Hereditary spherocytosis (HS) is a common form of haemolytic anaemia caused by defects or deficiencies in genes encoding erythrocyte membrane proteins, such as ANK1, SPTB, SLC4A1, EPB42, and SPTA1. Among these, ANK1 and SPTB mutations are the most frequent causes of HS worldwide. This study analysed 53 Indian HS patients, identifying 33 novel and 12 previously reported SPTB variants using targeted next-generation sequencing (t-NGS). The identified SPTB variants included frameshift (28%), missense (24%), nonsense (44%), and splicing (4%) types, with nonsense variants being the most common. These nonsense variants typically result in truncated proteins. The variants were widely distributed across the gene, with the highest density observed in the spectrin repeats and ankyrin-binding domain, while no variants were found in the tetramerization domain. All identified SPTB variants exhibited heterozygous inheritance, consistent with an autosomal dominant inheritance pattern of the gene causing HS. One patient, however, carried compound heterozygous variants, leading to severe anaemia, and five patients had de novo SPTB variants. This study expands the spectrum of SPTB variants, enhances the understanding of spectrin-related molecular defects, establishes genotype-phenotype correlations, and provides valuable insights for laboratories developing genetic tests for HS. The high number of identified variants highlights the importance of advanced technologies like NGS for accurate molecular diagnosis in HS disorder. This approach not only supports clinical diagnostics but also aids in family counseling for improved management of HS.

Biliary obstruction is a rare complication in patients with hereditary spherocytosis (HS). The clinical course and optimal treatment strategies for HS patients complicated by biliary obstruction remain unclear.

We conducted a retrospective review of 16 pediatric HS patients complicated by biliary obstruction who were treated at our hospital between January 2018 and October 2024. Based on previously published clinical severity classifications, patients were divided into Group A (non-severe group: trait, mild, and moderate) and Group B (severe group).

The study included 16 patients with a mean age of 9.0 ± 3.2 years, evenly distributed between the two groups (8 patients each). Preoperative routine blood tests showed no significant differences between the groups; however, Group B exhibited higher bilirubin levels and lower liver enzyme levels. Genetic testing was performed in 12 patients, revealing SPTB gene mutations in 7 (58.3%). Conservative management effectively resolved biliary obstruction in 10 patients (62.5%) within 14 days. Invasive interventions, such as endoscopic retrograde cholangiopancreatography (ERCP) or cholecystostomy, were required in 6 patients, with conjugated bilirubin levels normalizing within five days post-procedure. Complications occurred in two patients with prolonged intervals between diagnosis and surgery (> 3 months): one required stent replacement due to blockage after ERCP, and the other developed a gallbladder-skin fistula and coagulation disorder following laparoscopic cholecystostomy. Of the 14 patients who underwent subsequent splenectomy and/or cholecystectomy, 12 recovered without complications. Notably, the 6 patients who underwent splenectomy alone without cholecystectomy did not experience biliary colic during a mean follow-up period of 3.4 years (range: 0.5 - 5.5 years).

Biliary obstruction can complicate HS in pediatric patients regardless of anemia severity, particularly in those with SPTB gene mutations. Conservative management is effective in most cases, while invasive procedures are required for refractory cases. Shortening the interval between diagnosis and subsequent surgery may help prevent complications. Splenectomy alone appears to be a viable option once biliary obstruction is resolved.

Diamond-Blackfan Anemia (DBA) is a rare congenital disorder characterized by macrocytic anemia, physical abnormalities, and growth delays. Although RPS19 mutations have been more extensively studied in DBA compared to other ribosomal protein genes, the pathological mechanisms of genes such as RPS17 remain largely unexplored. This study aimed to investigate the role of RPS17 haploinsufficiency in DBA, focusing on its downstream effects on erythropoiesis and the involvement of SLC4A1, a critical erythrocyte membrane protein essential for red blood cell stability. Transcriptomic analysis of publicly available RNA sequencing data from DBA patients revealed significant downregulation of SLC4A1 in RPS17-mutated cases. To validate these findings, we generated a zebrafish model of DBA by knocking down rps17 using morpholino injections. Zebrafish embryos with rps17 knockdown exhibited reduced erythropoiesis, impaired hemoglobin synthesis, consistent with DBA. Further analysis confirmed decreased slc4a1a expression in rps17-morphants. Independent knockdown of slc4a1a in zebrafish resulted in similar erythropoietic defects, highlighting its critical role in red blood cell membrane integrity and function. This study identifies slc4a1 as a key downstream target of RPS17 haploinsufficiency and provides novel insights into the molecular mechanisms of DBA. By establishing zebrafish as an effective in vivo model, this research offers potential therapeutic targets for treating DBA and related erythropoietic disorders.

Management of mitral regurgitation (MR) in patients with hereditary spherocytosis (HS) poses unique challenges due to increased haemolysis risk. While surgical mitral valve repair is the standard treatment, the optimal strategy for recurrent MR after initial repair remains unclear, particularly regarding the safety and durability of transcatheter interventions in this high-risk population.

A 57-year-old woman with HS developed severe recurrent MR 4 years after initial surgical repair that intentionally omitted annuloplasty to minimize haemolysis risk. Given the risks of redo surgery and mechanical valve replacement, mitral valve transcatheter edge-to-edge repair (M-TEER) was performed. The procedure achieved successful MR reduction without causing haemolysis. At the 5-year follow-up, the patient maintained improved functional status with stable moderate MR and no evidence of haemolysis, despite her underlying condition.

This case demonstrates successful long-term outcomes of M-TEER for post-surgical recurrent MR in a patient with HS. The strategic approach-initial ring-less surgical repair followed by M-TEER-suggests a viable treatment pathway for patients with inherited haemolytic disorders, particularly when minimizing prosthetic material exposure is crucial.

