bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypomethylation Therapy of Decitabine in Patients with Myelodysplastic Syndromes (MDS) Induces Apoptosis and Reduces Proliferation.
Authors: Jilani I. , Kantarjian H. , Gorre M. , Issa J.-P. , Bennett J. , Raza A. , Ravandi F. , Saba H. , Albitar M.
Published: April 2020

Background: MDS is characterized by cytopenias believed to be the direct result of increased apoptosis of the hematopoietic cells in the bone marrow despite an increase in proliferation. Aberrant methylation resulting in leukemogenesis is frequent in MDS and is a potential target for pharmacologic therapy. Decitabine (Dacogen™; DAC), which has been shown to be effective in treating patients with MDS (Saba et al, Kantarjian et al), indirectly depletes methylcytosine, resulting in hypomethylation of target genes. However, it is not known whether this correction of methylation leads to reducing the apoptosis, allowing normal cells to grow, or by increasing the apoptosis killing off the tumor cells.

Methods: We studied apoptosis and proliferation in patients with MDS treated on a randomized protocol to receive either DAC or best supportive care (SC). Apoptosis as measured by annexin V and mitochondrial potential was studied in various subpopulations of cells using multiparameter flow cytometry. Proliferation was also measured in a similar fashion using BrdU incorporation. Bone marrow (BM) and peripheral blood (PB) samples were collected from patients at baseline and at various times on therapy.

Results: At baseline, the DAC group showed no significant difference in apoptosis, proliferation or percent of CD34+ cells from the SC group in BM (n = 39 and 33 patients, respectively) or PB (n = 51 and 50, respectively). After three months on treatment a significant increase in apoptosis (annexin V) in CD34+ cells was noted in the DAC arm but not in the SC arm (Wilcoxon test, P=0.01). Similar results were obtained when disturbance in mitochondrial potential was measured in the blast population (P=0.02). Interestingly, a similar increase in apoptosis was observed in CD8+ cells in the DAC arm (P=0.02). The increase in apoptosis was augmented with additional courses of DAC therapy. A greater reduction in proliferation (BrdU incorporation) in the CD34+ cells in the decitabine arm compared to SC (P=0.01) was also observed. Those DAC-treated patients who had a higher proliferation rate (BrdU incorporation) at diagnosis were more likely to achieve a CR than those with low level of proliferation (P=0.03).

Conclusions: Decitabine therapy in patients with MDS leads to hypomethylation, but the net effects include high levels of apoptosis and the death of the neoplastic cells and a complimentary reduction of proliferation of the leukemic cells.
Pyrimidine 5′ Nucleotidase Deficiency: Clinical and Molecular Characterization of Two New Italian Patients.
Authors: Fermo Elisa , Marcello Anna , Bianchi Paola , Viglio Simona , Chiarelli Laurent R. , Valentini Giovanna , Zanella Alberto
Published: April 2020

Hereditary pyrimidine 5′ nucleotidase deficiency (P5′N) is the most frequent abnormality of the red cell nucleotide metabolism causing hereditary non-spherocytic hemolytic anemia. The disorder is characterized by mild-to-moderate hemolytic anemia associated with reticulocytosis and hyperbilirubinemia and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. P5′N-1 gene is localized on 7p15-p14; eighteen mutations have been so far identified in 27 unrelated families, 6 of them of Italian origin. The aim of this study is to describe the hematological, biochemical and molecular characteristics of two new Italian patients affected by P5′N deficiency. Case1: The propositus was a 37 yrs old woman of Northern Italian origin affected by chronic hemolytic anemia with Hb levels ranging from 8.2 to 10.5 g/dL. At the time of the study Hb was 8.4 g/dL, reticulocytes 300x109/L, unconjugated bilirubin 3.2 mg/dL. Peripheral blood smear examination showed basophilic stippling and purines/pyrimidines ratio (OD260/280) was decreased (1, ref. values 1.4 – 2.98). P5′N activity, measured by capillary electrophoresis, was undetectable. Molecular analysis of P5′N-1 gene showed the presence of a new homozygous deletion of two bp (ag) at the splice junction between intron 7 and exon 8, which probably results in a splicing alteration and in the absence of a functional protein. Case2: The propositus, a 37 yrs old woman of Northern Italian origin carrying the hemoglobin variant HbD Punjab, had an history of chronic hemolytic anemia since childhood; at the age of 14 yrs splenectomy and colecystectomy were performed. The patient needed blood transfusions because of exacerbation of anemia (Hb 4.7g/dL) during parvovirus B19 infection. Iron status parameters were increased requiring desferrioxamine treatment. At the time of the study Hb was 9.3 g/dL, reticulocytes 752x109/L, unconjugated bilirubin 13.3 mg/dL. Serum ferritin was 1980 mg/mL and transferrin saturation 115%. The propositus was found to be homozygous for Gilbert’s syndrome and heterozygous for mutation H63D of HFE gene. Basophilic stippling (6%) was observed in peripheral blood smear. Pur/pyr ratio was 0.8 and residual P5′N activity was 40% of normal. Complete sequencing of P5′N-1 gene showed the presence of the frameshift mutation ins GG710-711, already described in Italian and Turkish patients, and the new in-frame aminoacidic deletion of Gln 143.
Mutations in the RNA Component of Telomerase, TERC, in Children with Aplastic Anemia and Myelodysplastic Syndrome: An NMDP Study.
Authors: Field Joshua J. , Mason Philip J. , Barnes Yvonne J. , King Allison A. , Bessler Monica , Wilson David B.
Published: April 2020

