THE INTRA-ASSAY PRECISION AND TIME STABILITY OF QUANTITATIVE βIII-TUBULIN EXPRESSION ASSESSMENT IN SOLID TUMOR TISSUE

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.

1. Gown A.M. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol 2008;(21Suppl 2):8–15. DOI: 10.1038/modpathol.2008.34.

2. Bogush T.A., Kaliuzhny S.A., Basharina A.A. et al. Immunofluorescence Analysis of the Vimentin Expression in Epithelial Cells. Moscow Univ Chem Bull 2019;74(6):290–5. DOI: 10.3103/S0027131419060063.

3. Friboulet L., Olaussen K.A., Pignon J.P. et al. ERCC1 isoform expression and DNA repair in non-small-cell lung cancer. N Engl J Med 2013;368(12):1101–10. DOI: 10.1056/nejmoa1214271.

4. Bogush T.A., Shaturova A.S., Dudko E.A. et al. Quantitative immunofluorescent estimation of estrogen receptor β expression in human solid tumors using flow cytometry. Moscow Univ Chem Bull 2011;66:305–12. DOI: 10.3103/S0027131411040031.

5. Mamichev I.A., Bogush T.A., Bogush E.A. et al. Microtubule protein βIII-tubulin: structure, expression and functions in normal and tumor cells. Antibiotiki i khimioterapiya = Antibiotics and Chemotherapy 2018;63(7–8):79–90. (In Russ.).

6. Selliah N., Eck S., Green C. et al. Flow cytometry method validation protocols. Curr Protoc Cytom 2019;87(1):53. DOI: 10.1002/cpcy.53.

7. Brown L., Green C., Jones N. et al. Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development. J Immunol Methods 2015;418:1–8. DOI: 10.1016/j.jim.2015.01.008.

8. Wood B., Jevremovic D., Bene M.C. et al. Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS – part V – assay performance criteria. Cytometry B Clin Cytom 2013;84(5):315–23. DOI: 10.1002/cyto.b.21108.