Splenectomy is a well-established therapeutic approach for pediatric hematologic disorders, especially in the case of hereditary spherocytosis (HS). In addition to the commonly acknowledged short- and long-term infectious and thrombotic complications, also splenosis represents a rare but noteworthy complication of splenectomy. Splenosis is characterized by the auto-transplantation and growth of splenic tissue in ectopic locations, following trauma or splenectomy. This condition can mimic malignancies, posing diagnostic challenges. We report the case of a 16-year-old girl with HS who presented with fever, abdominal pain, and a history of laparoscopic splenectomy ten years early. Imaging revealed a vascularized pelvic mass, initially suspected to be malignant. Diagnostic laparoscopy and histopathological analysis confirmed the mass as pelvic splenosis. The patient was asymptomatic, prompting a conservative management approach with regular follow-up. This case highlights the importance of considering splenosis in differential diagnoses for pelvic masses in patients with prior splenectomy, to ensure appropriate management and avoid unnecessary interventions.

The clinical manifestations of hereditary spherocytosis (HS) are often heterogeneous, spanning from asymptomatic to severe symptoms that may pose life-threatening risks. Genotype-phenotype correlations remain controversial in clinical research. This retrospective study evaluated the correlation between genetic variants and clinical characteristics in a cohort of 64 Chinese pediatric patients with HS. The predominant variants were found in the ANK1 (27 cases, 42%) and SPTB (26 cases, 41%) genes, while variants in the SPTA1 (6 cases, 9%) and SLAC4A1 genes (5 cases, 8%) were less common. No EPB42 variants were detected. A total of 71 variants were identified. Variation types included nonsense (21%), missense (27%), frameshift mutations (39%), splicing (8%), and large fragment deletions (4%). No statistical differences in hemoglobin levels, MCV, MCH, MCHC, or reticulocytes were observed across the various genetic variant groups. Bilirubin levels were remarkably elevated in patients with HS variants, and those with SPTB-HS had significantly higher bilirubin levels, including total bilirubin (p = 0.033) and indirect bilirubin (p = 0.018) compared to those with SPTA1-HS. Moreover, those with the ANK1 variants displayed reduced resistance to lysis at varying NaCl concentrations in comparison to those with the SPTA1 variants (p = 0.047). In short, patients with the ANK1 and SPTB variants had the most severe disease, while those with the SPTA1 variants had the mildest. Genetic testing is advised in patients without a family history or who are difficult to diagnose with routine laboratory tests, as this may also provide references for clinical treatment and genetic counseling.

The primary management of hereditary spherocytosis (HS) typically involves splenectomy to prevent hemolytic crises. However, splenic artery embolization (SAE) has emerged as a promising minimally invasive alternative. We report a case of a woman in her mid-20s with severe HS who presented with chronic fatigue, anemia resistant to iron supplementation, and splenomegaly. The diagnosis was confirmed through clinical findings, osmotic fragility test, and laboratory results. A partial embolization of the lower two-thirds of the spleen was performed while preserving the function of the upper third. Post-procedure, significant improvements were observed in hemoglobin levels (rising from 7.7 g/dL to 11.3 g/dL), hematocrit, and bilirubin levels, with no major complications reported. This case highlights that partial selective splenic artery embolization (PSE) is a safe and effective non-surgical procedure that can be considered as a potential alternative to splenectomy in the management of hereditary spherocytosis. Further studies are warranted to establish its long-term efficacy.

Background Hereditary spherocytosis (HS) is an erythrocytic membranopathy that belongs to a group of rare genetic disorders. Mutations in five genes, including ANK1, cause clinical manifestations of the disease. Identified variations in individual families provide a better understanding of the molecular basis of the disease. Methods In this study, we used two sequencing methods, whole exome sequencing (WES) and Sanger sequencing, analyzing gDNA and cDNA as templates, to detect and verify the variants putatively responsible for the clinical symptoms observed in a Polish family diagnosed with HS. Results We detected two variants that occur in cis in the ANK1 gene, a known missense mutation (NP_000028.3:p.V463I) and a novel frameshift mutation (NP_000028.3: p.V1626fs*64) that appears to be crucial for the probands. As shown by transcriptome studies, the mutant allele is not present at a detectable level. Conclusions We conclude that the molecular basis of this case is related to an unstable transcript of the mutant allele and that the direct cause of the HS is a deficiency of erythrocyte ankyrin leading to a disruption of the AE1-erythrocyte ankyrin-spectrin complex in the erythrocyte membrane.

Peripheral erythrophagocytosis appears to be a unique sign of acquired immune-mediated hemolytic anemia. It is said to be rare but its prevalence among patients with autoimmune hemolytic anemia has not been studied.

In this retrospective study from July 2014 to June 2024, the clinical and laboratory features, treatment and outcomes of children diagnosed with autoimmune hemolytic anemia were described. The prevalence of peripheral erythrophagocytosis was compared to a group of children with hereditary spherocytosis at the time of first diagnosis seen in the same period.

Twelve consecutive children with autoimmune hemolytic anemia were included. There were four female patients. The mean age was 6.7 (range 0.8 to 16.6) years. The mean hemoglobin was 6.0 (range 2.5 to 8.1) g/dL. Seven patients were positive by a direct antiglobulin test, three were positive with cold agglutinins and two were positive on both tests. In seven cases, an acute infection appeared to be the precipitating factor. Mycoplasma pneumoniae infection was documented in three and suspected in another two cases. Peripheral erythrophagocytosis was present in five cases (42%) but was not found at diagnosis in any of the 16 cases of hereditary spherocytosis (p = 0.0081). Six children had pre-existing diseases, including two with hereditary hemolytic anemia.

Peripheral erythrophagocytosis is a relatively common and characteristic finding in pediatric autoimmune hemolytic anemia and should be actively looked for in the evaluation of acute hemolysis, including in children with pre-existing hereditary hemolytic disorders.

This study aims to provide a comprehensive summary of the clinical phenotypic characteristics of children with anemia of unknown etiology, particularly focusing on the early detection of hereditary spherocytosis (HS) and exploring genetic screening strategies for this condition in childhood.

The study included children with anemia whose underlying cause could not be definitively identified through routine clinical diagnosis. Clinical data was collected and genetic diagnosis of HS was confirmed using next-generation sequencing. Statistical analysis was conducted to evaluate the clinical characteristics of children with HS.

A total of thirty children with unexplained anemia were included in the study, resulting in a gene detection diagnostic rate of 80%. This included the identification of five non-HS-related congenital anemia genes (16.66%, 5/30) and nineteen cases of hereditary spherocytosis (HS). Upon initial diagnosis, the clinical features of HS were not significantly distinct compared to other forms of anemia.