Mutations in TERC, the RNA component of telomerase, result in autosomal dominant dyskeratosis congenita (DC), a rare bone marrow failure syndrome. DC is clinically heterogeneous and TERC mutations have been detected in a subset of patients previously diagnosed with idiopathic aplastic anemia (AA) and myelodysplastic syndrome (MDS). Unrecognized TERC mutations are clinically relevant as patients with DC respond poorly to immunotherapy and have an increased risk of complications following conventional conditioning for stem cell transplant (SCT). We aimed to determine the frequency of TERC mutations in pediatric patients with AA and MDS who require a SCT. We obtained 315 blood or bone marrow samples from the National Donor Marrow Program Registry from children under age 18 with bone marrow failure who underwent an unrelated stem cell transplant. We screened these samples for mutations in the TERC gene using direct DNA sequencing. To exclude polymorphisms, we also screened 537 racially diverse healthy controls. The study group was composed of patients with MDS (n=151), AA (n=123), and juvenile myelomonocytic leukemia (JMML) (n=41), which may be difficult to distinguish from MDS. The mean age at the time of transplant was 9 years. We found sequence alterations in the promoter region of TERC in 2 patients. A 2 base pair deletion (-240delCT) was identified in a 4 year-old child with MDS and a 1 year-old child with JMML was found to have a point mutation (-99C→G), which was identified previously in an 18 year-old patient with paroxysmal nocturnal hemoglobinuria and is known to affect the Sp1 binding site. The pathogenicity of this mutation is unclear. In summary, our findings suggest that screening for TERC gene mutations is unlikely to diagnose occult DC in children with severe bone marrow failure who require a stem cell transplant but have no clinical features or history to suggest a familial bone marrow failure syndrome.
The “Thomas Plot”: Its Usefulness in the Assessment of Iron Status in Anemia.
Authors: Canals Carmen , Sardà Pilar , Piazuelo Jose María , Remacha Ángel F.
Published: April 2020

A diagnostic plot to assess iron status in anemia of chronic disorders (ACD) has recently been developed (Thomas C, Thomas L. Lab Hematol 2005). The plot is based on the relation between the ratio of the soluble transferrin receptor value to the logarithm of the ferritin value (sTfR/log sFt) and the RET-He parameter -reticulocyte hemoglobin equivalent-. A different cut-off point for sTfR/log sFt is proposed for patients with a CRP ≤5mg/L or with a CRP >5mg/L. The main aim of our study was to validate the usefulness of the “Thomas Plot” in assessing iron status. We also ascertained whether a modification on the sTfR/log sFt cut-off point improved the results. CRP was determined in 306 patients, 198 with iron deficiency (ID) and 108 with ACD. All of them were anemia cases studied in accordance with our standard protocol. RET-He, sTfR and sTfR/log sFt were calculated in all samples. The Thomas Plot was applied to all the cases and the results were compared. A discrepancy was considered relevant when it implied a change in patient treatment. Using logistic regression analysis, we investigated the optimum cut-off point of sTfR/log sFt in our series. Overall, the Thomas Plot correctly classified 223/306 patients (73%). There were clinically relevant discrepancies in 29 patients (9.5%), in 5 patients with CRP ≤5mg/L (5%) and in 24 patients with CRP >5mg/L (12%). It failed to identify ID in 12/198 patients (6%) but classified 17 cases with ACD (16%) as latent ID or IDA. The optimum sTfR/log sFt cut-off point in our series was 2.4 for patients with CRP ≤5mg/L and 2.39 for patients with CRP>5mg/L. As a consequence, we investigated a modified “Thomas Plot” using a sTfR/log sFt cut-off point of 2.4 for all the patients. The modified algorithm correctly classified 77% of the patients. There were clinical relevant discrepancies only in 17 patients (5%). It failed to identify ID in 12/198 patients (6%) and only classified 5 cases with ACD as latent ID or IDA (4.6%). The sensitivity of the Thomas Plot to identify ID was very high but the specificity was lower. A cut-off point of 2.4 for sTfR/log sFt is useful for patients with or without inflammatory traits and yields more accurate results.
A Nationwide Survey of Immunosuppressive Therapy for Idiopathic Acquired Pure Red Cell Aplasia in Japan and a Proposal for First-Line Treatment: Report of the PRCA Collaborative Study Group.
Authors: Hirokawa Makoto , Sawada Ken-ichi , Motegi Mutsuhito , Mamiya Shigeo , Teramura Masanao , Nakao Shinji , Ozawa Keiya , Urabe Akio , Omine Mitsuhiro
Published: April 2020

We have conducted a nationwide survey to define the first-line immunosuppressive treatment for idiopathic acquired pure red cell aplasia (PRCA) in Japanese adult patients diagnosed from January 1990 to December 2004. Questionnaires were sent to forty-seven medical centers and eighty-seven patients with acquired chronic PRCA from forty-seven institutions were registered in this retrospective study. Thirty-four patients (39%) were classified into idiopathic PRCA. Cyclosporine A (CsA), prednisolone (PSL) and cyclophosphamide (CY) were initially given to 20, 10 and 3 patients with idiopathic PRCA, respectively. One patient was treated with anabolic steroid as first-line therapy. The response rates with CsA and PSL were 80 and 60 %, respectively. All patients who had been resistant to PSL and two out of three CY-resistant patients were responsive to CsA. An anabolic steroid resistant patient also responded to CsA. Thus, CsA was used in twenty nine patients as a form of primary or salvage treatment with an 86 % clinical response rate. Nine out of 25 (36%) CsA-responders eventually relapsed and 12.5% of the patients relapsed during therapy. The median remission duration in the CsA responders was seven years. All PSL-responders were predicted to eventually relapse within 2 years, and four out of seven patients on prednisolone relapsed during maintenance therapy. All patients responded to CsA during their second and/or third remission induction with or without PSL. Overall survival was not different between the CsA- and PSL-responders. Although the difference in remission duration between both responders was not statistically significant (p<0.114), which was probably due to the limited number of patients included in the study, CsA may be a best first-line treatment option for Japanese idiopathic acquired PRCA.
Clinical Relevance of Cytogenetics Abnormalities in Adult Patients with Acquired Aplastic Anaemia.
Authors: Gupta Vikas , Brooker Carol , Tooze Jennifer A. , Yi Qi-long , Sage Deborah , Turner David , Kangasabapathy Pamela , Marsh Judith C.W.
Published: April 2020

