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J Histochem Cytochem. Mar 2010; 58(3): 221–228.
PMCID: PMC2825487

Proteomics Out of the Archive: Two-dimensional Electrophoresis and Mass Spectrometry Using HOPE-fixed, Paraffin-embedded Tissues

Abstract

Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:221–228, 2010)

Keywords: HOPE technique, two-dimensional electrophoresis, NSCLC, paraffin material, proteomics, mass spectrometry

Recently, many pathological challenges have evoked an increasing demand for confident molecular read-out techniques for elaborating diagnoses, as well as for discovering more specific disease-relevant marker molecules (Coleman 2000). Such a group of standard markers for lung carcinomas are members of the keratin family, ranging in molecular mass between 40 and 80 kDa, and keratin 10 can be used as a marker for squamous cell carcinomas (Moll et al. 1982; Elias et al. 1988; Nhung et al. 1999; Tsubokawa et al. 2002). As far as analysis of cellular macromolecules, i.e., proteins and nucleic acids, is concerned, large-scale studies of paraffin-embedded materials are still constricted due to the standard procedure of conserving tissue samples with formalin because of good morphological maintenance. This shows that semistandardized detection and characterization of molecular compounds in such archived materials does not supply the needs of detailed biochemical investigations. Protein cross-linking and degradation of nucleic acids hinder the achievement of high-quality results for molecular characterizations. Furthermore, the application of frozen tissues that provide good molecular read-out options is limited by cost-intensive problems such as the necessity for permanent nitrogen storage for long-term appropriation of specimens and the weak preservation of morphologic details. Moreover, after longer storage times, successive losses of molecular integrity occur (Srinivasan et al. 2002).

Since the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixation technique covers both formalin-like morphological maintenance of tissues and excellent conservation of nucleic acids, as well as antigenic structures, it has meanwhile enabled highly reproducible molecular analyses of fixed tissues by several protein- and nucleic acid-targeting methods (Olert et al. 2001; Goldmann et al. 2002,2003,2004; Wiedorn et al. 2002; Droemann et al. 2003; Sen Gupta et al. 2003; Umland et al. 2003; Uhlig et al. 2004). As to preservation of proteins, immunodetection techniques benefit from the avoidance of antigen retrieval procedures if tissues are conserved by HOPE technology instead of formalin (Goldmann et al. 2003). Therewith, the epitopes of protein antigens have been shown to persist even in longer-conserved tissues, and hence, any archived HOPE-fixed material can be adducted for high quality immunostaining analyses. Use of the HOPE technique allows an extended spectrum of analytical methods (immunohistochemistry, RT-PCR, DNA/RNA in situ hybridization, transcription arrays, and protein analyses by SDS-PAGE and immunoblotting) that are applicable for analysis of single-donor material. We have now additionally established a protocol which offers proteome analyses of paraffin-embedded HOPE-fixed tissues by two-dimensional electrophoresis (2DE) employing non-equilibrium pH gradient gel electrophoresis (NEPHGE) in the first dimension and conventional SDS-PAGE techniques in the second dimension to enlarge the methodical spectrum of the HOPE technology. Results were compared with those of corresponding formalin-fixed, ethanol-fixed, and frozen tissues from the same patient. We show in addition that 2DE gels obtained from HOPE-fixed tissue samples are applicable for immunostaining of disease-related marker antigens as well as for standard protocols of protein identification by tryptic digestion and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) analysis of single protein spots.

Materials and Methods

Tissues

The examined tissue samples were characterized as non–small-cell lung cancer, which required surgical resection. We analyzed 10 different cases using each of the three fixation techniques and corresponding frozen material. We show four adenocarcinomas, three squamous cell carcinomas, and one healthy lung tissue. After tumor or tumor-free parts were removed from fresh lobectomies, the tissues were fixed and embedded in paraffin, using the HOPE technology as previously described (Olert et al. 2001). Formalin fixation and frozen material work-up followed standard protocols using a Shandon Pathcentre unit for formalin fixation (Thermo Electron Corporation; Karlsruhe, Germany) and liquid nitrogen for frozen material (Srinivasan et al. 2002). Ethanol fixation was performed for 24 hr at room temperature, using 70% ethanol, and processed similarly to formalin-fixed material with the Shandon Pathcentre unit before paraffin embedding. The paraffin blocks were shelved at 4C (HOPE-fixed samples) or room temperature (formalin- and ethanol-fixed samples); frozen material was stored at −80C.

