The Research About The Effects Of Different Components Fixtives For Tissue Preservation

Background Tissue fixation is very important in tissue processing. In the past one hundred years Formaldehyde is a common international organizations fixative. However, formalin-fixed tissue following major shortcomings:①Volatile, toxic②Cause a wide range of protein cross-links within the tissue and hinder antigen exposure in the course of immuno-histochemical.③Lead to genomic DNA breaks, inhibits nucleic acid quantitative and limits the length of the PCR product of DNA fragment. With the development of molecular biological techniques and the studies of disease-related genes, molecular pathological detection has been an effective auxiliary approach used widely in clinical practice. Hence it is essential to improve the existing fixative or invent novel substitutes which are superior to formalin in morphology, immunohistochemistry and the extraction or purification of genetic substance.PurposesTo investigate the influence of morphology, histochemical,immunohistochemistry and DNA integrity in different fixative-fixed,paraffin-embedded tissue.Methods1.Fixatives preparation：Three non-formaldehyde fixatives were prepared in the laboratory:①（R）：Reference RCL-2fixative②（U）：Reference UMFIX fixative③（W）：Reference Weigners fixative2.Specimen Collection：48cases of fresh specimens for frozen section diagnosis were collected after surgical resection:1gastric cancers,8Uterine fibroids,5Colorectal cancers,10thyroids,6lung cancers,10Breasts. Accordance with the "clinical technology operation (Pathological Volume)" requirements, The junction of tumor tissue and normal tissue were selected in tumor specimens;The normal tissue were selected in normal specimens.Each tissue sample was divided into4 equal pieces (2cm×1.5cm×3mm).Then to be loaded in cassette and fixed in one of the fixatives at room temperature for12hours.10%neutral buffered formaldehyde (F) as a control. Convention Alde-hydrated, embedded in paraffin,3μm sections continuously. HE staining, IHC staining, special staining. five6μm sections kepts into1.5ml Eppendorf tubes for DNA extraction, PCR amplification3..Morphology：HE sections were given scores from six different aspects: overall morphology, overall staining, nuclear staining, cell outline, the integrity of red cells, cytoplasmic staining.4.Histochemical：PAS staining to evaluate the effect of different fixative for neutral mucin preservation; Masson, Reticular and Elastic fibers staining to evaluate the effect of different fixative for fibrous connective tissue preservation.5.Immunohistochemistry: CK、EMA、SMA、Desmin、P63、34βE12、TTF-1、Cadherin-E、CK7staining were given scores to evaluate the effect of different fixative for antigen preservation.6.Genomic DNA extraction and verification: OD260and OD260/OD280on DNA concentration, purity, integrity detection,and GAPDH gene PCR products by agarose gel electrophoresis, PCR products were observed to assess their integrity to compare the effect of different fixative for DNA preservation.Results1．Morphology：Of Overall morphology(3.44)，Overall staining(3.45) and Nuclear detail(3.25), Group R received the highest scores; Group F received the highest cell outline scores（3.26） and erythrocyte integrity scores（3.31）. Group W received the lowest scores。（P<0.05）2. Histochemical：PAS in stomach tissue：Fixative F strongest protects carbohydrate and Fixative W is the Worst; Fixative U stains colorful,; Fixative R appears signs of dissolved sugars and sugar distribution in cells have a very biased cell aggregation phenomenon. PAS in the thyroid colloid: Group F stained dark; Group R gum shrink significantly；Group U uniform. Masson: Group U and Group R results deep and bright colors, red in muscle fibers and green in collagen fibers comparative bright. Elastic fiber staining: Group F and Group U elastic fibers stain receive clear colored and clean background. But Group R and Group W receive messy, broken elastic fiber and obscure background. Reticular fiber staining：Group R receive clean background，Group F had dyed phenomenon；Group U receive the worst staining so that reticular fibers less illegible; Group W receive darker background, only a small amount of reticular fibers was showed.3. Immunohistochemistry:①CK、EMA、SMA、Desmin、P63、34βE12、TTF-1：Group F and Group R shows no significant difference (P>0.05), while Group F and Group U and group W had significant difference (P <0.05).②Cadherin-E： Group F and group R, Group U, group W had a significant difference (P <0.05).③CK7：There is no statistically significant difference among Group F, group R and Group U (P>0.05), Differences in group F and Group W was significantly (P <0.05).4. Genomic DNA: R group has the highest DNA conten（tOD260=0.029）(P <0.05). Order by U（OD260=0.025）, W（OD260=0.021）, F（OD260=0.020）. OD260/OD280ratios were not significantly different (P>0.05).5. PCR amplification: Breast tissue: group U, Group R receive significantly amplified target DNA bands; Group W target bands are not clear, Group F are almost no target bands; Thyroid tissue: no target bands from group W, Group F, Group U, Group R can be seen clearly target bands, but a lower rate of Group F. Gastric tissue: Group R, Group W had a clear target bands, Group F target band rather vague, Group U almost no target band. Intestinal tissue: Group U, Group R, Group W received clearly visible target bands, but Group F receives darkness. Uterine tissue: Group F is almost no target bands, Group U, Group R can see a clear target band, Group W rather vague; Lung tissue: Group R can see clear target band, Group U followed, Group F’ rate is very low, Group W target band is not clear and clutter.Conclusion1. Formaldehyde is still the best fixative for morphology preservation. 2. Formaldehyde and U of sugars and elastic fiber protection ability is good, and in the reticular fiber network protection, R is best.3. Of nuclear, pulp, membrane antigen Formaldehyde is still save the best effect, R is also.4. For genomic DNA extraction rate and the integrity, R stationary liquid is best, formaldehyde fixed liquid is the worst.