA I, 16 ultimately had another diagnosis, mainly PK deficiency, without erythroblast morphological differences from confirmed cases [65].

5.7. Prenatal and preimplantation diagnosis

Once familial causal variants are identified, prenatal diagnosis and preimplantation genetic testing are possible, as described in CDA I [9]. In GSS deficiency, prenatal diagnosis can use molecular analysis in chorionic villi, enzyme activity in amniocytes or villi and 5-oxoproline in amniotic fluid [8, 16]. For disorders with a biochemical marker, biochemical carrier detection and prenatal diagnosis are alternatives when the variant is unknown [51]. Ultrasound surveillance during pregnancy is discussed in section 8.

6. Differential Diagnosis

6.1. Comparative table

6.2. Main differentials

The differential between CDA II and spherocytosis is the most frequent in practice. CDA II may mimic spherocytosis, with microspherocytes and increased osmotic fragility, and some patients underwent splenectomy for presumed spherocytosis without complete remission [4]. They are distinguished by poikilocytosis and disproportionately low reticulocytes: in most CDA II patients reticulocytes are below 150×10⁹/L at the 90th percentile, and suspected spherocytosis with 120–180×10⁹/L reticulocytes requires differentiation from CDA II [4]. The spherocytosis GeneReviews highlights mild-phenotype CDA II, with mild anaemia, suboptimal reticulocytosis, jaundice and splenomegaly, distinguished by dyserythropoiesis and iron overload disproportionate to transfusions [10]. CDA II ektacytometry resembles atypical spherocytosis, and ICSH recommends SDS-PAGE or repeat testing after three months [4].

PK deficiency mimics CDA I. In the series of 36 Italian and Turkish patients with suspected CDA I, a molecular diagnosis was obtained in 89%: 16 had CDA I and 16 another diagnosis, predominantly PK deficiency; erythroblast morphology did not differ, and patients without CDA I were more severely affected (severe anaemia in 82% versus 18%; transfusion dependence in 100% versus 41.7%) [65]. The PKD consensus notes that marrow dyserythropoiesis is possible in PK deficiency and that PKLR heterozygosity may coexist with CDA, spherocytosis or xerocytosis and modify the phenotype [17]. An NGS panel including CDA and enzymopathy genes is therefore the most efficient approach in these cases [53].

Xerocytosis should be distinguished from hereditary haemochromatosis. In the Neapolitan cohort, suspected iron disease was the second reason for referral [40]. The case published by Molina-Arrebola and Bain describes a man referred in 1997 for suspected haemochromatosis, with haemoglobin of 166 g/L, MCV of 112.7 fL, transferrin saturation of 90.7%, ferritin of 956.5 ng/mL and grade IV/IV hepatic haemosiderosis with septal fibrosis, without HFE variants; the blood film showed stomatocytes and target cells, osmotic resistance was increased and the panel identified PIEZO1 c.5975C>T p.(Thr1992Met), classified as a VUS in ClinVar and considered pathogenic by the authors [33, 47]. The message is that not all hereditary iron overload is haemochromatosis and that the blood film is decisive [47]. The same reasoning applies to ATP11C deficiency [8].

Pyrimidine 5′-nucleotidase deficiency and lead poisoning share the blood film, low enzyme activity and pyrimidine nucleotide accumulation, so blood lead measurement is mandatory before accepting a genetic cause [8, 24]. BPGM deficiency erythrocytosis belongs to the low-P50 erythrocytosis group alongside high-affinity haemoglobins, after excluding polycythaemia vera (JAK2), and should not be treated with cytoreduction or indiscriminate phlebotomy [8]. Xerocytosis with high-affinity haemoglobin may also be referred as polycythaemia [13].

In forms with a muscular component, the differential is McArdle disease. The non-ischaemic forearm exercise test shows a flat lactate curve with a normal ammonia rise in both, but compensated haemolysis suggests PFK or PGK deficiency, and neurological manifestations suggest PGK [6, 71]. In acidosis with 5-oxoprolinuria, the acquired form associated with paracetamol and flucloxacillin in frail older adults and 5-oxoprolinase deficiency, an apparently benign biochemical condition, should be excluded [8, 16].

Pseudohyperkalaemia in xerocytosis must be distinguished from ABCB6-related familial pseudohyperkalaemia, which does not cause haemolysis, and from true hyperkalaemia; familial pseudohyperkalaemia has led to fludrocortisone treatment for presumed adrenal insufficiency and misdiagnosis of Gordon syndrome [4, 8]. ICSH also lists iron deficiency, thalassaemia, burns, myelodysplasia, heated samples and the disease-free Leach phenotype as exclusions for elliptocytosis [4].

In young children with non-haemolytic congenital anaemia, the differential includes UMPS deficiency, with megaloblastic anaemia unresponsive to iron, folate or vitamin B12 and orotic aciduria, adenosine deaminase 2 deficiency, which mimics Diamond-Blackfan anaemia, and Pearson syndrome [24, 62]. Elevated red cell ADA1 activity is a marker of Diamond-Blackfan anaemia and was historically described as a cause of nonspherocytic haemolytic anaemia [24]. GATA1-related cytopenia may be underdiagnosed in Diamond-Blackfan-type congenital hypoplastic anaemias without a ribosomal protein variant [8]. Acquired confounders, such as bleeding, nutritional deficiencies, alloimmunisation, infections, coexisting autoimmune anaemia or PNH and marrow, kidney or liver disorders, may mask or simulate congenital haemolytic anaemia [63].

