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Applied Immunology in Clinico-Pathologic Diagnostics
Path. Res. Pract, 164,17-23 (1979)
Dept. of Pathology, Un iversity of Basel, Basel, Switzerland
Applied Immunology in Clinico-Pathologic Diagnostics F.GUDAT
Immunopathology tries to yield a morphological basis for clinical immune phenomena within the framework of general and special pathology. Immune reactions take place between antigens and specific immunoglobulins or immune lymphocytes and lead to the secondary and tertiary activation of mediators of inflammation such as the complement or clotting system. Immunological methods are necessary to identify the antigens and specific molecules involved in these interactions. The major morphological tool of immunopathology is immune histology which is immunolog y applied to tissue sections allowing the demonstration of immunopathologically relevant components. But any high molecular substance which possesses antigenic sites to which specific antibodies can be attached can also be visualized. The diagram in Fig. 1 shows the basic technic of immune histology. In its simplest form, the direct method, the section containing the antigen in question is incubated directly with a labeled antigen-specific antibody which binds irre versibly to its specific antigen . Immunofluorescent technics use fluorescein or rhodamine as a marker which in the fluorescence
Direct method
Indirect method Species -specific anti globulin
n
~~
~
Antigen -speeitrc gommoglobulin
Fig. I. The principle of direct and ind irect immune histologic al incubation. The marker ("") may be fluorescein, rhodamine or an enzyme such as perox idase. 2 Path. Res. Pract. Vol. 164
18 . F. Gudat
microscope allows localization of the site of the specific antigen/antibody reaction. Alternatively, peroxidase or other enzymes may be used as a marker in immunohistochemistry (see article of Dr. Heitz in this series) or ferritin and peroxidase for immune electron microscopy. For detailed technical procedures the reader is referred to appropriate textbooks. It is the purpose of this contribution to illustrate the limitations and possibilities of immune histology in the particular field of kidney biopsy. Its diagnostic value is exemplified in the differential diagnosis of acute kidney failure since immune histology of unfixed biopsy material may yield the first evaluation after two to three hours, before light or electron microscopy is available. As indicated in Fig. 2, immune histology will allow a basic distinction between a glomerulonephritic or nonglomerulonephritic process. If there is no deposition of immunoglobulins, complement components, and fibrin Immune histological patterns in the differential diagnosis of acute kidney failure Immunofluorescence localization of immunoglobulins, complement and fibrin (-ogen): GN Interstitium Glomerula
+
~ granular
linear
Anti-BMantibody nephritis -+ Goodpasture?
GN:
glomerulonephritis
BM:
basement membrane
Fig.
2.
NON-GN
-+ extraglomerular? -+ extrarenal?
Immune complex glomeruloparhy: a) intrarenal: diffuse or focal Epimembraneous GN b) intraglomerular: global or segmental c) intralobular: peripheral BM, mesangial or mixed Endothelio-mesangial GN Membrano-proliferative GN Segmental-focally sclerosing GN IgA nephropathy Kidney transplants: - Glomerulopathy - Vasculopathy - Recurrent GN
Flow sheat for the evaluation of immune histological findings.
Applied Immunology· 19
(-ogen) within the glomeruli, an acute glomerulonephritis is highly unlikely and the clinician has to consider other extraglomerular or extrarenal causes for the acute failure. If such mediators of inflammation are present within the glomeruli, a second basic distinction between linear and granular depositions can be met. The linear pattern which indicates a diffuse, homogenous localization of immunoglobulins and complement along the glomerular basement membrane is diagnostic of anti-basement membrane glomerulonephritis which represents the analogue of the experimental Masugi type nephritis (Fig. 3). Since this glomerulonephritis may be part of Goodpasture's syndrome the clinician has to look for pulmonary symptoms. In the case of granular glomerulopathy, a more detailed analysis of the deposition pattern is necessary. When considering the overall involvement of the glomeruli we speak of diffuse and focal involvement, respectively. With respect to the individual glomerulum, the terms "global" or "segmental" are employed. Finally, at the same level of the single glomerulum the deposits may be localized in the peripheral basement membrane, within the mesangium, or in both locations. Based on these different deposition patterns and also considering the nature of the depositions (immune globulin classes, complement, etc.) the following tentative diagnoses may be recognized from immune histology: I.
