ZAP-70: practical aspects

ZAP-70 is a 70Kd protein kinase present in normal T Lymphocytes and natural killer cells (NK), but absent in normal B lymphocytes. Nevertheless, B cells in some B-cell Chronic Lymphocytic Leukemia (CLL) patients express ZAP-70 and it has been found to be associated with the unmutated status of immunoglobulin heavy chains (IgVH) genes, a strong prognostic factor in CLL. In addition, the ZAP-70 expression in CLL cells is stable over time, differently to the CD38 expression, another prognostic marker in CLL. Several studies have been performed to validate the ZAP-70 expression as a surrogate marker for the IgVH mutation status which uses a laborious and time consuming technique. Using monoclonal antibodies, anti-ZAP-70 is easily detected by a rapid and sensitive flow cytometric technique. Upstate Biotechnology developed the first unconjugated antibody (2F3.2 clone) and soon after other products were developed, including fluorochrome conjugated ones, facilitating the technical assays. ZAP-70 can be evaluated by flow cytometric methods and its expression predicts the prognosis in the great majority of CLL cases.A ZAP-70 é uma proteína kinase normalmente expressa nos linfócitos T e células NK, mas ausente nos linfócitos B. No entanto, ela está expressa nas células B de pacientes com LLC que não apresentam mutação nos genes da região variável da cadeia pesada de imunoglobulina (genes IgVH). Além disso, observa-se que a expressão da ZAP-70 é estável nestes pacientes ao longo do tempo, diferentemente da expressão de CD38, outro marcador considerado de prognóstico nesta doença. Estudos têm sido realizados para validá-la como marcador surrogate para o estado de mutação dos genes IgVH (um forte fator de prognóstico), uma vez que a sua avaliação molecular é laboriosa e demorada. O método mais rápido e simples para avaliar a expressão da ZAP-70 é através da citometria de fluxo utilizando-se anticorpos monoclonais já disponíveis no mercado. O primeiro produto comercial (clone 2F3.2 da marca Upstate) é puro, não conjugado com fluorocromos, o que torna o ensaio laborioso, apesar de resultados consistentes. Rapidamente surgiram outros Ac conjugados com fluorocromos (com FITC da própria Upstate, ou Alexa-fluor, FITC ou PE da Caltag) e estudos estão sendo realizados para a sua validação técnica.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Behavioral Alterations in Prairie Voles (Microtus ochrogaster) after Parent-Pup Separation

The prairie vole (Microtus ochrogaster), a highly social species, offers a unique opportunity to examine the effects of parent-pup separation in a biparental family system similar to humans. We hypothesized that 1) repeated separation from pups affects parental behavior and emotionality in parents, and 2) neonatal parental separation affects emotional and physiological development in pups, and thus induces altered adult parental, emotional, and social behaviors. During postnatal day (PND) 1-10, pups were removed from their parents for 0, 15, or 360 min and housed either individually or with siblings. Unhandled controls experienced only daily lid opening. Tests for parental responsiveness and emotionality were conducted on PND11 for parents and PND90-92 for their offspring. Emotionality tests included the elevated plus maze, open field, and forced swim tests. Starting at PND150, half of each litter was paired with an opposite-sex vole for 24 hours and tested for partner preference. Additionally, behavioral response to stress was measured in all animals 0, 30, or 60 min after exposure to a forced swim. Generally, the behavior of the parents and adult offspring was influenced by daily handling, the length of the separation, and presence of siblings. Parental behaviors in parents did not differ among groups, while their anxiety- and depression-like behaviors were influenced by pup separation. For the adult offspring, separation treatment altered parental behavior, emotionality, partner preference, and stress response. Our results demonstrated that parent-pup separation affects emotional and social behaviors in prairie vole parents and adult offspring

Multiple myeloma with cells typically seen in storage diseases

We report on a rare case of multiple myeloma with atypically large cells containing a great amount of azurophilic inclusions usually seen in storage diseases.Universidade Federal de São Paulo (UNIFESP) Hematology and Transfusion Medicine DepartmentHospital Alemão Oswaldo Cruz CICAP LaboratoryUNIFESP, Hematology and Transfusion Medicine DepartmentSciEL

Multiples aberrant phenotypes in multiple myeloma patient expressing CD56-, CD28+,CD19+

Universidade Federal de São Paulo (UNIFESP)Universidade Federal de PernambucoUNIFESPSciEL

Combined method for simultaneous morphology, immunophenotype and karyotype (MAC) in leukemias

