Microautoradiography Techniques: A Comparison of Frozen ... › ... › posters ›...
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Introduction Covance Laboratories Ltd., has conducted micro-autoradiography [MARG] investigations to study the distribution of radioactivity at a cellular level for many years. Based on experience, MARG images of fixed and wax-embedded tissues seldom turn out to be comparable with regards to location and density of silver grains. Covance has recently conducted two investigations to determine if fixed and wax-embedded tissue samples are appropriate for use in MARG by processing and comparing results from frozen and wax-embedded tissue samples obtained from the same subject. The test compounds used for these comparisons were two different large tritium labelled molecules and tissue-images with clear differences have been selected and presented in this poster. Objectives The objectives of this work were: ▶ to compare the distribution of radioactivity in selected frozen and wax-embedded tissues using qualitative MARG ▶ to determine the extent of radioactivity migration from tissue samples into solvents used during the wax-embedding process Methods Frozen tissue samples, collected from dosed and control animals, sectioned at 5μm, under darkroom conditions, were transferred to microscope slides pre-coated with nuclear emulsion (maintained at room temperature). Slides were placed in light tight cassettes and stored at -20°C to expose. Wax-embedded tissues, collected from dosed and control animals, sectioned at 5 μm, under normal laboratory lighting were mounted on glass slides and dewaxed. Slides were dipped in nuclear emulsion, under darkroom conditions, dried, placed in light tight cassettes and stored at -20°C to expose. Following exposure, all slides were photographically processed and the tissue sections histologically stained with haematoxylin and eosin. Slides were examined under a light microscope, with objectives up to x100, to assess the distribution of silver grains (radioactivity). The distribution in frozen and wax-embedded liver, kidney, testis, cerebellum and spleen from the rat, and brain and thymus from the marmoset were compared. Samples of rat liver, cerebellum, spleen, kidney and testis were placed in solvents used during the wax embedding processes as detailed in Figure 1. Aliquots of each solvent were subjected to liquid scintillation counting to determine if radioactivity had migrated from the tissue in to the solvents. Microautoradiography Techniques: A Comparison of Frozen and Wax-Embedded Tissue Procedures and Outcomes Phil Fernyhough 1 , Christine Farrell 1 , Lisa Wright 1* , John Kendrick 1 , Inga Bjørnsdottir 2 and Hans Helleberg 2 1 Covance Laboratories Ltd., Harrogate, UK; 2 Novo Nordisk A/S, Copenhagen, Denmark Presented at the Joint DMDG/GMP Open Meeting 2016 Microautoradiography Radioanalysis of Processing Solvents Radioanalysis of the different solvents (Table 1) used in wax-embedding procedures indicated that substantial radioactivity migrated from each tissue into formalin and into each subsequent solvent used in the process thereafter (ethanol, toluene and xylene). Advantages and Disadvantage of the Two Processing Techniques Conclusions ▶ Administered radioactivity was distributed into, and was observed, at the cellular level in frozen tissue samples. ▶ There was specific uptake of radioactivity noted in frozen tissues. This included elevated levels of radioactivity in the Kupffer cells of the liver, in the tubules of the medullary rays in the kidney and in connective tissue in interstitial spaces of the testis. A differential pattern of distribution in the spleen was also observed. ▶ In comparison, in wax-embedded tissues there was very little radioactivity associated with these tissues. Low levels of radioactivity were associated with the lumen in the testis and the renal papillae and outer renal cortex. ▶ Radioanalysis confirmed that radioactivity migrated from the tissue and into all investigated solvents used during the wax-embedding process. Significant levels of radioactivity were recovered in the 10% neutral buffered formalin used to fix the tissue samples. ▶ Frozen tissue MARG provides an idea of total radioactivity distribution. Whereas wax-embedded tissue MARG supports distribution investigations but may be limited unless radioactive material is highly bound. As demonstrated, the recovery of radioactivity, analyses