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Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 221-230
The effect of the alternative solutions to formaldehyde and xylene on tissue processing

1 Department of Pathology, School of Medicine, Dokuz Eylul University, Izmir, Turkey
2 Medical Biology and Genetic, School of Medicine, Dokuz Eylul University, Izmir, Turkey

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Date of Web Publication24-Oct-2013


Introduction and Aim: To assess the impact of new alternative solutions to formaldehyde and xylene on tissue processing, 13 different tissue processings were designed and performed on thirteen different tissues by using five different fixatives (formaldehyde, Glyo-Fixx® , FineFix® , Cell-block® , Green-Fix® ) and four different clearing agents (xylene, Sub-X® , Bio-clear® , Shandon Xylene Substitute® ). Materials and Methods: Hematoxylin and Eosine stained sections were compared by using qualitative histomorphological criterions. Histochemical and immunohistochemical (IHC) staining results were compared with qualitative and quantitative data obtained by a computer program, respectively. Tissue sections were tested for the availability of chromogenic in situ hybridization, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) extraction, and DNA quality by polymerase chain reaction. Results: The quality of sections was well for all tissue processings. All alternative solutions were suitable for histochemistry. IHC staining results showed that alternative solutions that contain glyoxal as active agent need optimization for this application. The clearance of signals with chromogenic in situ hybridization were nearly same and well for all tissue samples. Furthermore, tissue processes that do not contain formaldehyde were found to be superior on preservation of nucleic acids. Conclusion: Formaldehyde-free fixatives and alternative clearing agents have potential in routine pathology and research to replace formaldehyde and xylene.

Keywords: Deoxyribonucleic acid, formaldehyde, ribonucleic acid, tissue fixation, xylene

How to cite this article:
Aydin I, Yörükoglu K, Cingöz S, Agilkaya S. The effect of the alternative solutions to formaldehyde and xylene on tissue processing . Indian J Pathol Microbiol 2013;56:221-30

How to cite this URL:
Aydin I, Yörükoglu K, Cingöz S, Agilkaya S. The effect of the alternative solutions to formaldehyde and xylene on tissue processing . Indian J Pathol Microbiol [serial online] 2013 [cited 2021 Jul 25];56:221-30. Available from: https://www.ijpmonline.org/text.asp?2013/56/3/221/120371

   Introduction Top

There is no doubt that a good histomorphological review is not enough, although it has preserved its importance as the fundamental step for diagnosis. It is more than important to review the antigens, which are mostly composed of proteins, through immunological methods. For this reason, it is expected that the fixation method to be applied shall protect the proteins without changing their antigenic specifications and make them ready for immunophenotypic review methods. While discussing the fixation systems, nucleic acids and proteins are taken into the consideration. [1],[2],[3],[4],[5],[6],[7]

Formaldehyde and xylene that are used as main chemicals in the pathology laboratories have important toxic, allergic, and carcinogenic effects on the staff. These chemicals have been preferred because they are cheap, easily accessible, and convenient for the conventional methods. Nowadays, less toxic materials instead of these chemicals have been introduced into the markets. It is expected that these chemicals would not provide negative effects for advanced reviews such as, immunohistochemistry (IHC), in situ hybridization (ISH), polymerase chain reaction (PCR), and it is also expected that these will be adapted accordingly. In the literature, the few reports that survey this effect have analyzed one or more than one chemicals comparing with formaldehyde or xylene. [1],[2],[3],[4],[7],[8],[9],[10],[11],[12] However, there is not any study comparing formaldehyde and xylene alternative solutions together.

In our study, the effects of alternative tissue processing chemicals that have been sold commercially in Turkey have been reviewed under the framework of the above-mentioned expectations.