Preparation of Samples

From the HOPE-fixed paraffin blocks, samples of 8 to 10 sections were deparaffinized by two cycles of isopropanol treatment (5 ml; 5 min in a tube rotor) and centrifugation (13,000 rpm; 5 min) with respective removal of the supernatants. Subsequently, a single dehydration step of acetone treatment was performed, and the tissue samples were dried in a vacuum centrifuge for 10 min (Savant Speed Vac 110; Life Science International, Frankfurt, Germany). Ethanol-fixed specimens were processed using xylol and ethanol for deparaffinization.

Frozen material was crumbled in a dish filled with liquid nitrogen and placed directly into a reaction tube for protein extraction. Formalin material was processed adequately using a special retrieval kit (see below).

Protein Extraction

Two protocols of protein extraction arbitrarily designated as aqueous and complex protein extraction were compared within this study.

For aqueous protein extracts from the tissues, the dried sediments were resuspended in diethylpyrocarbonate-water at a sample concentration of 100 mg/ml by incubation at room temperature for 30 min with vortexing every 5 min. To remove insoluble material, this solution was ultracentrifuged at 50,000 rpm for 30 min, and the protein concentrations of the resulting aqueous tissue extracts were subsequently determined by the bicinchoninic acid assay (Pierce; Rockford, IL). After Speed Vac lyophilization, the dried samples were resuspended in ampholyte-phosphate buffer containing 9 M urea at a final protein concentration of 20 mg/ml by incubation at room temperature for 30 min with vortexing every 5 min and subsequent incubation at room temperature for another 30 min. After precipitated material was removed by ultracentrifugation (50,000 rpm, room temperature, 30 min), the clear supernatants were finally diluted at a ratio of 1:5 in ampholyte phosphate buffer containing 1% (w/v) of agarose that had been tempered at 65C.

For obtaining complex protein extracts from HOPE- and ethanol-fixed tissues and frozen material, we used an extraction buffer containing 7 M urea (Roth; Karlsruhe, Germany), 2 M thiourea (Merck; Darmstadt, Germany), 2% octyl phenol (Igepal; Sigma-Aldrich, Munich, Germany), 1% Triton-X (Merck), 100 mM dithiothreitol (Roth), 5 mmol PMSF (Roth), 4% CHAPS (Roth), and 0.5 mM EDTA.

Formalin-fixed material was deparaffinized and protein-extracted using a QProteome FFPE Tissue 2D-PAGE kit (Qiagen; Hilden, Germany) following the manufacturer's instructions to ensure adequate processing for formalin-fixed samples for 2DE. Fifty μg of protein from each sample was applied to the first dimension of the 2DE system.

2DE

The proteomes of the tissue-derived samples were analyzed by using a WitaVision high-resolution 2D gel electrophoresis system (WITA GmbH; Teltow, Germany). The charge-dependent initial separation of the tissue-derived protein solutions in the first dimension of 2DE was performed in pH gradient rod gels (pH range, 4–10) according to the protocol of the manufacturer using a set of standardized materials. Briefly, two gel solutions were cast in succession in a vertical device for preparation of the two-layered rod gels of the first dimension (quantities sufficient for a total of eight rod gels): 1.5 ml of separation gel solution plus 36 μl of 0.8% ammonium persulfate (APS) was prepared for polymerization of the first gel layer, and 600 μl of cap gel solution (WitaVision) was mixed with 15 μl of 0.8% APS for formation of the second gel layer of the rod gels (all solutions were degassed by sonication). For complete polymerization, the gels of the first dimension were held at room temperature for 30 min and then kept in a damp chamber for an additional 72 hr.