The clinical relevance of cytogenetics abnormalities in aplastic anaemia (AA) patients at time of diagnosis is unclear. We evaluated the clinical course of 81 AA patients with successful cytogenetics at diagnosis and treated with immunosuppressive therapy (IST) from January 1993 to March 2004. A cytogenetic study was considered to be successful if there were a minimum of 15 evaluable metaphases in the absence of a clonal abnormality. Response to IST, survival and later clonal complications in patients with an abnormal karyotype (n=10) was compared to those with a normal karyotype (n=71).

The cytogenetic abnormalities at diagnosis consisted of trisomy 6 (n=2), trisomy 8 (n=2), trisomy 15 (n=2), monosomy 7 (n=1), add(10) (n=1), t(3;11) (n=1) and t(4;6) (n=1). Four out of five evaluable patients with a trisomy responded to a first or subsequent course of IST. One patient with monosomy 7 achieved a complete response and later developed haemolytic PNH but with no recurrence of the monosomy 7. None of the patients with a non-numerical karyotypic abnormality responded to IST. No significant differences in 4-year event-free survival (EFS) (54% vs. 30%, p=0.15), overall survival (OS) (84% vs. 80%, p=0.33) or later clonal disorders (PNH, MDS and AML) were observed between the patients with a normal karyotype and those with an abnormal karyotype. Advanced age (≥60 years) was the only independent poor prognostic factor for survival in a multivariate analysis.

Among the patients with a normal karyotype (n=71), 6 patients later developed a clonal cytogenetic abnormality with a cumulative risk of 10% at 4 years. These abnormalities were trisomy 15 (n=2), trisomy 6(n=1), monosomy 7 (n=2) and t(13;15) (n=1). None of the three patients who acquired trisomies developed any clinically significant problem, while acquisition of monosomy 7 was associated with a transformation to MDS/AML.

Our data show that AA patients with a trisomy cytogenetic clone at diagnosis show a similar response to IST, evolution to later clonal abnormalities and survival, compared to those AA patients with a normal karyotype.
Results of Immunosuppressive Therapy in Children with Acquired Severe Aplastic Anaemia (SAA). Report Polish Paediatric Leukaemia and Lymphoma Study Group.
Authors: Pawelec K. , Matysiak M. , Niewiadomska E. , Rokicka-Milewska R. , Jackowska T. , Kowalczyk J. , Stefaniak J. , Balwierz W. , Zalecka-Czerpko E. , Chybicka A. , Szmyd K. , Sonta-Jakimczyk D. , Bubala H. , Krauze A. , Wysocki M. , Kurylak A. , Wachowiak J. , Kaczmarek-Karnold M. , Stolarska M. , Karolczyk I. , Krawczuk-Rybak M. , Leszczynska E. , Urasinski T. , Peregud-Pogorzelski J. , Wlazlowski M. , Balcerska A.
Published: April 2020

Introduction: Bone marrow transplantation (BMT) from HLA identical family donors is the treatment chosen for children with (SAA). When no donor is available, combined immunosuppressive therapy (IS) is given.

Material and method: SAA was recognised in 85 children (31 girls, 54 boys) aged 2–17.5 years in ten haematological clinic in Poland between 1993–2003 years. All patients received IS according SAA Working Party of the EBMT protocol: ALG or ATG, CsA, prenisolon and in 79 patients G-CSF or GM-CSF was additionally administered. Haematological response was evaluated on day 180 of therapy.

Results and conclusion: Complete remission (CR) occurred in 43 patients (40.5%), partial remission (PR) on 22 (25.8%), no response (NR) was obtained in 20 children (23.7%). Period of observation was from 12 months to 10.5 years. During this time relapse occurred in 10 patients (8.5%). Patients with NR and relapse were treated second course of IS or BMT from matched bone marrow donors. Unrelated BMT was performed in 11 patients with NR after IS. We observed 17 deaths (10 early during the first 3 months of IS) in all group. At present 54 children (63.5%) is in first remission (CR or PR) with lasts from 12 months to 10 years. During the 10.5 year of observation we notice one case with late clonal complication (PNH). Transformation to neoplasma was not observed in none of our patients after IS.
Phase II Study of Lenalidomide (CC-5013, Revlimid®) for Patients (pts) with Myelofibrosis (MF).
Authors: Cortes Jorge , Thomas Deborah , Verstovsek Srdan , Giles Francis , Beran Miloslav , Koller Charles , Kantarjian Hagop
Published: April 2020

Introduction: Thalidomide has been shown to induce responses in pts with MF but prolonged administration in these pts may be associated with neurotoxicity and other adverse events. Lenalidomide is the lead clinical compound in a new group of drugs called IMiDs®, which have immunomodulatory properties. It affects cytokine expression 200–5000 times more potently than thalidomide but lacks the teratogenecity, sedation and neurotoxicity.