In Jiangxi, China, our strategy of genetic screening for these children is feasible after excluding the common causes of anemia, such as nutritional anemia, G-6-PD deficiency, thalassemia, autoimmune hemolytic anemia, and myelopoietic abnormalities in children. This is an exploration to establish a genetic screening strategy for children with HS, and more detailed genetic screening strategies need to be further studied and explored. Next-generation sequencing remains the main method for the diagnosis and differential diagnosis of HS.

Defects in erythrocyte membrane proteins can cause the most common type of inherited hemolytic anemia, so called hereditary spherocytosis (HS). It is characterized by the appearance of spherocytes in peripheral blood, hemolytic anemia, splenomegaly, jaundice and gallstones. Due to difficulty of diagnosis solely based on aforementioned parameters, the addition of genetic testing seems to be effective and most acknowledged. Up to date, pathogenic variations in five genes encoding membrane proteins (ANK1, SPTA1, SPTB, SLC4A1, EPB42) are identified to cause HS. Here, we have studied the genetic spectrum in forty-one patients with clinically suspected HS and their families, as well as their genotype-phenotype correlations. Pathogenic mutations in ANK1, SPTB, SLC4A1 and SPTA1 were found in 17 (41.5 %), 12 (29.3 %), 7 (17.1 %) and 5 (12.2 %) patients, respectively. Deleterious variants include 12 missense, 15 nonsense, 12 frameshift, and 4 splicing variants. Among these variations 32 were novel. In our genotype-phenotype analysis, platelet levels in SPTB (p = 0.021) and SLC4A1 (p = 0.02) patients were found to be significantly lower than ANK1 patients. In addition, LDH levels in SPTB patients were remarkably lower than patients with ANK1 mutations (p = 0.025).

Hereditary spherocytosis is a rare genetic disorder of the red blood cell membrane that is characterized by anemia, jaundice, and splenomegaly; however, in the absence of family history and with unusual clinical presentation, the diagnosis might not be made until later in life.

Here, we present a challenging case of genetically proven hereditary spherocytosis that involves the SPTB gene in a 23-year-old female patient from Ethiopia who had repeated medical visits for episodic jaundice and hepatosplenomegaly, with unusual features of conjugated hyperbilirubinemia, pancytopenia, normal reticulocyte count, and lack of family history, where the delay in diagnosis led to several complications. The patient was successfully managed with simultaneous splenectomy and cholecystectomy.

This case underscores the importance of a thorough clinical examination, spending the time to review a case periodically without assuming the initial diagnosis is correct, and maintaining a healthy skepticism of inconsistent data to prevent misdiagnosis and mistreatment. The diagnostic delay highlights the need for increased awareness and familiarity with diagnostic modalities of hereditary spherocytosis among healthcare providers in Ethiopia.

Congenital dyserythropoietic anemia Ⅱ (CDA Ⅱ) is a rare inherited disorder of defective erythropoiesis caused by SEC23B gene mutation. CDA Ⅱ is often misdiagnosed as a more common type of clinically related anemia, or it remains undiagnosed due to phenotypic variability caused by the coexistence of inherited liver diseases, including Gilbert's syndrome (GS) and hereditary hemochromatosis.

We describe the case of a boy with genetically undetermined severe hemolytic anemia, hepatosplenomegaly, and gallstones whose diagnosis was achieved by targeted next generation sequencing.

Molecular analysis revealed a maternally inherited novel intronic variant and a paternally inherited missense variant, c.[994-3C > T];[1831C > T] in the SEC23B gene, confirming diagnosis of CDA Ⅱ. cDNA analysis verified that the splice acceptor site variant results in two mutant transcripts, one with an exon 9 skip and one in which exons 9 and 10 are deleted. SEC23B mRNA levels in the patient were lower than those in healthy controls. The patient was also homozygous for the UGT1A1*6 allele, consistent with GS.

Identification of the novel splice variant in this study further expands the spectrum of known SEC23B gene mutations. Molecular genetic approaches can lead to accurate diagnosis and management of CDA Ⅱ patients, particularly for those with GS coexisting.

Gallstones are among the most common complications of hereditary spherocytosis (HS). In previous treatments, gallbladder-preserving cholelithotomy (GPC) has remained a subject of significant debate due primarily to potential risks of stone recurrence. However, past studies have often overlooked the impact of specific disease conditions on GPC. In this study, we reviewed the clinical data of GPC in HS pediatric patients with concurrent gallstones over a period of seven years in a single center.

From December 2016 to April 2024, 32 pediatric patients with HS who underwent splenectomy and GPC surgery based on our inclusion criteria. Clinical pathological, and follow-up data of these patients were collected.

In terms of short-term complications, there were there were no cases of postoperative bleeding, bile duct injury. 3 cases (9.3%) experienced varying degrees of bile peritonitis. During long-term follow-up, only 2 cases (6.2%) showed recurrence of gallstones. One case of bile leakage occurred.

GPC demonstrates significant efficacy for pediatric patients with hereditary spherocytosis (HS) complicated by gallstones, showing a a low recurrence rate and high safety profile.

Hereditary spherocytosis (HS) is the most prevalent form of congenital hemolytic anemia, being caused by genetic mutations in genes encoding red blood cell cytoskeletal proteins. Mutations in the ANK1 and SPTB genes are the most common causes of HS.; however, pathogenicity analyses of these mutations remain limited. This study identified three novel heterozygous mutations in 3 HS patients: c.1994 C > A in ANK1, c.5692 C > T, and c.3823delG in SPTB by whole-exome sequencing (WES) and validated by Sanger sequencing. To investigate the functional consequences of these mutations, we studied their pathogenicity using in vitro culture erythroblast derived from CD34 + stem cells. All three mutations lead to the generation of a premature stop codon. Real-time PCR assay revealed that the two SPTB mutations resulted in reduced SPTB mRNA expression, suggesting a potential role for the nonsense-mediated mRNA degradation pathway. For the ANK1 mutation, gene expression was not reduced but was predicted to produce a truncated version of the ANK1 protein. Flow cytometry analysis of red blood cell-derived microparticles (MPs) revealed that HS patients had higher MP levels compared to normal subjects. This study contributes to the current understanding of the molecular mechanisms underlying mutations in the ANK1 and SPTB genes in HS.

