Esmeralda Cleto

Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis.

We studied 82 Portuguese individuals, 57 with hereditary spherocytosis (HS) and 25 unaffected controls. We performed standardized diagnosis tests, including electrophoretic membrane protein analysis to identify and quantify protein deficiencies underlying HS. Membrane bound hemoglobin (MBH) and band 3 profiles were determined as oxidative stress and aging markers. A protein of about 22 kDa, present in 21 of 57 HS patients, but not in controls, was identified as peroxiredoxin 2 (Prx2), by mass-spectroscopy and by immunoblotting. Human erythrocyte Prx2 is a peroxiredoxin with thiol-specific antioxidant activity. The presence of Prx2 in erythrocyte membranes was linked to higher levels of oxidative stress, as reflected by significantly increased MBH in those HS patients. No relation with HS clinical severity was observed and Prx2 was detected in all types of membrane protein abnormalities. Prx2 membrane linkage is associated with a higher oxidative stress susceptibility of HS erythrocytes.

Erythrocyte membrane protein destabilization versus clinical outcome in 160 Portuguese Hereditary Spherocytosis patients.

Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects – spectrin, ankyrin, band 3 or protein 4·2 – that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane‐linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied – 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde‐3‐phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.

Complementary markers for the clinical severity classification of hereditary spherocytosis in unsplenectomized patients

Hereditary spherocytosis (HS) is usually classified as mild, moderate or severe using conventional features, namely, hemoglobin (Hb) concentration, reticulocyte count and bilirubin levels, which do not always contribute to an adequate clinical classification. The aim of our study was to establish the importance of some laboratory routine parameters, as markers of HS clinical outcome, by studying a control group (n=26) and unsplenectomized HS patients (n=82) presenting mild, moderate or severe HS. We performed a basic hematologic study and evaluated the reticulocyte count, bilirubin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) levels; the osmotic fragility (OFT) and criohemolysis tests (CHT); the ratios Hb/MCHC (mean cell hemoglobin concentration), Hb/RDW (red cell distribution width) and MCHC/RDW, were calculated. Hb changed significantly in accordance with HS severity, but not reticulocytes or bilirubin. We found that MCHC, RDW, EPO, sTfR, OFT, CHT and the calculated ratios were significantly changed in patients, and, therefore, were valuable as complementary diagnostic tools for HS. Moreover, RDW, Hb/MCHC, Hb/RDW and MCHC/RDW changed significantly with worsening of HS; thus, they are also good markers for the clinical outcome of HS. In conclusion, we propose the use of these routine parameters as useful to complement the analysis of HS severity.

Hereditary spherocytosis and the (TA)nTAA polymorphism of UGT1A1 gene promoter region--a comparison of the bilirubin plasmatic levels in the different clinical forms.

Hereditary spherocytosis (HS) is the most common nonimmune hemolytic anemia in individuals of northern European ancestry, affecting 1 in 2000 [1]. HS is classified as mild, moderate or severe according to the severity of the symptoms, family history and analytical presentation—hemoglobin (Hb) concentration, reticulocyte count and serum bilirubin levels [1]. When performing the classification of HS in our patients during the last years, we observed that bilirubin levels were sometimes inconsistent with the other parameters defining the severity of this anemia. This was particularly evident in some mild HS cases that presented unexpectedly high bilirubin plasma concentration, which was not in agreement with the other analytical parameters. A potential cause for this discrepancy could be the co-inheritance of HS and

Erythropoiesis versus inflammation in Hereditary Spherocytosis clinical outcome

OBJECTIVES This study aimed to evaluate the relationship between erythropoiesis and inflammation, in Hereditary Spherocytosis (HS) clinical outcome. DESIGN AND METHODS We studied 26 controls and 82 HS patients presenting mild (n = 49) and severer (n = 33) HS forms. We evaluated plasma levels of EPO, sTfR, ferritin, iron, folic acid, vitamin B12, TNF-α, IFN-γ, elastase and lactoferrin; leukocyte and reticulocyte counts and RPI were determined. RESULTS All HS patients showed significantly higher EPO, sTfR, reticulocytes and RPI but only mild HS presented normal hemoglobin levels; the positive significant correlations between EPO and sTfR, reticulocytes and RPI observed in mild HS were not observed in severer HS patients. HS patients presented with higher levels of neutrophils, TNF-α, IFN-γ, elastase, lactoferrin and ferritin. CONCLUSIONS Our data show HS as a disease linked to enhanced erythropoiesis that is disturbed in the more severe forms, to which inflammation may contribute, at least in part.

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.

ESPLENOMEGALIA PERSISTENTE ASSINTOMÁTICA – UM ACHADO NÃO TRANQUILIZADOR

Introducao: A esplenomegalia em idade pediatrica esta frequentemente associada a infeccoes viricas, tendo um caracter transitorio. Em caso de esplenomegalia persistente, importa excluir doenca hematologica, congestiva, infiltrativa, de sobrecarga ou reumatologica. Caso Clinico: Adolescente 14 anos, sexo feminino, referenciada a consulta por hepatoesplenomegalia. Segunda filha de pais nao consanguineos, antecedentes pre, peri e neonatais irrelevantes com evolucao estaturoponderal e desenvolvimento psicomotor adequados. Aos 3 anos de idade, no decurso de um quadro clinico compativel com mononucleose infeciosa, foi objetivada esplenomegalia de novo. Manteve controlo ecografico regular, com persistencia da esplenomegalia, tendo aos 5 anos sido consta- tada hepatoesplenomegalia homogenea e aos 14 anos, imagem ecografica compativel com hemangioma hepatico. Nesta altura efectuou pela primeira vez hemograma que demonstrou trombocitopenia. Esta citopenia associada a hepatoesplenomegalia motivou a referenciacao a Hematologia Pediatrica. Referia equimoses faceis em contexto de trauma e gengivorragia esporadica. Ao exame objetivo, apresentava ar emagrecido (IMC <P3), equimoses e hematomas dispersos nos membros inferiores, sem adeno ou organomegalias palpaveis, sem estigmas de doenca hepatica cronica. A avaliacao imagiologica corroborou a hepatoesplenome- galia e excluiu hipertensao portal. O hemograma apresentava apenas trombocitopenia de 41000/uL e o estudo de coagulacao APTT prolongado. A investigacao inicial permitiu excluir as cau- sas congestivas, infecciosas, hematologicas e inflamatorias mais comuns. Apos discussao do caso com a Unidade de Doencas do Metabolismo, foi realizado o estudo de doencas de sobrecarga, em que o doseamento da glucocerosidade acida (1.5nmol/mg proteina), permitiu o diagnostico de Doenca de Gaucher. Perante o diagnostico de uma doenca de sobrecarga lisossomal foi encaminha para consulta especifica, onde ira iniciar terapia de substituicao enzimatica Comentarios: Este caso ilustra a importância da avaliacao multidisciplinar de causas secundarias de esplenomegalia per- sistente em idade pediatrica, ainda que assintomatica. O diagnostico presuntivo de esplenomegalia constitucional devera ser um diagnostico de exclusao, dada existencia de patologias que podem permanecer assintomaticas durante longos periodos de tempo, algumas com tratamento disponivel, modificador do curso e prognostico da doenca