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Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue
Journal of Immunological Methods 6 (1975) 283-289
© North-Holland Publishing Company
IMMUNOFLUORESCENT DETECTION OF HEPATITIS B ANTIGEN IN PARAFFIN-EMBEDDED LIVER TISSUE M.B. RAY and V.J. DESMET Laboratorium voor Histochernie en Cytochemie, Departement Medische Navorsing, Academisch Ziekenhuis Sint Rafa~l, B 3000 Leuven, Belgium
Received 23 August 1974,
accepted 6 September 1974
Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunofluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in paraffin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunofluorescence. The intensity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, including avoidance of freezing equipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its'immunological specificity and higher sensitivity.
1. Introduction HBAg has been detected in sera stored for 25 years (Zuckerman and Taylor, 1969) and has been found to be resistant to many physical and chemical treatments (Kim and Bissel, 1971). It is destroyed by heating only at 8 0 - 1 0 0 ° C (Zuckerman, 1972). Therefore it was thought that the antigenicity of HBAg might remain unaltered through the entire process o f paraffin-embedding using routine fixatives, and still remain detectable by immunofluorescence. Paraffin sections would be preferable since they give better preservation o f histological details and lack freezing artifacts which usually cause high background fluorescence in frozen sections.
2. Materials and methods 2.1. Biopsy selection
Twenty liver biopsies: 10 positive and 10 negative for HBAg by immunofluores. 283
284
M.B. Ray, V.J. Desmet, Detection of hepatitis B antigen
cence were selected from a group of 180 liver biopsies collected from patients submitted to needle biopsy for various hepatic dysfunctions. Both positive and negative biopsies were obtained during the same period of time. At the time of collection the biopsy cylinder was cut into a small (approx. 5 ram) part which was frozen immediately and used for immunofluorescence, and a larger part which was placed in Bouin's fixative (1.2% w/v (saturated) aqueous picric acid, 75 ml; formaldehyde, 25 ml and glacial acetic acid, 5 ml) and processed for routine histological studies. The maximum age of the paraffin blocks used in the present study was I year. 2.2. Immunofluorescence or frozen sections The procedure of freezing, cutting, processing and examining of the biopsies by immunofluorescence was reported earlier in detail (Ray et al., 1974). 2.3. Preparation o f paraffin sections The larger part of the biopsies was routinely dehydrated, cleared and embedded in paraffin using an automatic tissue processor. Ten serial sections of 4/~ thickness were cut from each block and mounted in a gelatin-water mixture. Finally the sections were dried at 37°C for at least 1 hr before deparaffinization. 2.4. Deparaffinization Sections were deparaffinized in two consecutive toluene baths and hydrated through two successive baths of alcohol. After a quick rinse in water the sections were placed in 1% borax solution to remove the picric acid of Bouin's fixative and finally rinsed in phosphate-buffered saline (PBS). At this stage, the sections are ready for haematoxylin-eosin, orcein and immunofluorescent staining. Haematoxylin-eosin was performed on the first serial section of the biopsies. 2.5. Immunofluorescence on paraffin sections The indirect fluorescent technique (Hadziyannis et al., 1972, Ray et al., 1974) was used with specific rabbit anti-HBAg (Behringwerk, Belgium) and fluoresceinconjugated goat antiglobulin antiserum (Hyland, Brussels). The specificity of both the specific and the antiglobulin antisera were carefully determined; the procedure of applying different types of controls has been described in detail in a previous communication (Ray et al., 1974). One additional control was used in the case of paraffin sections, i.e. incubation with normal rabbit serum instead of specific antiHBAg. The second serial section of each biopsy was washed in PBS for 15 rain (3 times, 5 min each), incubated with anti-HBAg for 1 hr at 30°C in a humid chamber, then washed for 15 rain, and reacted with conjugated antiglobulins for 30 rain at
M.B. Ray, KJ. Desmet, Detection of hepatitis B antigen
285
room temperature. After final washing, the slides were mounted, examined and photographed with a fluorescence microscope.
