Classification and clinical behavior of blastic plasmacytoid dendritic cell neoplasms according to their maturation-associated immunophenotypic profile.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of leukemia/lymphoma, whose diagnosis can be difficult to achieve due to its clinical and biological heterogeneity, as well as its overlapping features with other hematologic malignancies. In this study we investigated whether the association between the maturational stage of tumor cells and the clinico-biological and prognostic features of the disease, based on the analysis of 46 BPDCN cases classified into three maturation-associated subgroups on immunophenotypic grounds. Our results show that blasts from cases with an immature plasmacytoid dendritic cell (pDC) phenotype exhibit an uncommon CD56- phenotype, coexisting with CD34+ non-pDC tumor cells, typically in the absence of extramedullary (e.g. skin) disease at presentation. Conversely, patients with a more mature blast cell phenotype more frequently displayed skin/extramedullary involvement and spread into secondary lymphoid tissues. Despite the dismal outcome, acute lymphoblastic leukemia-type therapy (with central nervous system prophylaxis) and/or allogeneic stem cell transplantation appeared to be the only effective therapies. Overall, our findings indicate that the maturational profile of pDC blasts in BPDCN is highly heterogeneous and translates into a wide clinical spectrum -from acute leukemia to mature lymphoma-like behavior-, which may also lead to variable diagnosis and treatment.

Primer caso de hemoglobina Koln (codon98 GTC mayor ATG) en Costa Rica / First case of hemoglobin Koln (codon98 GTC mayor ATG) in Costa Rica

La exposición a ciertos medicamentos puede provocar anemia hemolítica con presencia de cuerpos de Heinz en sangre periférica. Esta anemia puede presentarse por sobredosis de medicamentos, tanto en individuos sanos como en personas con deficiencias enzimáticas como la glucosa-6-fosfato deshidrogenasa, o en presencia de hemoglobinas inestables. Este reporte muestra un caso de anemia hemolítica con cuerpos de Heinz, debido a la presencia de una hemoglobina inestable, cuyos estudios moleculares y HPLC confirmaron el primer caso descrito de hemoglobina Koln (Val98Met) en Costa Rica.

Exposure to certain drugs may result in hemolytic anemia with the appearance of Heinz-bodies in red blood cells. Thistype of hemolytic anemia may occur by simple drug overdosage in absence of any known abnormality, such as innormal persons or in patients with erithroid enzyme defects (e.g. G6PD deficiency) or unstable hemoglobins.1 The present report shows a Heinz-body hemolytic anemia because of an abnormal hemoglobin. High pressure liquid chromatography (HPLC) and molecular analysis confirmed the first case of Hemoglobin Köln in Costa Rica.

Molecular and epidemiologic findings of childhood acute leukemia in Costa Rica.

In Central America, nearly 70% of pediatric cancer is related to hemato-oncologic disorders, especially acute lymphoblastic leukemia (ALL). Preliminary studies have described a high incidence of childhood leukemia in these countries; however, no molecular analyses of these malignancies have yet been carried out. We studied diagnostic samples from 84 patients from the National Children's Hospital in San Jose, Costa Rica (65 precursor B-ALL, 5 T-cell ALL, and 14 acute myeloblastic leukemia). Our methodology included cytogenetic, fluorescent in situ hybridization, and polymerase chain reaction approaches. The observed rate of leukemia was 52.2 cases per million children per year. Twelve out of 65 (18.4%) precursor B-ALL tested positive for TEL-AML1 and 3 cases for BCR-ABL (4.6%). In addition, we detected 2 patients carrying an E2A-PBX1 transcript (3.1%) and 1 patient with an MLL-AF4 fusion gene (1.5%). None of the T-cell ALL cases were positive for either SIL-TAL1 or HOX11L2. Within 14 acute myeloblastic leukemia patients, we confirmed 2 cases with FLT3-internal tandem duplication+, 1 patient with AML1-ETO, and only 1 case carrying a PML-RARalpha rearrangement. The present study confirms the relatively high incidence of pediatric leukemia in Costa Rica and constitutes the first report regarding the incidence of the main molecular alterations of childhood leukemia in our region.

