Abstract
Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid α-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid α-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.
Keywords
The goal of this study was to achieve consistent tissue preservation and sharp staining contrast between the abnormal glycogen substrate and the surrounding subcellular structure, to support a histomorphometrically derived end point in clinical trials of Pompe disease therapeutics. A review of the recent literature suggested that further improvement of the morphology was warranted in order to apply morphometric analyses. Although the use of glycol methacrylate with Periodic acid-Schiff's (PAS) staining in recent studies can provide good preservation in many cases, it produces dark pink staining of lysosomal glycogen on an irregular, light pink background that does not provide the sharp contrast required for computer morphometry (Bijvoet et al. 1998,1999a,b; Winkel et al. 2003). In one study, the images required evaluation on a subjective 0- to 3-point scale (Winkel et al. 2003), a scoring method that is likely to be more prone to inter-observer variation than computer morphometry. Therefore, we set out to test the suitability of several fixatives, embedding media, and staining combinations that would yield a protocol that maximally and reproducibly preserved glycogen in Pompe tissue and was suitable for comprehensive evaluation of multiple histology samples by computer-assisted morphometry.
Glycogen is partially soluble in water (Kiernan 1999) and can be removed during prolonged exposure to aqueous solutions during formalin fixation, paraffin processing and embedding, and standard histochemical staining for routine light microscopy (LM). Alcoholic fixatives, such as Carnoy's, have been recommended for preservation of glycogen in normal mouse liver (Kinsley et al. 2000). However, our application of such methods to preserve the abnormal muscle glycogen in Pompe disease is a novel approach not yet addressed in the literature. Perfusion-fixation with traditional 10% neutral buffered formalin (NBF) was also tested to determine if rapid tissue penetration could improve the retention of glycogen in Pompe muscle. It was also important to optimize the staining of tissue sections to improve detection of glycogen. The PAS reaction is a carbohydrate stain that binds mucopolysaccharides, neutral mucins, and glycoproteins as well as glycogen. The addition of dimedone prevents the PAS reaction on mucopolysaccharides and glycoproteins in tissue sections, thereby restricting staining specifically to glycogen (Bulmer 1959).
The embedding medium can also affect the final appearance of tissue sections. Both glycol methacrylate and epon-araldite embedding were tested to eliminate the xylene and heating steps required for paraffin processing, steps that might contribute to glycogen loss. In addition, stronger fixation with glutaraldehyde and postfixation in osmium tetroxide were employed for tissues subsequently embedded in these plastic mediums. The combination of glutaraldehyde/osmium tetroxide fixation with epon-araldite embedding yielded the best results. This latter fixation method, traditionally employed for electron microscopy (EM), preserves glycogen well by using fixatives that provide better cross-linking of substrates and limit sample exposure to aqueous solutions. Submitting tissue samples for EM analysis on a routine basis is time-consuming, so we sought to adapt this methodology for analysis at the light microscopy level. One-μm sections from these blocks were successfully stained with PAS and Richardson's counterstain to produce high-resolution light microscopy (HRLM) sections in which the glycogen was maximally preserved and clearly visualized with a light microscope. The preservation of glycogen could be confirmed by EM in sections cut from these same blocks. Therefore, this single-processing methodology serves dual purposes, avoiding the need to embed tissue in both glycol methacrylate and epon as has been described previously (Bijvoet et al. 1998,1999a,b; Winkel et al. 2003). HRLM allows the tissue to be digitally analyzed and the glycogen to be quantified in any tissue affected by Pompe disease in a highly reproducible manner. We present our results using this methodology in heart and skeletal muscle, tissues that are severely affected by Pompe disease and most often contribute to the clinical manifestations of the disease.
Materials and Methods
Animals
The 6neo/6neo acid α-glucosidase deficient mouse model, developed by Nina Raben at the National Institutes of Health (NIH) (Raben et al. 1998), was used in all experiments. The mice were housed in a facility according to ALAAC accredited facility guidelines. The mice were sacrificed by CO2 asphyxiation or injection with EuthasolTM and diaphragm, quadriceps, heart, and liver (used as a control) were removed for histological processing according to each of the methods described below.
