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Ultrasound-accelerated Tissue Fixation/Processing Achieves Superior Morphology and Macromolecule Integrity with Storage Stability

Wei-Sing Chu, Qi Liang, Yao Tang, Randy King, Kondi Wong, Maokai Gong, Minqi Wei, Jilan Liu, Shaw-Huey Feng, Shyh-Ching Lo, Jo-Ann Andriko and Marshall Orr

Department of Scientific Laboratories (W-SC,QL,RK,MG,MW,JL), Neuropathology and Ophthalmic Pathology (KW), Infectious and Parasitic Diseases Pathology (S-HF,S-CL), Armed Forces Institute of Pathology, Washington, DC; Department of Pathology, Walter Reed Army Medical Center, Washington, DC (J-AA); Naval Research Laboratory, Washington, DC (MO); and Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland (YT)

Correspondence to: Wei-Sing Chu, MD, Department of Scientific Laboratories, Armed Forces Institute of Pathology, Washington, DC 20306-6000. E-mail: chu{at}afip.osd.mil

	Summary

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We demonstrate that high-frequency and high-intensity ultrasound (US) can be applied to both tissue fixation and tissue processing to complete the conventional overnight formalin-fixation and paraffin-embedding (FFPE) procedures within 1 hr. US-facilitated FFPE retains superior tissue morphology and long-term room temperature storage stability than conventional FFPE. There is less alteration of protein antigenicity after US-FFPE preservation so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. US-FFPE tissues present storage stability so that room temperature storage up to 7 years does not significantly affect tissue morphology, protein antigenic properties, RNA distribution, localization, and quantitation. In addition, during fixation, tissue displays physical changes that can be monitored and reflected as changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls. The mechanism of less alteration of protein antigenicity by US-FFPE was discussed. (J Histochem Cytochem 54:503–513, 2006)

Key Words: ultrasound • formalin fixation • tissue processing • immunohistochemistry • RNA extraction • storage stability

	Introduction

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CONVENTIONAL FORMALIN-FIXED, PARAFFIN-EMBEDDED (FFPE) tissue provides superior cellular morphology and the ease of long-term storage (Fox et al. 1985; Werner et al. 2000). The standard FFPE procedures require 8–24 hr fixation in 10% formalin followed by overnight (18 hr) processing to embed fixed tissues in paraffin. In addition, protein antigenic properties and nucleic acid integrity in FFPE tissues are affected by formaldehyde-induced cross-linking (Fetsch et al. 2002; Gillespie et al. 2002), impairing the application of newly developed molecular diagnostic techniques on FFPE tissues. For example, some cellular surface clusters of differentiation and muscular antigens are not detectable in FFPE tissues, even with advanced microwave antigen retrieval (MW-AR) (Shi et al. 1991). Cross-linking makes it difficult to extract proteins from FFPE tissue for SDS-polyacrylamide gel electrophoresis, Western blotting (Gillespie et al. 2002), and proteomic profiling (Fetsch et al. 2002). mRNA extracted from FFPE tissues are markedly shortened in length and reduced in amount than mRNA extracted from fresh tissues (Foss et al. 1994; Krafft et al. 1997; Liu et al. 2002).

Cross-linking causes proteins and nucleic acids to be less effectively extracted from FFPE tissues. Though methods are available to extract macromolecules from FFPE tissues (Chu et al. 2005b), traditionally, fresh and flash-frozen tissues are used for molecular testing (Emmert-Buck et al. 2000). However, frozen sections have relatively poor morphology inadequate for definitive pathologic diagnosis. Obtaining fresh/frozen tissues requires the cooperation of at least the OR nurse, surgeon, cell culture and pathology grossing technicians, clinician, and patient. Several physicians and technicians must work coordinately for an hour or more to ensure that a tissue specimen is handled and processed properly, compared with the two to three people needed to process several hundred FFPE tissue specimens daily. In short, the high cost, labor intensiveness, and low efficiency of handling and storage all limit the use of frozen tissues in current hospital practices.