Susceptibilidad de trypanosoma cruzi a diferentes venenos de serpientes de Costa Rica / Susceptibility of Trypanosoma cruzi to different Costa Rican snake venoms

Se evaluaron los efectos tóxicos de los venenos de cinco serpientes costarricenses en cuanto a su capacidad tripanocida contra dos cepas de trypanosoma cruzi y sus efectos en cuanto a los mecanismos de muerte celular. Los venenos de bothrops asper, bothriechis schlegelii, crotalus durissus durissus, atropoides nummifer y A. picadoi, mostraron actividad tripanocida contra las formas de epimastigoto, amastigoto y tripomastigoto. Los venenos de b. asper y de A. nummifer presentaron la más alta citotoxicidad para las células Vero. Los de b. asper y b. schlegelii presentaron la más alta actividad en los epimastigotos de la cepa CL, mientras que los venenos de b. asper y el de A. nummifer fueron más eficientes contra los epimastigotos de la cepa Jennifer. El veneno de b. schlegelii produce un efecto proliferativo en las células Vero; mientras que el de C. d. durissus produce el mismo efecto en los epimastigotos de la cepa CL, ambos a la concentración de 2,5 Ig/mL. Los valores de CI50 mostraron que se requieren menores cantidades contra los amastigotos en relación con los epimastigotos. Los venenos de b. asper y B. schlegelii presentan la más alta actividad contra los amastigotos de ambas cepas. Con los tripomastigotos sanguíneos de la cepa GA, los cinco venenos ocasionaron una disminución de la motilidad en los diferentes tiempos de exposición, pero el veneno de A. nummifer, en las concentraciones más bajas, mostró una actividad más marcada en comparación con los otros veneno. En cuanto a los efectos de los venenos, mediados por los grados de apoptosis, necrosis o proliferación celular, se observó que estos fenémenos se presentan y tienen relación con el tipo de veneno, su concentración y el tiempo de exposición.

The trypanocide effect of venoms from five Costa Rican species of snakes was evaluated against two strains of trypanosoma cruzi and their cellular toxic effects were likewise observed. The venoms of Bothrops asper, bothriechis schlegelii, crotalus durissus durissus, atropoide nummifer and A. picadoi showed evident trypanocide action against epimastigotes, amastigotes and trypomastigotes. The venoms of b. asper and b. schlegelii were shown to be the most active against the epimastigotes of the CL strain, whereas those of b. asper and A. nummifer were more effective against the epimastigotes of the Jennifer strain. The venoms of b. schlegelii and C.d. durissus, at the lowest concentrations of 2.5 Ig/mL, were able to trigger a proliferative effect on Vero cells and epimastigotes of the CL strain, respectively. The IC50 values showed that lower amounts of venoms are necessary in order to inhibit amastigotes as compared to epimastigotes. The venoms of b. asper and b. schlegelii exhibited the highest activity against amastigotes of both T. cruzi strains. All venoms were able to arrest motility of blood trypomastigotes of the GA strain at different times and the most active in this case was A. nummifer venom. The toxic effects of the venoms measured by the degree of apoptosis, necrosis and cell proliferation that they produced showed that all these events occur and are related to the type of venom, its concentration and exposure time.