Fixation
One of six tissue fixation protocols were used: (a) immersion in 10% neutral buffered formalin (NBF); (b) immersion in Carnoy's fixative; (c) immersion in 1% periodic acid in 10% NBF at 4C; (d) perfusion-fixation with 10% NBF followed by immersion in 10% NBF; (e) immersion in 3% glutaraldehyde in 0.2 M cacodylic acid buffer pH 7.3; (f) immersion in 3% glutaraldehyde in 0.2 M cacodylic acid buffer, pH 7.3, followed by 1% osmium tetroxide in 0.2 M cacodylic acid buffer, pH 7.3, for 1.5 hr (Table 1). All samples remained in the primary fixative for at least 24 hr before processing.
Embedding and Sectioning
The tissues were embedded in one of the following media: paraffin, glycol methacrylate (JB-4), or epon-araldite.
Paraffin. Paraffin embedding was used for fixation protocols: (a) immersion in 10% NBF, (b) immersion in Carnoy's fixative, (c) immersion in 1% periodic acid in 10% NBF at 4C, or (d) perfusion fixation with 10% NBF (Table 1). Using a Leica TP-1050 tissue processor (Leica Instruments; Oberkochen, Germany), the tissues were washed with PBS (two washes, 30 min each), dehydrated with ascending grades of reagent alcohol (70, 90, 90, 100, 100% for 45 min each), cleared in two changes of xylene (45 min each), and infiltrated with paraffin. Tissues measuring 3–4 mm in thickness were embedded in paraffin, cut at 5 μm, and mounted on charged slides.
Glycol Methacrylate. Glycol methacrylate (JB-4 Kit; Poly-Sciences, Warrington, PA) embedding was used for fixation protocols: (a) immersion in 10% NBF, (b) immersion in 3% glutaraldehyde, or (c) immersion in 3% glutaraldehyde with postfixation in 1% osmium tetroxide in 0.2 M cacodylate buffer (Table 1). All samples processed with this embedding medium measured 3–4 mm in thickness. Tissues were washed in PBS (two washes, 30 min each), and dehydrated in ascending grades of alcohol (70, 90, 90, 100, 100% for 30 min each) on a TP-1050 Leica Tissue processor. This was followed by overnight infiltration and embedding in JB-4 according to the manufacturer's directions. Two-μm sections were cut and mounted on charged slides.
Summary table of the different fixation, embedding, and staining methodologies employed to optimize preservation of glycogen in Pompe disease tissues; diaphragm, quadriceps, heart, and liver were all put through each of these protocols and compared
Epon-Araldite. Tissues were cut into 1-mm cubes (following standard procedures for EM), immersion-fixed in 3% glutaraldehyde in 0.2 M cacodylic acid, and postfixed in 1% osmium tetroxide in 0.2M cacodylate buffer (Table 1). Next, the tissues were washed in 0.2 M cacodylic acid buffer (pH 7.3, 10 min rinse; leave overnight in second wash at 4C; 10 min rinse the following day), and dehydrated in ethyl alcohol (30, 50, 70, 80, 90, 100% for 20 min each) followed by two baths of 100% propylene oxide (20 min each). Tissues were infiltrated overnight in a 1:1 mixture of resin [5 ml dodecenyl succinic anhydride, 5 ml LX-112, 5 ml Araldite 502, and 0.5 ml 2,4,6-tri (dimethylaminomethyl) phenol (Ladd Research Industries; Williston, VT)] to propylene oxide. After three changes of 100% resin the samples were left overnight in fresh resin. The samples were embedded the following day in fresh resin and polymerized at 60C for 48 hr. Polymerized blocks were cut at 1 μm on a Leica Ultracut E and mounted on charged slides.
Staining for Paraffin
To validate the staining throughout the paraffin experiments, a positive control sample of human liver, immersion-fixed in 10% NBF, was included in every staining cycle.
PAS Reaction on Paraffin-embedded Tissue
Slides were deparaffinized and hydrated to water. Slides were then incubated in 0.5% periodic acid at RT for 5 min followed by two or three quick rinses in deionized water (5–10 sec each). After 12 min in Schiff's reagent (SurgiPath; Richmond, IL) at RT, slides were washed in running tap water for 10–15 min. Slides were counterstained with hematoxylin 1 (Richard Allan Scientific; Kalamazoo, MI) for 10 sec, rinsed with water, and dipped in bluing reagent (Richard Allan Scientific) for 30 sec. Once dehydrated to xylene, the slides were coverslipped with Acrytol mounting medium (Surgipath).