To explore the comparative postoperative efficacy of partial splenectomy (PS) and total splenectomy (TS) in the treatment of children with hereditary spherocytosis (HS).

The relevant HS studies from databases were searched and screened, comparing the differences in hemoglobin concentration, reticulocyte percentage, bilirubin concentration before and after TS and PS surgery, and during the follow-up period, as well as the incidence of postoperative adverse events. Statistical analysis was performed using Review Manager 5.4.

A total of 5 studies were included in this meta-analysis, with a cumulative enrollment of 312 children, 130 in the PS group and 182 in the TS group. The meta-analysis results showed that both PS and TS groups had statistically significant differences in postoperative hematological outcomes compared to before surgery, with TS showing better improvement than PS. In the postoperative follow-up, the changes in hematological outcomes between PS and TS were statistically significant for hemoglobin concentration: within 1 year [MD = 1.85, 95%CI(1.09,2.60)], 1-2 years [MD = 1.74, 95%CI(0.25,3.24)], not statistically significant for 4-6 years [MD = 1.28, 95%CI(-1.75,4.32)]; for reticulocyte percentage: within 1 year [MD = 2.23, 95%CI(0.80,3.66)] was statistically significant, not statistically significant for 4-6 years [MD = 1.77, 95%CI(-2.04,5.59)]; for serum bilirubin concentration: within 1 year [MD = 1.55, 95%CI(0.91,2.18)] was statistically significant, not statistically significant for 1-2 years [MD = 1.77, 95%CI(-2.04,5.99)]. In the incidence of postoperative adverse events, the incidence of cholelithiasis [MD = 1.77, 95%CI(-2.04,5.99)] showed a statistically significant difference between PS and TS, while there were no statistically significant differences in other included events, such as postoperative infection rate, secondary surgery rate, thrombosis rate, postoperative hemorrhage rate, and transfusion therapy rate.

Splenectomy is a beneficial surgical strategy for children with moderate-to-severe HS, reducing; early hematological outcomes of TS are more robust than PS in the follow-up period, and there is no difference between the two in the later period; for postoperative adverse events, the incidence of cholelithiasis in children after PS is higher than after TS, and there is a risk of requiring a second surgery for total splenectomy due to hemolytic recurrences.

Clinical flow cytometry plays a vital role in the diagnosis and monitoring of various red blood cell disorders. The high throughput, precision, and automation potential of this technique allows for cost-effective and timely analysis compared to older and more manual test methods. Flow cytometric analysis serves as the gold standard diagnostic method for multiple hematological disorders, especially in clinical scenarios where an assay needs to have high sensitivity, high specificity, and a short turnaround time. In this review, we discuss the role of flow cytometric analysis in paroxysmal nocturnal hemoglobinuria, fetal-maternal hemorrhage, and hereditary spherocytosis.

A 6-year-old girl previously diagnosed with hereditary spherocytosis was admitted to our hospital with gallstones and cholangitis. Endoscopic retrograde cholangiopancreatography (ERCP) was performed, and fluoroscopy revealed a dilated common bile duct (CBD) without evident stones, possibly due to spontaneous excretion through the papilla of Vater. A 7-French plastic stent was inserted into the CBD. After the procedure, a marked increase in pancreatic enzyme levels was observed, and she was diagnosed with post-ERCP pancreatitis (PEP). Stent placement could have been a cause of pancreatitis; therefore, we removed the stent. Subsequently, recovery from pancreatitis was confirmed, although she suddenly complained of abdominal pain and was diagnosed with choledocholithiasis recurrence. ERCP was repeated, and fluoroscopy revealed a dilated CBD with a stone. A minimal endoscopic sphincterotomy (EST) was performed to reduce the risk of PEP, and a biliary dilation balloon placed across the papilla was gradually inflated until the waist of the balloon disappeared. Stones were extracted using a retrieval balloon catheter. The abdominal pain resolved immediately, and the patient recovered without developing PEP. To our knowledge, this is the first case report of a pediatric patient treated with minimal EST followed by papillary balloon dilation for choledocholithiasis.

Membranopathies encompass haemolytic disorders arising from genetic variants in erythrocyte membrane proteins, including hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anaemia type II (CDA II) is associated with the SEC23B gene and can exhibit phenotypic similarities to membranopathies. Current treatment options for these conditions, apart from splenectomy, are primarily supportive. Mitapivat, a novel pyruvate kinase (PK) activator, has demonstrated efficacy in increasing haemoglobin levels and reducing haemolysis in patients with PK deficiency, thalassemia, sickle cell disease and a mouse model of hereditary spherocytosis.

Safety and efficacy of mitapivat sulfate in adult patients with erythrocyte membranopathies (SATISFY) is a prospective, multicentre, single-arm phase two trial involving approximately 25 adult patients (≥18 years) diagnosed with a membranopathy or CDA II. During the 8-week dose escalation period, subjects will receive an initial dose of 50 mg mitapivat two times per day and may increase to 100 mg two times per day at week 4 based on the safety and changes in haemoglobin levels. Patients tolerating mitapivat well may be eligible to continue in two consecutive 24-week fixed dose periods.The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on haemoglobin levels, haemolysis, erythropoiesis, patient-reported outcome measures and spleen size.SATISFY aims to assess the safety and efficacy of mitapivat in adult patients with red blood cell membranopathies and CDA II, with the aim of establishing proof-of-concept in patients living with these rare conditions.

NCT05935202/CTIS:2023-503271-24-01. Findings will be published in peer-reviewed journals.

Clinicaltrials.gov, NCT05935202. CTIS:2023-503271-24-01. Registered 07-July-2023. Protocol number: 2.1. https://clinicaltrials.gov/study/NCT05935202.

This study aimed to investigate the clinical features, pathogenic gene variants, and potential genotype-phenotype correlations in Chinese patients with hereditary spherocytosis (HS).

Retrospective analysis of clinical data and molecular genetic characteristics was conducted on patients diagnosed with HS at Jiangxi Provincial Children's Hospital, the Second Affiliated Hospital of Nanchang University, Pingxiang People's Hospital and The Third People's Hospital of Jingdezhen between November 2017 and June 2023. Statistical analyses were performed to compare and analyze the red blood cell (RBC), hemoglobin (HB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) data between and within groups based on different mutations and age groups (< 14 and ≥ 14 years).

