Emília Costa

Two novel mutations in the tmprss6 gene associated with iron‐refractory iron‐deficiency anaemia (irida) and partial expression in the heterozygous form

Arceci, R.J., Sande, J., Lange, B., Shannon, K., Franklin, J., Hutchinson, R., Vik, T.A., Flowers, D., Aplenc, R., Berger, M.S., Sherman, M. L., Smith, F.O., Bernstein, I. & Sievers, E.L. (2005) Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33+ acute myeloid leukemia. Blood, 106, 1183–1188. Bornhauser, M., Illmer, T., Oelschlaegel, U., Schetelig, J., Ordemann, R., Schaich, M., Hanel, M., Schuler, U., Thiede, C., Kiani, A., Platzbecker, U. & Ehninger, G. (2008) Gemtuzumab ozogamicin as part of reducedintensity conditioning for allogeneic hematopoietic cell transplantation in patients with relapsed acute myeloid leukemia. Clinical Cancer Research, 14, 5585–5593. Burnett, A.K., Hills, R.K., Milligan, D., Kjeldsen, L., Kell, J., Russell, N.H., Yin, J.A., Hunter, A., Goldstone, A.H. & Wheatley, K. (2011) Identification of patients with acute myeloblastic leukemia who benefit from the addition of gemtuzumab ozogamicin: results of the MRC AML15 trial. Journal of Clinical Oncology, 29, 369–377. Cooper, T.M., Franklin, J., Gerbing, R.B., Alonzo, T.A., Hurwitz, C., Raimondi, S.C., Hirsch, B., Smith, F.O., Mathew, P., Arceci, R.J., Feusner, J., Iannone, R., Lavey, R.S., Meshinchi, S. & Gamis, A. (2012) AAML03P1, a pilot study of the safety of gemtuzumab ozogamicin in combination with chemotherapy for newly diagnosed childhood acute myeloid leukemia: a report from the Children’s Oncology Group. Cancer, 118, 761–769. de Lima, M., Champlin, R.E., Thall, P.F., Wang, X., Martin, T.G., 3rd, Cook, J.D., McCormick, G., Qazilbash, M., Kebriaei, P., Couriel, D., Shpall, E.J., Khouri, I., Anderlini, P., Hosing, C., Chan, K.W., Andersson, B.S., Patah, P.A., Caldera, Z., Jabbour, E. & Giralt, S. (2008) Phase I/ II study of gemtuzumab ozogamicin added to fludarabine, melphalan and allogeneic hematopoietic stem cell transplantation for high-risk CD33 positive myeloid leukemias and myelodysplastic syndrome. Leukemia, 22, 258–264. Petersdorf, S., Kopecky, K., Stuart, R.K., Larson, R. A., Nevill, T.J., Stenke, L., Slovak, M.L., Tallman, M.S., Willman, C.L., Erba, H. & Appelbaum, F.R. (2009) Preliminary Results of Southwest Oncology Group Study S0106: An International Intergroup Phase 3 Randomized Trial Comparing the Addition of Gemtuzumab Ozogamicin to Standard Induction Therapy Versus Standard Induction Therapy Followed by a Second Randomization to Post-Consolidation Gemtuzumab Ozogamicin Versus No Additional Therapy for Previously Untreated Acute Myeloid Leukemia. Blood (ASH Annual Meeting Abstracts), 114, 790. Sakaguchi, H., Watanabe, N., Muramatsu, H., Doisaki, S., Yoshida, N., Matsumoto, K. & Kato, K. (2010) Danaparoid as the prophylaxis for hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in childhood hematological malignancy. Pediatric Blood & Cancer, 55, 1118–1125. Sibson, K., Steward, C., Moppett, J., Cornish, J. & Goulden, N. (2009) Dismal long-term prognosis for children with refractory acute myeloid leukaemia treated with gemtuzumab ozogamicin and stem cell transplantation: where now? British Journal of Haematology, 146, 342–344. Wadleigh, M., Richardson, P.G., Zahrieh, D., Lee, S.J., Cutler, C., Ho, V., Alyea, E.P., Antin, J.H., Stone, R.M., Soiffer, R.J. & DeAngelo, D.J. (2003) Prior gemtuzumab ozogamicin exposure significantly increases the risk of veno-occlusive disease in patients who undergo myeloablative allogeneic stem cell transplantation. Blood, 102, 1578–1582.