PAS Reaction on Paraffin-embedded Tissue with Dimedone Block
Slides were deparaffinized and hydrated to water and incubated in 0.5% periodic acid as above. After rinsing the slides in two or three changes of deionized water (5–10 sec each) the slides were placed in a 5% solution of dimedone (5,5-dimethyl-1,3-cyclohexanedione; Sigma Aldrich, St Louis, MO) in absolute alcohol at 60C for 3 hr. After washing in three changes of water, the slides were stained by following the Schiff incubation and counterstaining as described above.
PAS Reaction on Paraffin-embedded Tissue with Diastase
Slides were deparaffinized and hydrated to water. Slides were incubated in diastase solution preheated to 37C for 1 hr (Rowley Biochemical; Danvers, MA). Slides were then washed in three changes of water. The slides were stained by following the Schiff incubation and counterstaining as described above.
Staining for Glycol Methacrylate (JB-4)
Slides were hydrated in deionized water for 5 min before being placed in 0.5% periodic acid at RT for 5 min. After a gentle 1- min rinse in tap water, slides were drained of excess water but not allowed to dry. Slides were then incubated in Schiff's reagent at RT for 10 min, followed by a rinse in tapwater for 30 min (or to desired color). Hematoxylin I was used for the counterstain, incubated for 15–20 min followed by a 1-min rinse in running tapwater. Slides were dipped into bluing reagent for 1 min. Finally, slides were rinsed in tapwater for 1 min. The slides were allowed to dry completely in a 60C oven (3 hr to overnight). Once dry, slides were coverslipped with Acrytol without the use of xylene.
Staining for Epon-Araldite
Slides were hydrated in deionized water for 5 min, followed by a 5-min incubation in 0.5% periodic acid at RT for 5 min. Slides were gently rinsed in tapwater for 1 min and carefully drained before being placed in the Schiff's reagent. Slides were incubated in Schiff's reagent at RT for 10 min, followed by a rinse in tapwater for 30 min or to desired staining intensity. Slides were counterstained with a 1:10 dilution of Richardson's stock solution, made up of methylene blue CI-52015, azure II, and borax (it is recommended to age the stock solution for at least 2 weeks before use). Each slide was counterstained by placing it on a warm plate at a low setting and adding a drop for 10–15 sec. Once stained, the slides were rinsed with running water until the water ran clear. After the slides were dried completely in the 60C oven (3 hr to overnight), the slides were coverslipped with Acrytol without the use of xylene.
MetaMorph® Digital Analysis
One representative field from each slide was photographed with a Nikon DXM1200 digital camera and acquired with the Nikon Act 1 photo image capture software for the DXM1200 digital camera, version 1.12 (Nikon Inc.; Instrument Group, Melville, NY). Each digital image was photographed with the ×40 objective and formatted at a fixed pixel density (8 × 10 inches at 150 dpi) using Adobe PhotoShop software (version 5.5). Each digital image was then opened using the MetaMorph® Imaging Processing and Analysis software (version 4.6; Universal Imaging) for histomorphometric analysis.
MetaMorph® was used to quantify the glycogen in each digital image; glycogen load was expressed as a percentage of total tissue area. The area of total tissue (designated as IMAGE A and represented by purple and blue staining together) and the area occupied by glycogen (designated as IMAGE B and represented by purple staining only) were calculated in terms of pixels. The co-localization function of MetaMorph® then calculated the percentage of pixels in common between IMAGE A and IMAGE B and automatically exported the calculation into an Excel spreadsheet. The calculation appears in the column “Area A over B” in an Excel spreadsheet and represents the percent area of tissue occupied by glycogen.
This assay was assessed for intra-observer reproducibility, inter-observer reproducibility, and equipment fidelity (Table 2). To assess intra-observer reproducibility, a single observer performed the MetaMorph® analysis on a single sample on five separate occasions to determine the intra-observer variability. To assess inter-observer reproducibility, five individual observers performed the MetaMorph® analysis on a single sample to determine the inter-observer variability. Finally, to assess equipment fidelity, a single slide was photographed on three identical digital camera/microscope set-ups (same make and model), and MetaMorph® analysis was performed on each separately captured image to determine equipment fidelity and variability. Percent coefficient of variation (%CV) was calculated for each assessment. Each of these assessments was carried out on three different samples (designated sample A, B, and C). In addition, all observers participating in this study had been fully trained on the use of MetaMorph® for histomorphometric analysis.
Electron Microscopy
Thin sections (70 nm) were cut from the same epon blocks described above and mounted on 200-mesh copper grids. Each grid was stained with 1% aqueous uranyl acetate solution for 20 min, followed by 0.4% lead citrate/NaOH solution for 30 sec. The grids were examined in a Philips EM 300 electron microscope. Images were collected on Kodak electron microscope film 4489 and printed on Kodabrome II photographic paper.