A total of 34 HS patients were included in this study, comprising 22 children (64.70%) and 12 adults (35.30%). The probands who underwent genetic testing were derived from 34 unrelated families. Thirty-two variants were tested and 9 of them are novel. Eighteen cases had ANK1 variants, 15 had SPTB variants, and 1 had SLC4A1 variant. 25 patients performed core family members underwent genetic testing, 17 (68.0%, 17/25) were de novo, 5 (20.0%, 5/25) were maternally inherited, and 3 (12.0%, 3/25) were paternally inherited. ANK1-HS patients exhibited more severe anemia compared to cases with SPTB-HS, showing lower levels of RBC and HB (P < 0.05). Anemia was more severe in patients diagnosed in childhood than in those diagnosed in adulthood. Within the ANK1-HS group, MCH levels in adult patients was significantly higher than those in children (P < 0.05), while there were no significant differences in RBC, HB, MCV, and MCHC levels between two groups. Adult patients with SPTB-HS had significantly higher levels of RBC, HB, and MCH than pediatric patients (P < 0.05), while MCV and MCHC levels showed no significant statistical differences.

This study conducted a comparative analysis of phenotypic characteristics and molecular genetics in adult and pediatric patients diagnosed with HS, confirming that pediatric ANK1-HS patients exhibit a more severe anemic phenotype compared to SPTB-HS patients, while the severity of HS in adults does not significantly differ between different causative genes.

Hereditary spherocytosis (HS) is a membranopathy that impacts the vertical junctions between the cytoskeleton and the plasma membrane of erythrocytes. The gold standard method for diagnosing it is osmotic gradient ektacytometry (OGE). However, access to this technique is scarce. We have devised a straightforward approach utilizing flow cytometry to quantify variations in an osmotic gradient, relying on FSC-H/SSC-H patterns. We studied 14 patients (9 pediatric, 5 adults) and 54 healthy controls (16 pediatric, 38 adults). After assessing the behavior of the samples in several osmolar gradients we selected for the study the 176, 308, and 458 mOsm/kg levels as hypo-osmolar, iso-osmolar, and hyper-osmolar references. We then selected the iso-osmolar point for assessment to determine its efficacy in discriminating between patient and control groups using a receiver operating characteristic curve. In the pediatric group, the area under the curve (AUC) was 1.0, indicating 100% sensitivity and 93.3% specificity. Conversely, in the adult group, the AUC was 0.98, with 80% sensitivity and 90.9% specificity. We introduce a method that is easily replicable and demonstrates high sensitivity and specificity. This technique could prove valuable in the diagnosis of spherocytosis.

The aim of this study was to investigate the complex heterozygous mutations of ANK1 and SPTA1 in the same individual and improve our understanding of hereditary spherocytosis (HS) in children. We also hope to promote the application of gene detection technology in children with HS, with the goals of identifying more related gene mutations, supporting the acquisition of improved molecular genetic information to further reveal the pathogenesis of HS in children, and providing important guidance for the diagnosis, treatment, and prevention of HS in children.

A 1-year and 5-month-old patient presented jaundice during the neonatal period, mild anemia 8 months later, splenic enlargement at 1 year and 5 months, and brittle red blood cell permeability. Genetic testing was performed on the patient, their parents, and sister. Swiss Model software was used to predict the protein structure of complex heterozygous mutations in ANK1 and SPTA1. Genetic testing revealed that the patient harbored a new mutation in the ANK1 gene from the father and a mutation in the SPTA1 gene from the mother. Combined with the clinical symptoms of the children, it is suggested that the newly discovered complex heterozygous mutations of ANK1 and SPTA1 may be the cause, providing important guidance for revealing the pathogenesis, diagnosis, treatment, and promotion of gene detection technology in children with HS.

This case involves an unreported complex heterozygous mutation of ANK1 and SPTA1, which provides a reference for exploring HS.

Hereditary spherocytosis (HS) is one of the most common causes of hereditary hemolytic anemia. The current diagnostic guidelines for HS are mainly based on a combination of physical examination and laboratory investigation. However, some patients present with complicated clinical manifestations that cannot be explained by routine diagnostic protocols. Here, we report a rare HS case of mild anemia with extremely high indirect bilirubin levels and high expression of fetal hemoglobin. Using whole exome sequencing analysis, this patient was identified as a heterozygous carrier of a de novo SPTB nonsense mutation (c.605G > A; p.W202*) and a compound heterozygous carrier of known UGT1A1 and KLF1 mutations. This genetic analysis based on the interpretation of the patient's genomic data not only achieved precise diagnosis by an excellent explanation of the complicated phenotype but also provided valuable suggestions for subsequent appropriate approaches for treatment, surveillance and prophylaxis.

Hereditary hemolytic anemia (HHA) is considered a group of rare hematological diseases in Korea, primarily because of its unique ethnic characteristics and diagnostic challenges. Recently, the prevalence of HHA has increased in Korea, reflecting the increasing number of international marriages and increased awareness of the disease. In particular, the diagnosis of red blood cell (RBC) enzymopathy experienced a resurgence, given the advances in diagnostic techniques. In 2007, the RBC Disorder Working Party of the Korean Society of Hematology developed the Korean Standard Operating Procedure for the Diagnosis of Hereditary Hemolytic Anemia, which has been continuously updated since then. The latest Korean clinical practice guidelines for diagnosing HHA recommends performing next-generation sequencing as a preliminary step before analyzing RBC membrane proteins and enzymes. Recent breakthroughs in molecular genetic testing methods, particularly next-generation sequencing, are proving critical in identifying and providing insight into cases of HHA with previously unknown diagnoses. These innovative molecular genetic testing methods have now become important tools for the management and care planning of patients with HHA. This review aims to provide a comprehensive overview of recent advances in molecular genetic testing for the diagnosis of HHA, with particular emphasis on the Korean context.