   Materials and Methods Top


In the fixation stage, in addition to the standard fixative formaldehyde, four alternative fixatives in the clearing stage, xylene and three respective clearing reagents that are available in Turkey have been used, accordingly [Table 1]. All fixation solutions and clearing reagents have been combined by the routine solutions. The fixation solutions and clearing reagents belonging to the same trade mark have been used together. Thus, through these five different fixatives and four different clearing reagents, 13 different tissue processing have been designed [Table 2].
Table 1: Fixatives and clearing agents used in the tissue processings

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Table 2: Thirteen different tissue processings used in the study

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Collection of tissue samples and fixation

Tissue samples were collected at the same day. In order not to effect the pathological reports negatively, radical resection materials were selected. Ten different nontumoral tissue (uterine cervix, uterine corpus, ovary, rectum, adipose tissue, gallbladder, skin, stomach, lung, and liver) and three different tumor tissues (sigmoid colon, lung, and breast cancer tissues) were studied. During the operation, the resected tissues were attempted to be transferred from operating room to pathology laboratory on due time (5-15 min). For 13 different tissue processings, 13 samples having 5-10-mm diameter and 1.5-2-mm thickness were taken from each tissue. The cassettes were immersed into appropriate fixatives and the fixation stage was applied in room temperature. The applied fixation process has changed between 15 and 24 h because of different transfer time of the tissues.

Tissue processing

First tissue processing was carried out in the rapid tissue processing device supported by full automatic microwave "Tissue-Tek X-Press x120 (Sakura Finetek USA, Torrance, CA)." The alcohol processing step of 2-13 numbered tissue processing were common and carried out in a full automatic closed tissue processing device (Leica® ASP300). On the other hand, manual processing was applied for clearing process and paraffin embedding steps. After tissue processing and paraffin embedding, 4-μm thick sections from each 169 tissue blocks were cut and stained with Hematoxylin and Eosine (H&E). Then, the quality of the sections was evaluated in terms of histomorphological parameters including cytoplasm, chromatin, and nuclear membrane. Each parameter was scored between 1 (poor) and 4 (optimal).

Histochemistry and immunohistochemistry

Five respective tissue microarrays (TMA) composed of breast cancer tissue, cirrhotic liver tissue, rectum mucosa and colon cancer, skin, stomach, liver, and uterus tissues were established (Tissue-Tek ® Quick-Ray™ 8018). Histochemical (HC) and IHC stainings were performed on 4-μm thick sections taken from these TMAs [Table 3]. Reticuline fibrils on silver impregnation, fibrosis in liver tissue on Masson staining, acidic mucin in colon mucosa and colon cancer on Alcian blue (AB), elastic fibrils in dermis on Verhoeff van Gieson (VVG), the eosinophilia of stomach mucous cells on periodic acid schiff (PAS)-stained sections were evaluated and scored between each 1 (poor) and 4 (optimal). IHC staining was applied by standard avidin-biotin complex method to the TMA sections, with a total of 30 containing six nuclear and 24 cytoplasmic primary antibodies [Table 3]. The stained sections were scanned in a digital virtual microscope (Mirax ® Pannoramic Midi, 3DHistech ® ) and representative fields were marked in the computer environment. Afterward, these marked fields were assessed with IHC analysis software (3DHistech ® Nuclear Quant ® , and MembraneQuant ® ) [Table 4].
Table 3: Histochemical and immunohistochemical stainings.

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Table 4: Parameters used for image analysis of immunohistochemical stainings

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Chromogenic in-situ hybridization
"Invitrogen ® , SPOT-Light HER2 CISH Kit" was used for the application of chromogenic in-situ hybridization (CISH). Within the framework of our study, 4-5-μm thick sections from TMA block of breast-, colon-, and lung cancer, positive control block (cerbB2++, fluorescence in situ hybridization positive breast tumor tissue), and negative control block were taken. The hybridizer device (Dako ® , Code S2451) was used for denaturation and hybridization stages. Denaturation stage was applied for 5 min in 95°C and hybridization stage was applied for 18 h in 37°C, accordingly.

