Largest comparison between onset and relapses of acquired thrombotic thrombocytopenic purpura reveals severe neurological involvement and worse analytic parameters at debut.

It has been proposed that the onset of Acquired Thrombotic Thrombocytopenic Purpura (iTTP) is more severe than subsequent relapses; however, existing studies have limitations. We conducted a retrospective observational study to compare analytical and clinical severity of onset and relapse aTTP cases between 2012 and 2023. A total of 370 episodes of aTTP were analyzed, comprising 272 at initial diagnosis and 98 relapses. At onset, analytical parameters indicative of severity (low hemoglobin, low platelet count, and increased LDH) were significantly worse; patients had severe neurological symptoms (p<0.001) and ≥ 3 points in the TMA mortality score (p<0.001). In conclusion, the onset of aTTP is associated with worse analytical parameters and severe neurological involvement.

Does size matter? Two new deletions in the HBB gene cause ß0-thalassemia.

Most ß-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions; in these cases, the loss ranges from a single nucleotide to even the entire HBB gene, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation. To clarify this, we present two new deletions in the ß-globin gene that cause ß0-thalassemia. The hematological parameters were determined with an automated cell counter; the Hb A2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion-exchange HPLC (BioRad Variant II ß-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit. We have characterized two new mutations-(1) 1-bp deletion [CD61/62(-G)] [HBB:c.186_187delG], (2) 105-bp deletion [IVS-2-nt767-CD111] [HBB:c.316-84_333del]-and we have described, for first time in Spain, the 25-bp deletion [ß nts 252 - 276 deleted] [HBB:c.93-22_95del] mutation. These mutations were classified as pathogenic by UniProt Variants confirmed according to the American College of Medical Genetics and Genomics guidelines. These mutations present a phenotype compatible with ß0-thalassemia, supported by hematological parameters that correlate the degree of reduction in the synthesis of the ß-globin chain. Identification of this type of mutation is important for genetic counselling of partners where both are carriers, so that they are aware of the genetic risk of having affected children, allowing them to take an informed decision about their reproductive choices.

Clinical and genetic features of congenital dyserythropoietic anemia (CDA).

INTRODUCTION: Congenital dyserythropoietic anemias (CDA) are characterized by hyporegenerative anemia with inadequate reticulocyte values, ineffective erythropoiesis, and hemolysis. Distinctive morphology of bone marrow erythroblasts and identification of causative genes allow classification into 4 types caused by variants in CDAN1, c15orf41, SEC23B, KIF23, and KLF1 genes. OBJECTIVE: Identify pathogenic variants in CDA patients. METHODS: Massive parallel sequencing with a targeted gene panel, Sanger sequencing, Comparative Genome Hybridization (CGH), and in silico predictive analysis of pathogenicity. RESULTS: Pathogenic variants were found in 21 of 53 patients studied from 44 unrelated families. Six variants were found in CDAN1: two reported, p.Arg714Trp and p.Arg725Trp and, four novel, p.Arg623Trp, p.Arg946Trp, p.Phe1125Ser and p.Ser1227Gly. Twelve variants were found in SEC23B: seven reported, p.Arg14Trp, p.Glu109Lys, p.Arg217Ter, c.835-2A>G, p.Arg535Ter, p.Arg550Ter and p.Arg718Ter and, five novel, p.Val164Leu, p.Arg190Gln, p.Gln521Ter, p.Arg546Trp, and p.Arg611Gln. The variant p.Glu325Lys in KLF1 was found in one patient and p.Tyr365Cys in ALAS2 in an other. Moreover, we identified genomic rearrangements by CGH in some SEC23B-monoallelic patients. CONCLUSIONS: New technologies for genetic studies will help to find variants in other genes, in addition to those known, that contribute to or modulate the CDA phenotype or support the correct diagnosis.

A novel antibody against human factor B that blocks formation of the C3bB proconvertase and inhibits complement activation in disease models.

The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.