Results
Immersion-fixed, Paraffin-processed Tissues
Immersion-fixation with 10% NBF followed by paraffin embedding and staining with PAS resulted in a considerable loss of soluble lysosomal glycogen. As shown in Figure 1A, the glycogen was not well preserved and created a lacey or bubbly meshwork within the tissue. In Figure 1B Carnoy's fixative was used. This fixative is made up of alcohol, acetic acid, and chloroform. The chloroform aids in a more rapid fixation that coagulates proteins and nucleic acids and extracts lipids. Most carbohydrate components are said to be preserved in liver sections by this method (Kiernan 1999). However, as shown in Figure 1B, there is lack of glycogen preservation in muscle with this fixative. Periodic acid was added to the 10% NBF in an additional attempt to improve muscle glycogen preservation (Figure 1C). This was recommended in the literature as the best fixative for glycogen preservation with optimal tissue morphology in liver sections. The method is believed to preserve glycogen and to enhance staining with PAS owing to the immediate formation of dialdehydes with the glucose rings during the fixation (Kinsley et al. 2000). The PAS reaction depends on the formation of these dialdehyde groups. However, there was little improvement in preservation and detection of glycogen in muscle sections from Pompe mice with this method.
MetaMorph® analysis of HRLM a
Each of the three assessments was performed on three different digital samples, “Sample A,” “Sample B,” and “Sample C.” Intra-observer reproducibility was assessed by having one individual measure each sample on five different occasions. Inter-observer reproducibility was assessed by having five individuals measure each of the three samples. Inter-equipment reproducibility was assessed by photographing and measuring each sample on three different microscope/digital camera/computer workstations. The percent coefficient of variation (%CV) for all sample assessments was found to be acceptable at less than 10%.
Immersion-fixation with 10% NBF, Carnoy's fixative, or 10% NBF in 1% periodic acid produces the classic “lace meshwork” artifact in Pompe tissue samples. (
Perfusion-fixation with 10% NBF improves the preservation of lysosomal glycogen in paraffin sections. (
Embedding in glycol methacrylate (JB-4) improves cell morphology but fails to improve preservation of lysosomal glycogen. (
Embedding in epon-araldite provides the most consistent glycogen preservation and best staining contrast. Quadriceps (
Perfusion-fixed, Paraffin-embedded Tissues
Perfusion-fixation with 10% NBF resulted in partial preservation of glycogen (Figure 2A). The lysosomal glycogen appears as dark pink PAS-positive beads, but these structures are not clear or crisp enough for digital quantification because of irregular light-pink background staining of other cellular elements. A dimedone block was suggested in the literature to eliminate this irregularity (Bulmer 1959). This method gave patchy results in which the background staining was eliminated in some areas of the tissue but not in others (Figure 2B). Additional serial sections were treated with diastase to verify that the dark-pink beads represented glycogen and were not artifacts (Figure 2C).
Glycol Methacrylate (JB-4)-embedded Tissues
Tissue was also processed with JB-4 embedding. Tissue immersion-fixed in 10% NBF and embedded in JB-4 yielded good cell preservation. However, there was still noticeable loss of glycogen, with persistence of the lace meshwork appearance (Figure 3A). Immersion-fixation of the tissue in 3% glutaraldehyde in conjunction with the JB-4 embedding failed to further improve glycogen preservation (Figure 3B) and imparted a hazy purple background stain to the tissue. Fixation of the tissue with 3% glutaraldehyde followed by postfixation in 1% osmium tetroxide and embedding in JB-4 resin yielded some improvement in glycogen preservation, as shown by the partial PAS staining of lysosomal glycogen vacuoles (Figure 3C). However, there remained lucent areas within the vacuoles, indicating incomplete glycogen preservation. Furthermore, the combination of JB-4, glutaraldehyde, and PAS stained the background tissue purple, which rendered the reddish-pink glycogen staining less distinct and unsuitable for digital imaging and analysis.
