Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

Abstract

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]^+ve), a formalin-free system (NBF^-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF^-ve, RNA from NBF^+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF^+ve and NBF^-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Keywords: Illumina; PFPE; ScreenTape; Xylene; degradation; degrade; fixative; formaldehyde; integrity; paraffin.

Figure 1.

Accumulation of formaldehyde in the final water flush of the tissue processor over 12 formalin-fixed, paraffin-embedded (FFPE) processing runs following replacement of all flush reagents.

Figure 2.

Immunohistochemistry using MLH1 antibody for PAXgene Tissue fixed, paraffin-embedded tissue (PFPE) normal colon (A, B), colon cancer (C, D), salivary gland (E, F), and uterus (G, H) tissue blocks that had been processed in a tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) (neutral buffered formalin [NBF]^+ve, images A, C, E, and G) and a formalin-free system (NBF^−ve, images B, D, F, and H). (A, B) In normal colon, MLH1 stained in the nucleus of epithelial cells and stromal cells, with NBF^+ve (A) staining a little weaker than NBF^−ve (B). (C, D) In colon cancer, MLH1 is negative in epithelial cells but is stained weakly in stromal cells. The immunohistochemical reactivity in stromal cells was a little lower in NBF^+ve (C) compared with NBF^−ve (D). (E, F) In normal salivary glands, MLH1 is expressed in the nucleus of acinar and ductal epithelial cells. The staining intensities were similar in both NBF^+ve and NBF^−ve. (G, H) In the uterus, stromal cells showed faint nuclear staining that was equivalent in NBF^+ve and NBF^−ve. The scale bar (B) applies in respect of all images and is 125 µM. Arrows denote positive staining cells.

Figure 3.

RNA yield from five 10-μm sections of PAXgene Tissue fixed, paraffin-embedded tissue (PFPE) tissue processed in a tissue processor also used for formalin-fixed, paraffin-embedded (FFPE) biospecimens (neutral buffered formalin [NBF]^+ve) or in a formalin-free system (NBF^−ve). RNA was extracted using the PAXgene Tissue RNA Kit (PAX kit) or the RNeasy FFPE Kit (FFPE kit). The boxes are first to third quartile intersected by the median and the whiskers are the range. The horizontal bars denote statistical significance (p < 0.001). The boxplot data are presented as individual data points in Supplemental Fig. 6A.

Figure 4.

RNA integrity numbers (RINs) from PAXgene-fixed tissue processed in a tissue processor also used for formalin-fixed, paraffin-embedded (FFPE) tissue (neutral buffered formalin [NBF]^+ve), in a formalin-free system (NBF^−ve), or remained unprocessed (PFnPE). RNA was extracted using the PAXgene Tissue RNA Kit (PAX kit) or the RNeasy FFPE Kit (FFPE kit). The boxplots are as in Fig. 3. *All pairwise comparisons between the groups are statistically significant (p < 0.012). The boxplot data are presented as individual data points in Supplemental Fig. 6B.

Figure 5.

Size reverse transcription polymerase chain reaction (RT-PCR) for four amplicons (943, 535, 266, and 66 bp) of the hydroxymethylbilane synthase (HMBS) gene. PAXgene-fixed tissue was processed using a tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) biospecimens (neutral buffered formalin [NBF]^+ve) or a formalin-free system (NBF^−ve). RNA was extracted using the PAXgene Tissue RNA Kit (PAXgene) or the RNEasy FFPE Kit (RNeasy).

Figure 6.

Cycle threshold numbers (Cq) following quantitative reverse transcription polymerase chain reaction (qRT-PCR) for hypoxanthine-guanine phosphoribosyl transferase (HPRT) (A), mucin 1 (MUC1) (B), and hydroxymethylbilane synthase (HMBS) (C) from PAXgene-fixed tissue processed in a tissue processor also used for formalin-fixed, paraffin-embedded (FFPE) tissue (neutral buffered formalin [NBF]^+ve), in a formalin-free system (NBF^−ve), or left unprocessed (PFnPE). RNA was extracted using the PAXgene Tissue RNA Kit (PAX kit) or the RNeasy FFPE Kit (FFPE kit). The boxplots are as in Fig. 3. The horizontal bars denote statistical significance (p < 0.001). The boxplot data are presented as individual data points in Supplemental Fig. 6C.

Figure 7.

DNA integrity measured using percentage double-stranded DNA (dsDNA) (A) or ΔCq in the Illumina formalin-fixed, paraffin-embedded (FFPE) QC assay (B) from PAXgene-fixed tissue processed in a tissue processor also used for FFPE tissue (neutral buffered formalin [NBF]^+ve), in a formalin-free system (NBF^−ve), or left unprocessed (PFnPE). The boxplots are as in Fig. 3. The horizontal bars denote statistical significance (p < 0.01). The boxplot data are presented as individual data points in Supplemental Figs. 7A and 7B.

Figure 8.

DNA integrity measured using DNA integrity numbers (A) and agarose gel electrophoresis (B) from PAXgene-fixed tissue processed in a tissue processor also used for formalin-fixed, paraffin-embedded (FFPE) tissue (neutral buffered formalin [NBF]^+ve), in a formalin-free system (NBF^−ve), or left unprocessed (PFnPE). The boxplot is as in Fig. 3. The horizontal bars denote statistical significance (p < 0.01). The boxplot data are presented as individual data points in Supplemental Fig. 7C.