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Background & objectives: Diagnosis of myelodysplastic syndromes (MDS) is subjective in low-grade cases with <5 per cent blasts or <15 per cent ring sideroblasts. Flow cytometry (FCM) has been used to diagnose MDS; but, it still has only an adjunctive role. This study was conducted to evaluate the role of FCM to diagnose MDS and correlate the number of aberrancies with revised international prognostic scoring system (R-IPSS). Methods: This study included 44 consecutive clinically suspected cases of MDS with refractory cytopenia(s) and 10 controls. Patients were divided into two groups: (i) proven MDS cases (n=26), and (ii) suspected MDS (n=18). Ogata quantitative approach, pattern analysis and aberrant antigen expression were studied. Results: Ogata score ≥2 correctly diagnosed 80.7 per cent (21/26) while aberrant antigen and pattern analysis with flow score of ≥3 could diagnose 92.3 per cent (24/26) patients with proven MDS. Combination of both with flow score ≥3 could diagnose 100 per cent patients. Eight patients in suspected MDS group with persistent cytopenia on follow up were labelled as probable MDS. Ogata score ≥2 was present in 5 of 8 and pattern analysis score ≥3 was present in six probable MDS patients. Combination of both with flow score ≥3 was present in seven of eight patients. Spearman's correlation between Ogata score and R-IPSS, pattern analysis and R-IPSS and combination of both scores and R-IPSS showed significant positive correlation in proven MDS as well as when proven and probable MDS patients were combined. Interpretation & conclusions: Our results showed that combined Ogata approach and pattern analysis, demonstration of ≥3 aberrancies in >1 cell compartment could diagnose most MDS patients. Patients with high flow scores had high R-IPSS scores. Patient with flow score ≥3 and borderline cytomorphology should be observed closely for the development of MDS.
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Síndromes Mielodisplásicas , Citometria de Fluxo , Humanos , Imunofenotipagem , Índia/epidemiologia , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/epidemiologia , PrognósticoAssuntos
Leucemia Eritroblástica Aguda/patologia , Cariótipo Anormal , Idoso , Antimetabólitos Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Aberrações Cromossômicas , Decitabina/uso terapêutico , Células Precursoras Eritroides/patologia , Evolução Fatal , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Eritroblástica Aguda/genética , Masculino , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Severe congenital neutropenia (SCN) is a rare disorder caused by heterogeneous genetic mutations. We describe here a rare association of SCN caused by a novel ELANE mutation and infantile hepatic hemangioendothelioma. In a 2-month-old infant, an abdominal ultrasound performed for omphalitis revealed a hepatic tumor, which was resected. Histopathology confirmed the diagnosis of hemangioendothelioma. Postoperatively, severe neutropenia was noted. Bone marrow examination showed myeloid maturation arrest, diagnostic of SCN. Mutation analysis for the neutrophil elastase gene identified a novel heterozygous de novo ELANE missense mutation in exon 2 (c.215T>A, p.Val72Glu). He was managed successfully with broad-spectrum antibiotics and high-dose granulocyte colony-stimulating factor.
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Hemangioendotelioma , Elastase de Leucócito/genética , Neoplasias Hepáticas , Mutação de Sentido Incorreto , Neutropenia , Hemangioendotelioma/enzimologia , Hemangioendotelioma/genética , Hemangioendotelioma/terapia , Humanos , Lactente , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Masculino , Neutropenia/congênito , Neutropenia/enzimologia , Neutropenia/genética , Neutropenia/terapiaRESUMO
Acute promyelocytic leukemia is a distinct subset of acute myeloid leukemia with characteristic clinical, morphological and genetic features. The gene product PML-RAR alpha resulting from reciprocal t(15;17) translocation, plays a pivotal role in the pathogenesis of acute promyelocytic leukemia and classified as favorable cytogenetic features. We are describing an unusual additional chromosomal abnormality t(2;3) in APL patient.