Saudubray also lists metabolic causes of haemolytic, dyserythropoietic or mixed-mechanism anaemia in the paediatric differential: abetalipoproteinaemia and other causes of acanthocytosis, erythropoietic porphyrias, galactosaemia, lecithin–cholesterol acyltransferase (LCAT) deficiency, sitosterolaemia, Wilson disease, Wolman disease, severe liver failure, GLUT1 deficiency with exercise-triggered haemolysis and Majeed syndrome as a dyserythropoietic cause [62]. In isolated transaminase elevation, haemolysis, myopathy and macrotransaminasaemia should always be excluded [62]. In children with purine nucleoside phosphorylase (PNP) deficiency immunodeficiency, approximately one-third have autoimmunity, including autoimmune haemolytic anaemia, an acquired rather than constitutional differential [24].

7. Treatments and Risk Management Strategies

7.1. Common supportive treatment

Treatment for most entities in the group is supportive. In PK deficiency, standard measures are transfusions as required, folate supplementation, neonatal phototherapy and exchange transfusion, splenectomy in selected patients with prophylaxis against encapsulated organisms, cholecystectomy and iron chelation [8]. The PKD Burden of Disease group recommends folic acid supplementation in virtually all patients, without specifying the dose outside pregnancy [11]. The same pattern applies to rare enzymopathies, stomatocytoses, symptomatic elliptocytosis and CDAs, with folate, transfusions during severe or aplastic episodes, gallstone surveillance and chelation when required [5, 8, 21]. Most patients with elliptocytosis need no treatment [8].

The decision to transfuse should be based on tolerance and lifestyle rather than an arbitrary haemoglobin threshold [11]. The PKD Burden of Disease group illustrates this with two splenectomised brothers with haemoglobin of 8–9 g/dL and 20–35% reticulocytes: one was transfused on demand and the other regularly to maintain haemoglobin above 10–11 g/dL, with oral chelation; unnecessary transfusion in a patient tolerating anaemia well is harmful because of the added iron [11]. In progressive extramedullary haematopoiesis, some experts use chronic transfusion to halt mass growth, without formal evidence, and malleolar ulcers are treated as in sickle cell disease and thalassaemia [11]. Regular exchange transfusion in severe cases did not achieve consensus [11].

For chronic jaundice with a psychosocial impact in PK deficiency, the PKD Burden of Disease group allows low-dose phenobarbital, 15–30 mg daily, as off-label use to induce hepatic bilirubin metabolism, after explaining adverse effects; in a 14-year-old adolescent, 15 mg daily reduced bilirubin from 3.9 to 2.0 mg/dL [11]. This indication does not apply to glutathione deficiencies, in which phenobarbital is listed among drugs to avoid [8, 16].

7.2. Splenectomy

The 2017 EHA recommendations are the reference across the group [3]. They were based on a literature review without randomised trials or case-control studies, so evidence comes from series and expert opinion, graded using GRADE (Grading of Recommendations Assessment, Development and Evaluation); consensus was defined as unanimous (100%) or majority (above 80%), and thalassaemias were excluded [3]. The document's summary tables, published as images, were not read; the recommendations below are those in the text.

In PK deficiency, the American Natural History Study experience is consistent with these data: splenectomy increased median haemoglobin by 1.6 g/dL and reduced transfusion burden in 90%, with thrombosis in 11%; patients with higher preoperative haemoglobin, lower indirect bilirubin and missense variants responded better, and 48% of those operated on without simultaneous cholecystectomy required it later [15]. The PKD Burden of Disease group adds that post-splenectomy sepsis reached 7%, approximately 15% continue to receive regular transfusions, non-responders tend to have two severe alleles and more intense preoperative haemolysis, and total splenectomy is preferable because response to partial splenectomy is poor [11]. Reticulocytosis rises markedly after surgery and haemolysis does not stop [3, 17]. Before surgery, PK deficiency should be confirmed and stomatocytosis or other thrombophilic states excluded, and PKLR sequencing is recommended particularly at this stage [17]. If haematopoietic cell transplantation is being considered, splenectomy should be avoided [11]. In 2020, the group suggested postponing splenectomy in patients with at least one missense variant while mitapivat was in trials [11].

In stomatocytoses, the contraindication is also supported by the French series: 16 patients had undergone splenectomy before xerocytosis diagnosis, with a mean delay of 15 years, and haemolysis persisted (haemoglobin 112±19 g/L); 12 of the 16 (75%) experienced 19 thrombotic events (5 portal thromboses, 5 strokes, 6 venous thromboembolisms and 3 cases of chronic thromboembolic pulmonary hypertension) a mean 13±8.6 years after surgery [13]. All eight splenectomised patients with PIEZO1 developed thrombosis and received prolonged anticoagulation, whereas none of the four with KCNN4 had thrombosis during a mean follow-up of 26.5 years, numbers too small to conclude that surgery is safe in Gardos channelopathy [13]. In this disorder, haemolysis persists after splenectomy and the GeneGuidance entry likewise advises against it [8]. If a patient with stomatocytosis has undergone splenectomy following misdiagnosis, EHA advises considering lifelong anticoagulation [3]. Failure to respond to splenectomy performed for presumed spherocytosis should prompt reassessment for stomatocytosis [4]. In elliptocytosis, splenectomy is reserved for severe anaemia or transfusion dependence, with mandatory prior vaccination, and should not be extrapolated to stomatocytoses [8]. In rare enzymopathies, benefit has been reported in severely affected patients with GPI, PGK1, ALDOA and AK1 deficiencies, without correction of haemolysis [8, 57].