Objective: The objective of this study was to pinpoint pathogenic genes and assess the mutagenic pathogenicity in two pediatric patients with hereditary spherocytosis. Methods: We utilized whole-exome sequencing (WES) for individual analysis (case 1) and family-based trio analysis (case 2). The significance of the intronic mutation was validated through a Minigene splicing assay and supported by subsequent in vitro experiments. Results: Both probands received a diagnosis of hereditary spherocytosis. WES identified a novel ANK1 c.1504-9G>A mutation in both patients, causing the retention of seven nucleotides at the 5' end of intron 13, as substantiated by the Minigene assay. This variant results in a premature stop codon and the production of a truncated protein. In vitro studies indicated a reduced expression of the ANK1 gene. Conclusion: The novel ANK1 c.1504-9G>A variant is established as the causative factor for hereditary spherocytosis, with the c.1504-9G site functioning as a splicing receptor.

Hereditary spherocytosis (HS), a rare familial extravascular haemolytic disorder, typically follows an autosomal dominant inheritance pattern with variable expressivity. Despite its classical presentation of anaemia, jaundice, and splenomegaly, HS is infrequently reported among individuals of Asian descent, contributing to its under diagnosis or delayed diagnosis. The primary objective of this case report is to underscore the pivotal role of the osmotic fragility test in diagnosing HS, emphasizing the importance of accurate and timely identification for effective clinical management and improved patient outcomes.

The patient, without known prior co-morbidities, presented with recurrent abdominal distension, early satiety, and easy fatigability persisting for 6 years. Physical examination revealed icterus, gnathopathy, left hypochondrium tenderness, and palpable splenomegaly. The osmotic fragility of red cells was significantly elevated. The patient underwent optimization before splenectomy, receiving immunization against encapsulated bacteria. Packed red blood cell transfusions were administered to achieve optimal haemoglobin levels. Follow-up showed symptom relief, significantly improving the patient's quality of life.

This case underscores the challenges of delayed HS diagnosis, with the patient enduring symptoms for years before seeking appropriate medical attention. Overlooking the simplicity and cost-effectiveness of an osmotic fragility test prolonged the diagnostic journey, emphasizing the impact on overall well-being.

HS remains underdiagnosed, especially in our regions. The osmotic fragility test emerges as an economical diagnostic tool in resource-limited settings, particularly when spherocytosis is absent in the peripheral blood smears. Its inclusion in diagnostic protocols can expedite accurate HS identification and enhance patient outcomes.

Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine mutations in genes associated with RBC membrane defect in patients with HS such as α-spectrin gene (SPTA1), β-spectrin gene (SPTB), ankyrin gene (ANK1), band 3 anion transport gene (SLC4A1) and erythrocyte membrane protein band 4.1 gene (EPB41). Blood samples were collected from 23 unrelated patients with HS. Patients were diagnosed according to the guidelines from the British Society for Hematology. All hematological examinations for the determination of RBC abnormalities and osmotic fragility tests were conducted. Genomic DNA were extracted from peripheral blood cells and coding exons of known genes for hereditary spherocytosis were enriched using Roche/KAPA sequence capture technology and sequenced on an Illumina system via next-generation sequencing (NGS). The data showed that most of the HS patients confirmed splenomegaly and showed elevated reticulocytes and abnormal bilirubin values. NGS analysis identified the heterozygous variant c.5501G > A in the exon 39 of SPTA1 gene, resulted in a Trp1834*, which leads to a premature stop codon and subsequent mRNA degradation (nonsense- mediated decay) or truncation in α spectrin. Moreover, our data also revealed conventional mutations in genes SPTB, ANK, SLC4A1 and EBP41 in severe patients of HS. In short, this is the first report that determined a novel mutation c.5501G > A in SPTA1 gene in the Saudi population. To the best of our knowledge, this variant c.5501G > A has not been described in global literature so far. This novel mutation in SPTA1 gene is unique in the Saudi population.

Children undergoing splenectomy for hemolytic anemia often have cholelithiasis, which may or may not be symptomatic. It is unclear whether concurrent cholecystectomy increases length of stay or morbidity after splenectomy. The purpose of this study was to compare morbidity among children undergoing laparoscopic splenectomy alone versus splenectomy with concurrent cholecystectomy in patients with hemolytic anemia.

We retrospectively evaluated children with hemolytic anemia undergoing non-traumatic laparoscopic splenectomy in the National Surgical Quality Improvement Program-Pediatric database (2012-2020). Outcomes were compared for patients undergoing splenectomy alone (n = 1010) versus splenectomy with cholecystectomy (n = 371). Pearson's Chi-square and Student's t-tests were utilized as appropriate. Propensity score-matching was completed, controlling for eight demographic and clinical variables.

1381 patients were identified, 73.1% undergoing splenectomy alone and 26.9% splenectomy with cholecystectomy. Splenectomy with cholecystectomy patients were older (10.9 years vs. 8.4 years, p < 0.01), more likely to have hereditary spherocytosis (56.1% vs. 40.8%, p < 0.01), less likely to have sickle cell disease (12.1% vs. 33.5%, p < 0.01), more likely ASA class 1 or 2 (49.3% vs. 42.1%, p < 0.01), and had similar preoperative hematocrit levels (29.6 vs. 29.3, p = 0.33). The splenectomy with cholecystectomy group was less likely to receive preoperative blood transfusions (13.5% vs. 25.4%, p < 0.01). There were 360 pairs selected on propensity score-matching, and splenectomy with cholecystectomy was associated with increased operative time (182 min vs. 145 min, p < 0.01) and decreased occurrences of a postoperative transfusion (4.2% vs. 8.9%, p = 0.01). Length of stay after surgery (2.5 days vs. 2.3 days, p = 0.13), composite morbidity (3.9% vs. 3.4%, p = 0.69), and 30-day readmission rates (3.3% vs. 7.4%, p = 0.08) were all similar.

Splenectomy with cholecystectomy is associated with similar postoperative morbidity, length of stay and readmission rates compared to splenectomy alone. These data support the safety of concurrent cholecystectomy with splenectomy for children with cholelithiasis in the setting of hemolytic anemia.

Retrospective Cohort Study.

Level III.