Clearly, a rapid tissue preservation technique that provides superior tissue morphology and improved macromolecular integrity with easy handling and long-term storage stability would benefit medical research and practices. Microwave-enhanced tissue fixation and processing represents one such improved breakthrough. As another physical force, ultrasound (US) has been widely used in diverse medical fields for over 50 years. Low-frequency (20–100 kHz) and high-intensity US is used for medical equipment cleaning, tissue and cell homogenization, and stone ablation. High-frequency (>0.5 MHz) and low-intensity (3 W/cm²) US is employed for physical therapy and medical imaging. US has been successfully applied to transdermal drug delivery (Mitragotri et al. 1995), transdermal monitoring of glucose (Kost et al. 2000), and gene transfer (Danialou et al. 2002). We have previously demonstrated that high-frequency and high-intensity US can greatly enhance the rate of tissue formalin fixation to achieve superior tissue morphology and improved macromolecule preservation (Chu et al. 2005a). Here we further demonstrated that US could be applied to formalin fixation as well as to processing procedures to achieve completion of the overall FFPE procedures within an hour. Details of the US apparatus were described, US-induced temperature increase in tissue was monitored, tissue physical changes (such as hardening) during the fixation process with and without US implementation were compared, the effect of US intensity and frequency on tissue preservation was addressed, and the preservation of tissue morphology, protein antigenicity, and RNA in US-FFPE tissues after 7 years storage was evaluated.

	Materials and Methods

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Ultrasonic Apparatus for Tissue Fixation and Processing
A prototypic US apparatus (Bio-Quick Inc.; Silver Spring, MD) to facilitate both fixation and processing is shown in Figure 1A . Unfocused plane transducers (EDO Corporation; Salt Lake City, UT) were used to provide large acoustic fields comparable to the size of a typical tissue sample (25 mm in diameter). A sliced tissue sample placed in a conventional tissue cassette (Figure 1A) was positioned 3–5 cm away from transducers in 10% neutral-buffered formalin (NBF) and immediately sonicated by 1.6–1.7 MHz US for 3–15 min at an intensity of 3–20 W/cm² acoustical power. After fixation, the tissue was immersed for 5 min each in graded ethanol and 15 min each in xylene and paraffin (preheated and kept at 58C in a water bath). US was continuously applied to the solution in each step.

View larger version (19K): [in this window] [in a new window]   Figure 1 Ultrasound (US)-facilitated formalin fixation and paraffin embedding (FFPE). (A) A prototypic apparatus showing the fixation and processing chamber. A tissue slice was placed in a tissue cassette 3 to 5 cm away from an US transducer and immersed in 500 ml of solution such as formalin, ethanol, xylene, or liquid paraffin. (B) Configuration of a monitoring–controlling system for temperature and transmission/reflection US. (C) Scheme for the monitoring–controlling system.

Routine FFPE and US-facilitated FFPE Procedures
A total of 86 (34 biopsy and 52 autopsy) tissues were fixed and processed with and without US. They were from 14 tissue types including arteries, brain, breast, colon, heart, kidney, liver, lung, lymph node, pancreas, prostate, skin, spleen, and uterus. All tissue specimens were obtained from Walter Reed Army Medical Center. Biopsy specimens were obtained 1–2 hr after excision, and autopsy specimens were obtained 15–32 hr postmortem. Samples were cut into 3- to 5-mm slices and stored either at –80C or processed by standard FFPE or US-facilitated FFPE procedures. Reagents and the working temperature (assuming the room temperature unless specifically stated) used for the two procedures were kept the same. Tissues preserved by standard FFPE were fixed in 10% NBF for 18–24 hr at room temperature, followed by 16–18 hr of automated processing (Tissue Tek; Miles Scientific, Naperville, IL) consisting of dehydration in graded ethanol, clearing in xylene, and infiltration in paraffin. Although apparatus for multi-sample US-FFPE was available and under further improvement, due to the availability of specimens and for better control of conditions, all tissues were preserved by the two methods, one at a time. Tissues preserved by US-FFPE were fixed in 10% NBF for 5 to 15 min, followed by dehydration, clearing, and paraffin infiltration under US for 10–15 min. US was continuously applied to the solution in each step. Fixation and processing were completed within 1 hr. For ethanol preservation, 10% formalin was replaced with 75% ethanol with all other steps unchanged. Blocks of fixed and processed tissues were either stored at room temperature for later use or cut into 4- to 5-µm sections and mounted onto glass slides. Tissue sections were deparaffinized in xylene and rehydrated in downgraded ethanol. Some were stained with hematoxylin and eosin (H and E), and others underwent immunohistochemical (IHC) procedures.