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BACKGROUND: Interpreting hemoglobin high performance liquid chromatograms with borderline HbA2 values is often problematic, especially in antenatal cases if the partner is a known thalassemia trait. METHODS: We tested for underlying ß-thalassemia mutations in 25 subjects with borderline HbA2 values (between 3.0%-4.0%). Amplification refractory mutation system (ARMS-PCR) was used to detect the five common Indian ß-thalassemia mutations: (IVS-I-5 (G>C), IVS-I-1 (G>T), codons 8/9 (+G), codons 41/42 (-TTCT) and 619 bp deletion). ß-Globin gene sequencing was performed if no mutation was detected. RESULTS: A ß-globin gene defect was identified in 8 (32%) of the 25 cases with HbA2 levels ranging from 3.5%-3.9%. ARMS-PCR revealed IVS-I-5 (G>C) in three, 619 bp deletion in two and codons 41/42 (-TTCT) in one case. Two cases had CAP +1 (A>C) mutation on gene sequencing. IVS-I-1 (G>T) and codons 8/9 (+G) were not found in this small cohort. CONCLUSIONS: Mutation analysis should be offered to all at-risk couples with borderline HbA2, especially those with values between 3.5% and 4.0% and microcytic hypochromic indices. Significant mutations different from those in other ethnic populations were seen in this small institution-based study.
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Hemoglobina A2/análise , Talassemia beta/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Humanos , Índia , Lactente , Pessoa de Meia-Idade , Mutação , Projetos Piloto , Fatores de Risco , Deleção de Sequência , Adulto Jovem , Talassemia beta/genéticaRESUMO
This retrospective analysis of 254 children less than 15 years of age treated with MCP-841 protocol from June 1992 to June 2002 was undertaken to identify the pattern of relapse and determine management lacunae. Two hundred twenty-three (87.8%) children achieved a complete remission of whom 40 (17.9%) relapsed. The mean age of relapsed patients was 6.5 years. The male/female ratio was 9:1. There were 23 (57.5%) isolated bone marrow (BM), 7 (17.5%) isolated central nervous system (CNS), 2 (5%) isolated testicular, 5 (12.5%) BM+testes and 1 each of BM+CNS, CNS+testes, and isolated bone relapses. Twenty-seven children (67.5%) relapsed on-therapy whereas 13 (32.5%) relapsed posttherapy. All 9 CNS relapses occurred on-therapy whereas 5/8 (62.5%) of testicular relapses occurred posttherapy. Lymphadenopathy was the only significant predictor for relapse. High-risk features such as age less than 1 year and greater than 10 years (P=0.047) and white cell count greater than 50.0 x 10(9)/L (P=0.044) were significantly more frequent in patients with early on-therapy relapse than in patients with off-therapy relapse. The overall survival in the entire study cohort was 67+/-3.5%. Modest survival outcome, relapse while on chemotherapy and the higher incidence of CNS and testicular relapse indicate the need for reappraisal of our treatment protocol. There is a need of identifying risk factors and high-risk groups in our set of patients and risk-stratified intensification of chemotherapy in them.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Medula Óssea/terapia , Neoplasias Encefálicas/mortalidade , Recidiva Local de Neoplasia/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Neoplasias Testiculares/terapia , Adolescente , Neoplasias da Medula Óssea/mortalidade , Neoplasias Encefálicas/terapia , Criança , Pré-Escolar , Estudos de Coortes , Terapia Combinada , Feminino , Humanos , Lactente , Masculino , Recidiva Local de Neoplasia/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Dosagem Radioterapêutica , Indução de Remissão , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias Testiculares/mortalidade , Resultado do TratamentoRESUMO