Running the First Dimension (NEPHGE Technique)

The first-dimensional separation of proteins in the rod gels was performed in a vertical electrophoresis device according to the operating instructions of the manufacturer (WitaVision). Briefly, the lower chamber of the device was filled with 400 ml of degassed cathode buffer (10-fold stock solution prepared on a 40C heating plate, containing 20 g of glycine, 216 g of urea, 200 ml of aqua dest, filled up to 380 ml; and the addition of 20 ml of ethylenediamine). Following fixation of the rod gels in the device, the freshly prepared sample solutions in agarose-supplemented ampholyte phosphate buffer were applied to the anodic sides of the capillary gels, and the remaining volumes of the capillary glass tubes were then covered with a sample stabilizing overlay solution (WitaVision). Subsequently, 400 ml of degassed anode buffer was applied (10-fold stock solution of 72 g of urea, 250 ml of aqua dest, filled up to 380 ml; addition of 20 ml of 85% phosphoric acid) to the upper chamber of the device, and the electrophoretic separation of the first dimension was started by using the following sequence of programmed running conditions: 100 V for 1 hr 15 min; 200 V for 1 hr 15 min; 400 V for 1 hr 15 min; 600 V for 1 hr 15 min; 800 V for 10 min; 1000 V for 5 min. After termination of electrophoresis, the rod gels were carefully pushed out of the glass tubes onto plastic rails, and adaptation to the conditions of the second dimension was achieved by a series of five 15-min equilibrations in a corresponding incubation solution. Optionally, the equilibrated rod gels of the first dimension were stored at −80C before application to the second dimension of the 2DE system.

Running the 2nd Dimension (SDS-PAGE)

For separation in the second dimension of 2DE, standard SDS-PAGE was performed with 15% (w/v) polyacrylamide gels, using a Mini Protean 3 Cell unit (Bio-Rad; Hercules, CA). Briefly, the rod gels of the first dimension were gently transferred from equilibration and storage rails to the top of the stacking gel zones and covered with 1% (w/v) agarose containing 0.1% (w/v) bromophenol blue to fix the rod gels and for visualization of the progress in sample migration during SDS-PAGE. The electrophoresis running conditions of the second dimensional separation were set as follows: 35 V for 5 min; 55 V for 10 min; 100 V for 15 min; and 150 V for 1 hr, until the bromophenol blue front reached the bottom of the gel.

Fixing and Staining of 2D Gels

After 2DE protein separation was complete, gels were fixed and silver stained according to Mortz et al. (2001). For protein identification by tryptic digestion and mass spectrometry, Coomassie staining of the corresponding 2DE gels, using a Simply Blue Safe system (Invitrogen; Karlsruhe, Germany) was performed according to the protocol of the manufacturer, until distinct spots began to appear.

Western Blotting and Immunodetection

Blotting was performed using 2DE gels produced from squamous cell carcinoma tissue lysates and an iBlot semidry electrotransfer system (Invitrogen) according to the protocol of the manufacturer (blotting in 7 min onto a nitrocellulose membrane). Immunodetection of pan-keratin representing a group of standard marker proteins for lung carcinomas was carried out by the following procedure: Blocking of unspecific antibody binding [1.5 hr, digoxygenin wash and block buffer (Boehringer Ingelheim; Ingelheim, Germany)]; primary antibody reaction [1.5 hr, anti-pan-keratin antibody (DAKO; Hamburg, Germany), concentration of 1:100]; three wash steps (10 min each, Tween-TBS-buffer, pH 7.4); secondary antibody reaction [45 min, mouse anti-human antibody (Dianova; Hamburg, Germany)], concentration of 1:10,000]; two wash steps (10 min each, Tween-TBS-buffer, pH 7.4); one wash step (10 min, TBS buffer, pH 9.5); determination of color reaction using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl system [(Boehringer Ingelheim), stopped after 5 min by washing with deionized water].

