Pre-analytical parameters associated with unsuccessful karyotyping in myeloid neoplasm: a study of 421 samples.

Cytogenetics is essential in myeloid neoplasms (MN) and pre-analytical variables are important for karyotyping. We assessed the relationship between pre-analytical variables (time from collection to sample processing, material type, sample cellularity, and diagnosis) and failures of karyotyping. Bone marrow (BM, n=352) and peripheral blood (PB, n=69) samples were analyzed from acute myeloid leukemia (n=113), myelodysplastic syndromes (n=73), myelodysplastic syndromes/myeloproliferative neoplasms (n=17), myeloproliferative neoplasms (n=137), and other with conclusive diagnosis (n=6), and reactive disorders/no conclusive diagnosis (n=75). The rate of unsuccessful karyotyping was 18.5% and was associated with the use of PB and a low number of nucleated cells (≤7×103/µL) in the sample. High and low cellularity in BM and high and low cellularity in PB samples showed no metaphases in 3.9, 39.7, 41.9, and 84.6% of cases, respectively. Collecting a good BM sample is the key for the success of karyotyping in MN and avoids the use of expensive molecular techniques.

Pre-analytical parameters associated with unsuccessful karyotyping in myeloid neoplasm: a study of 421 samples

Cytogenetics is essential in myeloid neoplasms (MN) and pre-analytical variables are important for karyotyping. We assessed the relationship between pre-analytical variables (time from collection to sample processing, material type, sample cellularity, and diagnosis) and failures of karyotyping. Bone marrow (BM, n=352) and peripheral blood (PB, n=69) samples were analyzed from acute myeloid leukemia (n=113), myelodysplastic syndromes (n=73), myelodysplastic syndromes/myeloproliferative neoplasms (n=17), myeloproliferative neoplasms (n=137), and other with conclusive diagnosis (n=6), and reactive disorders/no conclusive diagnosis (n=75). The rate of unsuccessful karyotyping was 18.5% and was associated with the use of PB and a low number of nucleated cells (≤7×103/µL) in the sample. High and low cellularity in BM and high and low cellularity in PB samples showed no metaphases in 3.9, 39.7, 41.9, and 84.6% of cases, respectively. Collecting a good BM sample is the key for the success of karyotyping in MN and avoids the use of expensive molecular techniques.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.(AU)

Leishmania braziliensis amastigotes stimulate production of IL-1ß, IL-6, IL-10 and TGF-ß by peripheral blood mononuclear cells from nonendemic area healthy residents.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1ß, IL-6, TGF-ß and IL-23 production, whereas IL-10 and TGF-ß are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1ß, IL-6 and TGF-ß. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

Multiple lymphoid nodules in bone marrow biopsy in immunocompetent patient with cytomegalovirus infection: an immunohistochemical analysis.

In Brazil, a high prevalence of cytomegalovirus (CMV) infection has been documented. In immunocompetent adults CMV infection is usually asymptomatic and therefore morphologic and immunophenotypic bone marrow changes have rarely been described. The authors report the case of a previously healthy patient who developed fever of undetermined origin. The diagnosis of acute CMV infection was based on serological testing. A computed tomographic scan showed mediastinal lymphadenopathy. A bone marrow biopsy revealed a hypercellular haematopoiesis with eosinophilia and large mixed T- and B-cell lymphoid aggregates. In spite of bcl-2 positivity, their reactive nature was demonstrated. Polymerase chain reaction (PCR) and immunohistochemistry were unable to detect CMV-DNA in paraffin-embedded bone marrow sections. Much like in other systemic disorders, the lymphoid nodules in this case seemed to be caused by immunological mechanisms, possibly due to cytokines released in response to the systemic infectious process.

Multiple lymphoid nodules in bone marrow biopsy in immunocompetent patient with cytomegalovirus infection: an immunohistochemical analysis

In Brazil, a high prevalence of cytomegalovirus (CMV) infection has been documented. In immunocompetent adults CMV infection is usually asymptomatic and therefore morphologic and immunophenotypic bone marrow changes have rarely been described. The authors report the case of a previously healthy patient who developed fever of undetermined origin. The diagnosis of acute CMV infection was based on serological testing. A computed tomographic scan showed mediastinal lymphadenopathy. A bone marrow biopsy revealed a hypercellular haematopoiesis with eosinophilia and large mixed T- and B-cell lymphoid aggregates. In spite of bcl-2 positivity, their reactive nature was demonstrated. Polymerase chain reaction (PCR) and immunohistochemistry were unable to detect CMV-DNA in paraffin-embedded bone marrow sections. Much like in other systemic disorders, the lymphoid nodules in this case seemed to be caused by immunological mechanisms, possibly due to cytokines released in response to the systemic infectious process.

Uma elevada prevalência de infecção pelo citomegalovírus (CMV) está documentada no Brasil. Em adultos imunocompetentes a infecção pelo CMV é, em geral, assintomática e, portanto, as alterações morfológicas e imunohistoquímicas na medula óssea têm sido raramente descritas. Relatamos o caso de um paciente previamente assintomático que desenvolveu febre de origem obscura. O diagnóstico de infecção aguda pelo CMV foi baseado em estudo sorológico. A tomografia computadorizada do tórax mostrou linfadenopatia mediastinal. A biópsia óssea revelou medula hipercelular com eosinofilia e grandes agregados linfóides mistos de células B e T. Apesar da positividade para bcl-2, a sua natureza reacional foi demostrada. A reação em cadeia da polimerase (PCR) e a imunohistoquímica não detectaram DNA viral nos cortes de medula óssea em parafina. Assim como em outros distúrbios sistêmicos, os nódulos linfóides no nosso caso parecem ser causados por mecanismos imunológicos, possivelmente relacionados a citoquinas liberadas em resposta ao processo infeccioso sistêmico.