Methods: We thus designed a phase II study for pts with myelofibrosis (primary or associated with myeloproliferative disorders) with a platelet (plt) count of at least 30 x109/L. Pts may have received prior therapies, but were excluded if they had known hypersensitivity to thalidomide. Pts received lenalidomide 10 mg/day orally (5 mg daily for patients with platelet count less than 100,000 at study entry).

Results: We have treated 41 pts. Their median age was 65 (range, 42 to 83). Thirty-six pts (88%) had received prior therapy for MF, including thalidomide 13 (32%), hydroxyurea 12 (29%), interferon 11 (27%) (pegylated 7), anagrelide 10 (24%), and erythropoietin 6 (15%). Baseline hemoglobin was <10g/dl in 19 (46%) (13, 32% transfusion dependent); platelets <100 x109/L in 12 (29%); neutrophils <1.5 x109/L in 2 (5%), and 20/34 (59%) had splenomegaly (median 10cm BCM, range 1 to 30) (7 prior splenectomy). Twenty pts are evaluable for response and toxicity (i.e., received at least 30 days of therapy, unless discontinued because of progression or toxicity). One pt discontinued therapy because of toxicity (grade 3 rash). Responses have been observed in 10 (50%) pts. These include CR in 2 pts (normalization of Hgb and WBC, respectively), PR in 2 pts (improvement in plts and hgb, ± spleen), hematologic improvement in 6 pts (improvement in plts 3, spleen 2, WBC 1). Three of 13 transfusion-dependent pts have become transfusion independent. The median time to response was 9 weeks (range, 2 to 22). Responses have been sustained for a median of 14 weeks (range, 2 to 28 weeks). Therapy has been well tolerated. The more common toxicities were rash in 12 (29%), and pruritus in 8 (20%). Grade ≥ 3 toxicities were thrombocytopenia (n=2), rash (n=1), fatigue (n=1), and neutropenia (n=1). Four (10%) pts have required dose reduction and 1 discontinued therapy because of toxicity (rash).

Conclusion: We conclude that lenalidomide has clinical activity in a subset of patients with myelofibrosis with an acceptable toxicity profile. Studies using lenalidomide in combination with other agents are justified.
Clinical and Laboratory Features of Homozygous Sickle Cell Patients in Tanzania; Malaria, Infections and Cerebral Blood Flow Velocity.
Authors: Makani Julie , Meda Elineema , Rwezaula Stella , Williams Thomas , Thein Swee Lay , Marsh Kevin
Published: April 2020

Background: Significant advances have been made in the management of patients with sickle cell disease (SCD), with resultant increase in survival, most notably in the USA where 1,000 children are born with SCD every year. In contrast, in Africa, where it is estimated that 450,000 children are born with SCD every year, there has been little change in the management and minimal reduction in mortality. There is no reason to doubt that similar interventions and consequent improvements in survival can be achieved in Africa. However, the lack of resources mean that it is vital that proposed interventions are based on a clear definition of the problem.

Aims: The study attempts to define causes of morbidity and mortality in SCD in Tanzania focusing on three major areas where it is important to establish clear answers. The aims of the study are to determine the role of Plasmodium falciparum infection in precipitating crises in SCD patients, to describe the major bacterial pathogens associated with admission in SCD patients and to establish the association of increased cerebral blood flow velocities (CBFv) and cerebrovascular accidents (CVA) in SCD patients in East Africa.

Study design: This is an ongoing hospital based, prospective, descriptive cohort study in Muhimbili national hospital, Dar-es-salaam, Tanzania. Patients are seen in outpatient clinic every three months, if there is any acute event requiring hospital attendance and when admitted to the hospital.

Results: From march 2004, there have been 3,650 visits during which clinical and laboratory data has been collected on 885 (male 51.4%) patients, all homozygous for HbS on cellulose acetate electrophoresis. The mean age of the patients is 10 ± 7.2 years, (range 9 months-54 yrs), with 22.7% being under the age of 5 years and 8.28% above 20 years. The frequency of major clinical events, including fits (5.2%), stroke (1.9%) is reported. The prevalence of malaria parasitaemia in outpatients and during admission was 2.9% and 7.7% respectively. 227 patients have been admitted during this period, presenting with pain (44%), anemia (17%) and fever (15%). Transcranial Doppler ultrasonography was done in 400 patients; with 25% having a time averaged maximal mean velocity of 150 cms/second or more, suggesting stenosis.

Conclusion: This study is the initial step in describing the causes of morbidity in patients with SCD disease in Tanzania, and will form the basis of longitudinal studies that will attempt to guide interventional strategies, target research and provide insight into natural history of SCD in East Africa.
Hemoglobin Ville-Marie: A New β-Globin Variant (del480C) Causing Oxydative Hemolysis.
Authors: Devaux Celine , Chagnon Pierre , Belisle Claude , Soulieres Denis , Busque Lambert
Published: April 2020

Introduction: A 26 year old male patient was investigated at Maisonneuve-Rosemont Hospital (Montreal, Canada) for chronic hemolysis. The patient was known for many years to have chronic hemolysis of unknow etiology and splenomegaly. He had previously undergone cholecystectomy. There was no family history of hemolytic anemia. The investigation revealed a normal automated CBC but the blood smear showed polychromatophilia, basophilic stippling and a few degmacytes (bite cells). The serum chemistry analysis revealed an increased LDH and bilirubin and a decreased haptoglobin level. The search for Heinz body was positive after exposure to acetyl phenylhydrazine. Unstable hemoglobin was demonstrated using the isopropanol test, but the heat instability test was negative. The dosages of erythrocyte enzymes (G-6PD, pyruvate kinase, gluthation reductase and acethylcholinesterase) were normal. Hb HPLC analysis was done using a VARIANT II, Bio-Rad. Results demonstrated the presence of normal HbA and an abnormal Hb migrating as Hb Hope at a level of 24% of total hemoglobin.