Immunohistochemistry
Rehydrolyzed tissue sections were pretreated with or without antigen retrieval by microwave (Shi et al. 1991) or pepsin digestion, as required for each antibody. The sections were then blocked with 10% horse and goat serum for 15 min to reduce nonspecific background. Primary antibodies (DAKO; Carpinteria, CA) were added onto slides at the appropriate concentrations (CD3 1:500, CD5 1:100, cytokeratin AE1/AE3 1:400) for specified time periods. Slides were washed three times in PBS and applied with biotinylated secondary antibodies for 5–30 min at concentrations specified by the providers. ABC kit (Vector Laboratories; Burlingame, CA) and chromagens diaminobenzidine (DAB) or aminoethyl carbazole (AEC) were used to develop color as previously described (Chu et al. 1999). Hematoxylin was used as the final counterstain.

mRNA In Situ Hybridization
Tissue sections were deparaffinized as described for IHC and subjected to enzymatic or MW-AR pretreatment prior to hybridization. ISH performed 7 years ago on the newly fixed/processed tissue sections was by manual protocols. Fluorescein isothiocyanate (FITC)-tagged synthetic oligonucleotide probes specific for mRNA of kappa and lambda immunoglobulin were from BioGenex (San Ramon, CA). FITC-labeled probe applied to the section was secured by a coverslip and denatured at 100C for 5 min in a steamer. Slide sections were allowed to cool and hybridize with the probe at room temperature for 1 hr. Sections were given two 3-min washes in 2x SSC, incubated for 30 min with monoclonal mouse anti-FITC, and followed by two 3-min washes in PBS. The slide section was incubated with biotinylated secondary antibody for 30 min at room temperature, followed by two 3-min washes in PBS, and then incubated with streptavidin–biotinylated peroxidase for 30 min. After PBS washing, the slide was treated with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium reagents for color development. Appropriate positive and negative controls provided by BioGenex were used with each reaction. ISH performed on 7-year stored tissue sections used INFORM kappa and lambda probes on a BenchMark Staining System (Ventana; Tucson, AZ) following the manufacturer's instructions.

Nucleic Acid and Protein Extraction from Tissue Sections
Four- to eight-5-µm sections per block were cut. Sections were placed in a 1.5-ml microcentrifuge tube and 800 µl of Hemo-De (Fisher Scientific) was added to dissolve the paraffin. After centrifugation, the supernatant was removed, the pellet was washed three times with 400 µl absolute ethanol, and then resuspended in 0.5 ml of buffer containing 20 mM Tris (pH 7.4), 20 mM EDTA, 1% SDS, and 1 mg/ml proteinase K (Sigma; St Louis, MO). After incubating 6 hr at 55C to solubilize cellular proteins, the tissue lysate was treated twice with TRIzol LS (Invitrogen; Carlsbad, CA) to extract DNA and proteins, according to the manufacturer's instructions. RNA was extracted with the Optimum FFPE RNA isolation kit (Ambion Diagnostics; Austin, TX) and DNase I treated. Extracted DNA or RNA was resuspended in 25 µl of molecular-grade water or appropriate buffer until PCR or RT-PCR reaction.

SDS-PAGE and Western Blot
Proteins extracted from paraffin blocks by TRIzol LS method (Invitrogen) as described above were first denatured at 70C for 10 min in a non-reducing loading buffer from NuPAGE kit (Invitrogen) and then separated by electrophoresis with a 4% to 15% gradient PAGE gel. For Western blot, the protein was transferred onto a polyvinylidene fluoride membrane (PVDF; BioRad, Hercules, CA). After washing in TBS and blocking overnight with 3% dry milk–TBS buffer, the membrane was incubated in TBS-diluted anti-cytokeratin monoclonal antibody (1:5000) for 1 hr, followed by 1 hr of incubation with 1:50,000 diluted secondary antibody, alkaline phosphatase-conjugated goat anti-mouse IgG. The signal was developed using the AP Color Development System (Promega; Madison, WI).

