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We describe the clinical/haematological characteristics of 446 patients with hereditary spherocytosis diagnosed in the last 40 years in a reference centre. The frequency of splenectomy decreased over time (44% before 1990 to 7% in 2011-2020), notwithstanding a confirmed good efficacy. Age at splenectomy progressively increased (63% in children before 1990 to 88% in patients aged ≥20 years in 2011-2020). Our real-life experience showed that even a fraction of patients in the trait/mild categories (19/92, 21%) were splenectomised, whilst 30/78 (38%) in the moderate/severe groups were not. Overall, these data pinpoint to the increasing awareness about post-splenectomy thromboses and infections.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disease marked by chronic hemolytic anemia of various severity and frequent complications including gallstones, splenomegaly, iron overload, and others. Disease phenotype is highly heterogeneous and changes over time with children, adolescents and adult patients displaying different transfusion requirement and rates of complications. The diagnosis relies on the initial clinical suspicion in a patient with chronic hemolysis and exclusion of other more common congenital forms of hemolytic anemias; it is supported by the demonstration of reduced PK enzyme activity, and further confirmed by the detection of (homozygous or compound heterozygous) mutations of PKLR gene. Therapy is mainly supportive, with vitamin supplementation and transfusions (based on symptoms and patient growth rather than on fixed Hb thresholds). Splenectomy is widely performed, although it is less effective than in membrane defects and carries thrombotic and infectious risk. In the last decade, the allosteric PK enzyme activator mitapivat showed dramatic clinical benefit in clinical trials and gene therapy is also being studied to substitute the defective enzyme. In this review, we provide an insight in the current challenges of PKD diagnosis and management and discuss the future application of novel drugs and gene therapy, including a focus on quality of life.

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 μg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.

Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

Hereditary hemolytic anemias are a group of rare and heterogeneous disorders due to abnormalities in structure, metabolism, and transport functions of erythrocytes; they may overlap in clinical and hematological features making differential diagnosis difficult, particularly in mild and atypical forms.In the present review, the main tools currently adopted in routine hematologic investigation for the diagnosis of hereditary hemolytic anemias are described, together with the new diagnostic approaches that are being to be developed in the next future. Available recommendations in this field together with a systematic review through MEDLINE, EMBASE, and PubMED for publications in English from 2000 to 2020 in regards to diagnostic aspects of hereditary hemolytic anemias have been considered.The recent development of specific molecules and treatments for hereditary hemolytic anemias and the increased interest in translational research raised the attention on differential diagnosis and the demand for novel diagnostic assays and devices. Automatic blood cell analyzers, omic-approaches including NGS technologies, and development of new automated tools based on artificial neural networks definitely represent the future strategies in this field.

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and β-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6–1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

BACKGROUND: The present study aimed to use the targeted capture and sequencing technique to diagnose adult hereditary spherocytosis (HS). These results were compared with clinical features and laboratory examinations to explore the diagnosis of HS. METHODS: Whole blood and clinical data from ten patients with HS were collected. Genomic DNA was extracted, and a library was prepared. Exomes of patients with ten HS-related genes encoding red cell membrane skeleton protein were captured and sequenced. Bioinformatics analyses were carried out throughout the 1000 Genomes Project, ExAC, dbSNP147, and 1000 Normal Han Population databases. RESULTS: Gene mutations were found in 9 out of 10 cases of HS. Our data validation showed 90% specificity. Three types of gene mutations were found, including 6 cases of SPTB, 3 cases of ANK1, and 2 cases of SLC4A1. There were 4 mutation forms, including nonsense mutation, missense mutation, shear mutation, and code shift mutation, all of which were new, heterozygous mutations. These variations were predicted to be pathogenic in four databases. CONCLUSIONS: Our data demonstrate that targeted gene enrichment and sequencing methods were an efficient tool for determining genetic etiologies of red blood cell (RBC) membrane disorders and can facilitate accurate diagnosis and genetic counseling. They are also in good agreement with the clinical results.