Methods and Results: Blood DNA sample was obtained to perform automated sequencing of the β-globin gene (HBB). The HBB gene was sequenced using primers designed to amplify coding sequences of the three exons, splice junctions, introns and the promoter region. Five samples (the patient and four controls) were analysed. Sequence were compared to the cDNA sequence of the HBB gene (NM_000518) and from the genomic sequence of the region (AC104389). In the case of the propositus, a 1 nucleotide deletion was detected at codon 142 (nucleotide 480 of GenBank entry NM_000518) in exon 3, causing a reading frameshift. This deletion (TGCCCTGGCC [C/del] ACAAGTATCA) was never detected before and eliminates the regular stop codon (TAA) at position 147. A new stop codon is therefore produced after the addition of 14 residues, identical to those observed in Hbs Saveme, Trento and TAK, all of which result from a similar reading frameshift secondary to a deletion. However, a threonine residu is present at position 142 in the case of the propositus.

Conclusion: Hemoglobin Ville-Marie is an elongated C-terminal hemoglobin variant causing mild compensated hemolytic anemia with some degree of hemoglobin instability. Others analysis are being performed to better characterise this new hemoglobin variant, the results of which will be presented at the meeting.
DNA Sequence of Hemoglobin Chico.
Authors: Fond Aaron M. , Neville Stephen , Gotchall John , Liu Enli , Ou Ching-Nan , Rognerud Cheryl L. , Mims Martha P.
Published: April 2020

We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient’s hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.
The Phosphatidylserine Receptor-Phosphatidylserine Axis Promotes Sickle Erythrocyte Retention in the Lung Microcirculation.
Authors: Ofori-Acquah Solomon F. , Obiako Boniface , Haynes Johnson
Published: April 2020

Pulmonary vascular occlusion is a major cause of morbidity and mortality in sickle cell disease. Adhesion between sickle erythrocytes, leukocytes and the endothelium are intimately involved in pulmonary vascular occlusion, which contributes to the pathogenesis of the acute chest syndrome. Despite this significance, specific molecules and cognate mechanisms responsible for trapping sickle erythrocytes in the lung microcirculation are poorly understood. In this study, we show for the first time that products released by activated neutrophils act through the phosphatidyserine and phosphatidylserine receptor axis to promote retention of sickle erythrocytes in the lung microcirculation. We demonstrated that co-incubation with autologous activated neutrophils increased 2-fold the proportion of phosphatidylserine exposed sickle erythrocytes. This effect was abrogated by prior treatment of neutrophils with zileuton a pharmacological inhibitor of 5-lipoxygenase. We next determined that primary endothelial cells derived from the pulmonary vasculature expressed the stereospecific receptor for phosphatidylserine. Transcripts for phosphatidylserine receptor (PSR) were 2-fold more abundant in endothelial cells from the pulmonary microvasculature compared to those from the pulmonary artery. To determine the relevance of neutrophil activation in PSR expression, cultures of PMVECs were treated with supernatants of activated neutrophils from patients with sickle cell anemia, and this resulted in 2-fold increase in PSR transcripts. To unravel the pathophysiological relevance of these findings, isolated rat lungs were perfused with 51Cr labeled sickle erythrocytes pre-incubated with annexin V, and retention of sickle erythrocytes quantified by 51Cr radioactivity. The number of sickle erythrocytes trapped in isolated lungs was 2-fold lower compared to the value in control experiments performed with untreated sickle erythrocytes. In agreement with our previous findings activated neutrophils significantly increased sickle erythrocyte retention in the lung (P=0.0004), however cloaking of sickle erythrocytes with annexin V reduced retention by 3-fold. Collectively, these data show that activation of neutrophils increases expression of PSR and its cognate ligand on endothelial cells and sickle erythrocytes respectively, providing the molecular basis for an adhesion complex that increases retention of sickle erythrocytes in the pulmonary endothelium. The PSR-phosphatidylserine adhesion complex therefore offers a novel therapeutic target to reduce pulmonary vascular occlusive events in sickle cell disease.
Survival of Children with Sickle Cell Disease: An Update from the Dallas Newborn Cohort.
Authors: Quinn Charles T. , Lee Nancy J. , Rogers Zora R. , Buchanan George R.
Published: April 2020

