RESULTS Figure 5. Morphology and Antigenicity

Figure 7. Total SNP Variant Calls.

Total SNP Variant Calls (TVC) were submitted to

cluster and heat map analysis by calculating mismatch

score = (variants in one) + (variants in the other) –

(variants in common). Lower and more negative

numbers indicate similar TVC between samples.

Similar sample, fixation and time point cluster along a

diagonal line. BF was not included in the analyses due

to poor performance.

Figure 7. Total SNP Variant Calls

Figure 8. Fixative Type affects NGS Metrics.

Results from two way ANOVA analysis results

qualitatively compare the effect of time and fixative

on the DNA NGS metrics. Cells shaded green

indicate that the experimental variable had a

statistically significant outcome on the

corresponding metric (p<0.05). Tukey multiple

comparisons of means with a 95% family-wise

confidence level performed on mismatch score data

were used to select optimal fixative and time point.

BF was not included in the analyses due to poor

performance.

Figure 8. Fixative Type affects NGS Metrics

DISCUSSION

Fixation method affects nearly all NGS metrics, whereas fixation time affects base

coverage depth and mapped reads for LCa tissues. Optimal IHC scores were observed at

24 hours for NBF, as well as 8 and 72 hours for ZBF. NBF performed sufficiently by H+E

stain at all time points. BF produced good H+E staining, but poor IHC scores and NGS

metrics as many samples could not produce NGS libraries. Clustering analysis suggests

that 8 and 24 hour time points outperform 72 hours. The clustering analysis also showed

that ZBF produced worse results compared to EtOH, MeOH, and NBF.

FUTURE DIRECTIONS

The results presented here represent preliminary data with LCa samples and additional

research is underway to expand this type of study to additional tissue types including

research samples from colon and breast cancer.

CONCLUSIONS

24 hour NBF fixation of lung cancer research samples had the best overall results for all

methods tested here: NGS, H&E staining and IHC.

REFERENCES

1.Paavilainen, L. et al. Journal of Histochemistry and Cytochemistry (2010)58.3

2.Tanca, A. et al. J Proteomics (2011)74.10

3.William, H. et al. Methods (2014)

TRADEMARKS/LICENSING

© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of

Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

Comparison of Type and Time of Fixation on Tissue DNA Sequencing Results

For Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com

Rob Brown1, Kirk Elliott1, Tanya Biorac2, Michael Allen2, Jennifer Freeland1, Ferda Filiz3, Shayna Donoghue1, Jim Veitch4, Cristina Van Loy2, and Jared Isaac1 Anatomical Pathology Division (APD) Kalamazoo, MI1, Clinical Sequencing Division (CSD) Carlsbad, CA2, APD Fremont, CA3, and CSD South San Francisco, CA4

Figure 5. Morphology and Antigenicity. H&E and EGFR IHC stained LCa slides for all fixatives and time points.

All images represent the same tissue with different fixative and time conditions and were taken at 40X.

Figure 2. Uniformity of Coverage

Figure 2. Uniformity of Coverage.

Analysis for both the uniformity of amplicon coverage and the uniformity of base coverage indicates that the type of

fixative affects results (two-way ANOVA p < 0.05). Bouin’s fluid samples were excluded from further analysis due to

poor performance. Tissues fixed in neutral buffered formalin for 24 and 72 hours showed the best results. Fresh

frozen controls values were 99.0% for uniformity of amplicon coverage and 99.1% for uniformity of base coverage.

Figure 3. Percent Reads

Figure 3. Percent Reads.

Analysis for both percent base reads on target and percent end-to-end reads indicates that the type of fixative affects

results (two-way ANOVA p < 0.05). Bouin’s fluid samples were excluded from further analysis due to poor

performance. Fresh frozen controls values were 82.8% for percent reads on target and 90.6% for percent end-to-end

reads.

Figure 4. Mapped Reads and Coverage Depth

Figure 4. Mapped Reads and Coverage Depth.

Analysis for both the average mapped reads and average base coverage depth indicates that both the time and type

of fixative affects the results (two-way ANOVA p < 0.05). Bouin’s fluid samples were excluded from further analysis

due to poor performance. Fresh frozen controls values were 346110 for average mapped reads and 1488.2 for

average base coverage depth.