2. 6. Examination The slides of both HBAg positive and HBAg negative biopsies were randomly coded and examined without prior knowledge of the results obtained with the frozen sections. After examination the code was broken and the results were matched with those obtained on the frozen sections of the same biopsies.
2. 7. Orcein staining for HBAg in paraffin sections Orcein staining on liver biopsies was performed according to Shikata et al. (1974). The third serial section of each biopsy was brought to PBS and oxidized with potassium permanganate solution for 5 min. After decolourization in 1.5% oxalic acid solution, the sections were placed in orcein (BDH) solution for 4 hr. The slides were dehydrated in absolute alcohol, cleared with toluene and mounted with DPX.
3. Results
3.1. HBAg in frozen sections As described previously (Ray et al., 1974) HBAg was found in the cytoplasm of the hepatocytes. At the cellular level, the fluorescence was usually focal or diffuse in pattern. Diffuse cytoplasmic fluorescence was either homogeneous or granular in character. Some hepatocytes showed distinct fine or course granular fluorescence. Homogeneous fluorescence appeared as fluorescent globules occupying a variable part of the cytoplasmic area (fig. 1). This corresponded to the 'ground glass' (Hadziyannis et al., 1973) appearance of the cytoplasm in haematoxylin-eosin sections. In some cases the fluorescence was mainly restricted to the sinusoidal pole of the liver cells. In other biopsies, or even in other areas of the same biopsy, the cell membrane covering the whole periphery of the liver cell was also fluorescent resulting in a honey comb appearance. At the lobular level, there was no definite relationship between the location of positive fluorescent cells and hepatic lobular topography. The number of scattered positive cells varied from very few to many. In cirrhosis sometimes a whole nodule or part of it was positive ; occasionally a few fluorescent cells were found in a single nodule. In some biopsies, 90-95% of the hepatocytes were fluorescent in one part of the section whereas no positive cells could be found in the remaining part. Mononuclear cells including Kupffer cells were rarely fluorescent.
286
M.B. Ray, V.Z Desmet, Detection of hepatitis B antigen
Fig. 1. Frozen section: HBAg fluorescence in the cytoplasm of numerous hepatocytes. Strong homogeneous fluorescence occupies variable portions of the cytoplasm. (× 264).
3.2. HBAg in paraffin sections The 10 biopsies previously shown to contain HBAg in frozen sections were also positive in paraffin sections even in cases where the fluorescence on frozen sections was rather weak. The HBAg-containing hepatocytes had a similar distribution pattern; the intensity of the fluorescence appeared slightly less than in the frozen sections. Histological localization o f the fluorescent cells was easier and more precise. Non-specific fluorescence either in the sinusoids or in the portal tracts was negligible in comparison to the frozen sections. No specific fluorescence was detected in the control sections. The 10 biopsies, previously found to be HBAg negative in frozen sections, were also negative in paraffin sections.
qD
It
8
41~
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II
4
B
b
Fig. 2: (a) Paraffin section of the same biopsy ~s in fig. 1. (processed 10 months after collection) shows a similar pattern of fluorescence in the cytoplasm. (× 264). (b) Orcein staining: serial section from the same biopsy cylinder as in fig. 2a: shows dark brown globules in the cytoplasm of the ~epatocytes. These cells correspond to the fluorescent hepatocytes in fig. 2a. (× 264).