Technique and complication prevention also follow EHA. Laparoscopy is standard for normal or mildly enlarged spleens, perioperative splenic artery embolisation reduces complications in large spleens and partial splenectomy removes 80–90% of the organ [3]. Splenectomy should generally not be performed before five years, below which the risk of overwhelming infection is highest; risk is greater during the first postoperative year and probably persists throughout life [3]. Overwhelming post-splenectomy infection is caused mainly by Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae, and asplenia also increases the risk of malaria, Capnocytophaga infection after animal bites, babesiosis and Bordetella holmesii infection [3]. Prevention relies on education to act immediately with fever, vaccination before and after surgery and antibiotic prophylaxis according to national guidelines, often lifelong [3, 11]. In a prospective German cohort of severe sepsis, pneumococcal aetiology accounted for 42% in splenectomised patients versus 12% in those with a functional spleen, and fewer than half of patients with overwhelming infection had received pneumococcal vaccination [3].

Acute portal and splenic vein thrombosis occurred in 11 of 89 patients (12.3%) operated on for haemolytic disease, compared with 1.7% in immune thrombocytopenia and none after trauma; massive splenomegaly is a risk factor and median time to asymptomatic thrombosis was six days [3]. EHA recommends at least one Doppler ultrasound on the seventh day after splenectomy (86%; grade 2C) [3]. Usual treatment is intravenous heparin followed by an oral anticoagulant for 3–6 months [3]. EHA considers the role of antithrombotic prophylaxis unknown; the PKD Burden of Disease group, as expert opinion, allows low-dose acetylsalicylic acid in adults with additional risk factors (advanced age, previous thrombosis, smoking) until platelets fall below 500×10⁹/L, and in children below 1000×10⁹/L [3, 11]. Long term, splenectomy is associated with increased risks of venous thromboembolism, ischaemic heart disease and pulmonary arterial hypertension, particularly in haemolytic disorders [3].

Concomitant cholecystectomy is disputed. EHA recommends it during the same operation in PK deficiency (86%; 2C), whereas the PKD Burden of Disease group states that it may accompany splenectomy, with counselling on the risk of intrahepatic cholestasis [3, 11]. Practice also differs between continents: splenectomy is more frequent in the USA, while in Europe more patients continue transfusions and chelation with an intact spleen [11].

7.3. Iron overload and chelation

Iron overload should be monitored in all patients, transfused or otherwise [11, 13, 15]. In xerocytosis, ferritin correlates poorly with tissue iron below 1000 ng/mL: eight patients with ferritin below 1000 ng/mL already had liver iron concentration (LIC) above 150 µmol/g, so the French series recommends assessing tissue iron by magnetic resonance imaging (MRI) at diagnosis even with moderate ferritin and reassessing iron annually [13]. Treatment included phlebotomy in 15 patients, deferasirox in 9, desferrioxamine in 3 and deferiprone in 1, and LIC fell from 200±103 µmol/g at diagnosis to 88±42 µmol/g at last follow-up [13]. In CDA I, chelation or phlebotomy applies to non-anaemic patients, and GeneReviews advises against any iron-containing preparation [9, 21]. In PK deficiency, a case illustrates hidden iron: a splenectomised patient with baseline haemoglobin of 7.0 g/dL, without transfusions and with excellent quality of life at 40 years, had ferritin of 670 ng/mL and LIC of 8.9 mg/g dry weight, and started deferasirox [11].

No chelator has an approved indication for non-transfusional overload in PK deficiency, xerocytosis or CDAs; use in these situations is extrapolated from non-transfusion-dependent thalassaemia (NTDT) and is off-label [8, 72, 73].

The consulted sources do not include European summaries of product characteristics for these chelators, so the table is restricted to US labels. Mitapivat reduced iron overload in PK deficiency (section 7.5) [34].

7.4. Mitapivat: mechanism, dosage and safety

Mitapivat (Pyrukynd®; Aqvesme® in the USA for thalassaemia) is an oral allosteric activator of red cell pyruvate kinase [8, 18]. In PK deficiency, it increases residual mutant enzyme activity, so response depends on the presence of at least one missense allele: in the phase 2 study, only patients with at least one missense variant responded, and response was associated with baseline PK-R protein level [35]. Patients homozygous for c.1436G>A p.(Arg479His) and those with two non-missense alleles did not respond in the dose-ranging study and were excluded from phase 3 trials [18]. Beyond its effect on haemolysis, mitapivat reduced erythroferrone, increased hepcidin and improved hepatic iron, making it the first drug with a demonstrated effect on ineffective erythropoiesis and iron overload in PK deficiency [7, 34].

Sources: [18–20, 75].

Tapering follows the US label table: from 5 mg twice daily, change to 5 mg once daily on days 1–7 and discontinue on day 8; from 20 mg twice daily, change to 20 mg once daily on days 1–7 and 5 mg once daily on days 8–14, discontinuing on day 15; from 50 mg twice daily, the sequence is 50 mg once daily, then 20 mg once daily and discontinuation on day 15 [18]. The European summary likewise provides for tapering over 1–2 weeks [19]. A missed dose is taken if up to 4 hours have elapsed and skipped if longer [18]. The dose may be reduced to 20 or 5 mg twice daily for adverse effects or haemoglobin above normal [18].

Mitapivat is metabolised by CYP3A. Strong CYP3A inhibitors and inducers should be avoided; with moderate inhibitors, titration should not exceed 20 mg twice daily, and with moderate inducers alternatives should be preferred or titration may exceed 50 mg twice daily without exceeding 100 mg twice daily [18]. Mitapivat itself induces CYP3A and reduces hormonal contraceptive efficacy, so a non-hormonal or barrier method is recommended; in vitro it also induces CYP2B6, CYP2C8/9/19 and UGT1A1 and inhibits P-glycoprotein [18]. It should be avoided in moderate or severe hepatic impairment [18].