DNA extraction and amplification

Invitrogen ® 's genomic DNA purification kit was used for DNA extraction (Invitrogen ® Pure Link Genomic DNA Kit, K1820). The colon tumor tissue was used for the application of DNA extraction. Kit procedures were implemented in the application. DNA concentrations were determined by using Nanodrop ND-1000 ultraviolet spectrophotometer. The DNA integrity in the samples to be isolated was defined by the gel electrophorese of DNA including 500 ng/μL for each sample. An adequate quality for PCR reactions of DNA obtained from the samples was searched in each sample by amplifying via PCR of DNA fragments in different sizes.

Polymerase chain reaction

Three primer pairs designed using Oligo primary analysis software (National Bioscience, Inc.) were used in the PCR reaction. PCR reaction was set up in sterile 0.2 μL PCR tubes purified from all enzymes with a total volume of 15 μL. Final reaction concentration was optimized to be as 1U DNA Taq polymerase (Ferments, EU), 25 ng genomic DNA, 1X DNA Taq polymerase tampon, 2.5 mM MgCl 2 , 0.6 pmol/μL reverse and forward primers and 0.2 mMdNTP. Thermal profile was adjusted as 7 min of starting denaturation at 95°C, denaturation 30 s at 95°C, annealing at 30 s in defined optimum temperature, the extension stage 30 s at 72°C, and the termination extension 7 min at 72°C. In each PCR group, contamination was checked where water was used instead of DNA sample as a negative control. Afterward, PCR products were implemented in 2% agarose gel and displayed by UV transilluminator.

RNA extraction
"Recover all total nucleic acid isolation" extraction kit was used (Ambion Ltd., Cambridgeshire, UK) for RNA extraction from fixed liver tissues. It was purified in accordance with kit protocol and the RNA quality that was purified was evaluated by performing 1.2% formaldehyde agarose gel electrophoresis together with RNA isolated from erythrocytes.

   Results Top

Odor analysis of the solutions

It was determined that current alternative fixatives do not have a strong odor as much as formaldehyde. Within the alternative clearing reagent, it was also defined that Shandon Xylene Substitute ® solution do not have any odor but others have less abrasive odor than xylene.

Histomorphological analysis results were compared with the routine processing (No.2) that is the tissue processing applied in many of the pathology laboratories worldwide. When each parameter was evaluated together in comparison with tissue processing No.2, similar or better qualified sections have been obtained accordingly in entire tissue processings. The sections obtained by the tissue processings performed with alternative fixatives were considered to be better for each of the parameters [Figure 1] and [Figure 2]. On the other hand, when reviewing tissue processings No. 1-5 where formaldehyde has been used as the fixative, the section No. 3-5, where alternative clearing solutions were used were better in terms of histomorphological detail, than section No. 1 and 2 where xylene was used. Another important point was the poor nuclear details of the tissue processing where microwave was used [Figure 3]. Nuclear details and other cellular morphological specifications were close to the optimal in the sections of the tissue processing where FineFix ® has been used, but there were some retraction artifact associated with tissue shrinkage and eosin over staining [Figure 4].
Figure 1: Histomorphology of the ti ssues processed with Glyo-Fixx® (x100). (a) Stomach mucosa, (b) Stomach mucosa, (c) Rectum mucosa, (d) Rectum mucosa, (e) Peripheral nerve, (f) Colon cancer

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Figure 2: Histomorphology of the ti ssues processed with Cell-block® (x1.25). (a) Stomach mucosa, (b) Stomach mucosa, (c and d) Vasculary structures, (e) Rectum mucosa, (f) Rectum mucosa, myenteric plexus, (g) Peripheral nerve, (h) Cervix uteri

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Figure 3: Comparing tissue processing in terms of nuclear morphology. (a) In the section belonging to tissue processing where microwave was used, it is difficult to read the nuclear details. Samples from the sections where the nuclear details could be read. (b) Routine tissue processing, No. 2. (c) with Glyo-Fixx® and Xylene Substitute®

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Figure 4: Samples belonging to the ti ssue processings where FineFix® have been used(a) FineFix®, (b) FineFix® and Xylene Substi tute®. As is seen in breast tumor tissue, FineFix® caused some amount of contraction artifact. However, cytomorphological details are optimal