Epon-embedded Tissues
Epon-araldite sections yielded the best preservation of glycogen, as shown for both skeletal and cardiac muscle samples (Figures 4A and 4B). These samples were fixed with 3% glutaraldehyde and 1% osmium tetroxide and processed into epon-araldite, resulting in sections with the highest resolution at the LM level. PAS staining led to distinct purple beaded structures that contrasted sharply against a sky-blue background with Richardson's counterstain. The identity of the purple beads was confirmed as lysosomal glycogen by EM of serial sections from the same blocks (Figures 5A and 5B). Electron microscopy demonstrated that the lysosomal glycogen was effectively preserved. The glycogen is present as small electron-dense β-particles surrounded by a lysosomal membrane. The sharp staining contrast between the lysosomal glycogen vacuoles and muscle tissue resulting from HRLM permitted accurate digital quantification of glycogen with the computer program MetaMorph® (Figure 6). Figures 7A and 7B show the marked improvement in morphology of the human form of the disease.
EM examination of serial sections from epon blocks confirms the presence of glycogen in membrane-bound vacuoles (lysosomes) At high magnification, occasional cell debris can be seen in lysosomes, along with the gray β-particles of glycogen. (
Digital Analysis Assay Validation
The reproducibility of computer-assisted morphometric measurement was assessed for intra-observer, inter-observer, and inter-equipment reproducibility. Three samples were used to evaluate each of these parameters. Table 2 shows high reproducibility for all three parameters, with confidence intervals (%CV) less than 10% in all cases, making this an effective methodology to employ when data, even among multiple users, are compared.
Discussion
In most hospital histology laboratories, 10% NBF is the standard fixative used for tissue preservation, followed by paraffin embedding (Carson 1997). However, histology texts make specific recommendations for the preservation of glycogen. Glycogen, a water-soluble molecule, is often lost by diffusion from the tissue when aqueous-based solutions such as NBF are used. Several standard histological texts suggest that alcoholic fixatives, such as Carnoy's fixative, be used to avoid dissolution of glycogen in aqueous fixatives (Sheehan 1980; Carson 1997; Kiernan 1999). Polysaccharides are believed to be best preserved by alcoholic fixatives (Kiernan 1999), and the addition of chloroform in Carnoy's fixative is said to accelerate the fixation process (Presnell and Schreibman 1997). To enhance PAS staining of NBF immersion-fixed tissues, the addition of sodium periodate (periodic acid) allows the periodate ion to oxidize the hydroxyl groups of glycogen to aldehydes, allowing linkage to Schiff's reagent (Kiernan 1999). Despite this recommendation in the literature for the preservation of liver glycogen (Kinsley et al. 2000), we did not observe improvement in preservation of the glycogen present in Pompe muscle tissue. In our hands, neither Carnoy's nor 10% NBF with 1% periodic acid immersion fixatives preserved the lysosomal glycogen as well as perfusion-fixation with 10% NBF.
Despite the improvement in glycogen preservation with perfusion-fixation (in the animal model) and paraffin embedding, the results from PAS staining were variable and unpredictable. Patchy background staining persisted despite the use of a dimedone block before PAS staining. In theory, the carefully timed treatment of sections with a dimedone solution before PAS staining should prevent the PAS reaction with mucopolysaccharides and glycoproteins present in the rest of the tissue (Bulmer 1959). The results shown in Figure 2B demonstrate that it is, in fact, very difficult to eliminate all background staining in a uniform manner. In addition, although a rapid delivery of 10% NBF through the vasculature provided marked improvement in glycogen retention, this method is not always practical or possible in every animal study design or in tissue recovered from autopsy. Many animal studies require the simultaneous collection of tissue for both histological and biochemical analysis, and perfusion-fixation may interfere with biochemical assays. In addition, perfusion-fixation of human biopsy specimens for analysis would be impossible.
PAS/Richardson-stained epon sections provide high resolution and sharp color contrast images suitable for accurate and reproducible digital analysis. (
Comparison of traditional vs HRLM-processed human Pompe samples. (
JB-4 plastic embedding provided enhanced preservation of tissue elements owing to the shorter dehydration schedule, and the thinner sections provide better cellular detail than paraffin sections (2 μm for JB-4 vs 5 μm for paraffin sections). In theory, the limited tissue exposure to aqueous solutions and the omission of xylenes should have improved the preservation of glycogen. However, despite the improvement in structural detail there was no dramatic improvement in glycogen retention, as demonstrated in Figure 3A.
Postfixation with glutaraldehyde followed by osmium tetroxide is commonly used for electron microscopy and preserves glycogen for viewing at high magnification (Hayat 2000). Therefore, we considered these fixatives for our LM applications.