ß-Thalassemia Intermedia: Interaction of α-Globin Gene Triplication With ß-thalassemia Heterozygous in Spain.

Objectives: To verify with hematimetric data that the diagnosis and clinical grade of ß-TI can be established when a triplication of alpha genes (αααanti 3.7) and heterozygous ß-thalassemia coexist. Materials and Methods: Retrospective study in which 73 patients of Caucasian origin participated, who simultaneously showed a triplication or quadruplication of genes α and ß-thalassemia.Screening for the most frequent α-thalassemia mutations as well as gene triplication (αααanti 3.7) was carried out by multiplex PCR followed by reverse hybridization with a commercial Alpha-Globin StripAssay kit and confirmed by MLPA (Multiplex ligation-dependent probe amplification). The molecular diagnosis of ß-thalassemia was carried out by automatic sequencing according to the Sanger method. Results: The genotypes have been classified into three groups according to the number of α globin genes and the severity of the alteration in the ß globin gene. All had a mutation in the HBB gene (ß0-thalassemia, ß+-thalassemia severe, and ß+-thalassemia mild). Group I patients who have coherent 6 α genes and groups II and III with 5 α globin genes. In group III, the patients were carriers of mutations affecting the ß and δ globin genes. The most significant hematological parameters were hemoglobin levels, MCV, RDW, and the percentage of Hb F. Conclusions: In group I, regardless of the distribution of the 6 α globin genes, homozygous triplication (ααα/ααα) or heterozygous quadruplication (αααα/αα), the association with heterozygous ß-thalassemia results in severe to moderate anemia that may or may not require transfusion therapy, is the severity of the HBB gene mutation that would determine the clinical variation. Group II patients phenotypically behaved like mild thalassemia intermedia, except for one case that presented thalassemic trait because it also presented an associated α-thalassemia (ααα/-α3.7). Finally, group III patients behaved as a thalassemic trait since all were carriers of mutations that increase the overexpression of γ genes.

[No deletion alpha thalassaemia in Spain. Abnormal hematological index and molecular study]. / Alpha talasemia no deleción en Espaia. indices hematológicos anormales y su estudio molecular.

The alpha thalassaemia diseases in most cases are caused by deletions that affect one or two of the alpha genes, being less frequent the cases due to punctual mutations, insertions or deletions of a few pairs of bases, which have been denominated no deletion a thalassaemias. The objective of this investigation was to determine the incidence of the no deletion alpha thalassaemia in patients with a thalassaemia using molecular biology techniques. We studied 517 individuals of the San Carlos Hospital (Thalassemia Molecular Research Center, Madrid-Spain) between January 2001 and December 2003, in whom iron deficiency anemia had been ruled out, that presented microcytosis and hypochromia and that presented normal HbA2, HbF and EEF from normal Hbs. The two types of no deletion a thalassaemia most frequently described in the Mediterranean were studied: 1) alpha Hph due to deletion of 5bp in the IVS I and 2) alphaNco due to a change in the initiation codon of the gene. Of the 517 cases studied, 40 (7.7% of the cases) represented a no deletion alpha thalassaemia. Of these cases, 28 were positive for alphaHph of the alpha2 gene, 24 in the heterozygote state, one homozygote and three double heterozygotes associated with the 3,7 kb deletion. The remaining 12 cases were positive for the alphaNco of the alpha2 gene, 10 heterozygotes, one homozygote and one double heterozygote associated with the 4,2 kb deletion. The no deletion alpha thalassaemias represent < 8% from the cases in our environment. The alphaHph is the most frequent type of no deletion a thalassaemia and its haematological abnormalities are more manifest that the ones present in the cases of alphaNco.

Why is the novel Hb Miguel Servet visualised by CE-HPLC newborn and not by the CE-HPLC ß-thalassaemia programme?

