Applications of Flow Cytometry in Diagnosis and Evaluation of Red Blood Cell Disorders.

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.

Precise diagnosis of a hereditary spherocytosis patient with complicated hematological phenotype.

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.

Coinheritance of hereditary spherocytosis with haemochromatosis: next-generation sequencing reveals.

We report the case of a man in his 50s with extravascular haemolysis, fluctuating indirect hyperbilirubinaemia, elevated transferrin saturation with hyperferritinaemia and normal liver enzymes. Spherocytes were detected in a blood smear and a mutation of unknown significance, c.1626+1G>A p.?, in intron 13 of the SLC4A1 gene, was identified by next-generation sequencing (NGS). The same mutation was found in his daughter, who presented with similar laboratory changes, confirming the diagnosis of hereditary spherocytosis. Abdominal MRI showed hepatosplenomegaly with hepatic iron overload. In this context of haemolysis (without anaemia) and iron overload, a diagnosis of haemochromatosis was presumed. NGS confirmed the presence of the variants p.(His63Asp) and p.(Cys282Tyr) in heterozygosity in the HFE gene. We report this case for the rarity of co-existing two haematological diseases counteracting each other.

Molecular characteristics of hereditary red blood cell membrane disorders in Thailand: a multi-center registry.

Red blood cell (RBC) membrane disorders represent a significant category of hereditary hemolytic anemia; however, information from Southeast Asia is limited. We established a national registry aiming to characterize RBC membrane disorders and their molecular features in Thailand. A total of 100 patients (99 kindreds) diagnosed with RBC membrane disorders between 2011 and 2020 from seven university hospitals were enrolled. The most prevalent disorders observed were hereditary elliptocytosis (HE; n=33), hereditary pyropoikilocytosis (HPP; n=28), hereditary spherocytosis (HS; n=19), Southeast Asian ovalocytosis (SAO; n=10 of 9 kindreds), and two cases of homozygous SAO. The remaining cases were grouped as unclassified membrane disorder. Seventy-six patients (76%) were molecularly confirmed by PCR, direct DNA sequencing, or hi-throughput sequencing. The primary causative gene for HE and HPP was SPTB, accounting for 28 out of 29 studied alleles for HE and 56 of 56 studied alleles for HPP. In the case of HS, dominant sporadic mutations in the ANK1 gene (n=4) and SPTB gene (n=3) were identified as the underlying cause. Notably, the four most common variants causing HE and HPP were SPTB Providence (c.6055 T>C), SPTB Buffalo (c.6074 T>G), SPTB Chiang Mai (c.6224 A>G), and SPTB c.6171__82delins TGCCCAGCT. These recurrent SPTB mutations accounted for 79 out of 84 mutated SPTB alleles (94%). In summary, HE and hereditary HPP associated with recurrent SPTB mutations are the predominant types of RBC membrane disorders observed in Thailand. These findings have significant implications for the clinical management and future research of RBC membrane disorders in the region.

Hereditary Spherocytosis: Can Next-Generation Sequencing of the Five Most Frequently Affected Genes Replace Time-Consuming Functional Investigations?

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 (ß-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.

[Clinical and genotypic analysis of hereditary spherocytosis combined with cholestasis among pediatric patients].

Objective: To understand the clinical and genetic characteristics of hereditary spherocytosis (HS) combined with cholestasis among pediatric patients. Methods: 12 cases of HS children accompanied by cholestasis at Hunan Children's Hospital were selected as the research subjects between January 2013 and December 2022. Clinical data were collected. Whole-exome sequencing was performed by second-generation sequencing. Suspected pathogenic mutation sites were verified by Sanger sequencing. Results: All pediatric patients were admitted to the hospital due to their yellow skin tone. Eight cases (66.67%) had a positive family history. The clinical manifestations were jaundice, splenomegaly (12/12), abdominal pain, anemia (4/12), and hepatomegaly (5/12). All pediatric patients had decreased hemoglobin, an increased reticulocyte ratio, total bilirubin and direct bilirubin, a positive erythrocyte fragility test, and remarkable spherical erythrocytes in their peripheral blood. Seven cases had elevated aminotransferase; four cases had severely elevated aminotransferase and bilirubin; eight cases had biliary calculi; and two cases had a dilated biliary tract. Liver pathological examination showed mild damage to the liver cells (G1S1) in three pediatric cases. Five children had a total of six unreported mutations: SPTB gene c.2431_2450del, c.4974-2A > G, c.2575G > A, and exon 22-35 deletion; ANK1 gene: c.2379-2380delC; and c .6dupC. Children still had abnormal bilirubin levels following treatment. Two pediatric cases underwent splenectomy. Bilirubin and hemoglobin levels returned to normal after surgery. Conclusion: Children with HS may experience cholestasis, and those with poor treatment results may consider undergoing a splenectomy. Six new types of variants have expanded the HS gene mutation spectrum.

