Comparative study of BCR-ABL1 quantification: Xpert assay, a feasible solution to standardization concerns.

The level of BCR-ABL1 reached after treatment with tyrosine kinase inhibitors is an effective marker of the therapeutic response and a good survival predictor in chronic myeloid leukemia (CML) patients. However, no agreement has yet been achieved about either the standardization of the technique to determine BCR-ABL1 or the interpretation of the results. The aim of this study was to compare the method currently recommended by the European Leukemia Net, which includes the application of a conversion factor to express the results in international scale, with an automated method (Xpert BCR-ABL™, Cepheid). BCR-ABL1 transcript quantification was performed in 117 samples from CML patients in two different laboratories by both methods, and the results were compared by statistical procedures. A high linear correlation was obtained in the results between the two methods. The concordance at logarithmic intervals reached 62 %. When the major molecular response (MMR) was analyzed, 85 % agreement was achieved. The automated method provides reproducible results and does not show significant differences compared with the traditional method. As a clinical tool, Xpert correctly classified the patients in MMR and can be considered a useful alternative for the molecular follow-up of CML patients.

Splenic marginal zone lymphoma--a clinicopathological study in a series of 16 patients.

Splenic marginal zone lymphoma (SMZL), characterized in the WHO classification of lymphoid tumors, is a rare disorder comprising less than 1% of lymphoid neoplasms; only a few series concerning this entity have been published. Although this type of lymphoma is well defined histologically, its histogenesis remains obscure. Moreover, specific biological markers are still lacking and immunophenotype profile is not specific. These and other reasons, such as the existence of cytogenetic subtypes, have led to some authors to suspect that SMZL constitutes a heterogeneous entity. We have analyzed a series of sixteen SMZL cases from four hospitals in our community, from a clinical, biological and pathological point of view. When compared with those reported in the literature, our findings show three main differences: our patients less frequently showed an intrasinusoidal bone marrow infiltration pattern; the presence of a serum monoclonal component was rarely seen; and CD5-positive SMZL cases appear to be more common than previously thought.

Monoclonal antibody fluorescence for routine lymphocyte subpopulation analysis with the Abbott CELL-DYN Sapphire haematology analyser.

Using previously described procedures, this study quantified T-cell, T-cell subset, B-cell and NK-cell populations with the CD-Sapphire haematology analyser in a series of patients with mild to moderate lymphocytosis. Lymphocyte counts ranged from 6.0 to 14.9 x 10(9)/l, with 86/97 being <10.0 x 10(9)/l. Immunophenotyping (CD3/CD19/HLA-DR, CD4/CD8 and CD16/CD56 combinations) was performed using EDTA-anticoagulated blood, automated CD-Sapphire analysis and subsequent software processing. Of 35 samples from younger (<12 years) patients, 22 (63%) had nonspecific lymphocyte changes, 4 (11%) showed specific increases in nonreactive T-Helper or T-Suppressor cells, and five showed a reactive T-cell lymphocytosis. The remaining four were classified as 'Transient/Persistent NK-associated (NKa) Expansion' (n = 3) and specific B-cell lymphocytosis (n = 1). For older patients (n = 59), 15 (25%) had an increase (>1.5 x 10(9)/l) in B-cells, and seven investigated for surface immunoglobulin expression were all found to be clonal. The remaining samples were categorized as 'Transient/Persistent NK-associated (NKa) Expansion' (13/59), Reactive Lymphocytosis (5/59), 'Reactive Lymphocytosis or Transient/Persistent NKa Expansion' (8/59), specific T-Helper cell (n = 8) or T-Suppressor cell (n = 3) lymphocytosis, and 'Lymphocytosis of Undetermined Significance' (n = 7). This study has demonstrated the feasibility of applying limited immunophenotyping protocols to the investigation of patients with abnormal lymphocyte counts in routine haematology. By using commercially purchased liquid monoclonal reagents to determine lymphocyte subpopulation profiles, haematology laboratories can provide more definitive information of potential clinical importance.

Analysis and enumeration of T cells, B cells and NK cells using the monoclonal antibody fluorescence capability of a routine haematology analyser (Cell-Dyn CD4000).

