Quantitative Evaluation of Tissue Preservation
Modern diagnostic pathology requires that both morphology and molecular integrity are preserved throughout processing and handling of the tissue. The major challenge for molecular analysis of breast cancer samples is to preserve the molecular integrity of the specimen while insuring the structural integrity needed for diagnostic pathology.
R. D. Cardiff, A. D. Borowsky, N. E. Hubbard, H.A. Velasquez-Garcia, J. P. Gregg, S. Liu, C.H. Miller, R. J. Munn, J.Q. Chen, K. Bell and J. Walls
Center for Comparative Medicine, Department of Pathology, School of Medicine, University of California, Davis, CA 95616
Standard Operating Procedures (SOP) usually require formalin fixation and paraffin embedding of samples. Several alcohol-based “Universal Fixatives” have been developed to optimize structural and molecular preservation. Each fixative has unique characteristics. An understanding of the characteristics of each fixative is imperative in any analysis. We developed a reproducible system for head-to-head quantitative evaluation of various preservation techniques. The basic data sets were obtained using mouse tissues to eliminate concerns of time between collection and processing.
Samples of tumors, liver, and uterus
were flash frozen or fixed in various fixatives and paraffin embedded. Samples
of normal and neoplastic tissues from mouse were examined.
Preservation of structure and molecular information was compared using commercially available cross-linking (formalin) fixatives including neutral-buffered formalin (NBF), Tellyesniczky’s (Telly’s) and coagulation fixatives (alcohol, PAXgene, Boon’s).
Coagulation fixatives resulted in the most severe cellular shrinkage in both mouse and human tissues resulting in distortion of nuclear/cytoplasmic ratios. Coagulation fixation also reduced, or eliminated, immunohistochemical staining of Ki67, ER and PR. Ki67, ER and PR staining was preserved with cross-linking fixatives. However, Her2 staining had higher Quantitative Image Analysis scores with coagulation fixation The RNA Integrity Number (RIN) scores were generally below 7 (10 is optimal) with most fixatives. However, fixation with Telly’s fluid (alcohol-glacial acetic acid-formalin) resulted in RIN numbers above 7. Further, expression analysis using microarrays suggest that expression profiles were altered with each of the fixative schedules but some of the key molecular profiles were retained.
1. Morphologic integrity was best maintained with both Telly’s and NBF fixation.
2. Immunohistochemical staining for ER, PR, Ki-67 was markedly reduced with coagulation fixative although Her-2 was high. All markers were preserved with the cross linking fixatives.
3. Coagulation fixation resulted in the greatest cellular shrinkage and distortion of the nuclear/cytoplasmic ratios in both mouse and human tissue.
4. RIN scores were useable (RIN greater than 5.5 ) with Telly’s fixation with both the fixed and the processed tissues. Boon’s was not useable. Both PAXgene and NBF produced variable RIN scores. NBF had the lowest percent recovery of nucleic acids.
Telly’s and NBF were adequate to preserve both morphology and nucleic acid integrity. Further studies are in progress evaluating other coagulation and cross-linking fixatives using.
For more information refer to Dr. Robert Cardiff, UC Davis ()