Next generation sequencing (NGS) interest in deciphering erythrocyte molecular defects' association in red cell disorders: Clinical and erythrocyte phenotypes of patients with mutations inheritance in PIEZO1, Spectrin ß1, RhAG and SLC4A1.

We report here an instructive case referred at 16 months-old for exploration of hemolysis without anemia (compensated anemia with reticulocytosis). The biology tests confirmed the hemolysis with increased total and indirect bilirubin. The usual hemolysis diagnosis tests were normal (DAT, G6PD, PK, Hb electrophoresis) except cytology and ektacytometry suggesting an association of multiple red blood cell (RBC) membrane disorders. This led us to propose a molecular screening analysis using targeted-Next Generation Sequencing (t-NGS) with a capture technique on 93 genes involved in RBC and erythropoiesis defects. We identified 4 missense heterozygous allelic variations, all of them were described without any significance (VUS) in the SLC4A1, RhAG, PIEZO1 and SPTB genes. The study of the familial cosegregation and research functional tests allowed to decipher the role of at least two by two genes in the phenotype and the hemolytic disease of this young patient. Specialized t-NGS panel (or virtual exome/genome sequencing) in a disease-referent laboratory and the motivated collaboration of clinicians, biologists and scientists should be the gold standard for improving the diagnosis of the patients affected with RBC diseases or rare inherited anemias.

A de novo ANK1 mutation in a childhood hereditary spherocytosis: a case report.

BACKGROUND: Due to the heterogeneity of the phenotype of Hereditary spherocytosis (HS) patients, some patients may have rare clinical complications such as biliary obstruction and ultra-high bilirubinemia. CASE PRESENTATION: A 8-y-old boy presented to the emergency with complaints of anemia for 6 years and worsened abdominal pain and scleral yellowing of the skin for 2 days. Physical examination showed tenderness in the middle and upper abdomen and splenomegaly. Abdominal CT revealed biliary obstruction. Genetic analysis revealed a de novo mutation in the gene ANK1, HS with biliary obstruction was diagnosed. The surgery of bile duct exploration and T-tube drainage, and splenectomy were performed successively. This patient was followed up for 13 months after splenectomy, and his condition was stable. CONCLUSION: The diagnosis of HS is not clinically difficult, and once a patient with HS is diagnosed, regular follow-up management and standardized treatment are required. Genetic testing is also needed to screen for other genetic disorders that may co-exist in patients with HS who do not have a good efficacy or who have a long-term chronic onset of jaundice.

Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

[Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene].

OBJECTIVE: To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis. METHODS: The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases. RESULTS: We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin. CONCLUSION: The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

[Hereditary Spherocytosis and Pregnancy: A Case Report]. / Esferocitose Hereditária e Gravidez: A Propósito de um Caso Clínico.

Even though it is a rare condition, hereditary spherocytosis (EH) is the main inherited cause of haemolytic anaemia and presents with a broad spectrum of symptoms. In the few reported cases of pregnancy and EH, maternal and foetal outcomes are controversial. Particularly, reports of pregnancies with EH associated with thrombosis or portal hypertension are scarce. We present a case of a woman who underwent splenectomy with EH and non-cirrhotic portal hypertension. Our patient presented polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) and plasminogen activator inhibit-1 that have a controversial impact on thrombotic risk. During pregnancy, the woman showed no signs of haemodynamical or cirrhosis deterioration. Concerning the foetus, late-onset foetal growth restriction was diagnosed but did not determine preterm delivery. Five weeks post-partum after an episode of acute abdominal pain, mesenteric venous thrombosis was diagnosed. In this case report, we describe our experience in managing pregnancy, labour and post-partum of a woman with EH, highlighting potential complications of this condition.