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288
M.B. Ray, V.J. Desmet, Detection of hepatitis B antigen
Table 1 Summary of results obtained by immunofluorescence in frozen and paraffin sections and by orcein staining. Frozen sections
Paraffin sections Immunofluorescence
Orcein
Number
Positive
Positive
Immunofluorescence positive
10
10
0
6
4
Immunofluorescence negative
10
0
10
0
10
TotN
20
Negative
Negative
3.3. HBAg localization by orcein staining Of the 10 biopsies which were positive in both frozen and paraffin sections, only six were positive by orcein staining. HBAg-containing hepatocytes and elastic fibers (the latter mainly in the centrilobular and portal area) appeared dark brown. The positive cells in serial sections were in the same location as the fluorescent cells in paraffin sections (fig. 2a, b). Smaller amounts of antigen, especially as detected by immunofluorescence in the cell membrane, did not stain with orcein, with which only intensely positive cells were visualized. However the exact histological location of the positive cells was easy to determine. Occasionally non-specific cytoplasmic binding of the orcein dye prevented definite reading of the biopsies. Sometimes macrophages and hepatocytes containing bile pigment or lipofuscin gave intense staining, resulting in false positive reactions. The results obtained are summarized in table f.
4. Discussion The present communication reports the detection by immunofluorescence of HBAg in paraffin-embedded liver tissue, fixed in Bouin's solution. The results obtained are identical to those obtained in frozen sections, except for a slight decrease in the intensity of the fluorescence. The immunofluorescent test on paraffin sections has several practical advantages. 1. Paraffin sections are easily cut with an ordinary microtome. There is no need for freezing equipment nor for a cryostat. 2. There are no freezing artifacts.
M.B. Ray, KJ. Desmet, Detection of hepatitis B antigen
289
3. The precise location of the antigen is easily delineated as there is slight shrinkage of the paraffin-embedded tissue. 4. The preservation of histological detail is better than in frozen sections. 5. Referred biopsies, mailed from distant hospitals, can be examined for HBAg by immunofluorescence. 6. Paraffin blocks at least one year old retain positivity. 7. There is less danger of spread of infection than with frozen sections from fresh, unfixed liver tissue from hepatitis patients. Hence, numerous complicating precautions are unnecessary when paraffin sections are used. 8. The use of serial paraffin sections allows a more easy comparative study of routine hematoxylin-eosin slides and slides stained for HBAg. Admittedly, the advantages enumerated above are also obtained with the orcein method (Shikata et al., 1974). However, the immunofluorescent technique applied in this study has the unquestionable advantage of immunological specificity. Moreover, from our results, it also appears to have a clearly higher sensitivity. These advantages have to be paid for by more expensive reagents, a somewhat more troublesome technique, and the use of a more expensive fluorescence microscope. Finally, it should be stressed that the paraffin sections used in this study were taken from routinely processed tissue, without any special manoeuvres such as have been prescribed previously (Sainte Marie, 1962).
Acknowledgements We would like to acknowledge the help and valuable suggestions of Dr. L. Broeckaert, Dr. J. Fevery and Prof. J. De Groote; and to thank Dr. C. De WolfPeeters and Dr. B. van Damme for their constant encouragement, Mrs. A. De Boeck for her technical assistance, Mr. M. Rooseleers for preparing the photographs and Mrs. M. Veulemans for typing the manuscript. This study was supported by a grant from the Nationaal Fonds Voor Wetenschappelijk Onderzoek (National Fund for Scientific Research) from Belgium.
R~ferences Hadziyannis, S., C.H. Vissoulis, A. Moussouros and A. Afroudakis, 1972, Lancet 1,976. Hadziyannis, S., M.A. Gerber, C. Vissoulis and H. Popper, 1973, Arch. Pathol. 96,327. Kim, C.Y. and D.M. Bissel, 1971, J. Infect. Dis. 123,470. Ray, M.B., B. van Damme and V.J. Desmet, 1974, J. Immunol. Methods 4, 47. Sainte-Marie, G., 1962, J. Histochem. Cytochem. 10, 250. Shikata, T,, T. Uzawa, N. Yoshiwara, T. Aratsuka and S. Yamazaki, 1974, Jap. J. Exptl. Med. 44, 25. Zuckerrnan, A.J., 1972, in: Hepatitis-associated antigen and viruses (North-Holland, Amsterdam) p. 73. Zuckerman, A.J. and P.E. Taylor, 1969, Nature 223, 81.