Polymerase Chain Reaction (PCR)
RNA extracted from FFPE and US-FFPE tissues was reverse transcribed into cDNA with SuperScript III First-Strand Synthesis System (Invitrogen), following manufacturer's instructions. Primers for ß-actin were synthesized as two forward primers: AF1 (5'-GAAGGATTCCTATGTGGGCG-3') and AF2 (5'-GCCAGCTCACCAT GGATGAT-3') and four reverse primers: AR1 (5'-GCGAGAAGATGA CCCAGATC-3'), AR2 (5'-CGGGAAATCGTGCGTGACAT-3'), AR3 (5'-TGCAGAAGGAG ATCACTGCC-3'), and AR4 (5'-A CAAGATGAGATTGGCATGGC-3'). PCR reaction using AF1 paired with one of the reverse primers generated amplicons of 220 bp, 489 bp, 811 bp, and 1062 bp, respectively. PCR reaction using AF2 and AR2 generated amplicons of 647 bp. PCR reaction was run for 30 to 40 cycles with 15 pmol primers, 1/10 total volume of DNA template, and 1 U Platinum Taq DNA polymerase (Invitrogen). PCR products were separated on 1% agarose gel and stained with ethidium bromide.

Real-time PCR
Real-time PCR was performed in a Roche LightCycler and the data analyzed by the accompanying software (version 1.1). The double-strand DNA probe SYBR Green I (Molecular Probes; Eugene, OR) was used to quantitate amplicons. All assays were carried out in 15 µl of PCR buffer (50 mM Tris, pH 8.3, 25 µg/ml BSA), 0.2 mM dNTP, 5 mM MgCl₂, 1 U Platinum Taq DNA polymerase (Invitrogen), 1 µM primer and 0.3x SYBR Green. Cycling conditions included initial denaturation of 2 min at 95C, followed by 35 to 45 cycles of 10 sec at 95C, 10 sec at 55C, and 20 sec at 72C. The threshold cycle (Ct) value was determined by the LightCycler (Roche) software.

	Results

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Technological Aspects of US Apparatus
The high-frequency, high-intensity US apparatus used in this study is shown in Figure 1 [any generator of high-frequency, high-intensity US, as specified here and in previous publications (Chu et al. 2005a), could be adapted to achieve similar results]. The apparatus generated US with a frequency of 1.6–1.7 MHz, and the intensity was adjustable in the range of 3 to 20 W/cm² acoustical power. Sonication time was in inverse proportion to the intensity of the US and the delicacy of the tissue being processed. Tissue fixation in formalin or 75% ethanol could be finished in 5–15 min, depending on US intensity. After fixation, the tissue was immersed 5 min each in graded alcohol and 15 min in xylene at room temperature and then in paraffin at 58C for 15 min, with US at 10–15 W/cm² acoustic power. Total fixation and processing time was 1 hr.

Limited Temperature Increase during US Implementation
A thermometer with dual needle probes registered no measurable difference between solution temperature and tissue temperature during US implementation. There was a slow increase in tissue/solution temperature along with US application, as shown in Figure 2A . Typically, tissues submerged in 500 ml of solution showed a temperature increase of <12C after 40 min of sonication. In general, the temperature increase gradually slowed down after solution temperature reached 32C, possibly due to normal environmental heat loss. Because no single step of fixation and processing exceeded 15 min, the temperature increase in any given tissue was <10C. Continued fixation and processing for multiple tissues would require a temperature regulation system.

View larger version (8K): [in this window] [in a new window]   Figure 2 Physical properties of tissues during fixation. (A) The change of temperature of the 250-ml fixative (and that of the tissue being fixed) during extended US implementation. The temperature inside tissues increased with US irradiation time but not substantially, <10C within 30 min. (B) Physical changes in the tissue during fixation with and without US could be reflected by changes in transmission US signals passing through the tissue. A slice of kidney tissue was fixed by conventional and US-facilitated formalin fixation. RMS is the root mean square of a waveform as detected by an oscillator induced by transmission US signals. The time required to reach RMS plateau, which indicated the completion of tissue physical changes, correlated well with the time for fixation completion.

