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Severe haemophilia carries an increased risk of life-threatening intracranial haemorrhages. Studies in adult survivors show a relatively high percentage of anterior pituitary hypofunction reported as the most frequent complication. We report the case of isolated growth hormone deficiency in a boy with severe haemophilia A. He experienced several intracranial haemorrhages in early childhood. At the age of seven years, growth hormone deficiency was diagnosed. The MRI scan of the pituitary gland was normal, but many focal changes in brain tissue were found. The function of pituitary-dependent hormonal axes beyond GH/IGF1 axis was sufficient. Therapy with rhGH was introduced and continued for over nine years. Growth velocity increased and the height normalised appropriately to parental height. We did not observe any complications besides sporadic subcutaneous bleedings. Patients with haemophilia should be considered as a high-risk group for hypopituitarism. Subcutaneous rhGH injections can be safe even in severe haemophilia.Ciężka hemofilia jest związana z wysokim ryzykiem wystąpienia zagrażających życiu krwawień wewnątrzczaszkowych. Badania u dorosłych wskazują na stosunkowo wysoki odsetek niedoczynności przedniego płata przysadki po incydencie krwawienia wewnątrzczaszkowego. W pracy przedstawiono przypadek nabytej izolowanej somatotropinowej niedoczynności przysadki u chłopca z ciężką postacią hemofilii A. We wczesnym dzieciństwie przebył kilka incydentów krwawienia wewnątrzczaszkowego. W wieku 7 lat u chłopca rozpoznano niedobór hormonu wzrostu. Badanie metodą rezonansu magnetycznego wykazało prawidłową przysadkę i liczne zmiany ogniskowe w tkance mózgowej. Czynność osi hormonalnych zależnych od przysadki poza osią GH/IGF1 była prawidłowa. Wdrożono leczenie rhGH i kontynuowano terapię ponad 9 lat. Uzyskano normalizację wzrostu chłopca stosownie do średniej wysokości rodziców. Nie obserwowano żadnych powikłań leczenia poza sporadycznym krwawieniem po iniekcjach. Pacjenci z hemofilią powinni być traktowani jako grupa zwiększonego ryzyka dysfunkcji przysadki. Podskórne iniekcje rhGH mogą być bezpieczne nawet u pacjentów z ciężką hemofilią.

Background Red cell pyruvate kinase deficiency (PKD) is a defect of glycolysis causing congenital non-spherocytic hemolytic anemia. PKD is transmitted as an autosomal recessive trait. The clinical features of PKD are highly variable, from mild to life-threatening anemia which can lead to death in the neonatal period. Most patients with PKD must receive regular transfusions in early childhood and as a consequence suffer from iron overloading. Patient Here, we report a Polish family with life-threatening hemolytic anemia of unknown etiology. Whole exome sequencing identified two heterozygous mutations, c.1529 G > A (p.R510Q) and c.1495 T > C (p.S499P) in the PKLR gene. Molecular modeling showed that the both PKLR mutations are responsible for major disturbance of the protein structure and functioning. Despite frequent transfusions the patients do not show any signs of iron overload and hepcidin, a major regulator of iron uptake, is undetectable in their serum. The patients were homozygous for the rs855791 variant of the TMPRSS6 gene which has earlier been shown to down-regulate iron absorption and accumulation. Conclusion The lack of iron overload despite a reduced level of hepcidin in two transfusion-dependent PKD patients suggests the existence of a hepcidin-independent mechanism of iron regulation preventing iron overloading.

Background The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. Methods Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. Results We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. Conclusion Our results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0428-1) contains supplementary material, which is available to authorized users.

Background: Osmotic Fragility Test (OFT) is widely considered as a sensitive indicator of red blood cells' sensitivity to the hypotonic solution. It is often used as a screening test for the diagnosis of hereditary spherocytosis (HS). Nowadays, the osmotic fragility test based on flow cytometric analysis (FCM OF) is widely used in laboratory practice. The purpose of this study was to optimize the assay sensitivity and to validate its clinical application in the diagnostic screening of childhood anemias. Methods: The study was conducted on 175 children suffering from various types of anemia (including 30 children with proven hereditary spherocytosis, HS) and 16 healthy subjects. All children were aged between 3 months and 17 years, including 94 boys and 97 girls. FCM OF was performed on every subject according to two different analysis time patterns (hemolysis was analyzed for 214 or 300 seconds) using Cytomics FC500 flow cytometer. Results: Significant higher sensitivity was demonstrated by the tests carried out according to the longer analysis time pattern (90.0% vs. 83.33%). The level of specificity of both the analysis patterns was similar. When an extended analysis time was used, the percentage of red cell survival levels in HS patients were significantly lowered compared to the same cases analyzed with shorter incubation times and all other non-HS anemic cases (9.31±4.69% vs. 35.59±15.30%, p

Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare disease in children resulted from clonal hematopoietic stem cell proliferation triggered by acquired somatic mutation of the PIGA gene. As a consequence, a deficiency of surface proteins, e.g. responsible for protection of the untouched myeloid progenitor cells from the complement aggression, is observed. It is characterized by intra and extravascular hemolysis, various degrees of bone marrow failure and a tendency to thrombophilia with multifocal thrombosis, especially affecting visceral circulation. The aim of the case description concerning a 16 years old boy with more than 4 years delay in proper diagnosis resulted from the abdominal mask of PNH, is to draw the attention of pediatrician to identifiable diagnostic problems, when the one has had to face with rare diseases. Many, even well-known and described disorders in adults had an unexpected course in children with significant differences resulting in various clinical masks leading to misdiagnosis. Overlapping symptomatology between popular and unique disorders of childhood may be responsible for misdiagnosis as well, what demand the specific clinical awareness and response. The other, but not less important aspect of presented case report, describing such a unique disease of childhood treated with one of the most expensive therapy in the world, is important question of the ethical and socio-economic borders of modern medical therapies. All the aspect of clinical and ethical problems comprises the subject of this publication.

Diagnosis of vitamin B12 deficiency could be difficult due to various and rather unspecific symptoms and often late manifestation in hematological findings. B12 has a crucial role as cofactor of many metabolic pathways and has essential role in many processes such as DNA synthesis, neuron myelination and gluconeogenesis. Blood smear has a huge role in early diagnosis and should be performed. B12 deficiency is not very common, however could be underestimated especially in developing countries. In children main cause of deficiency is exclusively breastfeeding by vitamin B12 depleted mothers. Consequences of low intake in children are more severe than in adults, because of lower liver storage. Early detection and treatment is very important, because long term deficiency could result in persistent neurological damage. Coexistence iron or folate deficiencies could result delay in diagnosis. In this article different manifestation and laboratory findings in group of children with B12 deficiency is described.

Paroxysmal cold haemoglobinuria (PCH) is a form of autoimmune haemolytic anaemia (AIHA) characterised by a sudden onset of haemoglobinuria, either spontaneously or following exposure to cold. In children, it is commonly seen following a viral illness or after immunisation. Diagnosis of PCH is confirmed by a positive Donath Landsteiner (DL) test in which biphasic haemolysins are detected. However, in a real clinical setting, the serological diagnosis of PCH is not always easy. PCH can cause tubular renal injury, which in turn can lead to renal impairment. We describe a case of a two-year-old boy who was admitted to the hospital with pallor, jaundice, dehydration, and dark urine. Two weeks before admission, the child had an upper respiratory tract infection. Laboratory tests showed severe anaemia (haemoglobin 4.5g/dl, haematocrit 11.5%, LDH 8525 U/l), hyperbilirubinaemia (104 μmol/l), haemoglobinuria, and acute kidney injury: GFR 43.9 ml/min/1.73 m2 (grade 2 according to Acute Kidney Injury Network). The direct antiglobulin test was positive for C3c and C3d complement components. The diagnosis of PCH was confirmed by the presence of biphasic antibodies in a DL test on the third day of hospitalisation. The patient received supportive treatment.

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with β-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the β-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality.

Introduction. Inherited hemoglobin disorders, including thalassemia and hemoglobinopathies, are frequently occurring diseases in the world. Thalassemias are autosomal recessive disorders of hemoglobin synthesis. Underlying molecular defects in the α-globin or β-globin gene clusters form the basis of defective hemoglobin synthesis and the various inherited forms of α- halasemias or β-thalasemias. Thalassemia is diagnosted where hemoglobin levels, volume of red blood cells (MCV) and mean cellular hemoglobin (MCH) are significantly reduced. Aim. We want to introduce atypical clinical falieure of hemoglobinopathy. Material and methods. We presented two children with anemia. In first case it was a boy with normal MCV and reduced MCH, next case it was a girl after splenectomy with increased MCV and MCH. In both cases the red cells were hypochromic and also target cells were present in peripheral blood smear. Hemoglobin electrophoresis, measurement of HbA2 and HbF levels were performed. Results. Test results were characteristic for thalasemia β. Extending the diagnostic panel with genetic tests helped to identify in both of children thalassemia α silience carrier (-α/αα). In first case unstabile hemoglobin Hb Köln (Codon 98(295G > A)) was also detected and in second unstabile hemoglobin Hb Bruxelles (deletions del 42/β+). Conclusion. Only the genetic tests made it possible to explain atypical blood smear for thalassemia.