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, the second reaction step of glycolysis. GPI deficiency, transmitted as an autosomal recessive trait, is considered the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase deficiency. Despite this, this defect may sometimes be misdiagnosed and only about 60 cases of GPI deficiency have been reported. GPI deficient patients are affected by chronic non-spherocytic hemolytic anemia of variable severity; in rare cases, intellectual disability or neuromuscular symptoms have also been reported. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far, about 40 causative mutations have been identified. We report the clinical, hematological and molecular characteristics of 12 GPI deficient cases (eight males, four females) from 11 families, with a median age at admission of 13 years (ranging from 1 to 51); eight of them were of Italian origin. Patients displayed moderate to severe anemia, that improves with aging. Splenectomy does not always result in the amelioration of anemia but may be considered in transfusion-dependent patients to reduce transfusion intervals. None of the patients described here displayed neurological impairment attributable to the enzyme defect. We identified 13 different mutations in the GPI gene, six of them have never been described before; the new mutations affect highly conserved residues and were not detected in 1000 Genomes and HGMD databases and were considered pathogenic by several mutation algorithms. This is the largest series of GPI deficient patients so far reported in a single study. The study confirms the great heterogeneity of the molecular defect and provides new insights on clinical and molecular aspects of this disease.

BACKGROUND Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS RBC antibody–bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p

A previously healthy 15-year-old girl was evaluated following five episodes of reddish urine discoloration after walking for approximately 30 min on a smooth roadway. In each episode, the discoloration lasted for four to five urinations and followed by normal urine dipstick tests. No other exercise-produced urine discoloration and no other symptoms were reported. Laboratory evaluation during the episodes revealed a reddish urine sample with 3+ hemoglobin/myoglobin and absence of hematuria. Full blood count, serum creatinine, liver function tests, and electrolyte levels were all within normal limits. Myoglobulinuria was excluded, since muscle enzymes were within normal limits. Blood smear analysis showed mild anisopoikilocytosis with stomatocytes and ovalocytes, leading to extended evaluation for erythrocyte disorders. This case is interesting in that the hemoglobinuria occurred after mild walking and was accompanied by erythrocyte morphological changes. This quiz discusses the differential diagnosis of hemoglobinuria with particular reference to the conditions of appearance (after walking) and emphasizes the importance of step-by-step investigations to reach a definitive diagnosis.

We report the first case of a child with recessive hereditary methemoglobinemia type II with demonstrated cerebellar atrophy. This very rare blood disorder results in mild cyanosis, profound mental and motor impairment, and movement disorders in infancy and childhood. We suggest that children with unexplained severe encephalopathy and cerebellar atrophy should also be tested for hereditary methemoglobinemia type II.

BACKGROUND: Febrile nonhemolytic transfusion reactions occur in 0.12% of transfusions, usually during transfusion or within 4 to 6 hours after transfusion and are not medically dangerous. CASE REPORT: A patient with thalassemia from Togo with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile nonhemolytic transfusion reaction, is presented. Thirty-two hours after transfusion of 2 O D− red blood cell (RBC) units, the patient (phenotype A2 D+) developed fever and multiorgan failure and was admitted to the intensive care unit. Direct and indirect antiglobulin tests were negative on posttransfusion samples. Blood cultures and infectious diseases testing were negative. No malaria parasites were found at thick blood smear microscopic examination on Days 1 and 2 and the malaria rapid diagnostic test gave inconsistent results. Plasmodium total antibodies were detected in the serum at high levels. On Day 5, routine microscopic examination of blood smear revealed the presence of parasites in a very small number of RBCs. This finding was almost simultaneous to the availability of polymerase chain reaction testing results that were positive for P. falciparum. The sequential agglutination with anti-A antiserum allowed patient's and donors' RBCs to be separated and revealed that the parasitized cells were almost exclusively those of donors (14.4% vs. 0.029%). Malaria infection in implicated donors was excluded. CONCLUSION: In this patient with thalassemia with asymptomatic malaria, the infusion of two normal RBC units provided a favorable environment for a rapid parasite replication leading to a dramatic acute malaria attack.