Screening of haemoglobinopathies is indicated for the detection of sickle cell anaemia; thus, neonates can benefit from early and adequate treatment that prevents neurological damage, reduces morbidity and mortality associated with the disease. These types of programmes sometimes lead to unexpected findings. We present a new haemoglobin (Hb) variant (Hb Miguel Servet) detected by newborn screening. During neonatal screening of haemoglobinopathies by cation-exchange high-performance liquid chromatography (CE-HPLC) newborn, an Hb variant was detected. An analysis at 8 months of age using capillary zone electrophoresis (CZE) confirmed the presence of this new Hb. The molecular characterisation was performed by automatic sequencing of the α and ß globin genes in an ABI PRISM 3100 Genetic Analyzer. Hb analysis by CE-HPLC ß-thalassaemia short programmedid not indicate the presence of abnormal Hbs. By CZE showed a peak in the zone 12 zone comprising 3.3% of the total Hb. A new analysis by CE-HPLC on a Tosoh G8-2 (Horiba) shown a peak, in the region of HbA1b, did not interfere with the quantification of HbA1c. Sequencing of the ß gene revealed the substitution of a guanine for a thymine (GGT >TGT) in codon 69 of the second exon, resulting in substitution of cysteine for the amino acid glutamine (HBB:c.208G>T). Hb Miguel Servet is a ß-chain globin variant detected by CE-HPLC newborn (BioRad), by CZE and by CE-HPLC-CE Tosoh G8-2 (Horiba), but no by CE-HPLC-CE ß-thalassaemia short programme (BioRad). In fact, for all the techniques that are visualised, what would be detected would be the glutathione variant of Hb (Miguel Servet).

Hb Maruchi [α165 (E14) Ala>Pro; HBA1: c.196G>C]: A new thalassemia hemoglobinopathy related to the alpha1 globin gene.

OBJECTIVE: To describe a new mutation causing alpha thalassemia and its mechanism of action. DESIGN AND METHODS: The propositus was a 37-year-old man who presented maintained microcytosis without iron deficiency. Molecular characterization was undertaken using automatic sequencing after testing negative for the most frequent α-globin mutations by multiplex PCR followed by reverse-hybridization. RESULTS: The mutation is a single base substitution at codon 65 of the α1 globin gene [α65(E14) Ala>Pro; HBA1: c.196G>C] and leads to the substitution of a proline residue in the E helix. The resulting hemoglobin variant has been named Hb Maruchi. This new variant cannot be separated from Hb A by electrophoretic and chromatographic techniques. CONCLUSIONS: The substitution α65(E14) Ala>Pro; HBA1: c.196G>C causes a α-thalassemia silent associated with a very mild phenotype. The diagnosis of this type of mutation is important because it may cause alpha thalassemia if inherited with other clinically relevant HBA1/HBA2 variants.

Sickle cell disease associated with thalassemia; description of a rare mutation.

Sickle cell disease (SCD) is a common hemoglobinopathy, secondary to alterations in the ß globin chain, resulting in an abnormal hemoglobin variant named as hemoglobin S. These disorders show a wide phenotypical spectrum, and the prevalence of these disorders has significantly changed over the time because of multiple factors such as migration. We report a case of a 17-year-old black male, born in Gambia, diagnosed with sickle cell disease, who presented an associated mutation only described in a Japanese family (Oshima et al., 1996).

Hb Moncloa: A new variant of haemoglobin that interferes in the quantification of Hb A1c.

BACKGROUND: In most routine laboratories in Spain, the commonly used method for evaluating HbA1c is ion-exchange high performance liquid chromatography (HPLC). The presence of a variant of Hb may interfere with the quantification of HbA1c. AIMS: Here, we report a novel haemoglobin variant, named Hb Moncloa, which was found during a routine health check at the Hospital Clínico San Carlos in Moncloa (Madrid), Spain. METHODS AND RESULTS: Molecular characterization of ß gene identified a novel transversion mutation [ß80(EF4)Asn>Ser; HBB:c.242A>G]. CONCLUSIONS: When there is no correlation between clinical, glycemic status and glycated haemoglobin of the patient, the chromatogram of HbA1c should be carefully checked to detect the possible presence of variants that cause interference in their measurement.

