Abstract
Introduction. The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks.Objective. Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue.Materials and methods. Method validation was carried out by quantitative estimation of βIII-tubulin (TUBB3) expression in single-cell suspensions of non-small-cell lung cancer obtained from surgical tumor samples. Primary antibodies to TUBB3 (ab7751) and secondary DyLight 650-conjugated antibodies (ab98729) were used for immunofluorescent staining. The «Navios» flow cytometer (Beckman Coulter) was used to measure the fluorescence. The validation parameters were assessed by the coefficient of variation calculated as the ratio of standard deviation of TUBB3 level to its mean value.Results. Two parameters were analyzed: intra-assay precision and time stability of the results of the TUBB3 expression assessment. It was demonstrated that the mean coefficients of variation of the marker expression level in the tumor tissue did not exceed 20 % for both parameters. According to recommendations on the analytical validation of methods based on flow cytometry, it proves the validity of the method for these parameters.Conclusions. The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors. The practical value of the time stability of immunofluorescence stain during 24 h storage of a stained cells suspension in the dark at 4 °C was highlighted. It shows the possibility of adjusting the time interval between completion of the analytical study part and flow cytometer measurement.
Highlights
The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks
Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue
The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors
Summary
Цель исследования – аналитическая валидация разработанного авторами иммунофлуоресцентного метода, ассоциированного с проточной цитометрией, для изучения опухолевых белковых маркёров в ткани сóлидных новообразований. Валидация метода проведена при количественной оценке экспрессии белка βIII-тубулина (TUBB3) в суспензиях клеток немелкоклеточного рака легкого, полученных из хирургических образцов опухоли. Для оценки валидационных параметров использован коэффициент вариации, рассчитанный как отношение среднеквадратического отклонения уровня TUBB3 к его среднему значению. Проведен анализ 2 параметров: сходимости и временнóй стабильности результатов оценки экспрессии TUBB3. Что средний коэффициент вариации уровня экспрессии исследованного маркёра в ткани опухоли при оценке сходимости и стабильности иммунофлуоресцентной окраски не превысил 20 %. Показана сходимость и временнáя стабильность результатов количественной оценки экспрессии прогностически и предиктивно значимого белка TUBB3 в ткани сóлидных опухолей при использовании разработанного авторами метода иммунофлуоресцентного окрашивания, ассоциированного с проточной цитометрией. Сходимость и временнáя стабильность количественной оценки экспрессии белка βIII-тубулина в ткани сóлидных опухолей. Lomonosov Moscow State University; 1 Leninskie Gory, Moscow 119991, Russia; 3Sechenov First Moscow State Medical University, Ministry of Health of Russia; Build 2, 8 Trubetskaya St., Moscow 119991, Russia
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