Desenvolvimento e validação de controle de qualidade interno in house para quantificação de células progenitoras hematopoéticas CD34+/CD45+.
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Data
2020-02-21
Autores
Rocha, Francielle Ramalho
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O sistema de qualidade é de suma importância em laboratórios clínicos para avaliação de processos analíticos de maneira que os resultados liberados sejam verdadeiros. Para a metodologia de imunofenotipagem celular por citometria de fluxo as amostras devem ser frescas e os exames realizados preferencialmente dentro de 48 horas. É relevante utilizar amostras de controle de qualidade internos (CQI) padronizadas, de modo que possam ser repetidas rotineiramente, como referencial de qualidade. No Brasil, poucos serviços comercializam amostras preservadas para uso como CQI. Deste modo, a padronização in house com validação de processo para obtenção de amostras que possam ser utilizadas para esta finalidade é relevante. O objetivo deste trabalho foi desenvolver controle de qualidade interno para as rotinas de quantificação de células progenitoras hematopoéticas (CPH), utilizando solução preservante e avaliar a reprodutibilidade e estabilidade ao longo do tempo. Foram preparadas soluções preservantes contendo diferentes concentrações de anticoagulantes e fixadores, e destas, foi selecionada uma composição, originalmente padronizada neste estudo. Foram utilizados 5mL de sangue periférico, sendo este acrescido da solução a ser testada. Imediatamente, realizou-se a quantificação das populações de CPH em tubo Trucount®, usando anti-CD45, anti-CD34 e 7-AAD, conforme indicado pelo fabricante. A leitura foi realizada em citômetro de fluxo modelo FACSCalibur®-BD, para obtenção dos valores absolutos das CPH no dia zero, 7, 21, 35 e 49. Durante este período, as amostras foram mantidas refrigeradas (2 a 8ºC). Os valores obtidos no dia zero foram definidos como referencial e fixou-se o percentual de variação aceitável em ±40%, o mesmo aceito nos CQIs comerciais. Ao avaliar o desempenho das amostras preservadas durante o período preconizado, observou-se eficiente preservação celular, com distribuição estável e boa reprodutibilidade, tendo as variações limitadas ao intervalo preconizado. O produto gerado trata-se de uma solução preservante/conservante exclusiva, utilizada como CQI, passível de registro ou patente, validada para uso em imunofenotipagem de CPHs para fins de transplante autólogo, com desempenho semelhante ao de um controle de qualidade comercialmente disponível.
The quality system is of paramount importance in clinical laboratories for evaluating analytical processes in order to consider true the released results. The samples must be performed fresh preferably within 48 hours for the cell immunophenotyping methodology by flow cytometry. It is relevant to use standardized internal quality control (IQC) samples, thus they could be repeated routinely, as a quality benchmark. In Brazil, only a few services commercialize preserved samples for use as IQC. Therefore, it is relevant to use in-house standardization with process validation to obtain samples that can be used for this purpose. The objective of this work was to develop an IQC for a daily routine quantification of hematopoietic stem cells (HSCs) by using a preservative solution and to evaluate the reproducibility and stability over time. Preservative solutions containing different concentrations of anticoagulants and fixatives were prepared, and from these, a composition was selected, which was previously originally standardized in this study. 5mL of peripheral blood were used, which was added to the solution to be tested. The HSCs populations were immediately quantified in a Trucount® tube, using anti-CD45, anti-CD34 and 7-AAD, as indicated by the manufacturer. The reading was performed in a flow cytometer model FACSCalibur®-BD in order to obtain the absolute values of HSCs on day zero, 7, 21, 35 and 49. During this period, the samples were kept refrigerated (2 to 8ºC). The values obtained on day zero were defined as a reference and the percentage of acceptable variation was set at ± 40%, the same accepted in commercial IQCs. It was observed an efficient cell preservation when evaluating the performance of samples preserved during the recommended period. In addition, it was noted a stable distribution and significant reproducibility, with variations being limited to the recommended interval. The generated product is an exclusive preservative/preservative solution, which was used as an IQC. Moreover, this finding is subject to be registrated or patented and validated for use in HSCs immunophenotyping for autologous transplantation purposes, with similar performance to the current commercially available quality control.
The quality system is of paramount importance in clinical laboratories for evaluating analytical processes in order to consider true the released results. The samples must be performed fresh preferably within 48 hours for the cell immunophenotyping methodology by flow cytometry. It is relevant to use standardized internal quality control (IQC) samples, thus they could be repeated routinely, as a quality benchmark. In Brazil, only a few services commercialize preserved samples for use as IQC. Therefore, it is relevant to use in-house standardization with process validation to obtain samples that can be used for this purpose. The objective of this work was to develop an IQC for a daily routine quantification of hematopoietic stem cells (HSCs) by using a preservative solution and to evaluate the reproducibility and stability over time. Preservative solutions containing different concentrations of anticoagulants and fixatives were prepared, and from these, a composition was selected, which was previously originally standardized in this study. 5mL of peripheral blood were used, which was added to the solution to be tested. The HSCs populations were immediately quantified in a Trucount® tube, using anti-CD45, anti-CD34 and 7-AAD, as indicated by the manufacturer. The reading was performed in a flow cytometer model FACSCalibur®-BD in order to obtain the absolute values of HSCs on day zero, 7, 21, 35 and 49. During this period, the samples were kept refrigerated (2 to 8ºC). The values obtained on day zero were defined as a reference and the percentage of acceptable variation was set at ± 40%, the same accepted in commercial IQCs. It was observed an efficient cell preservation when evaluating the performance of samples preserved during the recommended period. In addition, it was noted a stable distribution and significant reproducibility, with variations being limited to the recommended interval. The generated product is an exclusive preservative/preservative solution, which was used as an IQC. Moreover, this finding is subject to be registrated or patented and validated for use in HSCs immunophenotyping for autologous transplantation purposes, with similar performance to the current commercially available quality control.
Descrição
Palavras-chave
Controle de Qualidade Interno, Célula progenitora hematopoética, Citometria de fluxo, Solução preservante, Internal quality control, Hematopoietic progenitor cell, Flow cytometry, Preservative solution