Introduction. Immune thrombocytopenia (ITP) is an autoimmune disease that results in a reduction of blood platelets

Immune thrombocytopenia purpura (ITP) is a rare autoimmune disorder but it is one of the most common bleeding disorder in childhood. Most children with this disease have acute form of ITP with self-limiting thrombocytopenia. A small subset of children with ITP has clinically significant disease with severe thrombocytopenia. We presented the case of a boy with a severe form of chronic immune thrombocytopenia who was treated for the first time six years before presented. At first autoimmunohemolytic anaemia was recognized. Next in therapeutic procedure, multi-drug treatment, including intravenous corticosteroids, intravenous immunoglobulins, and cyclosporine were used to achieve permanent improvement. Due to a chronic ITP and significant decrease platelets counts and severe symptoms of nose bleeding and anaemia, eltrombopag, thrombopoiesis stimulating factor, was used. After 16 weeks of treatment, as a result of triple therapy consisting of eltrombopag, prednisone and azathioprine, the platelets counts were normalized and the symptoms of bleeding resolved.

Splenectomy is considered standard surgical therapy in hereditary spherocytosis. The procedure is indicated in patients with severe anemia, recurrent hemolytic, and aplastic crises. The aim of the study was to assess treatment outcomes in patients with hereditary spherocytosis who underwent total or partial laparoscopic splenectomy. Fifteen patients aged 4-17 yr underwent laparoscopic splenectomy from 2009 to 2012. Partial and total splenectomies were performed (five and 10 children, respectively). Hematologic parameters, liver function tests, and splenic volume before and after the surgery were analyzed retrospectively. Total follow-up was 1-30 months. Hospitalization and operating time were similar in both groups. In partial splenectomy group, branches of splenic arteries gave better blood supply than short gastric vessels. In both groups, hematologic parameters were improved. Postoperative markedly elevated platelet count was maintained up to 6 months, and after that, platelet count gradually decreased to normal values. Bilirubin level was decreased in early postoperative period; however, it increased later to achieve levels lower than in preoperative period. No severe general infections were observed in both groups. Laboratory parameters (hemoglobin and bilirubin concentrations and RBC) after the surgery improved in all patients, and the effect was maintained during 12 months of follow-up. Platelet count increased significantly after the surgery and was maintained at high levels during the next 6 months. However, it returned to preoperative levels within a year after the surgery. Our study showed that partial splenectomy was not inferior to total splenectomy. However, full assessment requires longer follow-up and larger group of patients.

Introduction Autoimmune hemolytic anemia (AIHA) is a rare disorder in which the immune system produces pathologic antibodies directed against its own red blood cells (autoantibodies). AIHA is most commonly diagnosed by a positive result of the direct antiglobulin test (DAT, Coombs test). Depending on the temperature at which autoantibodies react with red blood cells in vitro AIHA is classified as warm-type AIHA with incomplete IgG autoantibodies and cold-type AIHA with cold IgM agglutinins (CAS – cold agglutinin syndrome) or with biphasic hemolysins (PCH – paroxysmal cold hemoglobinuria). In mixed-type AIHA there are simultaneously warm autoantibodies and cold agglutinins. Aim The aim of this study was to find the number and types of AIHA diagnosed and treated in the Department of Pediatrics, Hematology and Oncology of Warsaw Medical University during the years 2004–2014. Material and methods The authors analyzed 54 children in which AIHA was diagnosed. The age of the child at diagnosis, the result of direct antiglobulin test and the type of autoantibodies in serum were taken into account. Results and discussion The most common type of AIHA in a group covered by the survey was cold-type AIHA, including paroxysmal cold hemoglobinuria (37%) and cold agglutinin syndrome (16%). Conclusions As far as the results are concerned, avoidance of cold is essential before the serological diagnosis is reached.

INTRODUCTION Hereditary haemolytic anaemias (HHA) encompass a heterogeneous group of anaemias characterized by decreased red blood cell survival. The aim of this study was to evaluate the status of red blood cell (RBC) surface molecules known or previously proposed to participate in preventing premature RBC clearance, analysing erythrocytes from patients with two types of HHA: hereditary spherocytosis (HS) and microcytosis. MATERIAL/METHODS Relative binding of five monoclonal antibodies (mAbs), anti-CD55, anti-CD59, anti-CD44, anti-CD47 and anti-CD58, was evaluated in erythrocytes of patients with HS and hereditary microcytosis, using flow cytometry. The amount of CD55 protein was assessed by semi-quantitative Western blots densitometry analysis. RESULTS The majority of both HS and microcytic patients demonstrated significant reduction of anti-CD55 binding by erythrocytes (average 23% and 19%, respectively, P

Sequences of the primers. (DOCX 13 kb)