Congenital defects of the erythrocyte membrane are common in northern Europe and all over the world. The resulting diseases, for example, hereditary spherocytosis (HS), are often underdiagnosed, partly due to their sometimes mild and asymptomatic courses. In addition to a broad clinical spectrum, this is also due to the occasionally complex diagnostics that are not available to every patient. To test whether next-generation sequencing (NGS) could replace time-consuming spherocytosis-specific functional tests, 22 consecutive patients with suspected red cell membranopathy underwent functional blood tests. We were able to identify the causative genetic defect in all patients with suspected HS who underwent genetic testing (n = 17). The sensitivity of the NGS approach, which tests five genes (ANK1 (gene product: ankyrin1), EPB42 (erythrocyte membrane protein band4.2), SLC4A1 (band3), SPTA1 (α-spectrin), and SPTB (β-spectrin)), was 100% (95% confidence interval: 81.5-100.0%). The major advantage of genetic testing in the paediatric setting is the small amount of blood required (<200 µL), and compared to functional assays, sample stability is not an issue. The combination of medical history, basic laboratory parameters, and an NGS panel with five genes is sufficient for diagnosis in most cases. Only in rare cases, a more comprehensive functional screening is required.

Subtotal or total splenectomy are recommended in severe and should be considered in intermediate forms of hereditary spherocytosis (HS). Data on laparoscopic subtotal splenectomy (LSTS) in HS patients are sparse.

Thirty three patients with HS (median age 10.7 years (yrs), range 1.8-15.5) underwent LSTS. Baseline and follow-up investigation included haematological parameters, microscopic analysis of pitted erythrocytes (pitE), and B-cell subpopulations assessed by flow cytometry. Results were compared to those of non-splenectomised HS patients, HS patients after total splenectomy (TS), and healthy individuals.

After LSTS, haemoglobin levels were normalised in all patients. During median long-term follow-up of 3.9 yrs (range 1.1-14.9), only four patients presented mild anaemia. Despite re-growing of the remnant spleen none of the patients required a second surgical intervention. As compared to TS, PitE in LSTS patients were significantly lower and indicated normal to only moderately decreased spleen function. Relative but not absolute IgM memory B-cell counts were reduced in both LSTS and TS patients.

LSTS is effective for the treatment of patients with HS. A small remnant spleen is sufficient to provide adequate phagocytic function and to induce a pool of IgM memory B-cells.

Hereditary Spherocytosis (HS) is a rare, congenital red blood cell disorder presenting with variable clinical manifestations ranging from mild hemolytic anemia to severe anemia with hypersplenism and hepatobiliary complications.

The objectives of the study were to evaluate the diagnostic pathway of HS, the presence and management of hepatobiliary complications in pediatric patients with HS followed in a tertiary care center. The demographic, clinical, hematological information were retrieved from medical records of patients having at least 1 hematology visit between 2010 and 2020.

Forty-two patients were enrolled, 23 M. Mean age at onset of symptoms was 2.8 years, at diagnosis was 3.5 years. Anemia was the first manifestation in 73%; suspect of HS arose for all patients in first or second level outpatient clinics. Only 64% of patients performed two confirmation tests in the reference center. 28/42 had familiarity for HS; of the 13/42 who did not, only 47% performed further analysis. Sixteen patients developed gallbladder stones (40%), visible at the first ultrasound (5.6 years). Hemolytic crises and parvovirus infections were more frequent in patients with stones (53.6% vs. 26.1% and 63.6% vs. 28.6%, respectively). 10/16 (62.5%) underwent elective cholecystectomy: 8 had concomitant splenectomy.

our study highlights the need to optimize the diagnostic pathway in networks of care involving general and specialized centers in order to reduce time to diagnosis and ensure that all patients receive confirmatory tests. A high frequency of hepatobiliary complications since young age was observed suggesting that screening with ultrasound should begin earlier.

Hereditary spherocytosis (HS), the most common inherited hemolytic anemia disorder, is characterized by osmotically fragile microspherocytic red cells with a reduced surface area on the peripheral blood smear. Pathogenic variants in five erythrocyte membrane structure-related genes ANK1 (Spherocytosis, type 1; MIM#182900), SPTB (Spherocytosis, type 2; MIM#616649), SPTA1 (Spherocytosis, type 3; MIM#270970), SLC4A1 (Spherocytosis, type 4; MIM#612653) and EPB42 (Spherocytosis, type 5; MIM#612690) have been confirmed to be related to HS. There have been many studies on the pathogenic variants and mechanisms of HS, however, studies on how to manage the transmission of HS to the next-generation have not been reported. In this study, we recruited a patient with HS. Targeted next-generation sequencing with a panel of 208 genes related to blood system diseases detected a novel heterozygous variant in the SPTB: c.300+2dup in the proband. Sanger sequencing of variant alleles and haplotype linkage analysis of single nucleotide polymorphism (SNP) based on next-generation sequencing were performed simultaneously. Five embryos were identified with one heterozygous and four not carrying the SPTB variant. Single-cell amplification and whole genome sequencing showed that three embryos had varying degrees of trisomy mosaicism. One of two normal embryos was transferred to the proband. Ultimately, a healthy boy was born, confirmed by noninvasive prenatal testing for monogenic conditions (NIPT-M) to be disease-free. This confirmed our successful application of PGT in preventing transmission of the pathogenic variant allele in the HS family.

Diagnosis of Hereditary spherocytosis (HS) often requires time-consuming and/or expensive tests. Cryohemolysis test (CHT) is a simple and easy to perform test with high predictive value for HS diagnosis. In this prospective study, we evaluated the diagnostic utility of CHT for the diagnosis of HS. We included 60 suspected HS patients, 18 patients with Autoimmune hemolytic anemia (AIHA) and 120 healthy controls. Among the 60 suspected cases, there were 36 HS cases and 24 with other hemolytic anemias. The mean CHT (%) ± SD for controls, AIHA, other hemolytic anemias, and HS was 6.63 ± 2.79, 6.79 ± 4.36, 6.61 ± 2.76 and 26.7 ± 8.9, respectively. The CHT % was significantly higher in HS group when compared to controls (p =  < 0.0001), AIHA (p =  < 0.0001) and other hemolytic anemia groups (p =  < 0.0001). At a CHT cut off of > 18.3%, the sensitivity, specificity, positive predictive value and negative predictive value for diagnosis of HS in our study were 97.1%, 94.4%, 97.2% and 90.3%, respectively. CHT is a simple and sensitive test for the diagnosis of HS but remains underutilized. The addition of CHT in the diagnostic workup of HS will be very useful, especially in a resource limited setting.