INTRODUCTION: Antiplatelet resistance is one of the urgent issues in current stroke care. One-third to one-half of the patients who experience a recurrent stroke is already on antiplatelet medications. We studied resistance to aspirin and clopidogrel in Indian stroke patients and its association with gene polymorphisms. METHODS: Platelet function testing by light transmission aggregometry was performed on 65 patients with ischemic stroke who were stable on dual antiplatelet therapy (clopidogrel 75 mg OD and aspirin 75 mg OD) along with 65 age-matched controls. Aspirin resistance was considered as mean platelet aggregation ≥70% with 10 µM adenosine diphosphate (ADP) and ≥20% with 0.75 mM arachidonic acid. Clopidogrel resistance was defined as <10% decrease from the baseline in platelet aggregation in response to ADP 10 µM and semi-response as <30% decrease from the baseline. Polymorphisms CYP2C19 * 2 and GPIIb/IIIa (PLA1/A2) were genotyped by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: We found 64.6% (42/65) patients with inadequate response to clopidogrel (15.4% [10/65] resistant and 49.2% [32/65] semi-responders) and 4.6% (3/65) patients with inadequate response to aspirin (3.1% [2/65] resistant and 1.5% [1/65] semi-responder). The frequency of CYP2C19*2 mutant genotype was significantly higher in clopidogrel nonresponders compared to responders (P = 0.014). Clopidogrel nonresponsiveness was much higher in small vessel stroke. CONCLUSION: Unlike aspirin, a high proportion of nonresponders to clopidogrel was identified. In an interim analysis on 65 Indian patients, a significant association was found between CYP2C19*2 and clopidogrel nonresponsiveness.
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INTRODUCTION: Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia, characterized by anemia, jaundice, and splenomegaly. The diagnosis of HS relies on symptoms of hemolysis, a family history of HS, and a positive laboratory test which is usually the osmotic fragility test (OFT). We conducted a study to assess the utility of mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean sphered cell volume (MSCV), and mean reticulocyte volume (MRV) in the diagnosis of HS and if these are helpful in distinguishing cases of HS from immune hemolytic anemia. METHODS: A total of 102 patients suspected to have HS were enrolled. In addition 10 cases of immune hemolytic anemia (IHA) were included in the study and performance of the above screening tests was evaluated. The diagnosis of HS was based on incubated OFT, eosin 5'-maleimide (EMA) dye binding test, and flowcytometric OFT. RESULTS: A total of 29 patients were diagnosed as having HS. The sensitivity and specificity for diagnosis HS by MCHC > 35â g/dL was 44.82%, and ΔMCV-MSCV > 10â fL has a sensitivity and specificity of 82.75% and 95.9% for diagnosis of HS. Using an algorithm of ΔMCV-MSCV > 10â fL and ΔMRV-MSCV < 25, for the differentiation of HS from IHA had sensitivity of 68.9% and specificity of 98.8%.
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Reticulócitos , Esferócitos , Esferocitose Hereditária/sangue , Esferocitose Hereditária/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Índices de Eritrócitos , Feminino , Citometria de Fluxo , Hemólise , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Outcome in multiple myeloma (MM) has improved substantially over recent years as a result of the availability of multiple novel agents with acceptable safety profile. STUDY DESIGN: Prospective observational study at a tertiary care institute. METHODS: Twenty-five newly diagnosed patients of MM were treated with bortezomib and dexamethasone induction with monitoring for response and safety, followed by peripheral blood autologous stem cell transplant (PBASCT) in eligible patients or maintenance. RESULTS: Out of 25 patients, 32% attained complete response (CR), 56% very good partial response (VGPR), 4% PR, and 8% showed no response. The overall response rate was 92%. In our study, 56% of patients showed hematological side effects, out of which thrombocytopenia was seen in 32%, anemia in 16%, and leukopenia in 8%. Six patients developed bortezomib-induced peripheral neuropathy, out of which four had grade 1 (66.66%), one had grade 2 (16.66%), and 1 (16.66%) had grade 3 toxicity. Sixteen patients were eligible for PBASCT, out of which eight patients received this therapy while as remaining eight patients opted for two more cycles of induction therapy followed by maintenance. After completing 18 months of maintenance, all the eight patients who underwent PBASCT were in CR. Out of the 15 patients who did not receive PBASCT five attained CR, eight VGPR while as two patients relapsed. CONCLUSION: Bortezomib plus dexamethasone is highly effective and well-tolerated regimen for frontline treatment of MM with a higher quality of response in an advanced stage and renal failure patients.