Tryptic Digest and Mass Fingerprinting

The protein spots separated by 2DE were excised, destained, and digested overnight with sequence-grade trypsin from bovine pancreas (Roche Diagnostics GmbH; Mannheim, Germany) as described previously (Shevchenko et al. 1996). For MALDI-TOF MS, the extracted tryptic fragments were mixed 1:1 (v/v) with saturated matrix solution [purified α-cyano-4-hydroxycinnamic acid, (Bruker Daltonics; Bremen, Germany)] and analyzed with a Reflex III spectrometer (Bruker Daltonics) in the reflector TOF configuration with an applied acceleration voltage of 20 kV. The mass spectra were processed by Biotools version 3.2 software (Bruker Daltonics), and mass lists were evaluated by a MASCOT database search, using a molecular weight search (MOWSE) scoring algorithm to create probability-based scoring as described under “Scoring Schemes.” (Matrix Science; http://www.matrixscience.com/help/scoring_help.html#PBM).

Results

In this study, a total of four adenocarcinomas, three squamous cell carcinomas, and one healthy lung tissue were applied to 2DE analysis, comparing HOPE-fixed tissue specimens with formalin-fixed and ethanol-fixed preparations and to frozen samples. Figure 1 shows hematoxylin/eosin (H and E) staining of all analyzed tissues to compare morphology among different tissue fixation methods and frozen material. The images show that HOPE fixation and formalin fixation display well-preserved morphology, while frozen material, as well as ethanol-fixed material, shows lower quality of morphologic details.

Figure 1
Hematoxylin/eosin -stained sections from all investigated tissue samples. (A, C–E) Adenocarcinomas; (B,F) squamous cell carcinomas; (G) tumor-free lung. H, HOPE-fixed tissue; FR, frozen; FF, formalin-fixed; ...

As shown in Figures 2formalin-fixed.formalin-fixed.44 the 2DE analysis of aqueous and complex protein extracts (50 μg of protein each) derived from lung tumor tissues and tumor-free lung, respectively, resulted in comparable spot patterns within each of the compared methods of tissue preservation. From two lung adenocarcinomas, aqueous protein extracts were separated and yielded high-resolution spot patterns with high numbers of protein spots for the HOPE-fixed samples (Figure 2). While frozen material showed lower protein yields in one sample and higher amounts of protein in the other sample compared with HOPE-fixed material, the two corresponding formalin-fixed samples subjected to a commercial standard protocol of adequate antigen retrieval for 2DE showed a lower degree of resolution and lower total numbers of protein spots within the gels than with HOPE-fixed and frozen tissue samples.

Figure 2
Comparison of 2DE results from adenocarcinoma (A) and squamous cell carcinoma (B) subjected to aqueous protein extraction. H, HOPE-fixed; FR, frozen; FF, formalin-fixed.
Figure 3
Comparison of 2DE results from three adenocarcinomas (C–E) subjected to complex protein extraction. H, HOPE-fixed; FR, frozen material; FF, formalin-fixed.
Figure 4
Comparison of 2DE results from one adenocarcinoma (F) with those from tumor-free lung tissue (G). H, HOPE-fixed; FF, formalin-fixed; ET, ethanol-fixed.

From the four tissue samples subjected to complex protein extraction, highly reproducible 2DE spot patterns were also obtained for each of the investigated forms of tissue preservation (Figure 3). Reproducibility within this set of complex protein extracts appeared to be highest in HOPE-fixed material; moreover, the total number of protein spots was higher in HOPE-fixed samples than with the frozen samples of these tissues. Once again, antigen-retrieved formalin-fixed samples resulted in a lower number of protein spots than with both the HOPE-fixed and the frozen material.

2DE analysis of HOPE-fixed vs ethanol-fixed tissues resulted in comparable spot patterns with higher resolution and total protein yield obtained from the HOPE-fixed samples (Figure 4). The 2DE gels of formalin-fixed tissue material again showed lower protein yields and spot resolutions than the corresponding ethanol-fixed and HOPE-fixed samples.