Congenital dyserythropoietic anaemia type II (CDAII) is a rare autosomal recessive disease characterized by ineffective erythropoiesis, haemolysis, erythroblast morphological abnormalities, hypoglycosylation of some red blood cell membrane proteins, particularly band 3, and mutations in the SEC23B gene. We report the analysis of 101 patients from 91 families with a median follow-up of 23 years (range 0-65); 68 patients are newly reported. Clinical and haematological parameters were separately analysed in early infancy and thereafter, when feasible. Molecular analysis of the SEC23B gene confirmed the high heterogeneity of the defect, leading to the identification of 54 different mutations, 24 of which are newly described. To evaluate the genotype-phenotype correlation, patients were grouped according to their genotype (two missense mutations vs. one missense/one drastic mutation) and assigned to two different severity gradings based on laboratory data and on therapeutic needs; by this approach only a weak genotype-phenotype correlation was observed in the analysed groups.

L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.

We report the hematological, biochemical, and molecular characteristics of a new defective adenylate kinase (AK) variant associated with chronic hemolytic anemia. The propositus was a 3-year-old girl of southern Italian origin with a history of severe anemia and occasional need for blood transfusion. The study of the most important red cell enzymes revealed low AK activity (22% of normal) in the propositus and intermediate values in the parents. The sequence of erythrocyte AK-1 gene showed a new homozygous mutation (delG138) determining a frameshift and a premature stop at codon 91.

We reviewed clinical and molecular data of 23 consecutive unrelated patients affected by paroxysmal nocturnal hemoglobinuria (PNH) (19 with hemolytic PNH, 3 with aplastic anemia/PNH, and 1 with myelodysplasia/PNH syndrome) with a mean follow-up of 11.8 years. Five patients had thrombotic episodes, and 10 needed regular blood transfusions; 2 died for cerebral hemorrhage and kidney failure, and 2 spontaneously recovered from PNH. Twenty different PIG-A gene mutations were detected in 21/23 patients: 15 frameshift, 1 splicing, 2 nonsense, and 2 missense mutations. Two mutations (DelG341 and IVS2 +1g-a) were detected twice. A PIG-A mutated clone was also revealed in the two patients in complete clinical remission. One patient with aplastic anemia/PNH syndrome was treated with two courses of antilymphocyte globulin and cyclosporin with partial sustained response. Six patients were given rHu-EPO 150 U/kg/day s.c. for at least 6 months: one became transfusion-independent for 8 months and then discontinued treatment for clinical complications; one displayed a mean rise of Hb of 1.5 g/dL and is currently maintaining Hb levels higher than 9 g/dL after 54 months of therapy. Mutation specific quantitative-competitive PCR showed that the rise of hemoglobin was related to an increase of PIG-A negative molecules, suggesting that the efficacy of rHu-EPO therapy may be due to the stimulation of the abnormal clone.

Summary. Pyrimidine 5′-nucleotidase deficiency is a rare autosomal recessive disorder characterized by haemolytic anaemia, marked basophilic stippling and accumulation of pyrimidine nucleotides within the erythrocytes. The gene encoding for this enzyme (P5′N-1) has been cloned recently, and seven mutations have so far been identified in 11 unrelated families. We describe the haematological and molecular characteristics of six unrelated Italian patients affected by pyrimidine 5′-nucleotidase deficiency (one from northern and five from southern Italy). The sequence of the complete P5′N-1 gene showed the presence of four different new mutations: a missense mutation AAT–AGT at codon 190 (Asn–Ser), one splicing mutation (IVS9-1 g-c) and two frameshift mutations, DelG576 and InsGG743. Although the molecular defect was homozygous in all patients but one, parents' consanguinity could be confirmed in only one case. InsGG743 was detected in two cases, and DelG576 was found in three patients originating from southern Italy, suggesting a possible geographical distribution of the genetic defect. Haematological data showed the presence of peripheral spherocytosis in all cases, although only one had a concomitant membrane defect. An increase in serum ferritin levels was observed in the splenectomized patients, suggesting that the iron status of these subjects should be monitored and that they should be investigated for potential additional risk factors for iron accumulation.

Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS.RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect.A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and β-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency.RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.

ABSTRACT We evaluated the iron status and searched for mutations C282Y and H63D in the hereditary hemochromatosis gene ( HFE ) in 34 pyruvate kinase (PK)-deficient patients from 29 unrelated families. Nine had received multiple transfusions. Thirteen of the 25 nontransfused patients displayed increased serum ferritin concentration, in the absence of conditions known to raise this parameter. HFE genotype was abnormal in 9 of 34 patients. The allele frequency was 1.8% for mutation 845G→ (C282Y) and 16.1% for mutation 187C→:G (H63D). Nontransfused subjects with abnormal genotype had serum ferritin and transferrin saturation values significantly higher than those with wild-type genotype. Of the 12 adult nontransfused patients with increased iron status parameters, 1 was C282Y homozygous, 1 compound heterozygous for C282Y and H63D, 3 H63D heterozygous, and 7 had a normal HFE genotype. Serum ferritin and transferrin saturation were not related to hemoglobin, reticulocytes, and bilirubin concentration. At multivariate analysis serum ferritin was independently associated with age and gender, but not with splenectomy and HFE genotypes. The retrospective evaluation of the iron status profile of 10 patients (3 with abnormal and 7 with wild-type HFE genotype) with at least 10 years follow-up showed that overt iron accumulation requiring iron chelation had occurred only in the 3 patients (2 of whom were splenectomized) with the mutated HFE gene.

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency.Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed.The molecular characterization of the PKLR gene showed a heterozygous mutation 994GA (Gly332Ser) associated with the promoter substitution -148CT, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS.The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype.

Background Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency. Methods Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed. Results The molecular characterization of the PKLR gene showed a heterozygous mutation 994G > A (Gly332Ser) associated with the promoter substitution -148C > T, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS. Conclusions The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype.