In ACTIVATE, serious adverse reactions occurred in 10% of treated patients (atrial fibrillation, gastroenteritis, rib fracture, musculoskeletal pain, one case each) [18]. The most frequent reactions were decreased oestrone (men), increased urate (15%), back pain (15% versus 8% with placebo), decreased oestradiol (men) and arthralgia (10% versus 5%); hypertriglyceridaemia occurred in 8% (grade ≥3 in 5%) [18]. In men, oestrone decreased in 56.3% versus none with placebo and oestradiol in 12.5% versus 6.7%, through mild aromatase inhibition, reversing approximately 28 days after discontinuation [18]. This aromatase inhibition prompted caution in paediatric use [11]. In the European summary, very common effects in PKD are insomnia, nausea and low oestrone in men [19]. The ACTIVATE publication reports nausea in 18% versus 23% and headache in 15% versus 33%, with grade ≥3 events in 25% versus 13% [76]. In ACTIVATE-T, the most frequent effects were increased ALT (37%), headache (37%), increased AST (19%), fatigue and nausea (19% each) [77]. In the phase 2 study, transient headache and insomnia occurred mainly initially [35].

Hepatic risk was identified in thalassaemia, at higher doses than those used in PKD. In thalassaemia trials, 2 of 301 patients (0.66%) in the double-blind phase and three in the extension had reactions suggestive of hepatocellular injury; two were hospitalised, one with peak bilirubin of 32 mg/dL, and all resolved with discontinuation [75]. If liver injury occurs with new jaundice or ALT ≥10 times baseline, the drug should not be restarted [75]. Since January 2025, the Pyrukynd® label has included the warning “hepatocellular injury in another condition”, describing reversible ALT elevations above 5 times the upper limit of normal in the first six months, with or without jaundice, and requiring monthly liver monitoring during this period in PKD too [18]. In the phase 3 sickle cell disease trial (RISE UP), the company announcement does not report a hepatocellular injury signal [78].

Abrupt discontinuation may cause acute haemolysis, observed in the dose-ranging study; patients and carers should know the warning signs (jaundice, dark urine, dizziness, confusion, fatigue, dyspnoea) [18]. Pregnancy data are insufficient; rat and rabbit studies showed no teratogenicity at up to 13 and 3 times exposure at the maximum recommended human dose, and the label notes that untreated PK deficiency may precipitate acute haemolysis, preterm delivery, miscarriage and severe anaemia, with estimated background risks of 18% for miscarriage, 24% for growth restriction and 56% for preterm delivery [18]. There are no data on passage into human or animal milk or effects on the infant, and breastfeeding decisions should weigh breastfeeding benefits against maternal clinical need [18]. Paediatric safety and efficacy are not established, and the ACTIVATE-Kids and ACTIVATE-KidsT paediatric trials had no published registry results at the consultation date [18, 79].

7.5. Regulatory status

Sources: [18–20, 75, 80–82]. The exact European Commission decision date was not confirmed in the Union Register; the European Medicines Agency updated product information on 21/05/2026 [20, 80]. The consulted sources do not include national access data in Portugal or Brazil. Mitapivat is not approved for HK1 deficiency, xerocytosis or other enzymopathies or membrane disorders [8].

7.6. Clinical trials

Interpretation of these trials has limitations. All phase 3 PK deficiency trials excluded patients without a missense allele, children and p.(Arg479His) homozygotes, and 73% of the ACTIVATE population were splenectomised, so results do not apply directly to the entire genetic spectrum [18]. The 40% response means that most treated patients did not reach the response threshold, and mean haemoglobin increase was 1.7 g/dL [18]. ACTIVATE-T had no control group, and the difference between publication and label (10 versus 9 responders) arises from different analysis criteria [18, 77]. A systematic review of 7 studies with 166 treated patients found haemoglobin responses of 38–80% and mean increases of 0.4–1.7 g/dL, with grade ≥3 events in 23.4% [89]. Thalassaemia and sickle cell disease trials used a higher dose and different populations, so should not be indirectly compared with PKD trials. Negative or partially negative trials form part of the picture: phase 3 RISE UP did not reduce painful crises or improve fatigue measured by the PROMIS (Patient-Reported Outcomes Measurement Information System) scale, and senicapoc, tested in sickle cell disease, did not reduce crises despite improving haemolysis [78, 86]. Individual compassionate-use experience in genotypes excluded from trials, as in the section 10 case, is anecdotal [46].

7.7. Targeted therapies in other entities

Interferon alfa is the only specific treatment for CDA I, discovered fortuitously in a patient treated for hepatitis C; the mechanism is unknown and use is off-label [9, 21]. GeneReviews reports interferon alfa-2a or alfa-2b intramuscularly or subcutaneously two to three times weekly, or peginterferon once weekly, prescribed by an experienced physician, with increased haemoglobin and reduced iron overload in most patients, including infants [9]. In a review of 33 published and 7 new cases, mean haemoglobin increase was 30.7 g/L (p<0.001), and one patient without CDAN1 or CDIN1 variants did not respond [21]. Availability varies: in the USA, the peginterferon alfa-2a label covers only chronic hepatitis B and C, and conventional and pegylated interferon alfa-2b have been withdrawn from the market [8].

Interferon adverse effects include fever, flu-like syndrome, headache, myalgia, diarrhoea, injection-site bruising, neutropenia, rash, arthralgia and polycythaemia, hindering adherence; a British study proposed a response classification with patient-reported measures in collaboration with the Congenital Anaemia Network [21]. In interferon-resistant transfusion-dependent CDA I, allogeneic marrow transplantation succeeded in 11 of 13 children [9]. In CDA I, splenectomy should be considered cautiously, as in non-transfusion-dependent thalassaemia; GeneReviews recommends laparoscopic cholecystectomy for gallstones, calcium and vitamin D for osteoporosis and, for extramedullary haematopoiesis, regular transfusions, surgical debridement or low-dose radiotherapy [9].