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Histochemical analysis

The HC color results applied to the TMA sections were scored between 1 (poor) and 4 (optimal). No difference was observed between the tissue processings in means of staining obtained accordingly with VVG of skin tissue, AB of rectum tissue, PAS of stomach tissue, and reticuline and Masson of liver tissue [Figure 5].
Figure 5: Histochemical staining samples. Alcian blue: (a) Microwave, (b) Formaldehyde and Xylene Substitute®, (c) Formaldehyde and Bioclear®, (d) Glyo-Fixx® and xylene, (e) Green-Fix® and Sub-X®; PAS: (f) Formaldehyde and xylene, (g) Glyo-Fixx® and xylene, and (h) Cell block® and xylene

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Immunohistochemical analysis

Together with entire tissue processings to be applied, good or optimal nuclear staining was obtained [Figure 6]. Any tissue section field with nonspecific background staining has not been observed that would negatively affect the assessment. Cytoplasmic staining by the antibodies toward the cytoplasm filamentous proteins such as actin, desmin, caldesmon, and cell surface proteins (CD markers) such as CD3, CD20, LCA, and CD38 was good or optimal as in nuclear stainings in all tissue processings. However, different and concerning results were obtained with cytokeratin (CK) antibodies (high molecular weight CK, Pan CK, CK7, and CK20) and hepatocyte specific antigen. By these antibodies, the results were optimal in the tissue sections where formaldehyde and FineFix ® were used as a fixative, but negative results were noted such as uncoloring, poor, or no staining where active material glyoxal was used as a fixative [Figure 7] and [Figure 8]. It is significant that the staining was applied by the TMA method in equal conditions and the results have been repeated by the similar cytoplasmic stains. The findings were consistent with the data obtained by IHC software program analysis results.
Figure 6: The immunohistochemical staining results with ER nuclear anti body in breast cancer. (a) Tissue-Tek Xpress x120 microwave, (b) Formaldehyde and xylene, (c) Formaldehyde and Xylene Substitute®, (d) Formaldehyde and Bio-clear®, (e) Formaldehyde and Sub-X®, (f) Glyo-Fixx®and xylene, (g) FineFix®and xylene, (h) Cell-block® and xylene, (i) Green-Fix®and xylene, (j) Glyo-Fixx®and Xylene Substitute®, (k) FineFix® and Xylene Substitute®, (l) Cell-block® and Bio-clear®, (m) Green-Fix® and Sub-X®, and (n) the data of the stainings obtained by immunohistochemical soft ware program

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Figure 7: Immunohistochemical staining by high molecular weighted cytokerati ne in breast cancer. As is seen in photos and data, the staining resulted negatively in the tissue processings where Cell-block® and xylene, Green-Fix® and xylene, Glyo-Fixx® and Xylene Substitute®, Cell-block® and Bio-clear®, and Green-Fix®, and Sub-X® were used. In the ti ssue processinsg where formaldehyde and Xylene Substi tute® and formaldehyde and Sub-X® were used, morphology of tumor cells and desmoplastic stroma and optimization of contrast staining is signifi cant. (a) Tissue-Tek Xpress x120 microwave, (b) Formaldehyde and xylene, (c) Formaldehyde and Xylene Substi tute®, (d) Formaldehyde and Bio-clear®, (e) Formaldehyde and Sub-X®, (f) Glyo-Fixx® and xylene, (g) FineFix® and xylene, (h) Cell-block® and xylene, (i) Green-Fix® and xylene, (j) Glyo-Fixx® and Xylene Substitute®, (k) FineFix® and Xylene Substi tute®, (l) Cell-block® and Bio-clear®, (m) Green-Fix® and Sub-X®, and (n) the data of the stainings obtained by immunohistochemical soft ware program