This communication details a method for the quantitative and qualitative analysis of blood T-, B- and NK-cell populations using the Abbott Cell-Dyn CD4000 haematology analyser. A series of 66 ethylenediaminetetraacetic acid (EDTA)-anticoagulated samples with lymphocyte counts between 0.2 and 33.3 x 10(9)/l were selected and analysed with CD3, CD19, Ia and CD56 monoclonal reagents. The flow cytometry reference method utilized a lymphocyte gate defined by optical scatter, with phenotypic analyses referencing to this gate and the absolute lymphocyte count. The CD4000 method analysed all leucocyte events, set primary gates for specific immunophenotypic fractions, and then determined population counts by reference to the white blood cell (WBC) count. Comparisons of CD3+ T-cell and CD19+ B-cell numbers showed high coefficients of correlation (R(2) > 0.95) and agreement (y = 1.01x) between the CD4000 and flow cytometry reference methods. Lower coefficients of correlation were obtained for CD3-CD56+ (R(2) = 0.52) and CD3+CD56+ (R(2) = 0.83) components. No major discrepancies were observed, and the CD4000 procedures additionally provided qualitative insights into the possibility of T-cell activation. The potential to undertake immediate analysis of EDTA-anticoagulated blood samples to determine the nature of abnormal lymphocyte morphology or numbers represents a considerable advance in the capability of haematology laboratories.

Acute promyelocytic leukemia developing after radiotherapy for prostate cancer in a patient with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is rarely associated with secondary acute myelogenous leukemia (AML) usually due to chemotherapy or radiotherapy. No cases of concomitant CLL and acute promyelocytic leukemia (APL) have been found in the literature. Nevertheless, up to 12% of therapy-related AML cases are classified as APL. Of these latter, most are related to topoisomerase treatment, with a few acute cases occurring after radiotherapy. We report here a patient with an untreated CLL who developed APL 2 years after radiotherapy for prostate carcinoma.

t(2;5) associated with a histiocytic-monocytic neoplasm.

We describe a patient with a neoplasm derived from the histiocytic-monocytic lineage associated with t(2;5) detected by FISH. The patient presented with bone marrow involvement, no organomegaly and subsequently developed a leukaemic picture. The clinical course was aggressive and the patient died four months from diagnosis. Cell morphology, immunophenotype (CD30-, EMA-, Lisozyme+, cy CD68+ and CD45+) and DNA analysis showing germ-line configuration of the Ig/TCR chain genes ruled out the diagnosis of anaplastic large cell lymphoma (ALCL). This unusual case ilustrates that t(2;5) is not exclusive for ALCL but may be found in a few cases of rare neoplasms derived from the histiocytic-monocytic cells.

Citoquímica de la fosfatasa ácida. Consideraciones metodológicas / The citochemistry of acid phosphatase. Methodological considerations

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Signet-ring cells in a Waldenström's Macroglobulinaemia.

A case of signet-ring cell lymphoma affecting bone marrow is reported. The patient was diagnosed as Waldenström's Macroglobulinaemia on the basis of clinical and laboratory features including morphology, immunohistochemistry and gene rearrangement studies. Light microscopy examination showed cells contained large globular inclusions (signet-ring cells) that stained for kappa immunoglobulin light chain by immunohistochemistry. In addition, the neoplastic cells expressed the common leukocyte antigen CD45 and the B cell marker CD19. This to the best of our knowledge is the first report of a patient with Waldenström's Macroglobulinaemia with the presence of vacuolated signet ring- cells in the bone marrow. Differential diagnosis arises with non-haemopoietic tumours and this needs to be based on specific immunostaining. Tumours and this need to be based on specific immunostaining.

[Splenic lymphoma with circulating hairy lymphocytes lymphoma. Clinical cytological study of 4 patients]. / Linfoma esplénico con linfocitos vellosos circulantes. Estudio clínico citológico de cuatro pacientes.