Background: Congenital hemolytic anemias (CHAs) are a heterogeneous group of inherited RBC disorders including membrane and enzyme defects and dyserythropoietic anemias. Iron overload is a well recognized complication of hereditary hemoglobinopathies, both in transfusion-dependent and independent cases. However, little is known in congenital hemolytic anemias, with the exception of anecdotic reports in pyruvate kinase deficiency and dyserythropoietic anemias. Aim: to describe the clinical and hematological features at diagnosis and enrolment, to investigate iron overload by hepatic and cardiac T2* MRI, and to study inflammatory/regulatory cytokine profiles (IL-6, TNF-alpha, IFN-gamma, IL-10, IL-17) and hepcidin levels in patients with CHAs. Confounding factors such as hemocromatosis genotyping, metabolic syndrome, and hepatic viral profile were also considered. Methods: Between July 2015 and April 2016, 38 patients were enrolled (13 hereditary spherocytosis -HS, 3 hereditary stomatocytosis - HSt, 8 congenital dyserythropoietic anemia type II - CDAII, 13 pyruvate kinase deficiency - PKD, 1 glucose-phosphate isomerase deficiency). HS cases were enrolled on the basis of ferritin >300 ng/mL at diagnosis. Cytokine levels were detected in serum by ELISA. Comparisons were made by Students T test (continuous) and Fisher's exact test (categorical), and correlations by Pearson's linear coefficient. Results: The main clinical and hematological findings are shown in table. Median Hb values progressively decreased in the 4 groups considered, being close to normal in HS and moderately reduced in CDAII patients, whereas hemolytic parameters were comparable among groups. Consistently with clinical severity, ferritin values were particularly high in CDAII (together with transferrin saturation-TfS) and PKD patients, notwithstanding chelation in about half cases of both groups. Of note, only 2 PKD patients were transfusion-dependent, suggesting that other factors are involved in iron overload. Splenectomy had been performed in 17/38 (44.7%), mainly CDAII. Liver iron concentration (LIC) showed a great heterogeneity in all groups, with a trend towards higher values in CDAII; 16/36 (44%) patients had a LIC>4 mg/g DW (23%, 33%, 38% and 88% in HS, HSt, PKD and CDAII, respectively). Cardiac T2* value was normal in all subjects, with the exception of a HS and a CDAII case. Regarding possible cofactors, 12/16 displayed at least one of the following: 1 homozygous for HFE C282Y and 1 for H63D mutations, 3 HCV+, 4 BMI>25, 2 alcohol abuse, 3 heterozygous for HFE mutations. The following positive correlations were observed at enrolment: LIC and ferritin (r=0.68, p4), of whom 5 with the above listed cofactors. As regards cytokine levels, IL-10 was significantly increased in HS, PKD and CDAII groups compared with normal cases; TNF-alpha was decreased in HS and PKD, and IFN-gamma increased in HS and CDAII. Ferritin values were positively correlated with IL-6 and IFN-gamma, and TfS negatively with IL-6 (r= -0.38, p Conclusion: Iron overload is highly prevalent in CHAs, particularly in PKD and CDAII, is independent from transfusion support, and is also observed in cases with ferritin Table Table. Disclosures Barcellini: Agios: Consultancy.

Febrile nonhemolytic transfusion reactions occur in 0.12% of transfusions, usually during transfusion or within 4 to 6 hours after transfusion and are not medically dangerous.A patient with thalassemia from Togo with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile nonhemolytic transfusion reaction, is presented. Thirty-two hours after transfusion of 2 O D- red blood cell (RBC) units, the patient (phenotype A(2) D+) developed fever and multiorgan failure and was admitted to the intensive care unit. Direct and indirect antiglobulin tests were negative on posttransfusion samples. Blood cultures and infectious diseases testing were negative. No malaria parasites were found at thick blood smear microscopic examination on Days 1 and 2 and the malaria rapid diagnostic test gave inconsistent results. Plasmodium total antibodies were detected in the serum at high levels. On Day 5, routine microscopic examination of blood smear revealed the presence of parasites in a very small number of RBCs. This finding was almost simultaneous to the availability of polymerase chain reaction testing results that were positive for P. falciparum. The sequential agglutination with anti-A antiserum allowed patient's and donors' RBCs to be separated and revealed that the parasitized cells were almost exclusively those of donors (14.4% vs. 0.029%). Malaria infection in implicated donors was excluded.In this patient with thalassemia with asymptomatic malaria, the infusion of two normal RBC units provided a favorable environment for a rapid parasite replication leading to a dramatic acute malaria attack.

We report the clinical, haematological and molecular characteristics of two triose phosphate isomerase deficient patients affected by haemolytic anaemia and neuromuscular impairment. The sequence of complete TPI gene showed the presence of two previously undescribed mutations: c.722 TC (Phe240Ser) and c.28 insG; each of the two unrelated patients showed the new mutation in compound heterozygosity with the most common variant Glu104Asp. The association of Glu104Asp with c.28 insG resulted in a very severe clinical pattern.

We report the clinical, haematological and molecular characteristics of two triose phosphate isomerase deficient patients affected by haemolytic anaemia and neuromuscular impairment. The sequence of complete TPI gene showed the presence of two previously undescribed mutations: c.722 T>C (Phe240Ser) and c.28 insG; each of the two unrelated patients showed the new mutation in compound heterozygosity with the most common variant Glu104Asp. The association of Glu104Asp with c.28 insG resulted in a very severe clinical pattern.