of the images and the conclusions drawn are affected by the choice of preparation method. The amount of radioactivity recovered in the different solvents may be related to compound type. It is therefore recommended to evaluate how much is lost from wax-embedding and if this will influence the study evaluation prior to selecting sample preparation technique. Acknowledgments The authors would like to extend their appreciation to Craig Swithenbank and Ian Williams-Davis, Histology Specials, Covance Laboratories Ltd., Harrogate for their excellent technical support with the sectioning and staining of the tissue samples. Frozen Rat Tissue Distinct levels of radioactivity were noted in: ▶ Peri-portal and centrilobular regions of the liver (moderate levels); Kupffer cells (high levels) (Figure 2) ▶ Differential distribution in the spleen (higher levels associated with the marginal region of the white pulp) ▶ In the kidney, there was an apparent accumulation and retention of drug-related radioactivity in the tubules of the medullary rays ▶ Elevated levels in the interstitial connective tissue of the testis (Figure 3) ▶ Distribution in the cerebellum was generally only at background levels; however, levels in the cellular region of the choroid plexus were elevated ▶ In all tissues, the numbers of silver grains detected in the evaluated tissues were higher than in the control samples Wax- Embedded Rat Tissue Generally only background levels of radioactivity in examined tissues: ▶ Only the lumen of the testis and the renal papillae contained detectable radioactivity Frozen Marmoset Tissue Distribution of radioactivity across the brain and thymus with low levels present in the majority of the tissue at each sampling time: ▶ Moderate levels of radioactivity were associated with the choroid plexus (Figure 4) and blood vessel walls in the brain ▶ In sections of the thymus, high levels of radioactivity were detected in the cortex, reticular cells and trabeculae ▶ Moderate levels of radioactivity in the thymus were associated with lymphocytes, blood vessel walls and the medulla and low levels with Hassall’s corpuscles. Background levels of radioactivity were associated with blood ▶ The numbers of silver grains detected in the evaluated brain and thymus were higher than in the control samples Wax- Embedded Marmoset Tissue Generally background levels of radioactivity in the majority of regions: ▶ Exceptions to this were the choroid plexus (Figure 4) and blood vessel walls (low to moderate levels) in the brain ▶ In thymus sections, the pattern of distribution was similar to that in frozen sections, but levels were generally lower in all regions 10% neutral buffered formalin (72 hours) 10% neutral buffered formalin (72 hours) Ethanol (72 hours) Toluene (72 hours) Xylene (72 hours) Whole ssue Sliced ssue Sliced ssue Sliced ssue Sliced ssue 25 mL 25 mL 25 mL 25 mL 25 mL Figure 1. Investigated solvents. Frozen Tissue: (1) portal tract / (2) Kupffer cells Wax-embedded Tissue: (1) portal tract Frozen Tissue: (1) seminiferous epithelium / (2) lumen / (3) connective tissue Wax-embedded Tissue: (1) seminiferous epithelium / (2) lumen Frozen tissue: (1) Blood vessel / (2) Ependyma cells / (3) CSF / (4) Brain tissue Wax embedded: (1) Blood vessel / (2) Ependyma cells / (3) CSF / (4) Brain tissue Figure 2. Example of rat liver depicting frozen and wax-embedded sample differences (x40 objective). Figure 3. Example of rat testis depicting frozen and wax-embedded sample differences. Figure 4. Example of marmoset brain (choroid plexus) depicting frozen and wax-embedded sample differences (x40 microscope objective). Table 1. Radioactive Content of Investigated Solvents dpm/sample % radioactivity lost Tissue/ solvent Formal saline Ethanol Toluene Xylene Liver (whole) 5868633 ----- ----- ----- Liver sliced 2485583 3611003 10228 4843 >99 Spleen (whole) 71385 ----- ----- ----- Spleen sliced 117645 15375 5522 2845 25 Kidney (whole) 733553 ----- ----- ----- Kidney sliced 585177 73929 5660 2913 60 Cerebellum (whole) 78353 ----- ----- ----- Cerebellum sliced 12421 982 42.4 47.3 >99 Testis (whole) 989888 ----- ----- ----- Testis sliced 1383085 227630 770 442 >99 Frozen Wax Loss of radioactivity during embedding ++++ + Ease of sectioning and mounting ++ ++++ Morphology ++ ++++ A rating of + (poor) to ++++ (excellent) was used
Transcript of Microautoradiography Techniques: A Comparison of Frozen ... › ... › posters ›...