Phenotype of mutations in the promoter region of the ß-globin gene.

BACKGROUND: ß+-Thalassaemia is characterised by reduced production of ß chains, which decrease can be caused by mutations in the promoter region (CACCC or TATA box), and is classified as mild or silent depending on the extent of ß-globin chain reduction. In both cases, homozygotes or compound heterozygotes for these mutations usually have thalassaemia intermedia. Frequently the diagnosis is made in adulthood or even in old age. A total of 37 alterations in the promoter region have been described so far. AIMS: In this report we describe the mutations found in the promoter region of the ß-globin gene in a single hospital in Madrid. METHODS: Between 1998 and 2015, more than 9000 blood samples were analysed for full blood count and underwent haemoglobin electrophoresis and high performance liquid chromatography. Genetic analysis of the ß and Gγ-globin genes was carried out by automatic sequencing and, in the case of α genes, by multiplex PCR. RESULTS: 35 samples showed mutation in the promoter region of the ß-globin gene, with a total of six different mutations identified: one in the distal CACCC box, two in the proximal CACCC box, three in the ATA box. CONCLUSIONS: Any alterations in the proximal CACCC and TATA boxes lead to a moderate decrease in synthesis of the ß-globin chain, which has been demonstrated in cases of thalassaemia intermedia that have presented in the second decade of life with a moderate clinical course.

Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia.

AIMS: To analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δß-TT), beta thalassaemia trait (ß-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological features of these three types of microcytoses. METHODS: We performed a descriptive study of 428 samples (43 δß-TT, 179 ß-TT and 206 IDA) that were run on Advia 2120 analyser (Siemens). The following reticulocyte indices were assessed: absolute reticulocyte count (ARC), percentage of reticulocytes, mean corpuscular volume of reticulocytes (MCVr), haemoglobin content of reticulocytes (CHr), mean corpuscular haemoglobin concentration of reticulocytes, red blood cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)), MCV ratio and MCHC ratio. Correlation between fetal haemoglobin (HbF) and RDWr in patients with thalassaemia was evaluated. RESULTS: RDWr was significantly higher in δß-TT compared with ß-TT (15.03% vs 13.82%, p<0.001), and so were HDWr (3.65% vs 3.27%, p<0.001), CHr (23.68 vs 22.66 pg, p<0.001) and MCVr (88.3 vs 85.5 fL, p<0.001). A good correlation was observed between HbF and RDWr (r=0.551, p<0.001). IDA subjects have more immature reticulocytes, but less ARC than ß-TT, suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with ß-TT. CONCLUSIONS: Previously described differences between δß-TT, ß-TT and IDA in the corpuscular indices of mature red blood cell can also be observed in reticulocytes. The degree of anisocytosis in reticulocytes from patients with thalassaemia is correlated with HbF.

Laboratory parameters provided by Advia 2120 analyser identify structural haemoglobinopathy carriers and discriminate between Hb S trait and Hb C trait.