Assessment of markers of bone formation under controlled environmental factors and their correlation with serum minerals in adult sheep as a model for orthopaedic research

Summary Eighteen healthy skeletally mature (3 years old) ewes, with an average weight of 45 kg, of the Portuguese Churra da Terra Quente breed were used to evaluate the normal values of total and bone-specific isoform of alkaline phosphatase serum activities (ALP and BALP, respectively) and serum osteocalcin (OC) and their correlation with the serum minerals - calcium (Ca), phosphorus (P), magnesium (Mg) and ionized calcium (Ca2+). The sheep were maintained under controlled environmental conditions (constant diurnal photoperiod cycle and identical husbandry and feeding) for six weeks before the collection of the blood samples. The measurement of the total ALP and serum minerals was performed with automated biochemistry analysers using the BioMérieux® kits, the serum electrolyte Ca2+ Diametrics Medical, Inc® specific cassettes and the BALP and OC METRATM kits from QUIDEL® Corporation. The mean ± standard deviation values obtained were: total ALP 90.17 ± 85.72 U/L, BALP 15.0 ± 5.44 U/L, ratio BALP/ total ALP 29.28 ± 24.22, OC 13.02 ± 1.87 ng/mL, Ca 2.57 ± 0.37 mmol/L, P 2.13 ± 0.42 mmol/L, Mg 1.04 ± 0.13 mmol/L, Ca2+ 1.29 ± 0.04 mmol/L. Significant correlations were observed between the total ALP and Ca (r = 0.5939; P = 0.05) and OC and Ca (r = 0.5706; P = 0.05). Reference to the serum values of bone turnover parameters in sheep could be of great value in research and could provide complementary non-invasive information on the bone healing process, particularly with regard to obtaining an early prognosis of fracture healing.

Epigenetic Alterations in Fanconi Anaemia: Role in Pathophysiology and Therapeutic Potential.

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3Tβ and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population.

Refractory iron-deficiency anemia and autoimmune atrophic gastritis in pediatric age group: analysis of 8 clinical cases.

Introduction: Refractory iron-deficiency anemia with no obvious etiology in pediatric age can be a puzzling problem. Screening of iron malabsorption conditions, including autoimmune atrophic gastritis (AAG), is emerging as a priority in the investigational procedure. Materials and Methods: Retrospective analysis of clinical process of children/adolescents with the diagnosis of AAG. Results: Eight patients (aged between 4.7 and 18 years old) were identified. The diagnosis was triggered on the basis of high serum gastrin levels and strong positivity of antiparietal cell antibodies. Upper endoscopy and biopsy revealed atrophic gastritis in all patients, with 2 of them with intestinal metaplasia. Four patients presented with Helicobacter pylori infection object of eradication therapy. After a medium follow-up of 36.6 months, antiparietal cell antibodies and hypergastrinemia did not show evidence of regression. Of the 3 patients who underwent endoscopic reevaluation, a similar anatomo-pathologic pattern was observed in 2 and intestinal metaplasia in 1 patient. Normalization of hematological parameters was achieved, using alternative iron formulas. Conclusions: AAG must be recognized as a pathology affecting pediatric patients. Gastric autoimmune lesion is a chronic process with potential evolution to malignancy. Management guidelines in childhood are not available. Their elaboration is important considering an important risk factor in these age group: a long life expectancy.

Iron deficiency anemia due to lymphocytic gastritis with Helicobacter pylori infection in childhood: case report.

Lymphocytic gastritis (LG) is a chronic inflammatory process of poorly understood pathogenesis. We report the case of a 12-year-old premenstrual girl with refractory iron deficiency anemia in which the oral iron absorption challenge suggested iron malabsorption. Laboratory studies ruled out celiac disease and autoimmune gastritis, and carbon-13 urea breath test for Helicobacter pylori was also negative. Upper endoscopy with gastric body and antral mucosa biopsies revealed a LG with focal intestinal metaplasia and H. pylori infection. H. pylori eradication was undertaken with success and 3 months later her hematologic parameters normalized. Histologic reevaluation showed disappearance of LG. This case shows that investigation of malabsorption disease in the presence of refractory iron deficiency anemia can lead to the diagnosis of important gastric diseases, even in the absence of gastrointestinal symptoms. This nonceliac child was diagnosed with a severe histopathologic pattern of LG, with potential risk of malignant transformation, which was completely reverted with adequate H. pylori eradication treatment.