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IntroductionCovance Laboratories Ltd., has conducted micro-autoradiography [MARG] investigations to study the distribution of radioactivity at a cellular level for many years. Based on experience, MARG images of fixed and wax-embedded tissues seldom turn out to be comparable with regards to location and density of silver grains. Covance has recently conducted two investigations to determine if fixed and wax-embedded tissue samples are appropriate for use in MARG by processing and comparing results from frozen and wax-embedded tissue samples obtained from the same subject. The test compounds used for these comparisons were two different large tritium labelled molecules and tissue-images with clear differences have been selected and presented in this poster.
Objectives
The objectives of this work were:
▶ to compare the distribution of radioactivity in selected frozen and wax-embedded tissues using qualitative MARG
▶ to determine the extent of radioactivity migration from tissue samples into solvents used during the wax-embedding process
Methods
Frozen tissue samples, collected from dosed and control animals, sectioned at 5µm, under darkroom conditions, were transferred to microscope slides pre-coated with nuclear emulsion (maintained at room temperature). Slides were placed in light tight cassettes and stored at -20°C to expose.
Wax-embedded tissues, collected from dosed and control animals, sectioned at 5 µm, under normal laboratory lighting were mounted on glass slides and dewaxed. Slides were dipped in nuclear emulsion, under darkroom conditions, dried, placed in light tight cassettes and stored at -20°C to expose.
Following exposure, all slides were photographically processed and the tissue sections histologically stained with haematoxylin and eosin. Slides were examined under a light microscope, with objectives up to x100, to assess the distribution of silver grains (radioactivity). The distribution in frozen and wax-embedded liver, kidney, testis, cerebellum and spleen from the rat, and brain and thymus from the marmoset were compared.
Samples of rat liver, cerebellum, spleen, kidney and testis were placed in solvents used during the wax embedding processes as detailed in Figure 1.
Aliquots of each solvent were subjected to liquid scintillation counting to determine if radioactivity had migrated from the tissue in to the solvents.
Microautoradiography Techniques: A Comparison of Frozen and Wax-Embedded Tissue Procedures and OutcomesPhil Fernyhough1, Christine Farrell1, Lisa Wright1*, John Kendrick1, Inga Bjørnsdottir2 and Hans Helleberg21Covance Laboratories Ltd., Harrogate, UK; 2Novo Nordisk A/S, Copenhagen, Denmark
Presented at the Joint DMDG/GMP Open Meeting 2016
Microautoradiography Radioanalysis of Processing SolventsRadioanalysis of the different solvents (Table 1) used in wax-embedding procedures indicated that substantial radioactivity migrated from each tissue into formalin and into each subsequent solvent used in the process thereafter (ethanol, toluene and xylene).
Advantages and Disadvantage of the Two Processing Techniques
Conclusions▶ Administered radioactivity was distributed into, and was observed, at
the cellular level in frozen tissue samples.
▶ There was specific uptake of radioactivity noted in frozen tissues. This included elevated levels of radioactivity in the Kupffer cells of the liver, in the tubules of the medullary rays in the kidney and in connective tissue in interstitial spaces of the testis. A differential pattern of distribution in the spleen was also observed.
▶ In comparison, in wax-embedded tissues there was very little radioactivity associated with these tissues. Low levels of radioactivity were associated with the lumen in the testis and the renal papillae and outer renal cortex.
▶ Radioanalysis confirmed that radioactivity migrated from the tissue and into all investigated solvents used during the wax-embedding process. Significant levels of radioactivity were recovered in the 10% neutral buffered formalin used to fix the tissue samples.
▶ Frozen tissue MARG provides an idea of total radioactivity distribution. Whereas wax-embedded tissue MARG supports distribution investigations but may be limited unless radioactive material is highly bound.
As demonstrated, the recovery of radioactivity, analyses of the images and the conclusions drawn are affected by the choice of preparation method. The amount of radioactivity recovered in the different solvents may be related to compound type. It is therefore recommended to evaluate how much is lost from wax-embedding and if this will influence the study evaluation prior to selecting sample preparation technique.
