Study On The Preservation Effects Of Zinc Salt Fixative On Morphology,Proteins And Nucleic Acids In Histopathological Specimens

Objective: In this paper,we studied the effect of zinc-salt fixative on the integrity of different components of pathological tissue specimens to explore a fixative that can stably preserve tissue morphology,protein antigen and nucleic acid.Thus,this result will provide a basis for the preparation of high quality samples for histopathology,especially molecular pathology diagnosis.Patients and Tissue Specimens: In total,12 pieces of histopathological specimens were collected according to clinical guidelines from patients(n=10)with colorectal,stomach,liver and uterine fibroids cancer tissues respectively between January 2015 and December 2015Experimental Design: The fixative experiments were performed at room temperature for 24 h in zinc-salt,4% neutral formaldehyde and carnoy fixative solutions by guidelines.Each subgroup contains 2-years and fresh 1-month paraffin-embedded tissues to evaluate the long period of paraffin–embedded fixation effects on the integrity of nucleic acid.3.Method for evaluation of preservation effects 1).H&E staining: The uniformity of the whole slides,color of staining and the integrity of RBCs(Red Blood Cells)were evaluated.2).Acid mucin staining: AB(Alicain Blue)staining was used to evaluate the preservation effect of fixatives on acid mucin 3)Neutral mucin staining: PAS(Periodic Acid-Schiff)staining was used to evaluate the preservation effect of fixatives on neutral mucin.4).Glycogen staining: PAS staining was used to evaluate the preservation effect of fixatives on glycogen.5).Net fiber staining: Gomori staining was used to evaluate the effect of fixatives onthe retention of reticular fiber.6).Muscle and collagen fibers staining: Masson trichrome staining was used to evaluate the effect of fixatives on tissue preservation of muscle fibers and collagen fibers.7).Membrane antigens staining: Immunohistochemical staining of EMA,Ber-Ep4,and CK8 was used to evaluate the preservation effect of fixatives on membrane antigens.8).Nuclear antigens staining: Ki67 and ER indexes were used for immunohistochemical staining to evaluate the preservation effect of fixatives on nuclear antigens.9).Cytoplasm antigens: Immunohistochemical staining of CK,Mucin-6,Desmin and Hepatocyte were used to evaluate the effect of fixatives on the preservation of cytoplasm antigens.10).Analysis of the content and purity of genomic DNA: DNA was extracted from indicated specimens.The values of OD260 and OD260/OD280 were measured to quantitate the content and purity of genomic DNA respectively.11).Analysis of the integrity of genomic DNA: PCR amplification of housekeeping gene GAPDH was performed to evaluate the integrity of genomic DNA.Results: 1.Effects of on overall tissue morphology 1)The preservation effects of zinc-salt fixatives on morphology,nucleic acid and the integrity of RBCs are as well as 4% neutral formaldehyde fixative but better than that of carnoy fixative solutions.2.Effects on the preservation of special histochemical components: 1).Zinc-salt fixative is not as good as carnoy fixative in preserving acid mucins of intestinal cancer tissues 2).Zinc-salt fixative is equivalent effects to carnoy fixative in preserving neutral mucins of stomach cancer tissues 3).Zinc-salt fixative is equivalent to that of 4% neutral formaldehyde fixative but it is not as good as carnoy fixative in preserving the glycogens of liver cancer tissues 4).Zinc-salt fixative is better than 4% neutral formaldehyde fixative and carnot fixative in preserving the reticular fibers of liver cancer tissues,5).Zinc-salt fixative is better than 4% neutral formaldehyde fixative in preserving the muscle fibers and collagen fibers of the uterus but it was not significantly different from that of carnoy fixative.3.Effects on the preservation of protein antigens:(1)Zinc-salt fixative is better than other groups in preserving the cell membrane antigen EMA,Ber-Ep4 and CK8 all of which are from intestinal,gastric and liver cancers respectively.However,there is no obvious advantage in SMA of uterine fibroid cancer tissues.(2)The zinc-salt fixative has the best preservation effect on the nuclear antigen Ki67 in gastric cancer tissues.However,the zinc-salt fixative has less effect on the preservation of ER in uterine cancer tissue than 4% neutral formaldehyde fixative.(3)There is no difference in the three solutions in terms of the preservation effect of the cytoplasmic antigens.4.Zinc-salt fixative groups were extracted the most amounts of DNA in all samples.However,the integrity of DNA among groups exhibited no obvious difference.5.Effects on the PCR amplification of housekeeping gene GAPDH: All of fresh paraffin-embedded specimens have clearly visible DNA amplification bands.The paraffin-embedded tissue after 2 years of archiving,the zinc salt fixative group has a clearer target band,the band of carnoy fixative groups is thin and dark,and the band of 4% neutral formaldehyde fixative groups is fuzzy.Conclusion: 1.The zinc-salt fixative is equivalent to the 4% neutral formaldehyde fixative in the preservation of tissue morphology and overall staining.In the preservation of histochemical substances,zinc-salt fixative has better preservation effect on glycogen and reticular fiber.2.Zinc-salt fixative has better preservation effect on the content and integrity of genomic DNA and antigens of cell membrane,nucleus or cytoplasmic.