Molecular fixative enables expression microarray analysis of microdissected clinical cervical specimens

Abstract

Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis.

Keywords: Cervical intraepithelial neoplasia; Gene expression microarray analysis; Immunohistochemistry; Microdissection; Molecular fixative.

Figure 1

Comparison between formalin (A, C) and molecular fixative (B, D) for H&E staining. The regions shown are benign (A, B) and CIN II (C, D) from case 0072. Scale bars are 20 μm. To get higher magnification than with the whole slide imager, these images were acquired using a QImaging MicroPublisher (Surrey, BC, Canada) camera mounted atop a regular brightfield transmission light microscope with a 60x immersion oil objective lens.

Figure 2

Comparison between formalin (A, B) and molecular fixative (C, D) for H&E (A, C) and CINtec immunohistochemical (B, D; brown = p16, red = Ki-67) staining in regions of CIN II in case 0072. Scale bars are 500 μm.

Figure 3

Comparison between formalin (A) and molecular fixative (B) for Ki-67 immunohistochemistry. The regions shown are benign from case 0072. IHC was performed using the CINtec kit as described in the text (red = Ki-67, brown = p16). Scale bars are 100 μm.

Figure 4

Example composite figure documenting microdissection of case 0028. A reference H&E section (A) is shown alongside photographs of an adjacent section before microdissection (B), after removing the epithelial top layer (C), after removing the epithelial bottom layer (D), and after removing the stroma (E). Microdissection was always performed in this order. See Supplementary Data for all other cases.

Figure 5

From left to right, 500 ng RNA each from one FFPE case (0008) and the two epithelial layers from an MFPE case (0043) run on a 0.8% agarose gel. The TrackIt 1Kb Plus DNA Ladder (Life Technologies) is on the right. In the MFPE samples, the 18S band appears distinctly, while the 28S band is less well-defined. The FFPE samples in comparison are completely degraded.

Figure 6

Plot of expression data from two aliquots of case 0043 basal layer. One aliquot had 200 ng RNA starting material in the labelling reaction (ordinate) while the other used only 50 ng (abscissa). Most data points lie along the linear regression line (R² = 0.96). Data from saturated spots have been removed (e.g., 200 ng > 2400).

Figure 7

Plot of expression data from two aliquots of case 0028 basal layer. One aliquot had 200 ng RNA starting material in the labelling reaction (ordinate) while the other used only 25 ng (abscissa). A distinct side branch in the data is visible, suggesting 25 ng may be too little starting material for reproducible data from MFPE tissue. Data from saturated spots have been removed.

Figure 8

Overlaid M-A plots of duplicate data for the two MFPE samples. Data have been adjusted so that the mean M is zero. The traces are manually fit double exponentials that can be used as thresholds separating data that is indistinguishable from technical variability and likely differentially expressed targets.

Figure 9

Cluster analysis of all log-transformed microarray data. Pearson distances and complete linkage were used. Each array is labelled by case number (abbreviated as per the legend at right), layer (T = Top, B = Bottom, S = Stroma), grade (Norm = Normal), and the amount of RNA used in the labelling reaction. Where given, the number after the @ symbol denotes the detector gain setting on the microarray scanner. In all other cases (plus the one where gain = 433), the detector gain was set automatically by the scanning software. Two samples that differ only by detector gain were the same hybridized array imaged twice with different settings. All other samples represent distinct arrays. Two 200 ng aliquots of top layer RNA were assayed from case 0044 (case 7 in this figure), so these are denoted A and B. Note that in this figure, cases 4 and 5 are from the same patient, as are cases 8 and 9.