Haematopoietic cell transplantation (HCT) is the only established curative option in PK deficiency, but is reserved for severe disease [8]. By 2018 only 16 patients had been transplanted worldwide, with a high rate of grade IV graft-versus-host disease and cumulative survival of 74% at two years, in patients heterogeneous in age, transplant type and conditioning; most experts prefer supportive care [11]. Outside PK deficiency, HCT has been described in isolated cases: severe transfusion-dependent HK1 deficiency, PGK1 deficiency (correcting only the blood defect), CDA II, CDA III in a Japanese family, CDA IV in a KLF1 compound heterozygote and severe GATA1-related cytopenia; in pyropoikilocytosis with near-complete α-spectrin deficiency, alternatives are lifelong transfusions with chelation or HCT [8, 60, 92].

In glutathione deficiencies, measures are based mainly on series. In GSS deficiency, acidosis is corrected with bicarbonate, citrate or trometamol, at doses potentially reaching 10 mmol/kg/day or more during acute infections; recommended antioxidant doses are lifelong vitamin E 10 mg/kg/day and vitamin C 100 mg/kg/day, and follow-up of 28 patients suggests that early supplementation prevents neurological damage [8, 16]. N-acetylcysteine is not recommended because it causes neurotoxic cysteine accumulation in fibroblasts; oral glutathione had no lasting benefit in two patients, glutathione esters are limited by toxicity and erythropoietin was effective in one patient [16]. Early diagnosis, acidosis correction and vitamins E and C are the most important prognostic factors [16].

In enzymopathies with a muscular component, treatment is preventive. In Tarui disease, intense exercise and carbohydrate-rich meals before exercise should be avoided, unlike McArdle disease; some patients benefited from a high-protein diet, a ketogenic diet was proposed in children with a severe variant and an adult followed for five years on a ketogenic diet experienced muscular symptom relief [6, 8]. In ALDOA deficiency, antipyretics during febrile episodes and avoidance of catabolic stress are recommended to prevent haemolysis and rhabdomyolysis [8]. In PGK1 deficiency, rhabdomyolysis requires immediate recognition because of acute renal failure risk, and levodopa is used off-label for parkinsonism [8]. In TPI deficiency, there is no effective therapy; ventilatory support appears to prolong life in some cases, and pharmacological stabilisation of dimer structure is a therapeutic hypothesis without clinical translation [6]. In GPI deficiency with hydrops, repeated intrauterine transfusion has been used successfully [8, 93]. In SLC4A1-associated renal tubular acidosis, lifelong oral alkali is used, with a median prescribed dose of 1.9 mEq/kg/day in 340 patients from 29 countries and adequate control in approximately half [8, 94].

7.8. Developments in the last 12 months and research

Other programmes remain under investigation. The PK activators tebapivat (AG-946) and etavopivat are in sickle cell disease trials, tebapivat also in anaemia of low-risk myelodysplastic syndrome, without an identified trial in PK deficiency or other hereditary haemolytic anaemias [95, 96]. SATISFY (NCT05935202) was designed as a single-arm phase 2 study with approximately 25 adults with spherocytosis, stomatocytosis or CDA II, using mitapivat 50 mg twice daily with possible increase to 100 mg, and safety as the primary outcome; the registry showed no results and was outdated [97]. The preclinical basis includes a murine spherocytosis model in which mitapivat improved anaemia [98]. A phase 1/2 senicapoc trial in dehydrated stomatocytosis, involving five patients, ended in February 2024 without registry results [99]. The registry also lists zoledronic acid in CDA (phase 1/2) and omeprazole to reduce iron absorption in CDA (phase 4, status unknown) [99]. CRISPR gene editing for PK deficiency is preclinical [87]. RAS–MEK pathway inhibition to restore hepcidin in xerocytosis is a preclinical hypothesis [44].

7.9. Agents and interventions to avoid

7.10. Pregnancy, perioperative care and transition

Pregnancy triggers haemolysis in PK deficiency, and haemodilution worsens anaemia; there is a tendency towards more preterm deliveries, miscarriages and transfusion requirements [11]. The PKD Burden of Disease group considers folate supplementation mandatory, at a minimum of 1 mg daily in pregnancy, recommends monitoring fetal growth and guiding transfusion by symptoms and ultrasound growth, and allows planned pregnancy to weigh in favour of prior splenectomy [11]. In the described case, a woman at 12 weeks of pregnancy with haemoglobin of 8.3 g/dL required 15 units during pregnancy and 4 at delivery [11]. In CDA I, a series of 28 women had one stillbirth, one first-trimester miscarriage and low birth weight in 42% of newborns, with more caesarean sections than controls, justifying follow-up in a high-risk pregnancy unit [9]. In xerocytosis and Gardos channelopathy, fetal surveillance is discussed in section 8.4 [13].

Perioperatively, splenectomy requires the vaccination, education and prophylaxis described in section 7.2, Doppler ultrasound on the seventh day and consideration of thromboprophylaxis in patients with risk factors [3, 11]. Surgery triggers haemolytic crisis in PK deficiency [11]. In patients with xerocytosis or ABCB6 deficiency, preoperative potassium values should be confirmed in an immediately processed sample [8]. In regulator-type Rh-null, transfusion requires Rh-null blood from relatives, frozen units or rare donor registries, and autologous donation and cryopreservation should be considered before elective surgery [8]. The consulted sources do not describe a formal paediatric-to-adult transition programme for these disorders; changing anaemia tolerance with age, increasing gallstones and progressive iron overload justify maintaining systematic surveillance in adulthood [11, 13].