US-FFPE Preserves Good Tissue Morphology with Storage Stability
So far, 86 tissues of 14 tissue types were subjected to H and E staining and one to three IHC staining using various antibodies, as represented in Figure 3 . Based on over 200 pairs parallel comparison, we concluded that compared with FFPE tissues, US-FFPE tissues constantly preserved similar superior morphology as evaluated on H and E staining, and provided improved protein antigenicity as evaluated on IHC staining. Under light microscopy, there were essentially no visible morphological differences in H and E staining between the FFPE and US-FFPE tissues in terms of evenness of stain, color balance, and visual sharpness of cellular membrane, subcellular organelles, and nuclei (Figures 3A and 3B). Improved antigen preservation resulted in increased IHC reaction and reduced the concentration of primary antibodies. The time for IHC reaction against CD3 was shortened from 1–2 hr to 10 min on US-FFPE preserved lung (brown DAB staining in Figure 3D). In contrast, under the same IHC conditions, no brown signal was detected in the FFPE tissue (Figure 3C). A 400x dilution of primary antibody against cytokeratin yielded greater staining intensity in US-FFPE tissue (brown staining in Figure 3J) than in FFPE tissues (Figure 3I), even after 7 years of storage at room temperature (Figure 3K vs Figure 3L). As another result of improved antigen preservation, antigen retrieval procedures were not necessary for CD5 (a membrane antigen) and p63 (a nuclear antigen of the p53 gene family) on US-FFPE tissue (brown in Figure 3F, red–purple AEC in Figure 3H), whereas routine FFPE tissue without antigen retrieval displayed no such signals (Figures 3E and 3G).

View larger version (83K): [in this window] [in a new window]   Figure 3 Comparison of general histology and IHC of tissues preserved by 36–48 hr of routine FFPE and 1 hr of US-FFPE. (A,B) Hematoxylin and eosin (H and E) stain of lung squamous cell carcinoma (x400); (C,D) anti-CD3 (1:500) of T lymphocytes in lung cancer tissue with pepsin digestion (x100); (E,F) anti-CD5 (1:100) in tonsil without microwave antigen retrieval (MW-AR) (x100); (G,H) anti-p63 (1:640) aminoethyl carbazole (chromagen) in prostate without MW-AR (x200); (I–L) anti-cytokeratin (1:1000) in lung squamous cell carcinoma with pepsin digestion (x400). (A-J) Staining within 1 year of preservation. (K,L) After 7 years of storage. US-FFPE tissues demonstrated high-quality H and E staining comparable to FFPE tissues and markedly increased immunostaining detection sensitivity.

View larger version (50K): [in this window] [in a new window]   Figure 4 Comparison of tissue morphology and protein extracts of well-preserved and underfixed kidney tissues. (A) Comparison of morphology of H and E-stained kidney tissues fixed and processed by R, routine FFPE (16-hr formalin fixation) and automatic (18-hr processing); UL, 30-min low-intensity (1.5 W/cm2 acoustic power) US-formalin fixation and normal US processing; UH, 30-min high-intensity (5 W/cm2 acoustic power) US-formalin fixation and normal US processing. (B) Coomassie blue staining of total proteins and Western blot analysis of specific keratin proteins extracted from tissue prepared in (A). Underfixation with low-power US did not increase extractable proteins.

View larger version (71K): [in this window] [in a new window]   Figure 5 Comparison of ISH signals on routine FFPE and US-FFPE tonsil tissues when newly preserved and after 7-year storage. (A) x40 magnified view of ISH showing peripheral staining in routine FFPE tissue and uniform peripheral and central staining in US-FFPE tissue. (B) x200 magnified view of ISH of routine FFPE and US-FFPE tonsil tissues when newly preserved and after 7-year storage. Compared with that on newly preserved tissues, the stronger staining on tissues after 7-year storage is due to different ISH staining protocols.

View larger version (16K): [in this window] [in a new window]   Figure 6 Comparison of PCR products using several primer sets for ß-actin DNA, corresponding to amplicons of 220, 489, 811, and 1062 bp, using extracts of lymph nodes preserved by different methods. (A) PCR products after 32 and 40 cycles using extracted DNA from frozen (F), routine FFPE (R), and US-FFPE (U) tissues. C, negative control using water as the template. (B) PCR products after 38 cycles using extracted DNA from tissues of 1, US-formalin fixed (3 min) and US-processed (total in 45 min); 2, US-formalin fixed (15 min) and routine processed (16 hr on an automatic processor); 3, US-ethanol fixed (10 min in 75% ethanol) and routine processed; 4, routine 16-hr ethanol fixed and routine processed; 5, conventional FFPE (16-hr formalin fixed and 16-hr routine processed).