A esferocitose hereditária (EH), embora rara, constitui a principal causa de anemia hemolítica hereditária, tendo uma apresentação clínica muito diversa. Raros casos de grávidas com EH estão publicados, tendo um impacto incerto nos desfechos maternos e fetais. Os relatos de gestações complicadas por EH e de complicações trombóticas ou hipertensão portal são particularmente escassos. Apresentamos o caso de uma grávida esplenectomizada, com EH e hipertensão portal não cirrótica. A utente apresentava polimorfismos da metiltetrahidrofolato redutase e fator inibidor do ativador do plasminogénio, mutações com impacto controverso no risco trombótico. Durante a gestação não ocorreu deterioração hemodinâmica ou hepática, diagnosticando-se restrição de crescimento fetal tardia que não condicionou término precoce da gravidez. Cinco semanas após o parto, surgiu um quadro de dor abdominal, tendo-se diagnosticado trombose de veia mesentérica. Descrevemos a nossa experiência de vigilância da gravidez, parto e puerpério de uma mulher com EH grave, com destaque para potenciais complicações associadas à EH.

De novo variations of ANK1 gene caused hereditary spherocytosis in two Chinese children by affecting pre-mRNA splicing.

BACKGROUND AND AIMS: Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic disorders. Here, two unrelated families with the probands displaying typical manifestations of HS were enrolled. Our study aimed to characterize the effect of two novel variants in HS patients on gene splicing to help minimize the rate of misdiagnosis of HS and enhance clinicians' understanding of the disease. PARTICIPANTS AND METHODS: A retrospective review was conducted. Peripheral blood samples were collected from all the family members, and genomic DNA was extracted for genetic diagnostics. First, high-throughput sequencing technology was used for the preliminary screening of candidate causative variants. Thereafter, the variants were verified via Sanger sequencing. Furthermore, a pathogenicity analysis of the detected variants was performed including in silico prediction and in vitro experiments. We constructed matched wild-type and mutant-type minigene plasmid of ANK1 based on HEK293T cells to address the effects of variants on mRNA splicing. RESULTS: The c.1305 + 2 T > A (family1) and c.1305 + 2del (family2) variants were detected in the ANK1 gene. These two de novo mutations described by us which have not been reported prior to this study. Moreover, the validation results of splicing reporter systems revealed that the intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the minigene plasmid expressing the c.1305 + 2 T > A variant transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 229 and r.1305_1306ins1305 + 1_1305 + 552. The minigene plasmid expressing c.1305 + 2del transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 228 and r.1305_1306ins1305 + 1_1305 + 551. CONCLUSION: The two de novo variants identified in the ANK1 gene were the genetic etiology of the probands with HS in our study. Our findings further enrich the HS genotype database and provide a basis for genetic counselling and molecular diagnosis.

Targeted next-generation sequencing identifies novel deleterious variants in ANK1 gene causing severe hereditary spherocytosis in Indian patients: expanding the molecular and clinical spectrum.

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.

A large family of hereditary spherocytosis and a rare case of hereditary elliptocytosis with a novel SPTA1 mutation underdiagnosed in Taiwan: A case report and literature review.

RATIONALE: Hereditary spherocytosis (HS) has a defect in the vertically connected proteins on the cell membrane of red blood cells (RBC). Hereditary elliptocytosis (HE) has a defect in proteins that connect the cell membrane horizontally. We reported two families of RBC membrane disorders in Taiwanese, one was HS and the other was HE. PATIENT CONCERNS: Case 1. A 19-year-old male student with chronic jaundice and splenomegaly. His mother, maternal uncle, grandmother, and many members of older generations also had splenomegaly and underwent splenectomy. Case 2. A 40-year-old man has experienced pallor and jaundice since the age of 20 and was found to have splenomegaly, and gall bladder stones in the older age. His younger sister also had pallor and jaundice for a long time. DIAGNOSES: In case 1, a peripheral blood smear showed 20% spherocytes. Eosin-5-maleimide labeled RBC by flow cytometry showed a result of 30.6 MCF (cutoff value: 45.5 MCF). He was diagnosed with HS. The gene analysis identified a heterozygous mutation with c.166A > G (p.Lys56Glu) in the SLC4A1 gene in this proband, his mother, and maternal uncle. In case 2, more than 40% of ellipsoid RBC present in the peripheral blood smear. He was diagnosed with HE. Genetic analysis of the SPTA1 gene identified a novel heterozygous exon2, c.86A > C, p.Gln29Prol mutation. INTERVENTIONS: The two patients had compensated anemia, clinical follow-up instead of splenectomy was done. OUTCOMES: The two patients had normal daily activities and lives. LESSONS: We reported two Taiwanese families, one was hereditary spherocytosis affected by a heterozygous mutation with c.166A > G (p.Lys56Glu) in SLC4A1, and the other was hereditary elliptocytosis caused by a novel heterozygous SPTA1 gene mutation, c. 86A > C, p.Gln29Prol. These 2 seemingly common hereditary red blood cell membrane protein defects induced by hemolysis are usually underdiagnosed or misdiagnosed.

