Estudo Pioneiro sobre a Presença de Clone de Hemoglobinúria Paroxística Noturna durante Acompanhamento Terapêutico de Leucemia Aguda / Pioneering Study about the Presence of Paroxysmal Nocturnal Hemoglobinuria Clone during Therapeutic Monitoring of Acute Leukemia / Estudio Pionero sobre la Presencia de un Clon de Hemoglobinuria Paroxística Nocturna durante el Seguimiento Terapéutico de la Leucemia Aguda

Introdução: O potencial de transformação maligna de células-tronco hematopoiéticas portadoras de mutações no gene glicosilfostatidilinositolclasse A (PIG-A) para leucemias agudas, embora raro, já é bem descrito na literatura. Objetivo: Neste estudo, porém, buscou-se evidenciar pela primeira vez na literatura o surgimento ou a manutenção de clones de hemoglobinúria paroxística noturna (HPN) em pacientes diagnosticados com leucemia aguda ou ainda após o início do tratamento quimioterápico. Método: A pesquisa de clones de HPN foi realizada por citometria de fluxo em blastos, hemácias, granulócitos ou monócitos de 47 amostras de sangue periférico e medula óssea de pacientes submetidos à investigação diagnóstica ou acompanhamento terapêutico, provenientes de dois hospitais oncológicos e públicos de Belém, no período de dezembro de 2017 a dezembro de 2018. Resultados: A presença de clones de HPN foi observada em 19/47 (40,4%) amostras de pacientes, em investigação diagnóstica ou acompanhamento terapêutico, que realizaram pelo menos um estudo de acompanhamento terapêutico e ainda tiveram o surgimento ou a manutenção do clone de HPN mesmo após iniciado o tratamento quimioterápico. Conclusão: Foi possível evidenciar, de forma primária, a presença de clones de HPN em pacientes diagnosticados com leucemia aguda tanto no período de investigação diagnóstica como durante o acompanhamento terapêutico, independentemente da ontogenia celular. Sem, porém, que se possa ainda avaliar a importância da presença desses clones de HPN para a evolução da doença primária, prognóstico ou necessidade de tratamento específico.

Introduction: The potential for malignant transformation of hematopoietic stem cells carrying mutations in theglycosylphosphatidylinositol class A (PIG-A) gene for acute leukemias, although rare, is already well described in the literature. Objective: In this study, however, it was attempted to show for the first time in the literature the emergence or maintenance of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients diagnosed with acute leukemia or even after the beginning of the chemotherapy treatment. Method: The search of PNH clones was performed by flow cytometry in blasts, erythrocytes, granulocytes or monocytes of 47 samples of peripheral blood and bone marrow from patients undergoing diagnostic investigation or therapeutic follow-up in two oncological and public hospitals in Belém, from December 2017 to December 2018. Results: The presence of PNH clones was observed in 19/47 (40.4%) patient samples, in diagnostic investigation or therapeutic follow-up, who participated of at least one therapeutic follow-up study and still experience the appearance or maintenance of the PNH clone even after the beginning of the chemotherapy treatment. Conclusion: Primarily, it was possible to demonstrate the presence of PNH clones in patients diagnosed with acute leukemia both during the diagnostic investigation period and therapeutic follow-up, regardless of cell ontogeny. However, the importance of the presence of these PNH clones for the evolution of the primary disease, prognosis or need for specific treatment was not evaluated yet.

Introducción: El potencial de transformación maligna de las células madre hematopoyéticas que portan mutaciones en el gen glicosofosfatidilinositol (GPI) clase A (PIGA) para las leucemias agudas, aunque raro, ya está bien descrito en la literatura. Objetivo: En este estudio, sin embargo, buscamos mostrar por primera vez en la literatura la aparición o mantenimiento de clones de HPN en pacientes diagnosticados de leucemia aguda o incluso después del inicio de la quimioterapia. Método: La investigación de clones de hemoglobinuria paroxística nocturna (HPN) se realizó mediante citometría de flujo en blastos, eritrocitos, granulocitos o monocitos de 47 muestras de sangre periférica y médula ósea de pacientes sometidos a investigación diagnóstica o seguimiento terapéutico de dos hospitales oncológicos y públicos de Belém, durante el período. de diciembre de 2017 a diciembre de 2018. Resultados: La presencia de clones HPN se observó en 19/47 (40,4%) muestras de pacientes, en investigación diagnóstica o seguimiento terapéutico, que realizaron al menos un estudio de seguimiento terapéutico y aún tenían la aparición o mantenimiento del clon HPN incluso después de iniciado el tratamiento de quimioterapia. Conclusión: Se pudo evidenciar, de forma primaria, la presencia de clones de HPN en pacientes diagnosticados de leucemia aguda tanto durante el período de investigación diagnóstica como durante el seguimiento terapéutico, independientemente de la ontogenia celular. Sin embargo, no podemos todavía evaluar la importancia de la presencia de estos clones de HPN para la evolución de la enfermedad primaria, el pronóstico o la necesidad de un tratamiento específico.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria / Avanços técnicos no diagnóstico e no monitoramento de hemoglobinúria paroxística noturna por citometria de fluxo

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

RESUMO Objetivo: Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. Métodos: Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. Resultados: Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. Conclusão: A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.