Western Blotting and Mass Spectrometry

Western blotting and immunodetection of keratin structures resulted in several signals, with the main signal in the 40- to 50-kDa range at an approximate pI of 4.9. The respective spot of the corresponding Coomassie-stained gel (Figure 5) was identified by tryptic digestion and mass spectrometry as “keratin 10” with high probability and significance [MOWSE score, 126 (Figure 6; also see supplemental data SD1–SD3)]. Similarly, three additional spots were identified as human serum albumin (which is a component of the blood and therefore present in tissues), tropomyosin, and calreticulin, respectively (Figures 5 and and6).6). For detailed information about the mass spectrometric results, see supplemental data SD1–SD9. The appearance of these molecules in lung (cancer) tissue is in accordance with previous studies (Tada et al. 1997; Nhung et al. 1999; Tsubokawa et al. 2002; Du et al. 2007). Thus, 2DE-separated samples of HOPE-fixed materials can be used for standard protocols of protein identification by tryptic digestion and mass spectrometry analyses.

Figure 5
Squamous cell carcinoma subjected to Western immunoblot targeting pan-keratin and the corresponding twin gel which was used for the tryptic digests. Enlarged section shows the exact positions of the chosen spots; the results of the mass spectrometric ...
Figure 6
Mascot search results with probability-based MOWSE scores of four protein spots excised from HOPE-derived 2DE. (A) Keratin 10; (B) human serum albumin; (C) tropomyosin; (D) calreticulin.

Discussion

The HOPE solution is hyperosmolar and consists of HEPES buffer, glutamic acid, glucose, and different amino acids (concentrated from 10–100 mM) exhibiting a pH range of 5.8–6.4 at room temperature. The exact mechanism of conservation has still to be elucidated in detail; however, it is known that HOPE functions as an immersion fixative affecting the cells via diffusion. Acetone is the only dehydrating agent within the HOPE technique (Olert et al. 2001). Deparaffinization is done by isopropanol treatment, the HOPE technology functions without any ethanol additive, which eliminates protein precipitating effects, and additional fixation with ethanol itself is a fixative. The aim of this study, therefore, was to investigate the applicability of lung tissue specimens fixed by the ethanol- and xylol-free HOPE fixation technique for proteome analyses by 2DE and mass spectrometric fingerprinting. In addition to HOPE-fixed samples, the protein extraction and 2DE results of ethanol- and formalin-fixed samples and frozen material of the same patients' tissues were analyzed to compare the feasibility of HOPE-derived 2DE with these types of tissue conservation for 2DE-mediated proteome analysis. Our study shows that fixation of tissues by the HOPE technique enables reproducible achievement of high-resolution 2DE results. The HOPE method therefore presents a convenient fixation protocol for large-scale proteome studies of archived, paraffin-embedded tumor tissues (Figures 26). The comparison of 2DE spot patterns of HOPE-fixed tumor samples to those of healthy tissue and to other carcinoma subtypes may allow for the assignment of novel diagnostic protein markers in future screenings integrating other molecular investigations using the same materials. The qualification of HOPE-fixed tissue samples for 2DE analysis further enlarges the analytical spectrum of the HOPE technique, in addition to its applicability to immunohistochemical and nucleic acid-based investigations shown in previous studies (Olert et al. 2001; Goldmann et al. 2002,2003,2004; Wiedorn et al. 2002; Droemann et al. 2003; Sen Gupta et al. 2003; Umland et al. 2003; Uhlig et al. 2004). Compared with the HOPE method, the conventional formalin fixation method was found to be largely less convenient for high resolution 2DE proteome investigations in our study. Formalin fixation restricts the application of expedient molecular protein-based and nucleic acid-based investigations due to the inherent, rather strong protein denaturation and nucleic acid degradation (Srinivasan et al. 2002). Use of a strong antigen retrieval procedure (with 20 min of heating at 100C and 2 hr of heating at 80C) with a Qiagen 2D-PAGE kit can result in obtaining 2DE data; however, that method shows lower resolution of protein patterns which would otherwise be required for comprehensive proteomic investigations. Additionally, we observed that frequently, in approximately one-third of preparations, no proteins were visible in 2DE gels, although the retrieval kit was used correctly. Scicchitano et al. (2009) have, meanwhile, established sample preparation for formalin-fixed paraffin-embedded material for mass spectrometric analyses.