7.11. Guidelines and disagreements

The most practice-relevant disagreements concern PK deficiency prevalence, test order (enzyme or genetics first), the post-transfusion interval (eight weeks for complete clearance in ICSH, 50 days as a safe period in the PKD consensus), concomitant cholecystectomy and splenectomy frequency, higher in the USA than Europe [3, 4, 11, 17]. The Blood reviews on CDAs (2020) and stomatocytoses (2025) were accessible only as abstracts; these provide CDA classification, erythroferrone's role in iron overload in CDA II, the genes PIEZO1, KCNN4 and ABCB6 and the pleiotropic nature of PIEZO1 gain of function [23, 45]. There are no specific guidelines for CDA I or elliptocytosis [8, 9].

8. Reproductive Options and Family Counselling

8.1. Recurrence risks by inheritance mode

In autosomal recessive disorders (PK deficiency and most enzymopathies, pyropoikilocytosis, CDA I and II, CDA IVb), siblings of a patient have a 25% probability of being affected, 50% of being asymptomatic carriers and 25% of inheriting neither variant [9]. Children of a patient are obligate carriers unless the other parent is also a carrier, which should be investigated in consanguineous or founder populations; the patient's uncles and aunts have a 50% carrier probability [9]. If only one parent is a carrier, a de novo variant, parental mosaicism and uniparental isodisomy should be considered [9].

In autosomal dominant disorders (xerocytosis, Gardos channelopathy, OHSt, elliptocytosis, SAO, CDA III and IVa), each child of a patient has a 50% probability of inheriting the variant. Expressivity varies: in Gardos channelopathy there are carrier parents without documented anaemia, and in elliptocytosis most heterozygotes are asymptomatic [8]. De novo variants are not rare in stomatocytoses, including KCNN4 p.(Arg352His) [4, 8]. In elliptocytosis, a proband more severely affected than affected relatives suggests a second allele in trans, such as αLELY, or a recessive mechanism, and ICSH recommends studying the entire family [4, 8].

In X-linked PGK1 deficiency, expression is full in hemizygous men; heterozygous women may have variable haemolytic anaemia and, rarely, neurological disease, and may have normal red cell activity, so carrier detection requires molecular analysis [6, 8]. The same principle applies to ATP11C deficiency and GATA1-related cytopenia [8].

8.2. Cascade testing and carrier detection

In PK deficiency, parental studies help confirm variant phase, particularly in transfusion-dependent patients; this is almost universal practice in European reference centres and less frequent in other health systems [17]. For family planning, partners should be tested by sequencing rather than enzyme activity: in 31 family studies, eight carriers had normal PK activity [11]. In CDA I, younger siblings should be assessed with a full blood count, blood film and molecular testing [9]. In pyrimidine 5′-nucleotidase and GCLC deficiencies, heterozygotes are haematologically normal and biochemical detection is unreliable [8]. Carrier testing, prenatal diagnosis and preimplantation genetic testing are possible when familial variants are identified; without a molecular diagnosis, storing the patient's DNA is recommended [9]. In the Amish population, the PKLR p.(Arg479His) carrier frequency of 1/151 justifies considering targeted partner testing [49].

8.3. Germline mosaicism and apparently de novo variants

The PKD Burden of Disease group describes a 24-month-old boy, transfusion-dependent and initially labelled as having xerocytosis and G6PD deficiency, with normal PK activity but a low PK/HK ratio and the PKLR variants c.1091G>A p.(Gly364Asp) and c.1529G>A p.(Arg510Gln) [11]. His mother had approximately 50% activity; his father showed no second variant in blood by Sanger sequencing, but massively parallel sequencing detected it in blood, buccal mucosa, urine and semen, with negative paternal grandparents, demonstrating paternal postzygotic mosaicism [11]. The practical consequence is that an apparently de novo variant should prompt parental mosaicism testing with sensitive methods, because recurrence risk is no longer negligible [9, 11].

8.4. Pregnancy with an affected parent

In xerocytosis, perinatal oedema recurred in all informative families, and the French series authors recommend ultrasound surveillance at least monthly during pregnancy when either parent is affected, regardless of genotype (PIEZO1 or KCNN4) [13]. The report of fetal loss from non-immune hydrops with a KCNN4 deletion reinforces this surveillance in Gardos channelopathy [8, 58]. In CDA I, severe fetal anaemia may require Doppler surveillance and fetal transfusions, and pregnancy in an affected woman should be followed in a high-risk unit [9]. In the Rh-null phenotype, anti-Rh29 alloimmunisation after a first pregnancy may cause fetal haemolytic disease in subsequent pregnancies [8]. Anti-Er4 and anti-Er5 antibodies, directed against PIEZO1-defined antigens, have also been implicated in severe haemolytic disease of the fetus and newborn [8].

8.5. Counselling minors and consultation content

Testing asymptomatic minors is clinically justified when results change surveillance: in CDA I, assessment of younger siblings is recommended, and in PK deficiency complications such as iron overload and gallstones occur even in mild forms [9, 15]. An SPTB heterozygous result in a newborn implies neonatal bilirubin surveillance [8]. Counselling should explain inheritance, variable expression within the same family, enzyme assay limitations in carrier detection, the possibility of mosaicism, prenatal and preimplantation diagnostic options and entity-specific transfusion and obstetric implications [9, 11, 13]. GeneReviews lists MedlinePlus as an informational resource for CDAs [9].