View larger version (40K): [in this window] [in a new window]   Figure 7 Comparison of RT-PCR products using RNA extracted from tissues preserved by conventional FFPE and US-FFPE methods after long-term storage. (A) RT-PCR by three primer sets corresponding to amplicons of ßß-actin 220-, 489-, and 811-bp fragments using extracted RNA from frozen lymph node tissue (F), routine FFPE tissue stored at room temperature for 1.5 years (R1, lymph node) and 7 years (R7, lung cancer), and US-FFPE tissue stored at room temperature for 1.5 years (U1, lymph node) and 7 years (U7, lung cancer). (B) Quantitative analysis of cDNA by real-time PCR. cDNA was transcribed from RNA extracts from frozen lymph node (F), routine FFPE tissue stored for 1.5 years (R1, lymph node) and 7 years (R7, lung cancer), and US-FFPE tissue stored for 1.5 years (U1, lymph node) and 7 years (U7, lung cancer). Each data point was averaged from two to three independent experiments. The threshold cycle value (Ct) determined by LightCycler Data Analysis software is defined as the PCR cycle number at which the fluorescence is above background. Thus, the lower the Ct value, the higher the initial cDNA concentration or copy number. A higher amount of RNA was consistently extracted from US-FFPE tissue than from routine FFPE tissue.

	Discussion

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We have demonstrated that, during fixation, tissue displays physical changes that can be reflected by changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls, which might be critical for standardized IHC studies (O'Leary 2001; von Wasielewski et al. 2002; Boenisch 2004). The use of transmission US signal to monitor tissue fixation is a new and useful concept. Until now, there is no means to verify and to standardize the fixation level during fixation. Because tissue preservation by routine FFPE or other fixatives is greatly affected by the type and size of samples, it is useful to control the degree of fixation to prevent over- or underfixation. We noticed significant changes in tissue color, size, and hardness upon fixation. These changes in tissue density and water content might affect the speed of sound waves passing through the tissue, which was reflected in transmission (and/or backscatter) US signal from the tissue. We found a promising correlation between transmission US signal and the level of tissue fixation (Figure 2). It is worth mentioning that this monitoring system is applicable to other tissue preservation methods using other fixatives, with or without implemented US for fixation. Further study is underway to confirm and to delineate the observed transmission US signal in correspondence to tissue fixation levels.

Compared with routine FFPE tissues, US-FFPE tissues seem to be less modified by formalin, so that protein antigenic properties and protein profiles are less altered, and more proteins and nucleic acids are extracted (Figure 3–Figure 7). It could be argued that US-FFPE tissues were simply underfixed compared with routine FFPE tissues. However, cellular morphologic disintegration was not observed in US-FFPE tissues, even after enzymatic or microwave treatment (data available). When we purposely underfixed a tissue by applying lower intensity US, we observed obvious tissue shrinkage, inferior staining, and lysis of erythrocytes (Figure 4A). Furthermore, this underfixed tissue generated fewer extractable proteins than the well-preserved US-FFPE tissue (Figure 4B). Underfixation causes both structural distortion and decreased amounts of extractable proteins and nucleic acids due to the possible loss of soluble components during processing.

Microwave irradiation can also speed tissue fixation and processing to within an hour (Leong and Duncis 1986; Morales et al. 2002). The intense heat generated by microwaves facilitates tissue fixation and aggregation (Leong and Duncis 1986; Login et al. 1987; Azumi et al. 1990; Morales et al. 2002) and necessitates using PBS as a fixative instead of formalin. For US-FFPE method, internal tissue temperature did increase with US in proportion to length and intensity of sonication but did not exceed 12C above room temperature during a continuous 40-min application to a 500-ml solution (Figure 2A). Because each step of fixation and processing could be completed within 15 min, the temperature increase is not significant. Thus, we believe that temperature may not be the most prominent factor in US facilitated by fixation and processing.

Though bulk temperature increase is not very remarkable, US may generate microscopically localized high temperature, high pressure, and cavitation so that a high-energy interface exists for unusual biochemical reactions such as free radical formation from water and organic solvent decomposition to occur (Henglein and Kormann 1985; Riesz and Kondo 1992). Intense US waves traveling through solvents and tissue generate cavitation and cyclic stresses that may concentrate energy and power, accelerating alcohol dehydration, xylene clearing, and paraffin impregnation reactions. As mentioned previously (Chu et al. 2005a), applying low frequency US of 20 kHz for 5 min resulted in severe tissue damage as demonstrated by cavitations throughout the tissue (data available). In contrast, high-frequency (1.6 MHz) US would not cause tissue cavitation even after 30 min of continued application. Chemical infiltration (Mitragotri et al. 1995) and permeability of tissues (Kost et al. 2000) are facilitated by US waves disrupting membrane cohesiveness and mutually exclusive forces along immiscible liquid surfaces. US-mediated emulsification into microscopic droplets favors both solvent penetration and crossover of paraffin molecules from the liquid phase into the tissue.