BACKGROUND: Haemoglobinopathies have spread owing to human migration, and the number of people needing diagnosis and management of these conditions is increasing. Clinicians need to accurately identify carriers and provide adequate genetic counselling in order to prevent the occurrence of homozygous or compound heterozygous offspring. OBJECTIVES: To identify red blood cell (RBC) laboratory parameters that discriminate between structural haemoglobinopathy carriers and healthy subjects, and to compare RBC laboratory indices between HbAS and HbAC individuals. METHODS: Samples of 500 variant Hb carriers (355 HbAS, 104 HbAC, 19 HbAD, 7 HbAE, 7 HbAO-Arab, 4 α-chain variants and 4 Hb Lepore) and 251 normal controls were run on an Advia 2120 analyser (Siemens). Classic haematological parameters and RBC populations were assessed in all subjects. A multivariable binary logistic regression model was created to predict the probability of a subject carrying any structural haemoglobinopathy. HbAS (n=355, 71%) and HbAC (n=104, 20.8%) subjects were compared. RESULTS: A clinical prediction rule was developed by assigning one point to each of the most efficient variables: mean corpuscular volume (MCV) <88.4 fL, RBC distribution width >13.4%, percentage of microcytic RBCs (%MICRO) >0.7% and the ratio of microcytic RBCs to hypochromic RBCs >0.8. A score of 0, 1, 2, 3 or 4, resulted in a probability of 9.6%, 36.3%, 66.7%, 85.2% or 98.3%, respectively. Among the most frequent variant Hb, HbAC subjects had lower values of parameters related to cell size (MCV, %MICRO) and higher values of parameters related to haemoglobin concentration (MCHC, %HYPER) than HbAS subjects. Coexistence of α-thalassaemia in both HbAS and HbAC individuals resulted in decreased Hb, MCV, MCH and MCHC. CONCLUSIONS: Structural haemoglobinopathy should be investigated in subjects belonging to ethnic groups with high prevalence of variant Hb and with a score of 3 or 4. Erythrocytes of HbAC subjects are smaller and denser than those of HbAS subjects.

[Hb Burgos (α1 CD64(E13)(Asp→Asn)): a new hemoglobin variant detected during follow-up of diabetic patients]. / Hb Burgos (α1 CD64(E13)(Asp→Asn)): una nueva variante de hemoglobina detectada durante la monitorización de pacientes con diabetes.

BACKGROUND AND OBJECTIVE: The glycated hemoglobin (HbA1c) test by high performance liquid chromatography is a useful tool for the follow-up of diabetes mellitus patients. Some structural hemoglobin (Hb) variants are known to cause interference in the analytical measurement of HbA1c. PATIENTS AND METHODS: In this study, it has been characterized a new Hb variant in 4 patients during their regular control of HbA1c. RESULTS: Selective α1 gene sequencing showed a mutation GAC>AAC at codon 64 within exon 2. This produces a change of aspartic acid (Asp) by asparagine (Asn) that does not produce any functional alteration so the resultant molecule behaves as a silent hemoglobinopathy. CONCLUSION: The structural Hb variants can be detected during the analysis of HbA1c and may alter its values. Though rare, this occurrence signals the need to being aware when measuring HbA1c.

δß-Thalassemia trait: how can we discriminate it from ß-thalassemia trait and iron deficiency anemia?

OBJECTIVES: To analyze the differences not only in classic hematologic parameters but also in RBC subpopulations among δß-thalassemia trait (δß-TT), ß-thalassemia trait (ß-TT), and iron deficiency anemia (IDA) and to evaluate the role of fetal hemoglobin (HbF) in elevated RBC distribution width (RDW). METHODS: Samples from 553 patients with microcytosis (74 δß-TT, 272 ß-TT, and 207 IDA) were run on an Advia 2120i analyzer (Siemens Medical Solutions Diagnostics, Tarrytown, NY). Classic hematologic parameters and RBC subpopulations were assessed. The correlation between HbF and RDW in patients with thalassemia (both ß and δß) was evaluated. An independent sample t test was used to compare classic hematologic parameters and RBC subpopulations among ß-TT, IDA, and δß-TT and receiver operating characteristic curves performed in the significant comparisons. RESULTS: RDW was significantly higher in δß-TT compared with ß-TT (18.79% vs 16.04%, P < .001), as was mean corpuscular volume (66.39 vs 64.82 fL, P < .001), mean corpuscular hemoglobin (20.73 vs 20.04 pg, P < .001), and mean corpuscular hemoglobin concentration (31.16 vs 30.66 g/dL, P = .03). Pearson coefficient showed a good correlation between HbF and RDW. The values obtained for all the parameters were significantly different (P < .001) between patients with thalassemia (ß and δß) and IDA. CONCLUSIONS: RDW is the best parameter to discriminate δß-TT from ß-TT. The degree of anisocytosis in patients with ß-TT and δß-TT is strongly correlated with HbF.