The Dallas Newborn Cohort is the largest newborn inception cohort of individuals who have sickle cell disease (SCD), and it has provided modern pediatric SCD survival data (Blood2004;103:4023–7). The Cohort includes subjects who were diagnosed at birth by universal newborn screening and followed at our center up to 18 years of age. All subjects with sickle cell anemia (SS) or sickle-β° thalassemia (Sβ°) were prescribed prophylactic penicillin until 5 years of age. The first report from the Cohort showed an overall survival of 85.6% at 18 years for individuals with SS or Sβ°. The standard error of this estimate was high because only 8 individuals remained at risk at 18 years of age at the time of the analysis, which included follow-up until July 2002. Accrual and follow-up of cohort members has continued. We therefore sought to update the survival estimates through age 18 by including three further years of follow-up. By definition the Dallas Newborn Cohort includes all individuals with SS, Sβ°, sickle-hemoglobin C disease (SC), or sickle-β+-thalassemia (Sβ+) who were born in Texas after November 1, 1983, diagnosed by the newborn screening program of Texas, and seen at least once in our center. New members of the Cohort who came to our center between July 2002 and July 2005 were identified. Follow-up of existing members was updated. All deaths and their causes were determined. Subjects were analyzed in two separate groups because of known clinical similarities: SS/Sβ° and SC/Sβ+. Overall survival was analyzed by the Kaplan-Meier method. Subjects were censored at the time of their last clinical encounter. We identified 115 new subjects, and included 1627 additional patient-years of follow-up. The cohort now includes 826 subjects (SS 503, Sβ° 18, SC 247, Sβ+ 58; male:female 427:399) and it provides 7275 patient-years of follow-up. Mean follow-up was 8.9 years (range 0.9–19.5 years). 62 patients (7.5%) were lost to follow-up. There were 25 deaths in the cohort; none was new and all were previously reported. Of the deaths, 15 were likely related to SCD (5 sepsis, 3 acute chest syndrome, 2 multi-organ failure syndrome, 5 other) and 10 were apparently unrelated to SCD (4 trauma or accidental death, 6 other diseases). There were 22 and 3 deaths in the SS/Sβ° and SC/Sβ+ groups, respectively. All the SC/Sβ+ deaths were apparently unrelated to SCD. Overall survival at 18 years for SS/Sβ° and SC/Sβ+ subjects was 92.4% (standard error [SE] 1.9; 52 at risk) and 98.1% (SE 1.3; 12 at risk), respectively. The overall incidence of death through 18 years of age was 0.46 and 0.12 per 100 patient-years, respectively. In conclusion, this updated survival analysis of the Dallas Newborn Cohort now shows that over 90% of children with SS/Sβ° survive childhood, and nearly 100% of children with SC/Sβ+ become adults.
Hemoglobin H Hydrops Fetalis Associated with Homozygous Hemoglobin Constant Spring. Case Report.
Authors: Gallivan Monica V.E. , Vlastos Emmanuel J. , Hanson Robin D. , Bettinoti Maria P. , Schonberg Steven A. , Pratt Victoria M. , Ou Ching-Nan , Mikula Mario , McGinniss Matthew J. , Sun Weimin , Strom Charles M.
Published: April 2020

While Barts hydrops is predicted when all 4 α globin genes are absent or non functional, hydrops has rarely been described with a hemoglobin H (Hb H) genotype. Hb H disease has a varied hematologic phenotype ranging from asymptomatic to transfusion dependant. The severest clinical expression is non immune hydrops and may result in fetal demise. The commonest form of Hb H disease is deletional-type. This occurs when there is deletion of α2 and α1 genes in cis and deletion of either α2 or α1 in trans. The less frequent type, but clinically more severe, is non-deletional. This is usually due to a mutation in either α2 or α1 in cis and a deletion of both α2 and α1 in trans. There are rare reports of non-deletional Hb H due to homozygous α2 mutations without any α1 deletions or mutations. Heretofore, there have been no reports of hydrops with homozygous hemoglobin Constant Spring (Hb CS). A 31-year-old Laotian woman presented at 22 weeks with polyhydramnios. Besides this, the pregnancy was uneventful for the mother. On ultrasound the fetus had a pericardial effusion and signs of congestive heart failure. The hematological and genotypic parameters of the parents and fetus are outlined in table 1. Intrauterine transfusions were performed at 22 and 29 weeks. A male infant was born vaginally at 36 weeks gestation weighing 2.3 kg. The hemoglobin was 15.9 g/dL and bilirubin was 8.4mg/dL. There was no evidence of maternofetal alloimmunization. G6PD screen was negative. By 4 weeks post-partum, the bilirubin had decreased to 0.9 mg/dL but the hemoglobin had progressively fallen to 6.9 g/dL, requiring transfusion. He had two additional transfusions at 6 and 8 weeks of life but has subsequently maintained a hemoglobin above 10 g/dL and has steadily gained weight. As in this patient, the genotypes of the other reported cases of Hb H hydrops are usually associated with a thalassemia intermedia phenotype. Thus the α globin genotypes alone do not explain why hydrops occurs. This suggests that there may be unknown genetic and/or acquired modifiers contributing to the severity of Hb H disease that results in hydrops as in this fetus with homozygous Hb CS.

[Table 1] Mother Father Fetus at 22 weeks Hemoglobin g/dL 10.0 12.5 5.2 MCV fL 86.3 81.8 143.8 MCH pg 26.5 25.6.8 46.4 RDW % 16.7 13.6 21.4 Reticulocyte % 9.9 ND 19.0 NRBC/100WBC 1 0 4756 Hemoglobin Fractions by HPLC Hb A 73.3% 97.8% 3.4% Hb F 0.8% 0.0% 77.7% Hb A2 0.9% 2.1% 0.0% Barts 0.0% 0.0% 17.7% Hb Constant Spring 2.8% 0.1% 1.2% Hb E 22.2% 0.0% 0.0% αGlobin Gene Dosage Assay by Linear PCR 4 4 4 αGlobin Gene Deletions by PCR - 3.7, - 4.2, _SEA, - 20.5, _MED,_FIL,_THAI None detected None detected None detected α2 Globin Gene Sequencing α-2CS/α-2CS α-2CS/WTα-2CS/α-2CS α1 Globin Gene Sequencing WT/WT WT/WT WT/WT βGlobin Gene Sequencing Heterozygous Hb E WT/WTWT/WT E
Allogeneic Hematopoietic Stem Cell Transplantation for Pesaro IIIβ-Thalassemia Major Using Fludarabine-Based Myeloablative or Non-Myeloablative Conditioning Regimen.
Authors: Zhu Kanger , Gu Jian , Zhang Tao , Zhong Juan
Published: April 2020

Objective : To explore the efficacy of Fludarabine-based myeloablative or non-myeloablative conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for classIII thalassemia major with regard to regimen toxicity, graft rejection, and disease-free survival (DFS).