Paroxysmal nocturnal hemoglobinuria in childhood and adolescence--a retrospective analysis of 18 cases.

OBJECTIVE: To assess the clinical and hematological profile of PNH in children. METHODS: Clinical and laboratory features of children with PNH diagnosed in the past six years at our centre were reviewed. Various investigations done included a complete blood count and peripheral smear examination, plasma hemoglobin, urine hemosiderin, acid ham test, sucrose lysis test, immunophenotyping of erythrocytes by sephadex column gel card and of granulocytes by flow cytometry. There were 18 children with a marked male predominance (M 14: F 4). RESULTS: Pallor, jaundice, dark urine and bleeding manifestations were the major presenting complaints. One girl suffered an arterial stroke. All children had cytopenia in at least one cell line. Children were treated with danazol, stanazolol, prednisolone and cyclosporin A variously. Overall response rate was 61%. Children with classical PNH performed slightly better with response rates of 66% (6/9) as compared to aplastic anemia-PNH group which has a response rate of 55% (5/9). Amongst various variables only danazol correlated with better response (p=0.029). CONCLUSION: PNH is an uncommon disease in children and should be included in the differential diagnosis of children presenting with cytopenia.

Estudio ultraestructural de las plaquetas de pacientes con hemoglobinuria paroxística nocturna / Ultrastructural study of platelets patients with paroxysmal nocturnal hemoglobinuria

Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia characterized by intravascular hemolysis, cytopenias and venous thrombosis. Previous studies in patients with PNH have shown platelet abnormalities; however, their association with the clinical development of the sickness has not still been determined. Material and methods. In this study, we compared the morphology and distribution pattern of actin, myosin, tubulin and p-selectin in resting and activated platelets from 22 PNH patients and healthy donors by transmission electron microscopy and immunofluorescence. Results. The PNH platelet ultrastructure of resting and activated with different agonists (ADP, collagen and thrombin) showed morphological changes which suggested the presence of circulating platelets. The developed structures during the adhesion process (filopodia and lamellipodia formation), as well as the pattern distribution of actin, myosin, tubulin and p-selectin in PNH platelets were not modified in relation to control platelets. Conclusion. Morphological changes in resting platelets were related with p-selectin expression suggesting its determination as thrombosis indicator.

Introducción. La hemoglobinuria paroxística nocturna (HPN) es una anemia hemolítica caracterizada por hemolisis intravascular, citopenias y trombosis venosa. Estudios previos en pacientes con HPN han revelado anormalidades plaquetarias; sin embargo, no se ha determinado su asociación con el desarrollo clínico de la enfermedad. Material y métodos. En el presente estudio se comparó la morfología y el patrón de distribución de actina, miosina, tubulina y P-selectina en plaquetas en reposo y activadas provenientes de 22 pacientes con HPN y de individuos sanos por microscopía electrónica de transmisión e inmunoñuorescencia. Resultados. La ultraestructura de las plaquetas de individuos con HPN en reposo y activadas en suspensión con diferentes agonistas (ADP, colágena y trombina) mostró cambios morfológicos que sugirieron la presencia de plaquetas activadas circulantes. Las estructuras desarrolladas durante el proceso de adhesión (formación de filopodios, lamelipodios), así como el patrón de distribución de actina, miosina, tubulina y P-selectina, no se modificaron en las plaquetas de los pacientes con HPN en relación con el testigo. Conclusión. Los cambios morfológicos en plaquetas en reposo fueron relacionados con la expresión de P-selectina, por lo que se sugiere su determinación como un parámetro indicativo de un posible riesgo trombótico.

XmnI polymorphism is associated with fetal hemoglobin levels in hypoplastic syndromes