9. Special Considerations: Population Diversity and Epidemiology

9.1. Prevalence and what each figure measures

Prevalence figures for this group derive from very different methods and should not be compared as though measuring the same thing.

Underdiagnosis characterises the group. In the United Kingdom, the national CDA I registry had only 11 patients versus 300–400 expected from carrier frequency [21]. In PK deficiency, haematologically normal parents, transfusions confounding testing, compensated forms and technically difficult assays explain the difference between genetic prevalence and diagnosed cases [17]. Published case counts are not rates and cannot estimate prevalence.

9.2. Geography, ancestry and founder effects

Malaria selection pressure has shaped the distribution of several of these disorders. Elliptocytosis is more frequent in West Africa and Southeast Asia, with predominantly spectrin alleles, and spectrin defects are more frequent and severe in Black populations, whereas protein 4.1 deficiency is more frequent in Caucasians [4, 8]. SAO protects against malaria [8]. PK deficiency protects against parasite replication in experimental models, without clear demonstration of increased frequency in endemic areas [17]. Red cell dehydration through PIEZO1 gain of function is associated with malaria protection, and the GeneGuidance entry reports a common PIEZO1 allele in populations of African ancestry, making a single worldwide prevalence value indefensible [8, 13]. Recessive SLC4A1-related distal tubular acidosis is concentrated in the tropics, from Oman to Papua New Guinea [4].

The founder effects described in section 2.8 have clinical and regional implications. In the USA, the PKLR p.(Arg479His) variant in the Amish of Pennsylvania and Ohio, with a carrier frequency of 1/151, produces a cluster of severe PK deficiency ineligible for mitapivat [18, 49]. In Europe, p.(Arg510Gln) predominates in the North and p.(Arg486Trp) in the South, and the PKLR exon 11 deletion affects Roma communities [17]. CDA I has its highest prevalence among Israeli Bedouins, with approximately 70 published consanguineous families versus approximately 100 isolated cases mainly in Europe [9, 21]. CDA III is concentrated in Västerbotten, Sweden [8]. PFKM deficiency is more frequent in American Ashkenazi Jews and Japanese people [6]. GPI p.Arg347His is common in Indian patients [8]. In Thailand, 0.86% of newborns are heterozygous for common SPTB variants [8]. The carrier frequency of KLF1 variants was 1.25% in 3839 people in southern China versus 0.08% in the north [8].

9.3. Data from Portugal and Brazil

In Portugal, the 2019 international PKD diagnostic consensus had co-authorship from Hospital Universitário de Coimbra [17]. Hospital de Santa Maria in Lisbon published a 2025 case of an infant with TPI deficiency, with haemolytic anaemia, progressive neurological deterioration and respiratory failure, genetically diagnosed and treated symptomatically [108]. The consulted sources do not include Portuguese prevalence estimates for these disorders.

In Brazil, the Universidade Estadual de Campinas Blood Centre published the first comprehensive genetic analysis of hereditary anaemias in the Brazilian population in 2020 [68]. A 35-gene panel applied to 36 patients identified potentially pathogenic variants in 26 (72%), 20 novel, with three confirmed de novo variants; SPTB was the main spherocytosis gene in southeastern Brazil (34.6% of cases), there were two cases of dominant spherocytosis with a null variant in trans with αLELY, two patients with PKLR (including homozygous NM_000298:c.1532G>A p.(Gly511Glu)) and two with CDA I from CDAN1 variants [68]. The consulted sources do not include Brazilian prevalence estimates.

9.4. Special populations

In pregnant women, haemolysis worsens and management requires folate, fetal growth surveillance and, in stomatocytoses, monthly ultrasound (sections 7.10 and 8.4) [11, 13]. In older adults, anaemia tolerance decreases, spherocytosis may be diagnosed between the seventh and ninth decades after an infection and xerocytosis has been diagnosed after 88 years [4, 11, 13]. Athletes with xerocytosis may develop haemolysis after each training session [4]. Patients with the Rh-null phenotype or rare alloantibodies should be enrolled in rare donor programmes [8]. Splenectomised patients travelling to malaria areas, or exposed to animals or ticks, have specific infectious risks [3].

9.5. Reference networks and patient organisations

The relevant European reference network is ERN-EuroBloodNet, dedicated to rare haematological disorders, which endorsed the PKD diagnostic consensus and incorporates the former ENERCA (European Network for Rare and Congenital Anaemias) [17, 22]. The ENERCA White Book defines requirements for centres of expertise, including ISO 15189 accreditation and, for PKD, mandatory PK activity and PKLR analysis [17]. Active registries include the global PKD PEAK registry (NCT03481738, up to 500 patients), the International CDA II Registry, based in Naples, and the CDA registry (CDAR, NCT02964494) [50, 99, 109, 110]. An international registry has been recommended for xerocytosis [8, 111]. Patient organisations include the National Organization for Rare Disorders (NORD), with a PKD page, the Thalassaemia International Federation, relevant to mitapivat in thalassaemia, and the Congenital Anaemia Network in the United Kingdom, supporting patients with congenital anaemias and funding research [21, 112, 113].

10. Illustrative Clinical Case

Ceglie and colleagues published in 2025 the case of a 20-year-old Latin American man, originally from Venezuela and followed in Italy, regularly transfused since childhood for chronic haemolytic anaemia of unknown cause [46]. He had undergone splenectomy at 12 years, with initial improvement. Next-generation sequencing identified homozygous PKLR NM_000298.6:c.1528C>T p.(Arg510Ter); the authors describe abnormal splicing with a truncated enzyme lacking 63 C-terminal residues, and PK activity was 5–6 IU/g Hb against a normal interval of 11.6–16.1 [33, 46]. In September 2022, he was receiving one transfusion every two months, low-dose acetylsalicylic acid and folic acid 5 mg daily; haemoglobin was 9 g/dL, total bilirubin 9.1 mg/dL (indirect 8.3 mg/dL), ferritin 1650 ng/mL and transferrin saturation 83%, so he started deferasirox; computed tomography excluded an accessory spleen [46].

