Comparison of ethylene carbonate and formamide as components of the hybridization mixture in FISH

The protocols for in situ hybridization (ISH) techniques can vary considerably; however, they usually include denaturation and hybridization steps. Denaturing compounds are used to reduce denaturation and hybridization temperature, which keeps the proper morphology of the preparation. Formamide is the most commonly used reagent in in situ hybridization to lower the melting temperature. The substitution of toxic formamide for a non-toxic ethylene carbonate at 20 % and 50% concentration in the hybridization mixture helped obtain a high quality in situ hybridization result with two sequences characteristic for rye, JNK, and Bilby. The results after hybridization, with a duration of 90 min and 16 h, were identical when formamide or ethylene carbonate were used in the mixture. In addition, the toxic formamide was eliminated from the post-hybridization steps and specific hybridization signals for both probes were still obtained.

Comparison of ethylene carbonate and formamide as components of the hybridization mixture in FISH

The protocols for in situ hybridization (ISH) techniques can vary considerably; however, they usually include denaturation and hybridization steps. Denaturing compounds are used to reduce denaturation and hybridization temperature, which keeps the proper morphology of the preparation. Formamide is the most commonly used reagent in in situ hybridization to lower the melting temperature. The substitution of toxic formamide for a non-toxic ethylene carbonate at 20 % and 50% concentration in the hybridization mixture helped obtain a high quality in situ hybridization result with two sequences characteristic for rye, JNK, and Bilby. The results after hybridization, with a duration of 90 min and 16 h, were identical when formamide or ethylene carbonate were used in the mixture. In addition, the toxic formamide was eliminated from the post-hybridization steps and specific hybridization signals for both probes were still obtained.(AU)

Guidelines for HER2 testing in breast cancer: a national consensus of the Spanish Society of Pathology (SEAP) and the Spanish Society of Medical Oncology (SEOM).

Identifying breast cancers with HER2 overexpression or amplification is critical as these usually imply the use of HER2-targeted therapies. DNA (amplification) and protein (overexpression) HER2 abnormalities usually occur simultaneously and both in situ hybridisation and immunohistochemistry may be accurate methods for the evaluation of these abnormalities. However, recent studies, including those conducted by the Association for Quality Assurance of the Spanish Society of Pathology, as well as the experience of a number of HER2 testing National Reference Centres have suggested the existence of serious reproducibility issues with both techniques. To address this issue, a joint committee from the Spanish Society of Pathology (SEAP) and the Spanish Society of Medical Oncology (SEOM) was established to review the HER2 testing guidelines. Consensus recommendations are based not only on the panellists' experience, but also on previous consensus guidelines from several countries, including the USA, the UK and Canada. These guidelines include the minimal requirements that pathology departments should fulfil in order to guarantee proper HER2 testing in breast cancer. Pathology laboratories not fulfilling these standards should make an effort to meet them and, until then, are highly encouraged to submit to reference laboratories breast cancer samples for which HER2 determination has clinical implications for the patients.

Determinação da infecção do papilomavírus humano por captura híbrida II: correlação com achados morfológicos / HPV infection detected by hibrid capture II: correlation with morphological findings

Introdução: o HPV é causa essencial, mas não suficiente, para o desenvolvimento de lesões pré-neoplásicas e neoplásicas do colo uterino. Sua identificação é fundamental para a avaliação de risco, sendo que o método de Captura híbrida apresenta reconhecida eficácia para esse fim. Por sua vez, os métodos morfológicos, colposcopia, citologia e histopatologia, demonstram as alterações celulares e tissulares sugestivas da ação desse vírus. Objetivo: comparar o desempenho dos métodos morlológicos, colposcopia, citologia e histopatologia, sugestivos de infecção pelo HPV, com o teste da Captura Híbrida II (CHII). Métodos: analisaram-se 2871 amostras cervicovaginais consecutivas de pacientes com suspeita de infecção pelo HPV em pelo menos um dos métodos morfológicos de diagnóstico. A coleta de amostra para o estudo biomolecular foi feita conforme as recomendações do fabricante, utilizando-se a escova cervical e o STM (specimen collection medium) como meio de preservação, tanto para o DNA quanto para o RNA. A CHII foi realizada utilizando-se sondas complementares de RNA para pool de vírus considerados de baixo risco (6,11,42,43 e 44) e para aqueles de alto risco oncogênico ( 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). A sensibilidade, a especificidade, a acurácia e os valores preditivosnegativos e positivos dos métodos morfológicos foram determinados considerando-se o método biomolecular como padrão-ouro. Resultados: em 1.761 casos com diagnóstico colposcópico sugestivo de infecção pelo HPV, não se identificou a presença de DNA viral. A citologia sugeriu corretamente HPV em 503 casos e a seguinte análise/porcentagem: especificidade-56, sensibilidade-62, acurácia-57,7, VPN-78,9 e VPP-35,7. O estudo histopatológico apresentou 709 casos com suspeita de alteração HPV-induzida confirmados pela determinação biomolecular, mostrando na análise estatística/porcentagem: especificidade-15,1, sensibilidade-87,4 , acurácia-35,6, VPN-75,4 e VPP-28,9. Juntos, os métodos morfológicos apresentaram os índices/porcentagem: sensibilidade - 94,1, sensibilidade- 10,3 e acurácia-34. Conclusão: entre os exames morfológicos, a citologia, apesar de baixa, mostrou o melhor desempenho. A sensibilidade aumenta com a associação dos métodos morfológicos, mas, com isso, a especificidade fica reduzida e observa-se grande o número de resultados falso-positivos. Por isso, a suspeita morfológica de infecção pelo HPV deve ser confirmada por método biomolecular.

[Genital human papillomavirus infection identification by molecular biology among asymptomatic women]. / Identificação do papilomavírus humano por biologia molecular em mulheres assintomáticas.

OBJECTIVE: To evaluate whether epidemiological factors may be associated to genital human papillomavirus (HPV) infection. METHODS: A cross-sectional study was carried out among 975 women seen at a public health service for cervical cancer screening in Porto Alegre, Brazil. Women were considered infected if tested positive to HPV either by Polymerase Chain Reaction (PCR) or Hybrid Capture II (HC-II) methods. Women with genital HPV infection were compared to women without infection drawn from the same population. RESULTS: The study enrolled 975 women. The HPV prevalence (both methods combined) in this population was 27%. However, when each diagnostic method is analyzed separately, HPV prevalence was 15% and 16% for HC-II and PCR, respectively. Unconditional multiple logistic regression was used to correlate disease status to women characteristics. A positive association was found with HPV infection for the following variables: years of schooling (11 years: OR=2.05; 95%CI =1.31; 3.20), married (OR=1.69; 95%CI=0.78; 2.00), number of lifetime sexual partners (2 partners: OR=1.67; 95%CI=1.01; 2.77; 4 or +: OR=2.18; 95%CI=1.15; 4.13), age at first intercourse (15-16 years: OR=4.05; 95%CI=0.89; 18.29). CONCLUSIONS: Various factors may contribute to genital HPV infection, especially those related to sexual behavior (young age at first intercourse, high number of lifetime sexual partners, and marital status), and those related to social and economic status (years of schooling).