CONTEXTO E OBJETIVO: O aumento adquirido da hemoglobina fetal (HbF) já foi implicado como fator prognóstico em distúrbios diseritropoiéticos. Nossos objetivos foram de examinar elevações adquiridas na HbF em pacientes com anemia aplástica (AA) e hemoglobinúria paroxística noturna (PNH), e de avaliar se há associação entre a presença de polimorfismos XmnI e de região de controle de locus gênico 5' (LCR-HS2) e os níveis de HbF. TIPO DE ESTUDO E LOCAL: Estudo longitudinal no Serviço de Hematologia e Transfusão de Sangue da Universidade Federal de São Paulo – Escola Paulista de Medicina.MÉTODOS: Estudamos um grupo de 37 pacientes com AA e/ou PNH. Reação de polimerase em cadeia (PCR) e digestão enzimática foram usadas para analisar polimorfismos XmnI; e PCR para clonagem e sequenciamento automático dos polimorfismos HS2. RESULTADOS: O nível médio de HbF foi de 2,32%, mas não houve diferença significativa entre o nível de HbF dos pacientes AA e PNH (p = 0.46). Os níveis de HbF menores que 1,0% mostraram correlação estatisticamente significativa com ausência do polimorfismo XmnI (+) (p = 0.007). CONCLUSÕES: Ausência de polimorfismo XmnI está associado com diminuição de HbF. Mais estudos são necessários para confirmar estas observações e fazer comparações sobre tratamento, prognóstico e sobrevida.

Paroxysmal nocturnal hemoglobinuria in childhood: an uncommon presentation.

An eight year old boy presented with severe anemia and bleeding spots. Complete blood count showed pancytopenia. There was mild reticulocytosis. Bone marrow was hypocellular with normoblastic erythroid hyperplasia. Ham's test (acidified serum test) was positive which confirmed the diagnosis of Paroxysmal nocturnal hemoglobinuria (PNH). Although PNH is rare in childhood, it should be considered as a diagnostic possibility in cases of aplastic anemia as the two conditions can coexist. The presence of PNH in association with aplastic anemia can influence the outcome of the latter.

Paroxysmal nocturnal hemoglobinuria with onset in childhood: a case report.

A twelve-year-old boy presented with recurrent episodes of anemia. Complete blood counts showed pancytopenia. Bone marrow was hypercellular with erythroid hyperplasia and depleted stores of iron. Positive Ham's test and sucrose lysis test revealed that he had paroxysmal nocturnal hemoglobinuria. There was a delay of nearly two years in the diagnosis in this patient. Paroxysmal nocturnal hemoglobinuria is rare in childhood. It must however be considered in a child who presents with unexplained anemia or bone marrow failure so that an early and accurate diagnosis is reached.

PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria.

Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.

Cytometric analysis of paroxysmal nocturnal hemoglobinuria erythrocytes.

Erythrocytes from six cases of paroxysmal nocturnal hemoglobinuria (PNH) were analysed by the H*1 hematology analyser using laser inspection for each individual red cell. The computer program categorized the red cells into 9 groups based on the data of red cell volume and intracellular hemoglobin concentration. The only 46.3 +/- 10.9 per cent (Mean +/- SD) of PNH red cells were normochromic normocytic. Hypochromia (hemoglobin concentration less than 28 g/dL) with normal red cell volume (between 60 to 120 fL) were found in 22.8 +/- 19.7 per cent and with large cell volume (greater than 120 fL) = 14.9 +/- 4.5 per cent of total red cells. Large cells with normal hemoglobin concentration were found in 12.9 +/- 13.2 per cent. The red cells had increased heterogeneity in red cell volume measured as red cell distribution width (RDW), Mean +/- SD = 23.6 +/- 2.3 per cent and increased heterogeneity in their hemoglobin concentration distribution with (HDW) (3.4 +/- 0.5 g/dL). The high RDW was resulted from increased number of varying size of macrocytes and the high HDW was caused by the increased number of hypochromic red cells. Heterogeneity in PNH red cell population described in this study has been postulated to relate with the clonal abnormality of the PNH red cells.

A clinico-haematologic profile of paroxysmal nocturnal haemoglobinuria.

Clinico-haematological parameters in sixteen patients of paroxysmal nocturnal haemoglobinuria (PNH) are presented. Their modes of presentation included recurrent episodes of cola-coloured urine (6/16), refractory anaemia (9/16) and predominant thrombotic manifestations (1/16). Laboratory investigations revealed the presence of anaemia (16/16), reticulocytosis (14/16), thrombocytopenia (11/16), leucopenia (5/16) and cellular bone marrow (14/16). Two patients had hypoplastic bone marrow initially but subsequently developed PNH. The patients were treated with haematinics, prednisolone (16/16) and oxymethalone (2). Prednisone was effective in suppressing haemolytic episodes. Oxymethalone given to the 2 patients with hypoplastic bone marrow resulted in amelioration of anaemia in one but no effect in the other patient.

Paroxysmal nocturnal hemoglobinuria: a study of 17 cases.

Retrospective analysis of 17 cases of paroxysmal nocturnal hemoglobinuria (PNH) was carried out. The presenting feature of anaemia and hemoglobin of less than 9 gm% were observed in all cases. Fever, jaundice and bleeding tendency were observed in 8, 8 and 7 cases respectively. Bone marrow examination revealed megaloblastic features, erythroid hyperplasia and hypocellularity in 8, 7 and 2 cases respectively. Fourteen cases were refractory to the treatment, while only 3 showed response.