Erythrophagocytosis by neutrophils in paroxysmal cold haemoglobinuria

A previous healthy 1-yr-old girl presented with a 3-d history of asthenia, fatigue, skin pallor, jaundice and dark urine. Laboratory tests revealed severe anaemia (Hb – 4.3 g/dL) with mild reticulocytosis (7.3% – 113 9 10/L), elevated levels of serum LDH (2734 U/L), total bilirubin (3.53 mg/dL) and aspartate aminotransferase (118 U/L), but normal value of alanine aminotransferase (19 U/L); the direct Coombs’ test was positive using poli-specific serum and had specificity for C3d. A peripheral blood smear showed polymorphonuclear neutrophils that had phagocytized erythrocytes (erythrophagocytosis). The diagnosis of paroxysmal cold haemoglobinuria was suspected based on the evidence of intravascular haemolysis and C3d positivity of the Coombs’ test. The positivity of Donath-Landsteiner antibody test confirmed the diagnosis. On the emergency room, the child was given a blood transfusion and after that was kept on a warm environment. She had a progressive and sustained normalization of her Hb values and had her symptoms improved. Paroxysmal cold haemoglobinuria is a rare disorder that characteristically has an acute and self-limited nature. As a consequence, this may result on an underdiagnosis of this pathology. Moreover and unlike the presented case, direct Coombs’ test can be negative, which may also mislead the diagnosis. Erythrophagocytosis by granulocytes in the peripheral blood is an unusual phenomenon. Generally, it is associated with haemato-oncology diseases or with some forms of haemolytic anaemia’s, in particular paroxysmal cold haemoglobinuria. Therefore, the observation of erythrophagocytosis by polymorphonuclear neutrophils on the blood smear can increase the diagnostic accuracy.

Infantile pyknocytosis: an under-recognized form of neonatal hemolytic anemia?

Infantile pyknocytosis (IP) is an under-recognized hematological entity of newborns that can cause a severe neonatal hemolytic anemia. A careful, prompt, and accurate peripheral blood smear examination is essential to establish the diagnosis. Here we describe the clinical features and histological parameters of 1 case of IP. Spontaneous resolution usually occurs by 4 to 6 months, but red blood cell transfusion may be needed if the anemia is severe.

Severe anaemia with reticulocytopenia: When watchful waiting is the best attitude: Severe anaemia with reticulocytopenia

A previously healthy 3-year-old boy, with physical growth and psychomotor development normal for age, was referred to our institution due to severe anaemia. There was no family history of haematological disorders. He presented with a history of progressive pallor and lethargy of a duration of 1 week. Two weeks before presentation, he suffered from febrile upper respiratory tract infection. There was no history of toxin exposure or acute blood loss. There was no previous complete blood count. Upon admission, he was pale, afebrile and tachycardic (138 bpm) with normal blood pressure and had a grade II/VI systolic flow murmur. No rash, lymphadenopathy or hepatosplenomegaly was noted, and his physical examination was otherwise unremarkable. A complete blood count demonstrated severe normocytic normochromic anaemia – haemoglobin (Hb) 3.6 g/dL, mean corpuscular volume (MCV) 77.9 fL, mean corpuscular haemoglobin 24.8 pg, with reticulocytopenia (28 300/mm). White blood cell (WBC) and platelet counts were normal (WBC 7520/mm, neutrophils 3400/ mm, lymphocytes 3360/mm and platelets 559 000/mm). The peripheral blood smear was non-specific. Direct antiglobulin test was negative, and the biochemical markers of haemolysis were absent (total bilirubin 0.34 mg/dL, lactate dehydrogenase 228 U/L and aspartate transaminase 25 U/L). Fetal Hb level (HbF) was normal (0.4%), and serum erythropoietin was increased up to 3250 U/L (<29 U/L). Serum ferritin value was normal (52 ng/mL). No laboratory evidence of occult blood loss in the stool was found. Serology for parvovirus B19, cytomegalovirus (CMV) and Epstein– Barr virus (EBV) showed no evidence of acute infection. He received a red blood cell transfusion on admission (day 1), increasing the Hb concentration to 6.4 g/dL on day 2. On day 4, absolute reticulocyte count increased significantly (245 000/ mm), with the improvement of Hb (7.1 g/dL). He was discharged on day 6 with Hb of 7.8 g/dL and reticulocyte count of 618 300/mm. Spontaneous recovery was observed 3 weeks later, with normalisation of haematological findings (Hb 12 g/dL, reticulocyte count 56 900/mm). One year later, he maintained stable haematological parameters. In this case, the findings of severe normocytic normochromic anaemia with low reticulocyte count for the degree of anaemia, otherwise normal blood count and no morphological abnormalities in peripheral blood smear, together with normal HbF, high erythropoietin and negative serology for parvovirus B19, in a child in the characteristic age range allowed the suggestion of a diagnosis of transient erythroblastopenia of childhood (TEC). Subsequent normalisation of haematological findings confirmed this diagnosis.