Detección molecular del gen BCR-ABL por RT-PCR en niños costarricenses con leucemia

Molecular detection of the BCR-ABL gen by RT-PCR In Costa Rican children with leukemia. Many leukemias could have chromosomic translocations and according to the transcripts formed by the genes involved, the patients present an specific phenotype of the leukemia. We show the first results of the investigation of the gen BCR-ABL using RT-PCR, in order to look for the t(9;22)(q34;q11) in pediatric leukemic children. We studied in total 55 patients, 6 (10.9%) of them were positive for that translocation. Two (3.63%) of the positive children had ALL and the other 4 (7.27%) presented CML, the genotyping analysis of the transcript was studied in these children. With the introduction of this methodology as part of the routine studies, the leukemic children could get in the future an specific diagnosis, that will be important to classify them in prognostic categories and to improve the detection of minimal residual disease. Rev. Biol. Trop. 56 (4): 1613-1618. Epub 2008 December 12.

Muchas leucemias pueden presentar traslocaciones cromosómicas, las cuales, de acuerdo a los transcriptos formados por los genes involucrados, originará un fenotipo leucémica variable. En este trabajo se muestran los primeros resultados de pacientes pediátricos con leucemia, a los cuales se les hizo el estudio molecular por RT-PCR y el genotipaje para el gen BCR-ABL producto de la t(9;22)(q34;q11). De las 55 muestras estudiadas, 6 (10.9%) fueron positivas para el transcripto mencionado. De las 6 positivas, 2(3.63%) de esos pacientes tenían LLA y 4 (7.27%) eran LMC. La introducción de esta metodología en el manejo rutinario de los niños con leucemia, servirá para establecer un diagnóstico más preciso, un pronóstico más certero y un seguimiento adecuado de la enfermedad mínima residual.

[Molecular detection of the BCR-ABL gen by RT-PCR in Costa Rican children with leukemia]. / Detección molecular del gen BCR-ABL por RT-PCR en niños costarricenses con leucemia.

Many leukemias could have chromosomic translocations and according to the transcripts formed by the genes involved, the patients present an specific phenotype of the leukemia. We show the first results of the investigation of the gen BCR-ABL using RT-PCR, in order to look for the t(9;22)(q34;q11) in pediatric leukemic children. We studied in total 55 patients, 6 (10.9%) of them were positive for that translocation. Two (3.63%) of the positive children had ALL and the other 4 (7.27%) presented CML, the genotyping analysis of the transcript was studied in these children. With the introduction of this methodology as part of the routine studies, the leukemic children could get in the future an specific diagnosis, that will be important to classify them in prognostic categories and to improve the detection of minimal residual disease.

Biopsia por aspiración con aguja fina en el Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera" / Fine needle aspiration biopsy at the National Children’s Hospital "Dr. Carlos Sáenz Herrera

Objetivo: Evaluar y describir la experiencia con la biopsia por aspiración con aguja fina (BAAF) en el Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera" (HNN) de San José, Costa Rica durante los primeros 21 meses de funcionamiento de dicho servicio. Métodos: Estudio observacional y retrospectivo del material de BAAF obtenido en el HNN desde octubre de 2004 a junio de 2006 (extendidos del material, inmunofenotipo, citogenética, cultivos microbiológicos). En aquellos casos en que se realizó cirugía, se hizo correlación entre material de biopsias convencionales anteriores o posteriores y la BAAF. La evolución de los pacientes se obtuvo por medio del expediente clínico. Resultados: Se realizó un total de 142 diagnósticos en 132 pacientes. La clasificación de dichos diagnósticos fue: benigno 86 (61%), maligno 36 (25%), sospechoso 5 (3.5%), atípico 5 (3.5%); se obtuvo material insuficiente para diagnóstico en 10 (7%) de las BAAF. De los diagnósticos benignos 74 (86%) consistieron en linfadenopatía reactiva-infecciosa. Los diagnósticos malignos fueron variados, siendo el linfoma el más frecuente 14 (39%); con el linfoma de Hodgkin como el mas común 9 (25%), detectándose tanto enfermedad de novo como recurrencias. Se determino el inmunofenotipo por citometría de flujo en 17 (12%) casos y se realizó cariotipo en 2 (1.4%) casos. Conclusiones: La BAAF es un procedimiento seguro, rápido y eficaz en el diagnóstico de múltiples patologías de la población pediátrica atendida en HNN. Los dos escenarios clínicos más frecuentes en que la BAAF cumplió un rol importante fueron el diagnóstico de recurrencia del linfoma de Hodgkin y el tamizaje de linfadenopatías en niños sin historia de malignidad. En algunos casos la BAAF hace innecesaria la realización de una biopsia convencional.