Because his homozygous nonsense genotype excluded him from trials requiring at least one missense allele, mitapivat was administered through a managed access programme approved by the ethics committee, starting at 5 mg twice daily and titrating to 50 mg twice daily over two months [46]. After 12 months, total bilirubin fell from 6.7±1.3 to 5.5±0.8 mg/dL (p=0.002) and transfusions decreased from 9 to 5 units (−44.4%), while haemoglobin did not change significantly (8.8±0.4 versus 9.1±0.4 g/dL; p=0.07) [46]. Liver and renal function remained normal, initial daytime somnolence and insomnia resolved within two months and the patient reported subjective improvement [46]. The article's figure 1, consulted in the PMC version (https://pmc.ncbi.nlm.nih.gov/articles/PMC12591939/figure/f1-blt-23-555/), compares the area under the curve for total bilirubin and haemoglobin in the preceding year and during treatment and shows transfusion requirements in the upper panel [46].

The case brings together several themes of this article: late diagnosis of an enzymopathy in a transfused splenectomised patient, iron overload and genotype-defined eligibility for targeted therapy. Partial response in a homozygous nonsense genotype is an individual observation and does not change the approved indication, restricted to adults and supported by trials in patients carrying at least one missense allele [18, 35]. Mitapivat use in this context lies outside the studied populations and should not be presented as routine practice.

11. Prognosis and Long-Term Follow-up

11.1. Prognosis

Prognosis depends on the entity, genotype and management of chronic complications. In PK deficiency, anaemia tends to improve with age, but iron overload (48%) and gallstones (45%) affect almost half the American cohort regardless of baseline haemoglobin, and anaemia tolerance decreases in older adults [11, 15, 17]. With mitapivat, 13 of the 15 evaluable ACTIVATE responders maintained response in the extension, and the six ACTIVATE-T patients who became transfusion-free remained so, with a median duration of 17 months [18]. The consulted sources provide no validated overall survival estimates in PK deficiency.

In other entities, data come from series. In xerocytosis, most patients have compensated haemolysis, neonatal anaemia improves with age and morbidity is dominated by iron overload, increasing with age, and thromboses after splenectomy [13]. In CDA I, 5 of 21 patients (23%) in one series died as adults, mainly from iron overload, and 9% of 32 Israeli adults died between 46 and 59 years from sepsis or pulmonary hypertension, all splenectomised [9]. TPI deficiency is the most severe glycolytic enzymopathy, with frequent childhood death from respiratory failure [6, 8]. In GSS deficiency, 7 of 28 patients in one series had died at the study date [8]. PGK1 deficiency has a variable prognosis and the fatal infantile form is very rare [6]. Tarui disease, outside the infantile form, does not progress to severe disability [6]. Heterozygous elliptocytosis has an excellent prognosis, whereas HPP with near-complete α-spectrin deficiency entails lifelong transfusions or HCT [8]. CDA III is associated with excess monoclonal gammopathy and multiple myeloma, and GATA1-related cytopenia may progress to myelodysplastic syndrome or aplasia [8].

11.2. Surveillance

Table 1 of the PKD Burden of Disease group, with screening intervals for iron overload, extramedullary haematopoiesis, osteopenia, gallstones and pulmonary hypertension, and the 2024 international guideline surveillance recommendations were inaccessible in the consulted sources, so the PKD intervals given are restricted to what textual sources support [11, 12]. At initial CDA I assessment, GeneReviews also recommends haemoglobin and bilirubin at diagnosis, ferritin from 10 years in non-transfused patients, hepatic and cardiac T2* MRI and genetic counselling [9].

12. Summary and Final Considerations

Non-immune hereditary haemolytic anaemias comprise membrane defects, enzymopathies and congenital dyserythropoietic anaemias sharing chronic haemolysis, jaundice, gallstones, splenomegaly and iron overload occurring even without transfusions through hepcidin suppression. Diagnosis relies on a stepwise algorithm confirming haemolysis, excluding immune and acquired causes, assessing the blood film and indices, testing membrane, haemoglobin and enzymes and increasingly using panel sequencing early, with yields ranging from 24% to 72% depending on population and study design. Enzyme assays have known limitations, with false-normal results from reticulocytosis and transfusion, and sequencing misses deletions, deep intronic variants and mosaicism, so a negative result does not exclude diagnosis.

PK deficiency is the most frequent glycolytic enzymopathy and the only entity in the group with approved targeted therapy: mitapivat, authorised in adults in the USA and European Union since 2022, achieved a haemoglobin response in 40% of patients not regularly transfused and reduced transfusion burden in approximately one-third of regularly transfused patients, with effects on ineffective erythropoiesis and iron, but only in patients with at least one missense allele. The thalassaemia extension brought a hepatocellular injury signal requiring liver surveillance. In stomatocytoses, splenectomy is contraindicated because of severe late thrombosis risk, and ektacytometry, which does not identify Gardos channelopathy, should be supplemented with genetics. In CDAs, interferon alfa is the only specific CDA I therapy, used off-label. Other rare enzymopathies require attention to neurological and muscular manifestations and their individual preventive measures. Long-term management depends on systematic surveillance of iron, gallstones and splenectomy complications, counselling accounting for assay limitations in carrier detection and parental mosaicism, and recourse to ERN-EuroBloodNet centres and international registries.

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