© North-Holland Publishing Company
IMMUNOFLUORESCENT DETECTION OF HEPATITIS B ANTIGEN IN PARAFFIN-EMBEDDED LIVER TISSUE M.B. RAY and V.J. DESMET Laboratorium voor Histochernie en Cytochemie, Departement Medische Navorsing, Academisch Ziekenhuis Sint Rafa~l, B 3000 Leuven, Belgium
Received 23 August 1974,
accepted 6 September 1974
Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunofluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in paraffin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunofluorescence. The intensity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, including avoidance of freezing equipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its'immunological specificity and higher sensitivity.
1. Introduction HBAg has been detected in sera stored for 25 years (Zuckerman and Taylor, 1969) and has been found to be resistant to many physical and chemical treatments (Kim and Bissel, 1971). It is destroyed by heating only at 8 0 - 1 0 0 ° C (Zuckerman, 1972). Therefore it was thought that the antigenicity of HBAg might remain unaltered through the entire process o f paraffin-embedding using routine fixatives, and still remain detectable by immunofluorescence. Paraffin sections would be preferable since they give better preservation o f histological details and lack freezing artifacts which usually cause high background fluorescence in frozen sections.
2. Materials and methods 2.1. Biopsy selection
Twenty liver biopsies: 10 positive and 10 negative for HBAg by immunofluores. 283
284
M.B. Ray, V.J. Desmet, Detection of hepatitis B antigen
cence were selected from a group of 180 liver biopsies collected from patients submitted to needle biopsy for various hepatic dysfunctions. Both positive and negative biopsies were obtained during the same period of time. At the time of collection the biopsy cylinder was cut into a small (approx. 5 ram) part which was frozen immediately and used for immunofluorescence, and a larger part which was placed in Bouin's fixative (1.2% w/v (saturated) aqueous picric acid, 75 ml; formaldehyde, 25 ml and glacial acetic acid, 5 ml) and processed for routine histological studies. The maximum age of the paraffin blocks used in the present study was I year. 2.2. Immunofluorescence or frozen sections The procedure of freezing, cutting, processing and examining of the biopsies by immunofluorescence was reported earlier in detail (Ray et al., 1974). 2.3. Preparation o f paraffin sections The larger part of the biopsies was routinely dehydrated, cleared and embedded in paraffin using an automatic tissue processor. Ten serial sections of 4/~ thickness were cut from each block and mounted in a gelatin-water mixture. Finally the sections were dried at 37°C for at least 1 hr before deparaffinization. 2.4. Deparaffinization Sections were deparaffinized in two consecutive toluene baths and hydrated through two successive baths of alcohol. After a quick rinse in water the sections were placed in 1% borax solution to remove the picric acid of Bouin's fixative and finally rinsed in phosphate-buffered saline (PBS). At this stage, the sections are ready for haematoxylin-eosin, orcein and immunofluorescent staining. Haematoxylin-eosin was performed on the first serial section of the biopsies. 2.5. Immunofluorescence on paraffin sections The indirect fluorescent technique (Hadziyannis et al., 1972, Ray et al., 1974) was used with specific rabbit anti-HBAg (Behringwerk, Belgium) and fluoresceinconjugated goat antiglobulin antiserum (Hyland, Brussels). The specificity of both the specific and the antiglobulin antisera were carefully determined; the procedure of applying different types of controls has been described in detail in a previous communication (Ray et al., 1974). One additional control was used in the case of paraffin sections, i.e. incubation with normal rabbit serum instead of specific antiHBAg. The second serial section of each biopsy was washed in PBS for 15 rain (3 times, 5 min each), incubated with anti-HBAg for 1 hr at 30°C in a humid chamber, then washed for 15 rain, and reacted with conjugated antiglobulins for 30 rain at
M.B. Ray, KJ. Desmet, Detection of hepatitis B antigen
285
room temperature. After final washing, the slides were mounted, examined and photographed with a fluorescence microscope.
