Formalin-fixed and paraffin-embedded tissues present particular challenges for proteomic analysis. However, most archived tissues in pathology departments and tissue banks worldwide are available in this form. Different approaches to removal of the embedding medium and protein digestion have been developed, thus releasing tryptic peptides, which are suitable for analysis by liquid chromatography-mass spectrometry. Peptide identifications made using this approach are comparable to those from matched fresh frozen tissue. Apparently, a high level of sequence coverage can be seen for proteins under study (3).
The effect of fixation on the degradation of nuclear and mitochondrial DNA in different tissues has been examined (4). Samples of different tissues were preserved in seven fixatives for periods extending from 1 to 336 days to determine which fixatives reduce the time-dependent degradation of DNA and preserve the histologic structure. For long-term storage in combination with amplification of nuclear and mitochondrial DNA, consistent results were obtained with Carnoy’s solution and glutaraldehyde. Variable results were observed for buffered formalin. In regard to comparison of the different tissues, the quantities recovered from skeletal muscles and kidneys were larger than from other tissues. These fixative studies will become even more important as molecular methods applied to fixed tissues become more common.
Molecular characterization of morphologic change requires precise tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses of fixed tissues, morphologic and RNA integrity in rat liver was assessed when sections were fixed in 70% neutral buffered formalin, modified Davidson’s II medium, 70% ethanol, UNIFIX, modified Carnoy’s solution, modified methacarn, Bouin’s medium, phosphate-buffered saline, or 30% sucrose. Each sample was treated with standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity (5). Modified methacarn, 70% ethanol, and modified Carnoy’s solution resulted in tissue morphology providing a reasonable alternative to formalin. Modified methacarn and UNIFIX, best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was somewhat improved. Modified methacarn, 70% ethanol, and modified Carnoy’s solution provided acceptable tissue morphology and RNA quality with both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens. In another study, the PAXgene Tissue System preserves histology similarly to formalin, but does not chemically modify RNA. RNA purified from PAXgene fixed tissues performs as well as RNA from fresh frozen tissue in real-time PCR regardless of amplicon length (6).
PCR has also been used to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of the rRNA gene of the ascarid parasites (ITS1 and ITS2) were compared with those registered in GenBank. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions (7).
Although formalin fixation is the most common storage, transportation, and preservation method for stool samples, it dramatically reduces the ability to extract DNA from stool samples for PCR-based diagnostic tests. Apparently, the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification. This has been seen in studies of the effect of formalin fixation on PCR of Entamoeba histolytica (8). The TOTAL-FIX stool collection kit is a single-vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, most fecal immunoassays, and some molecular methods can be performed from a TOTAL-FIX preserved specimen. TOTAL-FIX is a mercury-, formalin-, and PVA-free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories (www.med-chem.com; accessed 7/24/13). TOTAL-FIX is similar to UNIFIX and Zinc PVA (Z-PVA), commonly used fixatives that have been commercially available and used in many laboratories since 1992 (9, 10).
Parasites found in feces can be preserved in one of two ways (9, 10). One procedure involves thorough mixing of the fecal specimen directly in the fixative. This mixture can then be stored at room temperature without any additional processing. There are several disadvantages of this method: (i) if mixing is not complete, some of the organisms will not be well preserved and will disintegrate during storage; (ii) if the number of organisms is small, a random aliquot may not reveal the parasites; and (iii) the proper ratio of fixative to specimen may not be correct when one is working with larger volumes of material.
The second approach is to clean and separate the organisms from excess debris and concentrate them before fixation. The specimens can be mixed with a large volume of water, passed through a series of screens (large to small pore size), and finally allowed to sediment in a conical container (pilsner glass). After approximately 1 h, the sediment can be washed several times, resedimented, and finally fixed with hot (60 to 63°C) fixative to quickly preserve and stop any further development of certain helminth (Ascaris and Trichuris) eggs. The main disadvantage of this method is extensive washing and manipulation, which may lead to some organism distortion or destruction before fixation. Also, this approach is not very practical unless specimens are being processed for teaching/holding purposes.
The adult forms of most helminths must be relaxed prior to fixation. If this preliminary step is not performed, the worms often contract and curl up when they come in contact with the fixative, thus preventing visual examination of some of the key morphologic characteristics. Several methods, including those involving tap water, physiologic saline, and dilute menthol, are available for this purpose.
Definitive morphology needed for identification of protozoan trophozoites and cysts is best seen on the permanent stained smear, which can be prepared from fresh fecal specimens or from stool that has been submitted to the laboratory in fixative. No washing techniques should be used before bulk fixation, because the trophozoites will be destroyed. Immediately after collection or submission to the laboratory, the specimens should be thoroughly mixed with the fixative of choice. The ratio of fixative to stool should be at least 3 parts fixative to 1 part stool, and the fixation time should be at least 4 h (or less, depending on the amount of specimen used). The normal collection vial systems usually require a fixation time of at least 30 min. If an entire fecal specimen is used, 4 h is recommended. Individual slides can then be prepared from bulk-fixed material by a technique described by Scholten and Yang (11). This method involves centrifugation and smear preparation from the sediment. Although formalin-preserved specimens (except for sodium acetate-acetic acid-formalin-preserved fecal material) are not recommended for the preparation of permanent stained smears, formalin fixation for cyst preservation is recommended for the preservation of teaching specimens that will be examined as wet mounts only. The morphology of cysts can often be seen in formalinized wet mounts sufficiently well to identify organisms to the species level or certainly