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia, caused by mutations in five genes that encode erythrocyte membrane skeleton proteins. The red blood cell (RBC) lifespan could directly reflect the degree of hemolysis. In the present cohort of 23 patients with HS, we performed next-generation sequencing (NGS) and Levitt's carbon monoxide (CO) breath test to investigate the potential genotype-degree of hemolysis correlation.

In the present cohort, we identified 8 ANK1,9 SPTB,5 SLC4A1 and 1 SPTA1 mutations in 23 patients with HS, and the median RBC lifespan was 14(8-48) days. The median RBC lifespan of patients with ANK1, SPTB and SLC4A1 mutations was 13 (8-23), 13 (8-48) and 14 (12-39) days, respectively, with no statistically significant difference (P = 0.618). The median RBC lifespan of patients with missense, splice and nonsense/insertion/deletion mutations was 16.5 (8-48), 14 (11-40) and 13 (8-20) days, respectively, with no significant difference (P = 0.514). Similarly, we found no significant difference in the RBC lifespan of patients with mutations located in the spectrin-binding domain and the nonspectrin-binding domain [14 (8-18) vs. 12.5 (8-48) days, P = 0.959]. In terms of the composition of mutated genes, 25% of patients with mild hemolysis carried ANK1 or SPTA1 mutations, while 75% of patients with mild hemolysis carried SPTB or SLC4A1 mutations. In contrast, 46.7% of patients with severe hemolysis had ANK1 or SPTA1 mutations and 53.3% of patients with severe hemolysis had SPTB or SLC4A1 mutations. However, there was no statistically significant difference in the distribution of mutated genes between the two groups (P = 0.400).

The present study is the first to investigate the potential association between genotype and degree of hemolysis in HS. The present findings indicated that there is no significant correlation between genotype and degree of hemolysis in HS.

Parvovirus B19 (B19V) infection varies clinically depending on the host's immune status. Due to red blood cell precursors tropism, B19V can cause chronic anemia and transient aplastic crisis in patients with immunosuppression or chronic hemolysis. We report three rare cases of Brazilian adults living with human immunodeficiency virus (HIV) with B19V infection. All cases presented severe anemia and required red blood cell transfusions. The first patient had low CD4⁺ counts and was treated with intravenous immunoglobulin (IVIG). As he remained poorly adherent to antiretroviral therapy (ART), B19V detection persisted. The second patient had sudden pancytopenia despite being on ART with an undetectable HIV viral load. He had historically low CD4⁺ counts, fully responded to IVIG, and had undiagnosed hereditary spherocytosis. The third individual was recently diagnosed with HIV and tuberculosis (TB). One month after ART initiation, he was hospitalized with anemia aggravation and cholestatic hepatitis. An analysis of his serum revealed B19V DNA and anti-B19V IgG, corroborating bone marrow findings and a persistent B19V infection. The symptoms resolved and B19V became undetectable. In all cases, real time PCR was essential for diagnosing B19V. Our findings showed that adherence to ART was crucial to B19V clearance in HIV-patients and highlighted the importance of the early recognition of B19V disease in unexplained cytopenias.

Gallstones are common in hereditary spherocytosis (HS) and other chronic hemolytic diseases, with most affected patients being asymptomatic. Whether and how asymptomatic gallstones should be treated is controversial.

We conducted a retrospective cohort study of pediatric patients with HS and asymptomatic gallstones to compare the clinical outcomes between the observation group (followed up with gallstones in situ) and the intervention group (cholecystectomy or cholecystolithotomy). The primary outcome was the composite of adverse outcomes, including gallstone-associated, gallbladder surgery-associated, and splenectomy-associated events. Secondary outcomes included the changes in the size and number of gallstones and the recurrence of gallstones.

Fifty-two patients were included (38 in the observation group and 14 in the intervention group), with a mean follow-up length of 5.2 years. Patients in the intervention group had a lower incidence of primary outcomes (7.1% vs. 42.1%) than patients in the observation group (OR, 0.12; 95%CI, 0.01-0.99). Patients with gallstones >5 mm in diameter had the highest risk of adverse events (47.4%). In the observation group, gallstone growth, disappearance, and stability were observed in 19.4%, 29.0%, and 51.6% of splenectomized HS patients, respectively. Small gallstones (diameter ≤5 mm) or sludge were more likely to be associated with disappearance (P = 0.04).

Most asymptomatic gallstones grow or persist in splenectomized HS patients for an extended period. Surgical treatment of asymptomatic gallstones in HS patients requiring splenectomy is associated with a lower risk of adverse outcomes.

III (retrospective comparative study).

Hereditary Spherocytosis (HS) is a common cause of hemolytic anemia varying from mild to severe hemolysis due to defects in red cell membrane protein genes, namely ANK1, SPTB, SPTA1, SLC4A1, and EPB42. These genes are considerably very large spaning 40-50 exons making gene-by-gene analysis costly and laborious by conventional methods. In this study, we explored 26 HS patients harboring 21 ANK1 variants identified by next-generation sequencing (NGS), characteristics and spectrum of the detected ANK1variants were analyzed in this study. Clinically, all the HS patients showed moderate to severe transfusion-dependent hemolytic anemia, some requiring splenectomy. We identified 13 novel and 8 reported variants, mainly 9 frameshifts, 2 missense, 6 nonsense, and 4 splice site ANK1 variants, using NGS technology. Frameshifts were remarkably the most common variant type seen in Indian HS patients with ANK1 gene defects. We have also explored expression levels of red cell membrane ankyrin protein by flow cytometry in 14 HS patients with ANK1 gene defects and a significant reduction in ankyrin protein expression has been found. This report mainly illustrates the molecular and phenotypic heterogeneity of ANK1 variants causing HS in Indian patients. Ankyrin-1 mutations are a significant cause of loss of function in dominant HS in the Indian population. Comprehensive genetic and phenotypic evaluation assists in implementing the knowledge of genetic patterns and spectrum of ANK1 gene variants, providing molecular support for HS diagnosis.