2. 6. Examination The slides of both HBAg positive and HBAg negative biopsies were randomly coded and examined without prior knowledge of the results obtained with the frozen sections. After examination the code was broken and the results were matched with those obtained on the frozen sections of the same biopsies.
2. 7. Orcein staining for HBAg in paraffin sections Orcein staining on liver biopsies was performed according to Shikata et al. (1974). The third serial section of each biopsy was brought to PBS and oxidized with potassium permanganate solution for 5 min. After decolourization in 1.5% oxalic acid solution, the sections were placed in orcein (BDH) solution for 4 hr. The slides were dehydrated in absolute alcohol, cleared with toluene and mounted with DPX.
3. Results
3.1. HBAg in frozen sections As described previously (Ray et al., 1974) HBAg was found in the cytoplasm of the hepatocytes. At the cellular level, the fluorescence was usually focal or diffuse in pattern. Diffuse cytoplasmic fluorescence was either homogeneous or granular in character. Some hepatocytes showed distinct fine or course granular fluorescence. Homogeneous fluorescence appeared as fluorescent globules occupying a variable part of the cytoplasmic area (fig. 1). This corresponded to the 'ground glass' (Hadziyannis et al., 1973) appearance of the cytoplasm in haematoxylin-eosin sections. In some cases the fluorescence was mainly restricted to the sinusoidal pole of the liver cells. In other biopsies, or even in other areas of the same biopsy, the cell membrane covering the whole periphery of the liver cell was also fluorescent resulting in a honey comb appearance. At the lobular level, there was no definite relationship between the location of positive fluorescent cells and hepatic lobular topography. The number of scattered positive cells varied from very few to many. In cirrhosis sometimes a whole nodule or part of it was positive ; occasionally a few fluorescent cells were found in a single nodule. In some biopsies, 90-95% of the hepatocytes were fluorescent in one part of the section whereas no positive cells could be found in the remaining part. Mononuclear cells including Kupffer cells were rarely fluorescent.
286
M.B. Ray, V.Z Desmet, Detection of hepatitis B antigen
Fig. 1. Frozen section: HBAg fluorescence in the cytoplasm of numerous hepatocytes. Strong homogeneous fluorescence occupies variable portions of the cytoplasm. (× 264).
3.2. HBAg in paraffin sections The 10 biopsies previously shown to contain HBAg in frozen sections were also positive in paraffin sections even in cases where the fluorescence on frozen sections was rather weak. The HBAg-containing hepatocytes had a similar distribution pattern; the intensity of the fluorescence appeared slightly less than in the frozen sections. Histological localization o f the fluorescent cells was easier and more precise. Non-specific fluorescence either in the sinusoids or in the portal tracts was negligible in comparison to the frozen sections. No specific fluorescence was detected in the control sections. The 10 biopsies, previously found to be HBAg negative in frozen sections, were also negative in paraffin sections.
qD
It
8
41~
•
II
4
B
b
Fig. 2: (a) Paraffin section of the same biopsy ~s in fig. 1. (processed 10 months after collection) shows a similar pattern of fluorescence in the cytoplasm. (× 264). (b) Orcein staining: serial section from the same biopsy cylinder as in fig. 2a: shows dark brown globules in the cytoplasm of the ~epatocytes. These cells correspond to the fluorescent hepatocytes in fig. 2a. (× 264).