Time Fixative

Amplicons reading end-to-end - +*

Amplicons with no strand bias - -

Base coverage depth +* +*

Mapped Reads +* +*

Percent Base Reads on Target - +*

Percent end-to-end reads - +*

Percent Reads on Target - +*

Target bases with no strand bias - +*

Uniformity of Amplicon Coverage - +*

Uniformity of base coverage - +*

*p<0.05

Figure 6. IHC and H&E Scoring

Figure 6. IHC and H&E Scoring. IHC micrographs shown in Figure 5 were scored by pathologist by a digitized

3+ scale with 3.5 being the highest value. H&E micrographs shown in Figure 5 were scored by pathologist

microscopic review based upon nuclear stain intensity, nuclear stain clarity, mucin staining, and morphology each

on a scale from 0-6, where 0 is poor and 6 is excellent. The highest possible H+E score was 24.

ABSTRACT

The effects of type and duration of tissue fixation were studied using three different

lung (LCa) cancer research samples. Each tissue sample was fixed in five different

fixatives, for three different time points in each fixative. Next generation sequencing

(NGS), tissue morphology analysis (H+E), and antigenicity (IHC) were performed

for each of the resulting samples. The analysis indicates that both time and type of

fixation impact NGS results.

INTRODUCTION

Variability in tissue fixation leads to variability in tissue morphology and antibody

based protein profiling of tissues (1, 2), whereas this effect is not widely reported

on NGS results. Tissue fixatives in clinical practice are classified as denaturing

fixatives, cross-linking fixatives, or a combination. Fixatives have different rates of

penetration and fixation mechanisms, and combination with fixation time will lead to

variability in the protein cross-linking or denaturation (3). This pre-analytical

variability could lead to differences in analysis of morphology, IHC, and NGS. In

order to understand tissue fixation effects on anatomical and molecular assays, five

different fixatives were used to fix LCa tissues for 3 different time points. Tissues

were processed and sectioned under routine conditions, and tissue morphology

and protein antigenicity were analyzed and scored based upon pathologist

microscopic review of H+E and IHC stained slides. NGS DNA data were obtained

with an FFPE DNA preparation kit, library construction, and sequencing.

MATERIALS AND METHODS

Sample Preparation

Three fresh frozen (FF) human LCa samples were purchased from Asterand

Bioscience in Detroit, MI (Adenocarcinoma > 95%, RIN > 9.5, and mass >2g for

each tissue). Each FF sample was grossed into six equal sections. One of the six

pieces was saved as a FF control, and the remaining five were placed into pre-

labeled Shandon biopsy cassettes and cups, and fixed for eight hours with 50mL of

either Thermo ScientificTM Richard-Allan ScientificTM Neutral Buffered Formalin

(NBF), Zinc Buffered Formalin (ZBF), Bouin’s Fluid (BF), methanol (MeOH), or

ethanol (EtOH). After eight hours, each of the 15 pieces of fixed tissue was grossed

into three equal pieces, one of which was placed into a new tissue cassette and

processed with the Thermo ScientificTM ExcelsiorTM AS under standard conditions,

then paraffin embedded, and stored at +4ºC. The remaining pieces were fixed for

24 and 72 hours, followed by processing, embedding, and storage. 4 and 7 µm

sections were cut and mounted to Thermo ScientificTM Superfrost PlusTM glass

slides for H+E, IHC and NGS, respectively.

IHC and H&E Staining

IHC was performed on the Thermo ScientificTM Lab VisionTM Autostainer 360-2D

and PT Module and Thermo ScientificTM UltraVisionTM Quanto Detection System

HRP DAB per manufacturer’s recommendations. H&E staining was performed

using the Thermo ScientificTM Richard-Allan ScientificTM Signature Series

Hematoxylin 7211 and the Thermo ScientificTM

Richard-Allan ScientificTM Signature Series Eosin –Y

7111 routine on the Thermo ScientificTM GeminiTM AS

Automated Slide Stainer.

NGS

DNA preparations were generated using the Thermo

ScientificTM Ion AmpliSeqTM Direct FFPE DNA Kit

(performed in duplicate by separate users). Libraries

were made using the AmpliSeqTM Cancer Hotspot

Panel v2 with amplicon lengths of 125-175bp and the

HTP liquid handler Tecan® Freedom EVO®.

Templating was performed on a Thermo ScientificTM

Ion ChefTM and sequencing on the Ion S5TM

instrumentation.