The main effect of US may be to induce more rapid cross-linking reactions among macromolecules, increase cell membrane permeability, and accelerate the exchange rate of water and reagents in subcellular compartments. Mitragotri et al. (1995) reported that US accelerated transdermal drug penetration and diffusion by 1000-fold. However, we believe that increased penetration is not the most prominent effect of US just as slow penetration is not the main reason for slow fixation. Using a thin layer of tissue or even using dissolved protein solution did not substantially shorten the fixation time. mRNA in a 5-µm tissue section after 5-min formalin fixation was not preserved but degraded (Fend et al. 1999). Without any need for penetration, it still required between 6 and 16 hr for complete formalin fixation of a molecular layer of peptides on the glass microscope slide (Sompuram et al. 2004). Rait found that inhibition of RNase A in formalin solution requires 15 hr or more (unpublished data), as measured by enzymatic activity and indicated by structural and thermal property changes (Rait et al. 2004a,b). As presented here and published previously, we observed that 5–15 min formalin fixation with US preserved RNA better than 16–24 hr of routine formalin fixation (Figure 5). We believe the mechanism of much accelerated fixation by US is primarily due to a similar sonochemical reaction as described by Stephanis et al. (1998) that US treatment accelerated the cross-linking between formaldehyde and macromolecules.

How to explain less modification of protein antigenic properties? Our hypothesis is that US-facilitated formaldehyde fixation greatly accelerates cross-linking reaction similar to snap-frozen macromolecules and their conformation. As summarized by Shi et al. (2000), formaldehyde is capable of forming cross-linking to lysine, arginine, tyrosine, asparagines, histidine, glutamine, and serine. In general, free amine (as in lysine and arginine) is more active than primary amides, even more so than secondary amides (Riesz and Kondo 1992; Stephanis et al. 1998; Masuda et al. 1999; Shi et al. 2000). In routine formalin fixation, cross-linking formation is a slow process, allowing methyl (from formaldehyde) to form between optimal reaction sites. For this kind of cross-linkage to occur, proteins may have to alter their local conformation (Mason and O'Leary 1991; Rait et al. 2004a,b), thus altering protein antigenicity as a result. In contrast, when formalin solution is exposed to high-frequency, high-intensity US, deprotonation of formaldehyde, amine, and amide may be much accelerated. These intermediates have high energy, so that covalent bonds between some of the less-optimal sites that are most adjacent may also be formed. Cross-links between these less-optimal sites will be easier to reverse and have less effect on conformation than that between optimal sites. Thus, US-accelerated formalin fixation is somewhat similar to the snap-frozen method, as it tends to preserve tissues close to their original conformation. Furthermore, less energy is needed to disrupt chemical bonds that form between non-optimal sites, facilitating the process of protein extraction. A study of the mechanisms of US-accelerated formalin fixation is currently underway in our laboratory.

In summary, US-accelerated FFPE is a powerful tool for the preservation of biological specimens. Treating fixatives and other processing solvents with US is simple and safe and can be easily integrated into hospital procedures. This technology combines the superior morphological preservation provided by traditional FFPE with the rapid, superior preservation of macromolecules provided by flash freezing. We believe that US-FFPE has great potential to facilitate intraoperative diagnosis and to be broadly applicable to molecular diagnostic techniques. With this technology, pathologists can provide rapid and accurate diagnosis at the morphological and molecular levels, while supporting molecular research with time and cost efficiencies.

	Acknowledgments

 
This work was supported by a National Institutes of Health/National Cancer Institute Grant 1R21 CA-091166-01A1 and an American Registry of Pathology/Armed Forces Institute of Pathology Grant (UBKL) 300-1012-3029-0, both to W-SC. The opinions expressed in this article are the personal views of the authors and are not to be construed as representing the views of the Department of the Army or the Department of Defense.

Special thanks to Bonnie L. Casey for excellent editorial assistance and to Drs. Christopher R. Owner, Douglas J. Wear, Susan L. Abbondanzo, and Stephen I. Fisher for professional expertise and technical support in the preparation of this manuscript.

	Footnotes

 
Received for publication August 2, 2005; accepted November 8, 2005

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