~*~
~aP
f
-o
288
M.B. Ray, V.J. Desmet, Detection of hepatitis B antigen
Table 1 Summary of results obtained by immunofluorescence in frozen and paraffin sections and by orcein staining. Frozen sections
Paraffin sections Immunofluorescence
Orcein
Number
Positive
Positive
Immunofluorescence positive
10
10
0
6
4
Immunofluorescence negative
10
0
10
0
10
TotN
20
Negative
Negative
3.3. HBAg localization by orcein staining Of the 10 biopsies which were positive in both frozen and paraffin sections, only six were positive by orcein staining. HBAg-containing hepatocytes and elastic fibers (the latter mainly in the centrilobular and portal area) appeared dark brown. The positive cells in serial sections were in the same location as the fluorescent cells in paraffin sections (fig. 2a, b). Smaller amounts of antigen, especially as detected by immunofluorescence in the cell membrane, did not stain with orcein, with which only intensely positive cells were visualized. However the exact histological location of the positive cells was easy to determine. Occasionally non-specific cytoplasmic binding of the orcein dye prevented definite reading of the biopsies. Sometimes macrophages and hepatocytes containing bile pigment or lipofuscin gave intense staining, resulting in false positive reactions. The results obtained are summarized in table f.
4. Discussion The present communication reports the detection by immunofluorescence of HBAg in paraffin-embedded liver tissue, fixed in Bouin's solution. The results obtained are identical to those obtained in frozen sections, except for a slight decrease in the intensity of the fluorescence. The immunofluorescent test on paraffin sections has several practical advantages. 1. Paraffin sections are easily cut with an ordinary microtome. There is no need for freezing equipment nor for a cryostat. 2. There are no freezing artifacts.
M.B. Ray, KJ. Desmet, Detection of hepatitis B antigen
289
3. The precise location of the antigen is easily delineated as there is slight shrinkage of the paraffin-embedded tissue. 4. The preservation of histological detail is better than in frozen sections. 5. Referred biopsies, mailed from distant hospitals, can be examined for HBAg by immunofluorescence. 6. Paraffin blocks at least one year old retain positivity. 7. There is less danger of spread of infection than with frozen sections from fresh, unfixed liver tissue from hepatitis patients. Hence, numerous complicating precautions are unnecessary when paraffin sections are used. 8. The use of serial paraffin sections allows a more easy comparative study of routine hematoxylin-eosin slides and slides stained for HBAg. Admittedly, the advantages enumerated above are also obtained with the orcein method (Shikata et al., 1974). However, the immunofluorescent technique applied in this study has the unquestionable advantage of immunological specificity. Moreover, from our results, it also appears to have a clearly higher sensitivity. These advantages have to be paid for by more expensive reagents, a somewhat more troublesome technique, and the use of a more expensive fluorescence microscope. Finally, it should be stressed that the paraffin sections used in this study were taken from routinely processed tissue, without any special manoeuvres such as have been prescribed previously (Sainte Marie, 1962).
Acknowledgements We would like to acknowledge the help and valuable suggestions of Dr. L. Broeckaert, Dr. J. Fevery and Prof. J. De Groote; and to thank Dr. C. De WolfPeeters and Dr. B. van Damme for their constant encouragement, Mrs. A. De Boeck for her technical assistance, Mr. M. Rooseleers for preparing the photographs and Mrs. M. Veulemans for typing the manuscript. This study was supported by a grant from the Nationaal Fonds Voor Wetenschappelijk Onderzoek (National Fund for Scientific Research) from Belgium.
R~ferences Hadziyannis, S., C.H. Vissoulis, A. Moussouros and A. Afroudakis, 1972, Lancet 1,976. Hadziyannis, S., M.A. Gerber, C. Vissoulis and H. Popper, 1973, Arch. Pathol. 96,327. Kim, C.Y. and D.M. Bissel, 1971, J. Infect. Dis. 123,470. Ray, M.B., B. van Damme and V.J. Desmet, 1974, J. Immunol. Methods 4, 47. Sainte-Marie, G., 1962, J. Histochem. Cytochem. 10, 250. Shikata, T,, T. Uzawa, N. Yoshiwara, T. Aratsuka and S. Yamazaki, 1974, Jap. J. Exptl. Med. 44, 25. Zuckerrnan, A.J., 1972, in: Hepatitis-associated antigen and viruses (North-Holland, Amsterdam) p. 73. Zuckerman, A.J. and P.E. Taylor, 1969, Nature 223, 81.