Aim: To evaluate the performance of Fine Needle Aspiration Biopsy (FNAB) and describe the experience at Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera" (HNN) in San José, Costa Rica during the first 21 months since introduction of this procedure. Methods: Retrospective review of all FNAB material collected at HNN from October 2004 to June 2006 (glass slides, flow cytometric analyses, cytogenetics, and cultures). When available, previous or later surgical biopsy material was also reviewed and results were correlated with FNAB. Patient followup was assessed from clinical charts. Results: A total of 142 diagnoses in 132 patients were performed. These were classified as follows: benign 86 (61%), malignant 36 (25%), suspicious 5 (3.5%), atypical 4 (3.5%). Material insufficient for diagnosis was obtained in 10 (7%) cases. Reactive-infectious lymphadenopathy comprised 74 (86%) of benign diagnoses. Most common malignant diagnosis was lymphoma. New cases and recurrences of Hodgkin’s disease were the most common specific malignancy 9 (25%) detected. Flow cytometric analysis was performed in 17 (12%) of the cases and conventional cytogenetics in 2 (1.4%). Conclusions: FNAB demonstrated to be a fast, safe and efficient diagnostic tool at HNN. Diagnosis of recurrence of Hodgkin’s lymphoma and screening of lymphadenopathies in children without history of malignancy were the two most common clinical scenarios where FNAB played an important role. FNAB can obviate the need for surgical intervention in some cases.

Calcium plays a key role in the effects induced by a snake venom Lys49 phospholipase A2 homologue on a lymphoblastoid cell line.

A catalytically-inactive Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper induces diverse effects (necrosis, apoptosis and proliferation) in a lymphoblastoid cell line, depending on the toxin concentration. The increments in cytosolic Ca2+ levels induced by this toxin in this cell line were assessed. At high toxin concentration (100 microg/mL) the toxin induces drastic disruption of the plasma membrane, associated with a prominent Ca2+ influx and necrosis. Previous incubation of the cells with the chelating agent EGTA or with ruthenium red, an inhibitor of the uniporter mitochondrial Ca2+ transport, greatly reduced necrosis. At a toxin concentration of 12.5 microg/mL, apoptosis is the predominant response, being associated with lower increments in cytosolic Ca2+. This effect was inhibited by preincubation with ruthenium red and the cytosolic Ca2+ chelator BAPTA-AM. The proliferative response, which occurs at a low toxin concentration (0.5 microg/mL), is associated with a small and oscillatory increment in cytosolic Ca2+. It was inhibited by EGTA, ruthenium red and BAPTA-AM, by inhibitors of the endoplasmic reticulum Ca2+ -ATPase (SERCA) and by blockade of the ryanodine receptor. It is concluded that necrosis and apoptosis induced by this toxin are associated with increments in cytosolic Ca2+ levels following plasma membrane perturbation, together with the involvement of mitochondria. The cellular proliferative response depends on a limited Ca2+ influx through the plasma membrane, being associated with a concerted functional unit constituted by SERCA, the ryanodine receptor and mitochondria, which regulate the observed oscillations in cytosolic Ca2+ concentration.

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2.

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 microM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase-8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteases.

A Lys49 phospholipase A(2) homologue from Bothrops asper snake venom induces proliferation, apoptosis and necrosis in a lymphoblastoid cell line.