AcknowledgmentsThe authors would like to extend their appreciation to Craig Swithenbank and Ian Williams-Davis, Histology Specials, Covance Laboratories Ltd., Harrogate for their excellent technical support with the sectioning and staining of the tissue samples.
Frozen Rat Tissue
Distinct levels of radioactivity were noted in:
▶ Peri-portal and centrilobular regions of the liver (moderate levels); Kupffer cells (high levels) (Figure 2)
▶ Differential distribution in the spleen (higher levels associated with the marginal region of the white pulp)
▶ In the kidney, there was an apparent accumulation and retention of drug-related radioactivity in the tubules of the medullary rays
▶ Elevated levels in the interstitial connective tissue of the testis (Figure 3)
▶ Distribution in the cerebellum was generally only at background levels; however, levels in the cellular region of the choroid plexus were elevated
▶ In all tissues, the numbers of silver grains detected in the evaluated tissues were higher than in the control samples
Wax-Embedded Rat Tissue
Generally only background levels of radioactivity in examined tissues:
▶ Only the lumen of the testis and the renal papillae contained detectable radioactivity
Frozen Marmoset Tissue
Distribution of radioactivity across the brain and thymus with low levels present in the majority of the tissue at each sampling time:
▶ Moderate levels of radioactivity were associated with the choroid plexus (Figure 4) and blood vessel walls in the brain
▶ In sections of the thymus, high levels of radioactivity were detected in the cortex, reticular cells and trabeculae
▶ Moderate levels of radioactivity in the thymus were associated with lymphocytes, blood vessel walls and the medulla and low levels with Hassall’s corpuscles. Background levels of radioactivity were associated with blood
▶ The numbers of silver grains detected in the evaluated brain and thymus were higher than in the control samples
Wax-Embedded Marmoset Tissue
Generally background levels of radioactivity in the majority of regions:
▶ Exceptions to this were the choroid plexus (Figure 4) and blood vessel walls (low to moderate levels) in the brain
▶ In thymus sections, the pattern of distribution was similar to that in frozen sections, but levels were generally lower in all regions
10% neutral buffered formalin
(72 hours)
10% neutral buffered formalin
(72 hours)Ethanol
(72 hours)Toluene
(72 hours)Xylene
(72 hours)
Whole tissue Sliced tissue Sliced tissue Sliced tissue Sliced tissue
25 mL 25 mL 25 mL 25 mL 25 mL
Figure 1. Investigated solvents.
Frozen Tissue: (1) portal tract / (2) Kupffer cells
Wax-embedded Tissue: (1) portal tract
Frozen Tissue: (1) seminiferous epithelium / (2) lumen / (3) connective tissue
Wax-embedded Tissue: (1) seminiferous epithelium / (2) lumen
Frozen tissue: (1) Blood vessel / (2) Ependyma cells / (3) CSF / (4) Brain tissue
Wax embedded: (1) Blood vessel / (2) Ependyma cells / (3) CSF / (4) Brain tissue
Figure 2. Example of rat liver depicting frozen and wax-embedded sample differences (x40 objective).
Figure 3. Example of rat testis depicting frozen and wax-embedded sample differences.
Figure 4. Example of marmoset brain (choroid plexus) depicting frozen and wax-embedded sample differences (x40 microscope objective).
Table 1. Radioactive Content of Investigated Solventsdpm/sample
% radioactivity lostTissue/
solventFormal saline
Ethanol Toluene Xylene
Liver (whole) 5868633 ----- ----- -----
Liver sliced 2485583 3611003 10228 4843 >99
Spleen (whole) 71385 ----- ----- -----
Spleen sliced 117645 15375 5522 2845 25
Kidney (whole) 733553 ----- ----- -----
Kidney sliced 585177 73929 5660 2913 60
Cerebellum (whole)
78353 ----- ----- -----
Cerebellum sliced
12421 982 42.4 47.3 >99
Testis (whole) 989888 ----- ----- -----
Testis sliced 1383085 227630 770 442 >99
Frozen Wax
Loss of radioactivity during embedding ++++ +
Ease of sectioning and mounting ++ ++++
Morphology ++ ++++
A rating of + (poor) to ++++ (excellent) was used