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Figure 8: HEPA immunohistochemical staining in the liver. In some ti ssue processings (Glyo-Fixx® and xylene, Green-Fix® and xylene, Glyo-Fixx® and Xylene Substitute®, Cell-block® and Bio-clear®, Green-Fix® and Sub-X®), negati ve results have been obtained as in cytokerati n, and correlates with the data obtained by immunohistochemical soft ware program. (a) Tissue-Tek Xpress x120 microwave, (b) Formaldehyde and xylene, (c) Formaldehyde and Xylene Substi tute®, (d) Formaldehyde and Bio-clear®, (e) Formaldehyde and Sub-X®, (f) Glyo-Fixx® and xylene, (g) FineFix® and xylene, (h) Cell-block® and xylene, (i) Green-Fix® and xylene, (j) Glyo-Fixx® and Xylene Substi tute®, (k) FineFix® and Xylene Substitute®, (l) Cell-block® and Bio-clear®, (m) Green-Fix® and Sub-X®, and (n) the data of the stainings obtained by immunohistochemical soft ware program

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Chromogenic in-situ hybridization analysis

CISH method was applied to the three tumor tissues including breast, colon, and lung. The signal has not been observed in the cell nucleus in the negative control. In the positive control, the signals that were compatible with high amplification in the cell nucleus have been observed accordingly. On the other hand, in 13 different tissue processings, positive results were obtained in terms of chromogenic staining. There was no section without positive staining. In all tissue samples, two points were observed in more than 50% of the tumor cells. In almost all sections, the readiness of the signals and the contrast of the ground were similar and in good conditions [Figure 9].
Figure 9: Some examples showing the results obtained by CISH. Breast cancer. (a) Tissue-Tek Xpress x120 microwave, (b) Green-Fix® and xylene; Colon cancer, (c) Formaldehyde and xylene, (d) Formaldehyde and Sub-X®, (e) Cell-block® and xylene; Lung cancer, (f) Green-Fix® and Sub-X®

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DNA analysis

By means of gel electrophoresis, quality of DNA was good in all samples but No. 1 [Figure 10]. In the samples of the tissue processings No. 6-13 where alternative fixatives have been used, it was observed that DNA was at the same line as single bands and there were not any little fragments differing from tissue processings No. 1-5. In this respect, DNA quality was observed to be superior by gel electrophoreses in the tissues that were fixed with alternative fixatives. Three different-sized DNA fragments were amplified by PCR in the samples of DNA [Figure 11]. Amplified DNA fragments have lengths of 232, 1608, and 2274 bp, where only 232 bp length fragments could be amplified in all samples. However, DNA obtained by tissue processings with alternative fixatives gave more powerful signal than others that have been fixed by formaldehyde [Figure 11]a. The 1698 bp fragments were amplified in tissue processings that Finefix was used as a fixative in accordance with similarity in genomic DNA of the control sample [Figure 11]b. On the other hand, there was a little amplification in the sample No. 2-5. In addition, the amplification of fragment at the size of 2274 bp was carried out in the sample No. 7 and 11, accordingly [Figure 11]c.
Figure 10: Comparison of DNA qualities by agarose gel electrophorese that was obtained after extracti on regarding the samples of colon tumor tissue where different tissue processings composed by different fixatives and clearing agents that have been applied in equal conditions. (a) Tissue-Tek Xpress x120 microwave, (b) Formaldehyde and xylene, (c) Formaldehyde and Xylene Substitute®, (d) Formaldehyde and Bio-clear®, (e) Formaldehyde and Sub-X®, (f) Glyo-Fixx® and xylene, (g) FineFix® and xylene, (h) Cell-block® and xylene, (i) Green-Fix® and xylene, (j) Glyo-Fixx® and Xylene Substi tute®, (k) FineFix® and Xylene Substitute®, (l) Cell-block® and Bio-clear®, (m) Green-Fix® and Sub-X®