Marked acanthocytosis in the setting of Klippel-Trenaunay syndrome: A case report

KlippelTrenaunay syndrome (KTS) is a rare congenital disorder defined by the classic triad of capillary malformations, venous malformations with or without lymphatic malformations and limb overgrowth.1-3 Although the diagnosis of KTS is mostly based upon the evidence of the key clinical features, laboratory and imaging studies may be crucial for further characterization of the extent of disease and the presence of complications.3 The management of individuals with KTS should be individualized and involve a multidisciplinary medical and surgical approach. Acanthocytes are hyperchromic red blood cells (RBC) that exhibit 220 irregularly spaced thornlike spicules of variable length, thickness and shape, frequently with drumstick ends.4-6 Since the integrity of RBC membrane, which consists of a lipid bilayer, a set of transmembrane proteins and a cytoskeleton, depends on proteinprotein and proteinlipid molecular interactions,7 the mechanism of acanthocyte formation is probably linked to abnormalities in the protein or lipid composition of this structure.4-6 Acanthocytes are rarely seen in otherwise normal peripheral blood films but, in those circumstances, they correspond to older senescent RBC. The causes of acanthocytosis can be either inherited or acquired. Slight (1+) to moderate (2+) acanthocytosis can be found not only in the newborn and the elderly, but also in hypothyroidism, postsplenectomy, chronic kidney disease, enzymatic deficiencies (particularly pyruvate kinase deficiency), anorexia nervosa/chronic starvation and vitamin E deficiency.4-6 Marked (3+) acanthocytosis can be found in hereditary abetalipoproteinaemia and hypobetalipoproteinaemia, degenerative neurological disease with normal lipoprotein levels (particularly McLeod red cell phenotype), severe endstage chronic liver disease and myelodysplastic syndromes.4-6 The aim of this short report is to describe the case of a Portuguese young man with KTS and marked (3+) acanthocytosis with no other aetiological cause.

QUANDO O ACHADO SE REVELA A CAUSA

Introducao: A neutropenia define-se como uma contagem do numero de neutrofilos no sangue inferior a dois desvios-padrao da media para a idade. No recem-nascido (RN) neutropenico, a neutropenia aloimune devera ser considerada. Esta deve-se a producao de anticorpos maternos em resposta a antigenios de origem paterna presentes nos neutrofilos fetais. Tratam-se de anticorpos anti-neutrofilos (AAN) tipo IgG, que atravessam a pla- centa provocando a destruicao dos neutrofilos fetais. Caso Clinico: RN, sexo feminino, 10 dias, levada ao Ser- vico de Urgencia por sinais inflamatorios do coto umbilical. No estudo analitico apresentava neutropenia grave (490/uL), pelo que iniciou antibioticoterapia endovenosa para tratamento de onfalite. Por ausencia de resposta ao tratamento inicial e manu- tencao da neutropenia, efetuou-se pesquisa de AAN na RN, por citometria de fluxo, que foi inconclusiva pela gravidade da neu- tropenia. Realizou-se ainda pesquisa de AAN tipo IgG no soro materno contra os neutrofilos paternos, que se revelou positiva. Alem disso, fez-se a genotipagem do antigenio de neutrofilos hu- mano (HNA-1) nos pais, que mostrou: mae HNA-1a/HNA-1b e pai HNA-1a/HNA-1b/HNA-1c. Em face dos resultados, a neutro- penia aloimune e muito provavel, devendo ser confirmada a pos- teriori por genotipagem HNA-1 da RN. Apos alteracao da anti- bioticoterapia, verificou-se resolucao da onfalite, tendo tido alta sem alteracoes ao exame objectivo e com indicacao de realizar controlos analiticos seriados e medidas de isolamento. Durante o seguimento nao apresentou novas intercorrencias infeciosas, verificando-se normalizacao do valor serico de neutrofilos a partir dos 3 meses de idade. Comentarios: A neutropenia aloimune e uma patologia rara e, perante uma neutropenia no RN, a pesquisa de AAN deve- ra ser tida em conta. O caso clinico descreve uma neutropenia aloimune por AAN contra HNA-1c, o qual, segundo a literatura, esta raramente envolvido na doenca. Os autores querem realcar o papel dos estudos imunologicos e da biologia molecular na confirmacao diagnostica e na probabilidade de ocorrencia numa futura gestacao para atuacao precoce.