Efficacy of cytochemical tests in gene analysis of hereditary spherocytosis: a case study of six patients with different disease subtypes.

OBJECTIVES: In this study, clinical and biochemical methods were utilized to predict the final diagnosis of hereditary spherocytosis (HS), correlate the diagnosis with splenectomy, and examine the usefulness of this approach. METHODS: We biochemically and cytochemically analysed erythrocyte membrane proteins before making a final HS diagnosis based on gene analysis to compare diagnostic approaches. The clinical features of six patients with various subtypes of HS and symptoms were observed by blood analysis using eosin-5'-maleimide staining, biochemical analysis using sodium dodecyl sulphate - polyacrylamide gel electrophoresis with western blotting, and mass spectrometry. Finally, diagnostic membrane gene analysis was performed. RESULTS: Five of the six patients showed mild to moderate or severe anaemia, and the other patient was non-anaemic; all six patients showed faint eosin-5'-maleimide staining. In western blotting of erythrocyte membrane proteins, all six patients (three with ß-spectrin, two with ankyrin, and one with SLC4A1 anomalies) showed low-molecular-weight peptide fragments, which were confirmed by mass spectrometry in the region corresponding to the band 3 protein. The two patients with an ankyrin gene anomaly exhibited severe anaemia, and two patients with simultaneous SLC4A1, SPTB, and UGT1A1 anomalies exhibited mild anaemia and hyperbilirubinemia. DISCUSSION: We determined the relationship among clinical features, cytochemical parameters, and gene anomalies in six patients with newly diagnosed HS while referring to previously published cases. CONCLUSION: These findings reveal a close relationship between clinical features and membrane characteristics in HS, which can facilitate diagnosis and inform treatment.

Analysis of erythrocyte membrane proteins in patients with hereditary spherocytosis and other types of haemolytic anaemia.

OBJECTIVES: In order to investigate the pathophysiology of erythrocyte membrane proteins, 10 patients (6 pre- and 4 post-splenectomy) with hereditary spherocytosis (HS) and other patients with haemolytic anaemia were examined. METHODS: The membrane proteins were analysed by biochemical and mass spectrometry. RESULTS: Reductions in the extracellular membrane of band 3 protein by eosin-5'-maleimide (EMA) binding test were greater in patients with pre-splenectomy HS than in patients with post-splenectomy HS, other types of haemolytic anaemia, and controls. Compared to patients with haemolytic anaemia and healthy controls, the band 3 protein of patients with HS pre- or post-splenectomy was more easily decomposed with N-glycosidase F and by mass spectrometry interactions with degraded low-molecular-weight spectrin and ankyrin. The resulting fragments were observed more frequently in pre-splenectomy than post-splenectomy HS. Haemoglobin-derived peptides were present in patients with haemoglobinopathy (Hb Evans, Hb Sabine) but not in those with haemolytic anaemia and healthy controls. CONCLUSION: Haemolysis in patients with HS occurred because the fragile proteins in erythrocytes (band 3, spectrin, and ankyrin) collapsed due to compression during blood circulation in the spleen. Further, haemolysis in patients with haemoglobinopathy occurred owing to membrane damage due to combined spectrin, band 3 with denatured haemoglobin in the vessel during blood circulation.

High-dose Cepharanthin for pediatric chronic immune thrombocytopenia in Japan.

BACKGROUND: A nationwide, multicenter and observational study was retrospectively conducted to evaluate the clinical utility of Cepharanthin (CEP) for pediatric patients with chronic immune thrombocytopenia (ITP). METHODS: Clinical and laboratory data for 46 Japanese patients aged <16 years who were diagnosed as having chronic ITP in 14 hospitals during 2001-2011, and were treated with CEP for >12 months, were analyzed. RESULTS: Median daily CEP dose was 1 mg/kg (range, 0.12-2 mg/kg). Median platelet count prior to CEP was 20.5 × 109 /L (IQR, 8.3-53.0 × 109 /L), and then significantly increased to 58.5 × 109 /L (IQR, 22.8-115.0 × 109 /L) and 69.0 × 109 /L (IQR, 23.0-134.0 × 109 /L) at 12 and 24 months of treatment, respectively. No life-threatening bleeds or moderate-severe adverse events were reported. Of 38 patients who received both corticosteroids (CS) and CEP, 17 patients (45%) were weaned from CS, and 15 patients (39%) attained the reduced dose of CS. The duration from the start of CEP to the stopping of CS was a median of 413 days (range, 49-1734 days) in patients who were weaned from CS. CONCLUSIONS: CEP alone or combined with CS was useful for the management of pediatric chronic ITPs.

Non-hodgkin lymphoma in a patient with cardiofaciocutaneous syndrome.