Glucose 6 Phosphate Isomerase Deficiency, a Rare Hemolytic Anemia Misdiagnosed as Hereditary Spherocytosis.

Hereditary hemolytic anemias are a heterogenous group of disorders that include membranopathies, enzymopathies, and hemoglobinopathies. Genetic testing is helpful in the diagnostic workup when the clinical and laboratory workup is not conclusive. Here, we present a case of a 21-month-old female who was initially diagnosed with hereditary spherocytosis based on the presence of a variant of unknown significance in the SPTB gene. Further genetic workup revealed a homozygous glucose 6 phosphate isomerase mutation and the patient was ultimately diagnosed with glucose 6 phosphate isomerase deficiency.

A Novel SPTA1 Mutation in a Patient with Hereditary Spherocytosis without a Family History and Coexisting Gilbert's Syndrome.

Most patients with hereditary spherocytosis (HS) have a family history of disease, while those without such a history are difficult to diagnose. We herein report a case of HS with no family history harboring a novel heterozygous mutation of SPTA1, c.2161G>A (p.E721K), and a homozygous polymorphism of UGT1A1*6. In silico analyses suggested that the mutation might contribute to the pathogenesis of HS. The coexistence of HS and Gilbert's syndrome increases the risk of gallstones. Therefore, splenectomy, alone or in combination with cholecystectomy, is recommended. The determination of genetic diathesis provides useful information for the management of hemolytic anemia.

[When HbA1c is unreliable : a case report of hereditary spherocytosis]. / Cas clinique. Quand le dosage de l'HbA1c n'est pas fiable : à propos d'un cas de sphérocytose héréditaire.

Glycated haemoglobin (HbA1c) is a biological parameter used in the management of diabetic patients. Independent of the daytime glycaemic variations, but complementary to the measurement of blood glucose or subcutaneous glucose concentrations, it allows both the clinician and the patient to have an appreciation of the glycaemic balance of the last weeks. In this way, anti-diabetic treatment can be adjusted if necessary to achieve the desired goal and hopefully delay or prevent diabetes-related micro- and macroangiopathic complications. Some conditions can alter the glycation of haemoglobin. In this case, the HbA1c level becomes difficult to interpret. Hereditary spherocytosis may be revealed by a dissociation between low HbA1c level and high blood glucose levels. A family history, Coombs-negative haemolytic anaemia, or a finding of spherocytes in the blood smear is suggestive of hereditary spherocytosis. Fructosamine testing may be an alternative. This article will present a patient with hereditary spherocytosis in whom the HbA1c level was not interpretable when compared to the elevated blood glucose measurements.

: L'hémoglobine glyquée (HbA1c) est une valeur biologique utilisée dans le suivi des patients diabétiques. Indépendante de la variation glycémique nycthémérale, mais complémentaire à la mesure de la glycémie ou de la concentration sous-cutanée de glucose, elle permet tant au clinicien qu'au patient d'avoir une appréciation de l'équilibre glycémique des dernières semaines. De cette manière, le traitement anti-diabétique peut être éventuellement adapté pour atteindre l'objectif escompté et espérer retarder, voire prévenir, les complications micro- et macroangiopathiques liées au diabète. Certaines affections peuvent altérer la glycation de l'hémoglobine. Dans ce cas, le taux d'HbA1C devient difficile à interpréter. La sphérocytose héréditaire peut se révéler par un tableau de dissociation entre un taux bas d'HbA1C et des valeurs élevées de glycémie. Des antécédents familiaux, une anémie hémolytique à Coombs négatif, ou une observation de sphérocytes dans le frottis sanguin sont en faveur d'un diagnostic de sphérocytose héréditaire. Le dosage de la fructosamine peut être une alternative. Le présent article abordera le cas d'un patient atteint d'une sphérocytose héréditaire chez qui le taux d'HbA1c n'était pas interprétable en regard des contrôles glycémiques.