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Figure 11: PCR-based analysis of DNA quality in the samples of colon tumor tissue where different tissue processings composed by different fixatives and clearing agents that have been applied in equal conditions. Agarose gel electrophoresis of the PCR products in diff erent sizes [(a) 232 bp, (b) 1608 bp, and (c) 2274 bp]. (a) Tissue-Tek Xpress x120 microwave, (b) Formaldehyde and xylene, (c) Formaldehyde and Xylene Substitute®, (d) Formaldehyde and Bio-clear®, (e) Formaldehyde and Sub-X®, (f) Glyo-Fixx® and xylene, (g) FineFix® and xylene, (h) Cellblock® and xylene, (i) Green-Fix® and xylene, (j) Glyo-Fixx® and Xylene Substitute®, (k) FineFix® and Xylene Substi tute®, (l) Cell-block® and
Bio-clear®, (m) Green-Fix® and Sub-X®

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RNA analysis

Together with the RNA isolated from the blood, RNA quality was assessed by electrophoreses of the samples. 28S and 18S subdivisions were observed clearly in total RNA isolated from blood, and RNA isolated from the tissues embedded in paraffin was observed in a smear pattern.

   Discussion Top

In addition to the acute and/or chronic toxic effects of the chemical materials that have been widely used in fixation and tissue processing, there are also mutagenic, teratogenic, and carcinogenic effects also. IARC (The International Agency for Research on Cancer) has divided the chemical materials into four groups in terms of the carcinogenic effects. [13] Xylene is located in the category of group 3 (the materials that has not been proven that has carcinogenic effect on human being). Until 1997, formaldehyde located in IARC group 2a category was widely used in laboratories and also in industry, cosmetic, clothing, chemistry, and food sector and by the results of the documentations obtained from secondary studies in 2004, [14] it was taken into group 1 (the materials that have carcinogenic effect on human) by IARC working group and called attention. [13] Formaldehyde is classified as a carcinogen that causes nasopharyngeal cancer and possibly leukemia in group 1 category. [15],[16] Nowadays, many European countries have limited the usage of formaldehyde.

Under normal conditions, the solutions that are used in fixation and tissue processing should have low toxicity and provide optimal histomorphology by H&E staining, adaptable with HC and IHC staining methods, as well as provide effective recovery of DNA - RNA for molecular genetic analyses. There are many commercial fixatives presented in the markets. Some of the formaldehyde alternative fixatives contain nonformaldehyde aldehyde, and some do not contain any aldehyde. [17] When reviewing the current studies in the literature, it could be observed that formaldehyde was compared with one or few alternative fixatives, and for one or few (mostly molecular) parameters. It could be seen that the researches are particularly concentrated on the molecular effects. In most of the studies, alternative fixatives are found to be superior to formaldehyde in terms of impact on molecular methods. However, research on the inter-relation to the routine pathology applications of the formaldehyde alternative fixatives is restricted. On the other hand, the research are limited to one single tissue type such as lung cancer, [1],[8] colon cancer, [3] effusion cytology and fine needle aspirations, [9] thyroid tissue, [4] liver tissue, [5] and colon mucosa. [6] Also, consideration of search on ideal fixative has pushed xylene alternative solutions into background.

Most of the researches in the literature dealt with FineFix ® , which was used as one of the alternative fixatives in our study. Its formula is patented. It is a water-based and concentrated solution without formaldehyde, and not ready to use. It is used by diluting with ethanol. In the studies that have been carried out, while the mark difference has not been observed with the formaldehyde in terms of histomorphology and stainings, it was superior to formaldehyde for molecular protection. [4] Glyo-Fixx ® is an aldehyde group fixative and it does not contain methanol and formaldehyde. Its active material is glyoxal. Glyoxal is an important commercial fixative, establishes intermolecular bridges, but not cross links as in formaldehyde. [18] There are also publications searching effects of Glyo-Fixx ® in the literature [Table 5]. On the other hand, similar to Glyo-Fixx ® , Green-Fix ® , and Cell-block ® are aldehyde group fixatives that do not contain formaldehyde. Acute irritant effect on the eyes and respiratory mucosa of formaldehyde are the disadvantages well known by the pathologists and technicians. Four alternative fixatives that we have used in our study are superior than formaldehyde due to the fact that they do not have any abrasive odor acute irritant effect in short term using. Apart from the alternative fixatives in our study, three different clearing solutions alternative to xylene that has less toxic effect were analyzed. The publications regarding xylene alternatives in the literature are very limited. Among the alternative clearing solutions that were used in our study, Shandon Xylene Substitute ® differed from others with being odorless.
Table 5: The studies regarding comparison of FineFix® and Glyo-Fixx® with formalin, and the obtained results