Methods: From June 2001 to October 2004, 8 patients underwent allo-HSCT in our BMT unit, including 5 male and 3 female, with median age 5 (3 ~ 19) years. Four patients received graft from sibling donor, including cord blood and peripheral blood stem cells, and the remaining 4 patients received graft from unrelated donors, including bone marrow and peripheral blood stem cells. Fludarabine (FDR) was added into the standard BU/CY regimen, consisting of FDR, BU, CY and ATG. Six patients received myeloablative stem cell transplantation and the remaining 2 patients with evidence of organ damage from iron-overload received nonmyeloablative unrelated donor stem cell transplantation. All patients received Cyclosporine A and Methotrexate for GVHD prophylaxis.

Results: Eight patients were successfully engrafted with the median time of absolute neutrophil count (ANC) more than 0.5 ×109 /L was day +13 (+9 ~ +14), and the median time of platelet count more than 20 ×109 /L was day +25 (+8 ~ +39). Two patients died of grade IV aGVHD. The regimen-related toxicity (gradeImucositis, gradeII hemorrhagic cystitis, and gradeIhepatic toxicity) occurred in 3 patients. At a median follow up of 24 (8~48) months, the probability of DFS was 75%, including the two patients given nonmyeloablative stem cell transplantation from unrelated donor.

Conclusion: Fludarabine-based conditioning regimen for allo-HSCT in Pesaro III thalassemia major was well tolerated, without increasing toxicity, and associated with durable engraftment and higher rate of DFS (75%). The successful transplantation from unrelated donors using nonmyeloablative conditioning showed that thalassemia clone can be eradicated by the reduced intensity HSCT, which relies upon immunosuppressive rather than myeloablative conditioning to facilitate engraftment of donor cells, and is a novel approach for the treatment of the patients with evidence of organ damage from iron-overload.
Multi-Centre Validation of the Cardiovascular Magnetic Resonance Multi Breath-Hold T2* Technique for Myocardial Iron Quantification in Thalassaemia Major.
Authors: Tanner Mark A. , He Taigang , Westwood Mark A. , Galanello Renzo , Matta Gildo , Cohen Alan R. , Fogel Mark , Piga Antonio , Forni Gianluca , Economides Charalambos , Firmin David N. , Dudley Pennell J.
Published: April 2020

Introduction: Beta thalassaemia major (TM) is a hereditary anaemia affecting 60 000 births worldwide each year. Survival is dependent upon lifelong blood transfusions resulting in iron overload. Cardiac siderosis can result in a cardiomyopathy which is the leading cause of death in TM. The validated cardiac magnetic resonance (CMR) T2* technique allows non-invasive and reproducible quantification of myocardial iron. Assessment of myocardial iron loading is essential in determining appropriate chelation therapy. This technique has the potential to become the new gold standard in the assessment of cardiac siderosis but is currently available at only a few sites worldwide. For maximal healthcare benefit its inter-scanner reproducibility must be demonstrated before being widely disseminated.

Objective: To demonstrate that CMR T2* quantification of myocardial iron can be reproducibly transferred to MR scanners of different manufacturers in different centres. This project was sponsored by the Thalassemia International Federation.

Methods: The previously described multi breath-hold gradient echo T2* technique was installed on MR scanners (all 1.5Tesla) at 6 different centres. Scanner details were as follows: Site 1, Phillips Intera (Turin, Italy), Site 2, Siemens Sonata (Philadelphia, USA), Site 3 GE Signa (Limassol, Cyprus) Site 4, Phillips Intera (Nicosia, Cyprus), Site 5, GE Signa (Cagliari, Sardinia) and Philips Intera (Genova, Italy). 34 patients (mean age 30+/− 5.7years) were scanned in total. All patients were subsequently re-scanned at the standardization centre in London, UK (Siemens Sonata, 1.5T) within 31 days of their original scan.

Results: The T2* sequence was successfully installed on all 6 scanners. Myocardial T2* values ranged from 3.6ms to 51ms (14.2 +/− 11ms). The overall inter-scanner reproducibility (SD/mean) was 5.3% (figure 1). The mean difference between T2* values at the standardization centre and visited sites was 0.32ms.

Conclusion: We have demonstrated that the multi breath-hold T2* technique for the quantification of myocardial iron can be reproducibly transferred to 1.5T MR scanners at different sites and of different manufacturers. There is therefore real potential to roll out this technique worldwide to facilitate maximal healthcare impact in the management of patients with iron overload conditions such as thalassaemia.

Figure Figure
Serum Ferritin and Liver Iron Concentration in Patients with Iron Overload.
Authors: Pakbaz Zahra , Fischer Roland , Fung Ellen , Nielsen Peter , Engelhardt Rainer , Harmatz Paul , Vichinsky Elliott
Published: April 2020