Lys49 phospholipase A(2) homologues are abundant in viperid snake venoms. These proteins have substitutions at the calcium-binding loop and catalytic center which render them enzymatically inactive; however, they display a series of toxic activities, particularly cytotoxicity upon various cell lines in vitro. In this study we explored whether myotoxin II (MT-II), a Lys49 phospholipase A(2) homologue from the venom of the snake Bothrops asper, is capable of inducing various effects in a single cell type, using the lymphoblastoid B cell line CRL-8062 as a model. Cells were incubated with varying concentrations of MT-II for 24 and 48 h, time intervals that are more prolonged than the usual incubation times previously used in the characterization of this toxin. Results indicate that MT-II induces proliferation at low concentrations (0.5-5.0 microg/mL). Apoptosis was predominant at higher toxin levels (5-25 microg/mL), whereas necrosis, associated with overt plasma membrane disruption, occurred at concentrations > or =25 microg/mL, and was the predominant effect at higher MT-II concentrations (50 microg/mL). It is concluded that a single phospholipase A(2) homologue can induce markedly different effects on a single cell line, depending on the concentration used, an observation that may have implications for the action of this type of venom component in vivo.

Evaluación de parámetros plaquetarios en trombocitosis pediátrica / Evaluation of parametrics platelet in pediatric thrombocytosis

El hallazgo de un recuento plaquetario elevado tiene importantes implicaciones para el diagnóstico, pronóstico y tratamiento del paciente. En el presente estudio, se pudo comprobar la utilidad de los índices plaquetarios como el VPM, (volumen plaquetar medio ) y el PDW (rango de la distribución plaquearia), en unión del cómputo de plaquetas, para el diagnóstico de trombocitosis reactiva en pacientes pediátricos. Un 45 por ciento de los pacientes estudiados presentaron alteraciones en el VPM, en el PDW o en ambos, en comparacón con los valores que se obtuvieron en el grupo control. Se observó en que los niños con trombocitosis reactiva, tendían a presentar plaquetas de menor tamaño y con mayor heterogeneidadd. Es importante considerar estos parámetros disponibles actualmente, como una orientación diagnóstica de primera línea para las trombocitosis. (Rev Cost Cienc Med 1999; 20(3,4): 185-191) Palabras claves: plaquetas, volumen plaquetar, heterogeneidad plaquetaria, trombocitosis

Leucemia mielomonocítica crónica: una mielodisplasia singular. A propósito de un caso / Chronic myelomonocytic leukemia: a singular. Myelodysplasia a purpose of one case

En un paciente varón, hebreo, de 86 años de edad, con antecedentes de cardiopatía isquémica y portador de un carcinoma gástrico, se demostró la existencia de una leucemia mielominocítica crónica (LMMC) por citomorfología, citometría de flujo y citoquímica. La médula ósea fue muy sugestiva del trastorno por el hallazgo de megacariocitos hipoploides, sideroblastosen anillo e hipercelularidad. La evolución del paciente ha seguido el patrón esperable en una displasia refractaria del tipo que se informa.

Diferenciación inmunofenotípica de un sindrome linfoproliferativo crónico y linfoma periférico / Differentiation immunophenotyping of a chronic lymphoproliferative syndrome and peripheral lymphoma

Paciente de 80 años, a quien cuatro años atrás se le trató un linfoma gástrico de células grandes. En la visita actual mostró un hemograma básicamente normal, salvo ligera leucocitosis linfocítica, en la que un 50 por ciento de sus elementos eran de aproximadamente 15 un de diámetro y mostraban características prolinfocíticas de tipo neoplásico, por la presencia de un prominente nucleólo inserto en una matriz cromatínica de textura intermedia. A fin de aclarar la naturaleza del larvado proceso leucémico, se utilizaron técnicas citoquímicas (ANAE, CAE, PX y FAC), y un panel de Ac monoclonales para los estudios en el citómetro de flujo. Los resultados demostraron la índole linfosarcomatosa del proceso, el cual inmunofenotípicamente resultó ser un linfoma periférico de células tumorales B diferenciadas (CD5+/CD19+) tipo kappa