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic-genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)-to-Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII.

We report the clinical and molecular characteristics of 6 new patients with recessive hereditary methemoglobinemia due to cytochrome b5 reductase deficiency. One patient was affected by Type-II disease with cyanosis and severe progressive neurological dysfunction, whereas the others displayed the benign Type-I phenotype. Methemoglobin levels ranged from 12.1% to 26.2% and cytochrome b5 reductase activity from 0 to 10% of normal. Eight different mutations were detected among the twelve mutated alleles identified, one splicing mutation, two stop codon, and five missense. Two mutations c. 82 C>T(Gln27STOP) and c. 136 C>T(Arg45Trp) are new. Prenatal diagnosis was performed in the family with Type-II disease.

Introduction: Hereditary hemolytic anemias (HAs) is a group of heterogeneous disorders mainly due to defects of red cell (RBC) membrane and metabolism. HAs of RBC membrane are classified according to the morphological appearance on a blood smear: the more common are hereditary spherocytosis (HS), elliptocytosis, followed by stomatocytosis (dehydrated, DHSt and overhydrated, OHSt). Among RBC enzyme defects associated with chronic hemolysis, pyruvate kinase (PK) deficiency is the most common, followed by glucose-6-phosphate isomerase (GPI), pyrimidin 5' nucleotidase (P5'N), and more rarely, other enzymes deficiencies. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a battery of laboratory tests. Ektacytometry, and more recently, laser-assisted optical rotational cell analyser (LoRRca MaxSis, Mechatronics Instruments, NL), able to measure RBC deformability in osmotic gradient conditions (Osmoscan), is reported to be a useful tool in the detection of RBC membrane disorders and in particular for the differential diagnosis of HSt. Few data are available in other haemolytic anemias. Methods: We evaluated the diagnostic power of laser-assisted optical rotational cell analyser in 84 cases with RBC membrane disorders (69 HS, 7 HE, 7 DHSt, 1 OHSt) and 26 enzymopathies: 19 PK deficiency (14 not splenectomised), 4 GPI deficiency and 3 P5N deficiency. Moreover, we analysed 8 congenital diserythropoietic anemia type II (CDA II), of whom 3 not splenectomised. A reference curve interval was obtained from 89 healthy controls. The evaluated parameters were: Omin (osmotic value at which the EI reaches its minimum, coinciding with the 50% hemolysis in osmotic fragility assays), EImax (maximal deformability obtained near the isotonic osmolality), and Ohyper (osmotic value at which the EI reaches half of its maximum value, representing cellular hydration status). Results: Considering membrane defects, all the 69 HS, regardless the biochemical defect, showed typical altered ektacytometry curves, with a decreased EImax (0.516±0.05 vs 0.595±0.008 for controls, p Concerning RBC enzymopathies, we found that all the GPI deficient cases showed altered enlarged Osmoscan curves associated with significant increased Ohyper (539±36 vs 462±13.4, p We separately analysed splenectomised and not splenectomised patients. Interestingly, for some diseases (PK, P5N, CDAII and OHSt) Osmoscan curve after splenectomy, falls in a defined atypical area, regardless the pathology (Fig 1d). Conclusion: The Osmoscan curves were diagnostic for all the HS and HE cases analysed, with Omin and EImax parameters significantly different from controls. DHSt cases always showed typical left-shifted curve. Interestingly, Ohyper was firstly described increased in GPI deficiency, offering a new diagnostic tool for this rare enzyme defect. The interpretation of Osmoscan requires caution in splenectomised cases as splenectomy may interfere with the RBC deformability. In conclusion, the Osmoscan analysis performed by LoRRca Maxsis represents a useful and feasible first step screening test in the diagnosis of RBC membrane disorders and other rare haemolytic anemias. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.