In the present study, a combined method (CM) for attaining simultaneous identification of leukemic cell morphology, karyotype and immunophenotype has been evaluated in 21 patients with acute leukemia and 1 with CML in blast crisis were studied for morphology, citochemistry, immunophenotype and karyotype. Karyotype was performed in a bone marrow sample by using conventional techniques. In each case, direct method (DM) and/or three cultures were tried. The CM consisted in separating a small part of the material resulting from any of the cultures or DM, preparing slides through cytospin and immunophenotyping through APAAP method using the same monoclonal antibodies (MoAb) as for diagnosis. In 14 cases, the metaphases proved positive to the MoAb: in 4, the cells with abnormality had their origin defined; in other 4 the karyotype was normal preventing any identification; 6 cases had minimal abnormalities not visible through CM; and in two cases abnormal karyotypes were detected only in the cultures with GM-CSF. This study showed that CM is feasible in cases where evident numerical or structural chromosomal abnormalties are present.Este trabalho avaliou um método combinado (MC) para identificação simultânea da morfologia, cariótipo e imunofenótipo da célula leucêmica. Foram estudados 21 pacientes com leucemia aguda e 1 com LMC em crise blástica através da morfologia, citoquimica, imunofenotipagem e citogenética. O cariótipo foi feito em material proveniente da medula óssea pelas técnicas convencionais e para cada caso, foi feito o método direto (MD) e/ou três culturas. O MC consistia em separar pequena parte do material de qualquer cultura ou MD, preparar as laminas através de citocentrífuga e fazer a imunofenotipagem pelo método do APAAP com os mesmos anticorpos monoclonais (AcMo) usados ao diagnóstico. Em 14 casos as metáfases mostraram positividade para os AcMo: em 4 as células com anormalidades tiveram sua origem definida, em 4 casos o cariótipo era normal impedindo identificações e 6 tinham aberrações mínimas impossíveis de serem vistas através do MC. Em dois casos o cariótipo anormal foi encontrado apenas nas culturas com GM-CSF. O estudo mostrou que o MC é válido para casos com alterações cromossômicas numéricas ou estruturais evidentes.Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM)UNIFESP, EPMSciEL

Toxic epidermal necrolysis as adverse effect of intermediate dose of cytosine arabinoside

Toxic epidermal necrolysis is a drug-induced dermatologic disease related to Lyell syndrome, erythema multiforme and Stevens-Johnson syndrome. PURPOSE: To report a fatal case of toxic epidermal necrolysis owing to intermediate dose of cytarabine. CASE REPORT: A 16 year-old female patient with acute lymphocytic leukemia (LLA-L1) treated with the Protocol of the Brazilian Group for Treatment of Leukemia of Childwood (GBTLI-85-AR). On the second day after the administration of intermediate dose of cytarabine (1.5g/m² IV every 12 hours for 3 days), she presented bullous lesions in the left buttock that disseminated envolving to necrosis, sepsis, and death on the 13th day. CONCLUSION: Cytarabine is frequently associated with dermatologic toxicity but, until now, there is no other case of toxic epidermal necrolysis described.A necrólise epidérmica tóxica é afecção dermatológica secundária ao uso de drogas e corresponde à síndrome de Lyell, relacionada ao eritema multiforme e à síndrome de Stevens-Johnson. OBJETIVOS: Relatar um caso de necrólise epidérmica fatal secundária à citosina-arabinosídeo (Ara-C) em dose intermediária. RELATO DE CASO: Paciente do sexo feminino, com 16 anos de idade, portadora de leucemia linfóide aguda - LLA-L1. Iniciou tratamento segundo o protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil/85, alto risco. Na fase II da indução, após o uso de Ara-C na dose de 1,5g/m², intravenoso, 12/12h x três dias, desenvolveu múltiplas lesões cutâneas bolhosas, que aumentaram rapidamente por progressão das bordas. As bolhas continham secreção serosa, evoluíram para ulceração superficial central, com infecção secundária múltipla. Faleceu por septicemia, no 13º dia após o início do quadro dermatológico. CONCLUSÃO: O Ara-C tem sido relacionado a diversas manifestações de toxicidade dermatológica; no entanto, até o momento, não há relato de necrólise epidérmica tóxica, sendo este o primeiro caso da literatura.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUNIFESP, EPM, Depto. de MedicinaSciEL

Septic arthritis as the first sign of Candida tropicalis fungaemia in an acute lymphoid leukemia patient

Fungal infections caused by Candida species have increased in incidence during the past two decades in England, North America and Europe. Candidal arthritis is rare in patients who are not intravenous drug users or are who not using a prostheses. We report the case of a 24-year-old man with acute lymphoid leukemia, who developed Candida tropicalis arthritis during an aplastic period after chemotherapy. This is the eighth case described in the literature of C. tropicalis causing arthritis without intra-articular inoculation. We call attention to an unusual first sign of fungal infection: septic arthritis without intra-articular inoculation. However, this case differs from the other seven, since despite therapy a fast and lethal evolution was observed. We reviewed reported cases, incidence, risk factors, mortality and treatment of neutropenic patients with fungal infections.Federal University of São PauloUNIFESPSciEL

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Disciplina de Hematologia e HemoterapiaUNIFESP, EPM, Disciplina de Hematologia e HemoterapiaSciEL