PURPOSE: The splenic lymphoma with circulating villous lymphocytes (SLCVL) is an infrequent disease included within the low grade non Hodgkin's lymphoma, B-cell type. The results of the study of four patients are reported. PATIENTS AND METHODS: Clinical, cytological, immunophenotypic, ultrastructural, evolutive and therapeutic data have been revised in all the cases. RESULTS: Two males and 2 females of 76, 66, 68 and 62 years, respectively were diagnosed as having SLCVL. The initial symptoms were scarce, basically asthenia, and a big spleen without significant lymphadenopaty was the most relevant physical finding in each of them. In peripheral blood leukocyte count was normal with a slight lymphocytosis and a variable percentage of villous circulating lymphocytes. The immunophenotype of peripheral blood obtained by flow cytometry was according with a mature B-cell lymphocyte population, CD 5 and CD 25 negative. The cells were positive to acid phosphatase with a diffuse pattern of variable intensity; the reaction was inhibited by tartaric acid. All the patients had BM infiltration, studied with aspiration and biopsy. One case (M,66) had an IgM monoclonal gammopathy. The ultrastructural study, performed in 3 cases, showed thin and short villous prolongations. After splenectomy, a low degree lymphoma therapy has been employed in all the cases. The follow-up ranges between, 4 years and 4 months, all the patients being alive. CONCLUSIONS: The SLCVL is a definitive entity regarding the clinical, morphologic and immunophenotype features. A long clinical evolution and a good prognosis after splenectomy are common.

DNA analysis of bronchoalveolar lavage in the diagnosis of pulmonary lymphoma.

Lung lymphomas frequently present a diagnostic challenge. The diagnosis of malignant lymphoma is possible when lymphocyte subpopulations show aneuploidy or a definable monoclonal surface marker or both. We present a case of lymphoma of the lung in which DNA analysis of bronchoalveolar lavage fluid was the first successful method in the time course of diagnosis.

Estudio comparativo de las técnicas de Machado Guerreiro y Elisa en el diagnóstico de infección chagásica / Compartive assay of Machado Guerreiro and Elisa techniques in diagnosis of chagas disease

Se procesaron 241 muestras de donantes voluntarios del banco de Sangre del Estado Zulia de sexo masculino y femenino, en edades comprendidas entre 18 y 30 años; a cada una de las cuales se le practicaron las pruebas de Elisa realizadas en el IVIC utilizando cepas puras del antígeno de T.cruzi, ELISA utilizando un kit comercial y Machado Guerreiro. Ambas técnicas realizadas en el Banco de Sangre del Estado Zulia. De los resultados obtenidos, con la técnica de Machado Guerreiro, 7 sueros resultaron positivos y 234 negativos, con ELISA (Banco de Sangre) 21 positivos y 220 negativos; con ELISA (IVIC) 32 positivos y 209 negativos. La comparación de Machado Guerreiro con ambas ELISA empleando el método de análisis Ji-Cuadrado modificado por Brand-Snedecor, reveló que las diferencias observadas entre los resultados de las técnicas fueron significativas. Evaluando los resultados ELISA (Banco de Sangre) y ELISA (IVIC) se demostró que no hubo diferencias significativas entre ambas pruebas. Los resultados de este estudio demostraron que ELISA es más sensible que Machado Guerreiro ya que detectó cantidades mínimas de anticuerpos específicos para antígenos chagásicos que no fueron detectados por Machado Guerreiro. El uso de la técnica ELISA amplía el rango de seguridad para las transfunsiones sanguíneas en relación a pacientes con infección chagásica

Value of DNA analysis in addition to cytological testing in the diagnosis of malignant pleural effusions.

BACKGROUND: Aneuploidy appears to be a highly specific marker for cancer, and measurement of cellular DNA content by flow cytometry is rapid and reliable. This study was undertaken to determine if the addition of DNA analysis improved the sensitivity of cytological diagnosis of malignancy in pleural fluid. METHODS: Pleural effusions from 92 patients were studied by cytological examination and flow cytometry. RESULTS: In 41 patients the final diagnosis was malignancy, there were 40 cases of benign effusions including 22 with pleural tuberculosis, and in the remaining 11 patients with biopsy proven cancer the presence of malignant cells was not found by cytological and histological means in the pleural fluid. Aneuploidy and cytological malignancy were found in 14 samples. There were seven cases with abnormal flow cytometry and negative cytological results. In 12 patients the cytological test results were positive but DNA analysis was normal. Thirty six samples of fluid were both diploid and cytologically negative. Of the 22 tuberculous effusions seven contained aneuploid cells. The sensitivity of DNA and cytological analysis was 51.2% and 63.4%, respectively. The specificity of DNA analysis was 74.5%. CONCLUSIONS: DNA analysis of cells in malignant pleural effusions is both less sensitive and specific than the cytological diagnosis. Flow cytometric analysis is not recommended for routine use in the diagnosis of pleural effusions.