When glutaraldehyde-fixed samples were embedded in JB-4, the tissues were brittle and difficult to section. This may have been due to the ability of glutaraldehyde to more strongly crosslink the proteins, causing over-hardening properties (Carson 1997). In addition, the unbound aldehyde group (within the glutaraldehyde) not bound during the fixation reaction caused nonspecific PAS staining throughout the tissue (Kiernan 1999), giving an odd purple color to the muscle fibers and rendering the glycogen vacuoles fuzzy and indistinct. The lack of a sharp color contrast compromises digital discrimination of margins in such sections and is a source of increased error in histomorphometric analysis. Glutaraldehyde- and osmium-fixed tissues, embedded in epon-araldite and stained with PAS, demonstrated the best preservation of lysosomal glycogen by both HRLM and EM.
When this method was combined with Richardson's counterstain, images with sharp margins and color contrast between the glycogen and background could be achieved. Although the literature cautions that PAS staining in the presence of glutaraldehyde can cause false-positive results by reaction of the Schiff's reagent with the extra aldehyde group (Carson 1997), we are confident of the specificity of our glycogen staining because the identity of the PAS-positive vacuoles has been repeatedly confirmed by EM as glycogen. Under the electron microscope, the glycogen appears as uniformly distributed gray β-particles within membrane-bound vacuoles (Figures 5A and 5B), consistent with the expected appearance of glycogen fixed with cacodylate-buffered glutaraldehyde (Hayat 2000) and similar to Pompe images published elsewhere (Dickersin 2000).
The use of cacodylate-buffered glutaraldehyde vs phosphate-buffered glutaraldehyde has been discussed in the literature, specifically for the perfusion-fixation of liver glycogen (Kuhn and Wild 1992). Standard EM texts state that either buffer is acceptable for general glycogen preservation (Hayat 2000), and both have been used in studies reported in the Pompe literature (Hudgson and Fulthorpe 1974; Griffin 1984; Bijvoet et al. 1998,1999a; Winkel et al. 2003). EM texts state that the choice of buffer may influence the intensity of the glycogen staining, ranging from gray (with cacodylate buffer) to black (with phosphate buffer) (Hayat 2000). For our purposes, a protocol that provided good immersion-fixation of muscle was needed because perfusion was not feasible for clinical specimens. In our hands, cacodylate-buffered glutaraldehyde performed better in this regard because immersion-fixation with phosphate-buffered glutaraldehyde led to incomplete glycogen preservation and inconsistent PAS staining in HRLM sections of Pompe tissues (data not shown).
The elegance of this methodology allows serial sections to be taken from a single block for both light and electron microscopy, to answer all questions regarding subcellular structure identity. In addition, the use of these HRLM sections for glycogen quantification by MetaMorph® analysis has been validated in multiple animal and human studies. The change in histological glycogen levels in Pompe animals treated with recombinant enzyme replacement therapy parallels the results from biochemical glycogen measurements performed on samples taken from the same animals (Genzyme, unpublished observations). Rigorous assessment of the assay for intra-observer, inter-observer and inter-equipment fidelity yielded %CVs well below 10%, thus demonstrating its utility for comparison of data generated by multiple trained users when necessary.
The use of optimal fixation, embedding, and staining methods to create HRLM provides consistent preservation of the abnormal glycogen present in Pompe disease. Digital images taken of these sections allow a reproducible quantitative assessment of glycogen accumulation by computer morphometry. With the use of HRLM it is possible to accurately and more easily evaluate larger areas of tissue than is possible by EM alone. Quantitative evaluation of the equivalent size area by EM can take hours, requires multiple image acquisitions, and is often performed manually with plastic grid overlays. Digital analysis of each HRLM field takes minutes and is performed automatically by the computer. These methods have been used successfully in multiple studies (Raben et al. 2003; and Genzyme, unpublished preclinical and clinical observations) to demonstrate glycogen clearance by enzyme replacement therapy in samples from mice and human subjects. Since the completion of this technical study, we have also applied this protocol to many non-muscle Pompe tissues, including brain, spinal cord, peripheral nerve, and vascular tissues, as well as other organs, with similar success. This methodology can be applied to semithin sections taken from the same epon blocks that are routinely produced for EM when a muscle biopsy is performed (Silverberg 1997), and should therefore be easily adopted by pathology laboratories that may find this additional analysis useful in following Pompe patients.
Footnotes
Acknowledgements
We thank Dr Nina Raben of the NIH for sharing her mouse model of Pompe disease. We would also like to thank Mike O'Callaghan for his suggestions and critical review, and all members of the Department of Pathology at Genzyme who have supported this project.