Cardiofaciocutaneous (CFC) syndrome is a multiple congenital anomaly/mental retardation syndrome characterized by a distinctive facial appearance, ectodermal abnormalities, and heart defects. Clinically, it overlaps with both Noonan syndrome and Costello syndrome. Mutations in KRAS, BRAF, and MAP2K1/2 (MEK1/2) have been identified in patients with CFC syndrome. BRAF mutations are involved in more than 80% of CFC syndrome patients, and we have reported earlier that 2 CFC patients with BRAF mutations developed acute lymphoblastic leukemia. Here we report a boy with CFC syndrome who developed non-Hodgkin lymphoma. At 2 months of age, he developed pneumonia with pleurisy and was diagnosed as having non-Hodgkin lymphoma (precursor T-cell lymphoblastic lymphoma) by cytopathologic examination of the pleural fluid. He was suspected of having Noonan syndrome because of his facial appearance, webbed neck, and cubitus valgus. Precursor T-cell lymphoblastic lymphoma was treated by the TCCSG NHL 94-04 protocol. At 9 years of age, he was clinically reevaluated and diagnosed as having CFC syndrome because of his distinctive facial appearance, multiple nevi, and moderate mental retardation. Sequencing analysis showed a germline p.A246P (c.736G>C) mutation in BRAF reported earlier in CFC syndrome. Molecular diagnosis and careful observation should be considered in children with CFC syndrome.

Method for calibrating system parameters of a multidirectional interferometers system.

A multidirectional interferometer system has been proposed and developed to measure the position and orientation of a positioning stage. In this method the system parameters, such as positions of the corner-cube reflectors and directions of the rays in the interferometers, must be determined beforehand. However, it is difficult to find ways to determine the system parameters for each different system with necessary accuracy. This paper proposes a systematic method for calibrating the system parameters when the number of the interferometers is larger than the degrees of freedom of the stage. The method is verified for a two-dimensional stage by both simulation and experiment.

Identification of simultaneous mutation of fibrinogen alpha chain and protein C genes in a Japanese kindred.

Afibrinogenaemia usually induces a bleeding tendency during infancy, whereas protein C deficiency increases susceptibility to thrombosis in children or adolescence. Mutations of these genes have been, therefore, established as independent risk factors for coagulation disorders. We describe the homozygous mutation of the fibrinogen alpha chain gene and additional heterozygous mutation of the protein C gene in a male infant who showed prolonged umbilical bleeding after birth. On examination, the plasma fibrinogen was undetectable, and the activity and antigen level of protein C were reduced. The patient showed no fibrinogen Aalpha chain as well as Bbeta and gamma chains by Western blotting. The sequencing analysis showed the homozygous deletion of 1238 bases from intron 3 at position 2008 to intron 4 at position 3245 in the fibrinogen alpha chain gene. Both parents were heterozygous carriers of this mutation. In this patient, an additional mutation was also detected in the protein C gene: the heterozygous deletion of exon 7 at position 6161-6163 or 6164-6166, resulting the deletion of one amino acid (Lys150 or 151). His mother was also a carrier of this mutation. As the simultaneous mutation of the fibrinogen alpha chain and protein C genes has not been previously reported, the influence of the interaction between these two mutations on the clinical manifestations of this patient should be carefully monitored for a long period.

Successful reduced-intensity stem cell transplant from one-locus HLA-mismatched unrelated cord blood after rejection of unrelated bone marrow in an infant with myelogenous leukemia.

An 8-month-old girl had acute myelogenous leukemia (EAB M2) that relapsed 5 months after diagnosis during intensive consolidation chemotherapy. She underwent bone marrow transplantation (BMT) from an HLA-A, -B, -C and -DR phenotypically matched, but one locus DRB1 genotypically mismatched unrelated donor, but rejection occurred.Subsequently, she received reduced-intensity transplant (fludarabine/cytosine arabinoside/cyclophosphamide) from one locus HLA-A-mismatched, but DRB1 genotypically matched unrelated cord blood stem cells and remission was induced by acute GVHD (grade II) that progressed to chronic GVHD with involvement of the skin, liver, and gastrointestinal tract. In this case, it seems that remission was induced by an adequate graft-versus-leukemia effect and mild chronic graft-versus-disease due to the HLA-A difference more than DRB1 matched between the patient and the cord blood stem cells.

Successful bone marrow plus cord blood stem cell transplantation in a girl who developed myelodysplastic syndrome from hepatitis-associated aplastic anemia treated with long-term immunosuppressants and growth factors.

A 9-year-old girl who had hepatitis-associated aplastic anemia was treated intermittently with methylprednisolone pulse therapy and growth factors (granulocyte-colony stimulating factor (G-CSF), recombinant human erythropoietin (rhEpo) and cyclosporin A (CyA) for over two years. At this time, there was hematological improvement, but chromosome analysis revealed monosomy 7. After six months, there was progression to myelodysplastic syndrome (MDS) (stage in refractory anemia of excess blasts (RAEB)) with monosomy 7, monosomy 6, marker chromosome and with hematological deterioration. She received bone marrow (1.57 x 10(5) cells kg(-1) (patient body weight)) plus cord blood cell (0.3 x 10(7) cells kg(-1) (patient body weight)) transplantation from her brother, 2 years and 7 months after the diagnosis of hepatitis-associated aplastic anemia. Engraftment was achieved after two weeks, and acute graft-versus-host disease occurred in a mild form after four weeks. Hematological remission has been continuous for 20 months after bone marrow transplantation. Transformation of hepatitis-associated aplastic anemia to MDS with the monosomy 7, monosomy 6 and marker chromosome in this patient was considered to have been related to the administration of high doses of immunosuppressive drugs plus growth factors.