Frozen tissue samples, the gold standard in proteomics, are also known to be applicable in 2DE analysis, although the loss of molecular integrity occurs after longer periods of storage. Furthermore, frozen materials display poor morphology in histological investigations (as shown in the H and E–stained sections), and after longer freezing periods, the loss of nucleic acid and protein components is frequent (Srinivasan et al. 2002).

In contrast to those protocols, the application of the HOPE technique (which does not need thermic treatment of antigens for retrieval) allows for hybridization of DNA and RNA (RT-PCR; DNA/RNA in situ hybridization; transcription arrays, and Northern blots) and protein analyses (immunohistochemistry; Western blots; 2DE; and MALDI-TOF MS) by use of one single-tissue source that can be stored long term and shows excellent preservation of morphologic details (Figure 1). These features represent a major advantage of this fixation technique with respect to molecular pathology analysis, which is gaining in importance.

In addition to the above-mentioned biochemical and molecular techniques in the analysis of tumor tissues based on the HOPE fixation results reported in previous studies, the implementation of 2DE and subsequent mass spectrometry analyses shown here can be expected to further complete protein analysis techniques common in the research of cancer, which means finding tumor-specific protein expression patterns supplemental to investigations of corresponding cellular transcripts, including detailed protein identification and sequencing. These multimethod gene expression analyses of tumor tissues may thus provide a powerful base for the identification of novel disease-relevant protein markers that in turn, could further improve diagnostic and treatment options in the future.

Several proteomic approaches using paraffin-embedded material have recently been reported by other groups. Ahram et al. (2003) used ethanol as a fixative prior to paraffin embedding, which provided good conditions for subsequent 2DE. Those results also showed the insufficient properties of formalin for proteome investigations, whereas the ethanol-fixed and frozen specimens resulted in multiple protein spots after 2DE. This can also be affirmed by our tests. The results of these latter methods were found to be comparable to each other in these studies. Similar results were achieved using another ethanol fixation method (FineFix; Milestone Medical, Kalamazoo, MI) by Stanta et al. (2006). Nevertheless, ethanol fixation is also known for lower preservation of morphologic details.

Taken together, replacing formalin fixation with HOPE-fixed, paraffin-embedded tissues seems to ensure better conditions for 2DE proteomic approaches besides the other-mentioned molecular methods as reported predominantly by Goldmann et al. (2002,2003,2004). According to the results from this study and our prior investigations, standardized HOPE technology represents an efficient and, analytically, most versatile fixation method, enabling multiple data to be obtained from (single) archived tissue materials. As this fixation method is comparably inert and as it has been shown to provide a powerful base for all standard types of molecular analyses tested so far, the HOPE technique appears to be well suited in future screenings of multiple tissues for the identification and characterization of novel disease-relevant molecules, as well as the feasibility of 2DE for describing changes in posttranslational protein modifications such as phosphorylation, acetylation, and glycosylation status of proteins.

The results obtained from Western blotting and mass spectrometry analyses in this study confirm our previous reports of the maintenance and immunoreactivity of protein structures in HOPE-fixed tissue samples. The observed suitability for proteomic investigations represents an additional advantageous feature of the HOPE technique compared with other fixation methods because, first, there is no ethanol and no xylol necessary (replaced through acetone and isopropanol) and, second, HOPE-fixed tissue samples are applicable to all common techniques of RNA/DNA and protein research for the respective analysis of single-donor materials, resulting in high inter-method comparability.

Acknowledgments

The authors thank Nina Grohmann, Jasmin Tiebach, Stefanie Fox, Maria Lammers, Helga Lütje, and Helge Meyer for excellent technical assistance.

Notes

This article is distributed under the terms of a License to Publish Agreement (http://www.jhc.org/misc/ltopub.shtml). JHC deposits all of its published articles into the U.S. National Institutes of Health (http://www.nih.gov/) and PubMed Central (http://www.pubmedcentral.nih.gov/) repositories for public release twelve months after publication.

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