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BACKGROUND: Cation exchange-high performance liquid chromatography (CE-HPLC) is most commonly used to evaluate hemoglobin (Hb) variants, which elute in the Hb A2 window. This study aimed to assess prevalence of an uncommon Hb variant, Hb D-Iran, and compare its red cell parameters and peak characteristics with those of Hb E that commonly elutes in the Hb A2 window. METHODS: Generally, we assess abnormal Hb using CE-HPLC as the primary technique along with alkaline and acid electrophoresis. All cases with Hb A2 window >9%, as assessed by CE-HPLCs during 2009-2013, were selected. RESULTS: Twenty-nine cases with Hb D-Iran variant were identified-25 heterozygous, 2 homozygous, 1 compound heterozygous Hb D-Iran/ß-thalassemia, and 1 Hb D-Iran/Hb D-Punjab. Overall prevalence of Hb D-Iran was 0.23%. Compared to patients with Hb E, those with Hb D-Iran had significantly higher Hb (12.1 vs. 11.3 g/dL, P=0.03), MCV (82.4 vs. 76.4 fL, P=0.0044), MCH (27.9 vs. 25.45 pg, P =0.0006), and MCHC (33.9 vs. 33.3 g/dL, P=0.0005). Amount of abnormal Hb (40.7 vs. 26.4%, P=0.0001) was significantly higher while retention time (3.56 vs. 3.70 min, P=0.0001) was significantly lower in Hb D-Iran than in Hb E. CONCLUSION: Hb D-Iran peak can be easily missed if area and retention time of the Hb A2 window are not carefully analyzed. To distinguish between variants, careful analysis of peak area and retention time is sufficient in most cases and may be further confirmed by the second technique-alkaline electrophoresis.
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A 74 years old male patient, presented with history of generalized weakness, fatigue, loss of appetite and breathlessness on exertion for past one and a half months. On examination, he was found to have significant pallor and generalized lymphadenopathy (cervical, axillary and inguinal). The skeletal survey showed punched out lytic lesions in skull and pelvic bones. The peripheral smear examination showed lymphocytosis with absolute lymphocyte count of 25,000/µL. The bone marrow aspirates revealed a hypercellular marrow with 74 % lymphocytes & 14 % plasma cells, suggestive of chronic lymphoplasmacytic disorder. The bone marrow biopsy had two morphologically distinct populations of lymphocytes & plasma cells. The immunohistochemical markers on bone marrow biopsy showed hat plasma cells were positive for CD138 with kappa light chain restriction. Flow cytometry showed B cell population with CD19/CD5 co expression, CD5/CD23 coexpression, were positive for CD22, CD20 and negative for FMC-7 and lambda light chain. In addition, plasma cells were also identified as CD45 negative cells and showed CD38/CD138 co-expression with variable CD19 and CD56 positivity. Serum protein electrophoresis revealed M band, serum immunofixation electrophoresis corresponded to IgA -Kappa. The final diagnosis of chronic lymphocytic leukemia with concomittant presence of plasma cell myeloma was concluded. This case imparts an important message to look for presence of coexisting entities in a single specimen and highlights the benefits of testing both plasma cell and B-cell compartments when the clinical features are not entirely consistent Flow cytometry together with protein electrophoresis can help to clinch difficult and rare dual diagnosis. These cases are rare and pose therapeutic challenge.