Despite its limitations, serum ferritin (SF) is commonly used to monitor chelation therapy in primary and secondary hemochromatosis. To better predict liver iron concentration (LIC), we prospectively investigated the relationship between SF and LIC in a total of 421 patients with primary (HFE-1 associated, n=241) or secondary hemochromatosis (n=180), consisting of chronically transfused thalassemia (Tx-Thal: n=89) or sickle cell disease patients (Tx-SCD: n=45) and transfusion independent thalassemia patients (nTx-Thal: n=26). In all patients, LIC was measured by SQUID biosusceptometry. SF correlated with LIC (RS = 0.51–0.83, p < 0.001) but was a poor predictor for LIC. SF was significantly lower (p < 0.001) in nTx-Thal and HFE-1 patients despite similar LIC (421 – 5524 μg/g-liver) and it was higher in Tx-SCD compared to Tx-Thal (p = 0.03). In order to improve the value of SF, we calculated the SF/LIC ratio for each group. SF/LIC remained stable over time in patients whose therapy did not change. In iron loaded patients without blood transfusion therapy (nTx-Thal and HFE-1), the median SF/LIC ratio was significant lower (0.32 and 0.43) as compared to transfused patients (Tx-Thal: 0.87, HCV-Thal: 0.99, Tx-SCD: 1.2), probably, indicating differences in the secretion of ferritin into plasma. We conclude that SF alone can mislead the iron unloading therapy as it underestimates LIC in nTx-Thal patients and overestimates LIC in Tx-SCD patients. Once the initial LIC value is obtained and the individual SF/LIC ratio is determined in a patient, the ratio together with SF may be more useful than SF alone to monitor iron overload and predict LIC.
Pregnancy in Thalassemia Patients and Their Partners in a Canadian Population.
Authors: Apostoli Anthony , Cipolla Amanda , Kinghorn Maureen , Kaufman Jacob , Muraca Giulia , Olivieri Nancy
Published: April 2020

Although successful pregnancy is not an uncommon outcome in patients with homozygous beta thalassemia over the last 20 years, most of these have been reported in case reports and small series, while many publications do not appear to distinguish between pregnancies in thalassemia intermedia and those in thalassemia major. For example, of the total of 150 pregnancies in women with thalassemia reported in the literature from 1984 to 2004, 74 pregnancies appear clearly to be documented in patients with thalassemia major, while in the other 76, a reportedly late onset of transfusions suggests a milder phenotype of thalassemia intermedia. We examined the records of all thalassemia patients managed at the Toronto General Hospital, Canada (n = 82 patients with thalassemia major; 23 patients with thalassemia intermedia; 7 patients with hemoglobin E thalassemia = 112 patients over 18 years, of whom 54 are female) to determine the incidence and complications of pregnancy in our population. A surprisingly high number (17) and proportion (31%) of all female thalassemia patients over age 18 years have attempted to become pregnant. A total of 14 (82% of those who attempted pregnancy) of patients (9 thalassemia major; 4 thalassemia intermedia; 1 Hemoglobin E thalassemia) successfully conceived 26 times. The maternal age at first attempt to become pregnant was 29.6 ± 4.9 (range 18–37) years; average age at delivery was 27.2 ± 4.1 (range 19–33) years. Of the 26 conceptions, 20 were carried to term; 19 live births resulted. Six abortions and one fetal death occurred. Deferoxamine was avoided in all patients during pregnancy; control of body iron was followed using hepatic iron concentration. Pre-pregnancy hepatic iron concentration, obtained 2.8 ± 1.8 (range 1–7) years prior to conception, was 9.3 ± 7.9 (range 0.9–31) mg/gram dry weight; post partum hepatic iron concentration, obtained 3 ± 3.2 (range 0.2–10) years following parturition, was 17.4 ± 9.6 (range 1.25–32.5) mg/gram dry weight. There were one reported significant worsening in cardiac function during pregnancy; diabetes (present in one woman prior to pregnancy) developed in another woman during pregnancy. Other complications occurring or worsening during pregnancy included immune thrombocytopenic purpura and post- transfusional thrombocytopenia. In 13 of the 58 men in this population, 13 (8 with thalassemia major; 4 with thalassemia intermedia and one with Hemoglobin E thalassemia) fathered 20 children at 33.5 ± 4.8 (range 24–44) years. These data report the largest North American series of pregnancy in thalassemics and their partners and while raising issues of the safety of pregnancy in thalassemic women offer encouragement to those patients managed with adequate control of body iron burden.
Application of the Luminex Platform for Multiplexed Genotyping of Non-Deletional Alpha Thalassemia Mutations.
Authors: Aslanian Sharon , Mahin Azimi , Hoppe Carolyn
Published: April 2020

Objectives: Certain point mutations in the alpha globin gene may lead to unstable hemoglobins such as Hb Constant-Spring and Hb Quong-Sze. These alpha chain variants, when inherited with deletional α-thalassemia mutations cause more severe forms of HbH disease. To evaluate the utility of the Luminex xMAP platform for rapid screening of non-deletional α-thalassemia mutations, we developed a multiplexed genotyping assay to detect six mutations in the α2-globin gene. that are clinically significant or, when coinherited with α-thalassemia-1, result in HbH disease.

Methods: A panel of positive controls with known heterozygosity for Hb Constant-Spring, Hb Quong-Sze, Hb J-Toronto, Hb Hasharon, Hb G-Philadelphia and Hb G-Norfolk, was obtained from anonymized whole blood or newborn dried blood spots. The α2 gene was amplified using nested PCR and single base chain extension (SBCE) was carried out using the PCR products, capture-tagged primers, thermosequenase, and one of four biotinylated ddNTPs. Biotinylated amplicons were hybridized to fluorescent microspheres containing allele-specific capture probes and analyzed by flow cytometry on the Luminex100.

Results: Median flourescent intensity (MFI) values uniformly discriminated between wild type and mutant alleles in three independent experiments. Mean background-subtracted MFIs ranged from 255 to 1000 for all positive alleles and genotypes were correctly identified in all of the samples tested.

Conclusions: As HbH disease is becoming more prevalent in the U.S., early detection of this disorder using accurate, affordable diagnostic methods will permit timely delivery of health care and prevention of severe clinical manifestations of disease. As the severity of many of these disorders has not been well-characterized, an understanding the phenotypic expression of these hemoglobin disorders is essential to providing adequate counseling and treatment options.