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The sections obtained through different fixatives and tissue processings in our study have provided similar or better results required for routine light microscopical morphology. The results were superior and close to the optimal with Glyoxal-based fixatives. In the studies that have been carried out by Glyo-Fixx ® , results were reported to be similar to formaldehyde. [4],[11],[19] Glyo-Fixx ® and Cell-block ® are aldehyde based fixatives containing the same active reagent, and could be the alternative to formaldehyde in terms of histomorphological features.

Only fixative that is not from the aldehyde group we have assessed in our study is FineFix ® . The sections had optimal specifications in terms of cytomorphological and nuclear detail, however, caused some amount of retraction artifact that was less than seen with ethanol. Due to the fact that ethanol does not fix the tissue through cross linking of the proteins, morphology occurred by these fixatives are not the same with the tissues fixed with formaldehyde.­[5] On the other hand, we have observed that particularly nuclear morphology was affected negatively with the fast tissue processing of Sakura using microwave, as mentioned in the literature. [20],[21]

Nowadays, a good histomorphological interpretation will not be sufficient alone and to review the antigens composed of the proteins by means of immunologic methods (immunologic phenotype and immunophenotype) is important. Today, IHC has a very important impact on diagnostic and research purposes. Good and optimal staining has been obtained in all our activities regarding the processing of the tissues by nuclear and cytoplasmic IHC stainings. On the other hand, while positive results have been obtained with CK7, CK20, high molecular weight CK, PanCK, and HEPA with the tissue processings where formaldehyde was used, poor results have been obtained from the sections of tissue processings where glyoxal has been used. It is significant that the staining has been applied in equal conditions by TMA method and the results have been repeated by similar cytoplasmic stainings. In addition to the negative staining results such as nonstaining or faint staining with the sections of the tissue processings that glyoxal was used as a fixative, another remarkable point was the deterioration of tissue morphology. When taking into the consideration of the last finding, the negative results obtained by the stainings without making any change in the routine procedure enable us to consider that the applied IHC procedure could be nonadaptive with the effects in the tissue as well.

Formaldehyde has been used as the gold standard fixative in pathology laboratories and hence, almost all IHC manufacturers have optimized their products for formaldehyde-fixed and paraffin-embedded tissues (FFPE). [17] In order to use the fixatives out of formaldehyde, our study has shown that the respective protocols must be optimized repeatedly for each antibody particularly for IHC laboratory. In some studies in this matter, [8] by taking into consideration that alternative fixative does not compose cross links between proteins, it is emphasized that the retrieval process must be respectively adapted and proteinase digestion process should be completely removed or applied only for several seconds. It is essential that the retrieval processes that have been planned toward the effects of formaldehyde must be reviewed. When the tissues have been fixed by alternative solutions, it is required that the prognostic or treatment-related factors such as cerbB2 must be optimized once again and the obtained score definitions must be taken into consideration and approved accordingly.