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PURPOSE: The purpose is to develop a real-time multiplex reverse transcription-PCR assay for detection and quantification of leukemia-specific chimeric transcripts that identify the genetic subgroups of acute lymphoblastic leukemias (ALLs) proposed by the WHO classification. EXPERIMENTAL DESIGN: Real-time multiplex assay for t(12;21), t(4;11), and t(1;19) with hypoxanthine phosphoribosyltransferase as internal standard used in tandem with a new real time quantitative-RT-PCR assay for the t(9;22). This new strategy was designed to yield an amplicon from each translocation with a distinct melting peak allowing dependable identification using only Sybr green I, without any need for expensive hybridization probes. RESULTS: We validated this method with 92 primary ALLs and identified 4 E2A-PBX1, 4 mBCR-ABL and 10 TEL-AML1. When compared with conventional RT-PCRs and Southern blot analyses, 100% concordance was obtained. During the course of these studies, we found marked variations in the levels of the TEL-AML1 transcripts in individual patients. We, therefore, extended the study to accurately and reproducibly determine TEL-AML1 mRNA levels in 47 additional patients with t(12;21). The results indicated that the level of expression of TEL-AML1 varied among individual patients, and it was independent of the WBC count. CONCLUSIONS: Our new real-time multiplex assay can be used for rapid, simple, and reliable classification of pediatric ALL. Its reproducible quantification results should also facilitate studies on minimal residual disease. The observed variation in TEL-AML1 transcript levels is of interest because it could reflect biological and/or clinical heterogeneity in the behavior of these leukemias.
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Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação Genética , Southern Blotting , Criança , Pré-Escolar , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 4/genética , Subunidade alfa 2 de Fator de Ligação ao Core , Primers do DNA/química , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Proteína de Leucina Linfoide-Mieloide , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/classificação , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismoRESUMO
CD20 is a B cell differentiation antigen with variable expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The significance of CD20 expression has been evaluated in BCP-ALL with conflicting results. There is paucity of data regarding CD 20 expression in BCP-ALL in Indian patients. We retrospectively analyzed 100 patients of BCP-ALL for CD20 expression. CD20 positivity was defined as expression of CD20 to be more than or equal to 20 % in the blast population. 62 % patients expressed CD20 while 38 % patients were negative for CD20. The positivity ranged from negative to dim (35.5 % patients), moderately bright (19.3 % patients) to bright (45.2 % patients). Additional prospective studies are needed to determine the optimal use of rituximab in treatment of CD20-positive BCP-ALL.
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Cation exchange high performance liquid chromatography (CE- HPLC) is an excellent tool for the diagnosis of various hemoglobin (Hb) disorders. HbD-Punjab is an uncommon structural Hb variant seen in North-India. Rarely, a compound heterozygous state for HbD-Punjab with high HbA2 has been described. We describe an index case whose CE-HPLC showed a compound heterozygous state for Hb-Punjab/HbD-Iran which was confirmed by family study, acid and alkaline electrophoresis and beta gene sequencing. This case highlights the role of alkaline and acid electrophoresis to resolve common peaks that elute with HbA2 on CE-HPLC. To the best of our knowledge, this compound heterozygous state of HbD-Punjab with HbD-Iran has not been reported earlier.
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Fanconi anemia (FA) is a genetically and phenotypically heterogeneous recessive disorder characterized by diverse congenital malformations, progressive pancytopenia and predisposition to both hematologic malignancies and solid tumors. We report, a 14-year-old boy who presented with clinical features of aplastic anemia (AA). Subsequent bone marrow examination and multiparametric flowcytometric immunophenotyping revealed an evolving hypoplastic acute myeloid leukemia. Chromosomal breakage studies using clastogenic agent mitomycin C showed 88% stress induced chromosomal/chromatid breaks, gaps and rearrangements revealing an underlying FA. The case emphasizes upon the role of a systematic clinico-investigative approach in diagnosing such patients who by clinical criteria appear to have idiopathic AA and appear phenotypically normal. A timely and accurate diagnosis becomes vital in these cases to implement appropriate therapy.
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We report an unusual case of bi-lineal mixed-phenotype acute leukemia (T/Myeloid, NOS) with complex cytogenetic abnormalities in a 2-year-old boy. Despite attaining complete remission with therapy, he succumbed to status epilepticus following febrile illness. Flow cytometry represents the current standard of care for the diagnosis of this malignancy and the approach adopted in our case is discussed.