When reviewing the tissue processings where formaldehyde was used and the best IHC staining results were achieved, clearing solutions that have been ignored since now come forward [Table 6]. These alternative clearing solutions that are less toxic than xylene seem as strong candidates to replace xylene.
Table 6: H score comparison in the tissue processings that formaldehyde and alternative clearing agents have been used

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In the studies that have been carried out to collect intact nucleic acid from tissue, generally one of three ways has been used. Rapid freezing of the tissue embedded in the OCT (optimal cutting temperature) requires protecting tissues at -70°C. It is more expensive, not practical, difficult to cut the samples by cryostat and it deteriorates the morphology. [5],[7],[22] The second approach accepts formaldehyde as the gold standard of diagnostic histopathology and histotechnology and tries to develop the methods that optimize nucleic acid isolation from FFPE tissues. This method harms the efficiency and integrity of nucleic acids. [23],[24] DNA could be sufficiently isolated from FFPE tissues. However, the size of the DNA to be analyzed could not exceed 300-500 bp. Similar restriction is also valid for RNA extraction. By means of this method, less than 50-99% RNA could be obtained and the amplicons provided in this RNA is generally less than 300 bp. [5],[25] The third approach attempts to find a balance between the integrity of tissue morphology and nucleic acids by using different tissue processing methods through the alternative fixatives. The most promising results until today have been obtained by alcohol based fixatives. [7]

DNA quality was good apart from microwave-based processed tissues, in our study. However, where formaldehyde has been used as a fixative, it was observed that significant DNA fragmentation and small fragments existed. We also evaluated DNA quality by PCR amplification. While amplification was successful in each tissue processing for PCR that has been made by small-sized amplicons, the case was not similar through big sized amplicons as expected. Certain amplification and strong signal was observed with 1608 bp amplicon similar with genomic DNA in the tissues processed where FineFix ® was used as a fixative. On the other hand, in the tissue processings where the xylene alternatives have been used, some amount of amplification was carried out. Additionally, by means of amplicons sized by 2274 bp, the amplification was performed only in the tissue processings where FineFix ® has been used. In the tissue processings where formaldehyde and FineFix has been used, RNA quality assessed through gel electrophorese was better. Hence, it was observed that molecular protection of FineFix ® is better than others. The result that we have obtained is similar with other studies' that have been carried out withFineFix ® . For example, in the study where FineFix ® and formaldehyde have been compared, the DNA fragment length obtained from the FineFix ® fixed tissues was 2400 bp while 350 bp from the formaldehyde fixed tissues. [3] At the same time, it was defined that degradation of RNA was less with FineFix ® fixed tissues than FFPE tissues and longer RNA sequences could be extracted. [3]

We have focused on DNA in this study and have not done reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the suitability of RNA for molecular diagnostics on paraffin embedded material. The quality of DNA and RNA should be evaluated in further studies searching the impact of alternative solutions of tissue fixing and processing on molecular techniques including RT-PCR, quantitative PCR, detection of single nucleotide variants, copy number analysis, exome sequencing using next generation sequencing technology.

When comparing the costs of solutions, it could be seen that alternative solutions are more expensive. The prices of alternative fixatives are approximately 15-80 times higher than formaldehyde, and the prices of alternative clearing reagents are 7-10 times higher than xylene. However, it must be taken into consideration that human life is priceless, and these alternative solutions' prices will probably cheapen by their utilization is expanded.

In summary, although most of the alternative solutions have been come into the forefront by their specifications, they also include components having toxic potential and combustible specifications. It is expected that ideal fixatives that will replace formaldehyde should give the similar histomorphological results and in this respect glyoxal-based fixatives were superior, in our study. Our HC-staining panel did not contain protocols for microbiological organisms and in this respect the effect of the alternative solutions should be taken into the consideration. It would be more than important to optimize the immunohistochemical protocols in accordance with the tissue processing to be applied by the alternative solutions. It is observed that particularly Bio-clear ® and Sub-X ® among the clearing agents provided the best results. In terms of molecular protection, FineFix ® was an ideal fixative but caused some amount of tissue shrinkage. All these results and results of other studies that have been carried out recently showed us that alternative fixatives and clearing agents have provided better results by means of morphology, conventional, and molecular techniques. If the negative aspects of these solutions could be eliminated by appropriate optimizations, it is assumed that alternative solutions are candidate to replace formaldehyde and xylene accordingly.

   References Top

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Correspondence Address:
Kutsal Yörükoglu
Department of Pathology, School of Medicine, Dokuz Eylul University, Izmir
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.120371

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

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