Epimembraneous glomerulonephritis
In this condition, there is a diffuse and global deposition of immune complexes which are found exclusively along the peripheral basement membrane in a beaded fashion (Fig. 4). 2. Endothelio-mesangial glomerulonephritis This is the glomerulonephritis of the post-streptococcal type, characterized by diffusely distributed granular depositions within the mesangium as well as in the basement membrane (Fig. 5). 3. Membrano-proliferative glomerulonephritis
In typical forms, there is a garland-like staining of the peripheral basement membrane with sparing of the distended mesangium. 4. Segmentally, focally sclerosing glomerulonephritis
In this type, there is a segmental deposition of complexes within the mesangium and bordering the basement membrane. 5. Kidney transplant
As a rule, acute cellular rejection does not give impressively positive immune histological findings. Depending on the duration of the transplant,
20 •
F. Gud at
Fig. 3. Linear staimng of the glomerular basement membrane for immunoglobulin (and complement) in anti-basement membrane glomerulonephritis.
Fig. 4. Diffuse (all glomeruli affected), global, and granular deposition of immunoglobulin (and complement) along the glomerular basement membrane in epimembraneous glomerulonephritis.
Applied Immunology .
21
Fig. 5. Granular deposition of immun e comple xes in mesangium and basement membrane as seen in ("poststreptococcal ") endorhelio-rnesangial glomeru lonephritis.
however, there might be a transplant glomerulopathy which is characterized by mesangial deposit s of IgM and complement. In addition, there might be a vasculopathy of the Vasa afferentia. These findings, however, are not the morphological substrate of acute, but rather of chronic rejecnon. In the rare event of recurrent glomerulonephritis in a transplant, the deposition of IgG in one of the above mentioned forms would be highly diagnostic. In some cases of apparently antibody-mediated vascular rejection, deposition of immunoglobulins, fibrinogen and complement may be apparent in arterial vessels larger than Vasa afferentia. Apart from these tentati ve immune histological diagnoses which should always be confirmed by light and electron microscopy there are some absolutely diagnostic immune histologic patterns which are listed in the following: 1. Linear fluorescence of the peripheral basement membrane in antibasement antibody glomerulonephritis (see above and Fig. 3). 2. Berger's glomerulonephritis: Preferential deposition of IgA within the mesangium (Fig. 6).
22 •
F. Gudat
Fig. 6. Mesangial deposition of IgA in glomerulonephritis of Berger.
3. Immune histology is the method of choice for the demonstration of the antigenic part of immune complexes which is possible in the minority of biopsies. Examples are the demonstration of nuclear antigens in systemic lupus erythematodes or of viral antigens as in hepatitis B-associated glomerulonephritis. It should be added that in many instances immune histology is less clear in cases which we prefer to call nonspecific glomerulopathy which is characterized by mesangial deposits of IgM and/or C3 but lack of IgG or IgA without light microscopic inflammatory reactions and clinical signs of glomerular damage. This is seen in hypertension, chronic pyelonephritis or in kidneys resected for urologic reasons. Identical findings, however, might be found in minimal-change nephropathy classifying it as secondary, i.e., glomerulonephritic minimal change. For the sake of completeness it might be mentioned that primary minimal-change nephropathy (i.e, "lipoid nephrosis") and eclampsia are immune histologically negative by definition. The examples mentioned briefly in this review illustrate that immune histology may yield additional insights into the immune pathogenesis of glomerulonephritis in addition to mere deposition patterns of biologically active components. The following aspects are worthy of consideration:
Applied Immunology· 23 I. The overwhelming majority of glomerulonephritic kidney diseases is due to immune complexes.
2.
Immune complex deposition may occur in diagnostic patterns.
3. The diversity in these patterns suggests different pathogenic properties of immune complexes: complexes of high molecular components, especially those of IgM and IgA are preferentially deposited in the subendothelial and mesangial area which behaves as a functional entity in this respect. Small complexes are localized to the peripheral basement membrane. It should be mentioned in parenthesis that primary local damage of the basement membrane or of the mesangium predisposes for the deposition of high molecular complexes leading to focal nonspecific depositions of IgM and/or complement in a segmental pattern in nonglomerulonephritic diseases such as Alpert's syndrome and the above mentioned conditions of nonspecific glomerulopathy. This brief review has been an attempt to delineate characteristic immune histological patterns which are useful in diagnostic kidney biopsy. In daily routine, however, the interpretation is of course more difficult. The following listing is meant to emphasize that besides methodological problems additional diagnostic procedures and cooperation of the clinician are essential for an efficient diagnostic approach. Essentials for diagnostic immune histology are: I •
Availability of purified specific antigen and antibody prepara-
tions. 2. Availability of a reasonable number of biopsies from normal and pathological tissue.
3. Availability of additional serological, light and/or electron microscopic methods for additional confirmation.
4. Cooperation of the clinician.
References For detailed literature on methodology and individual diseases see H. U. Zollinger and M. J. Mihatsch: Renal Pathology in Biopsy. Springer, Berlin-Heidelberg-New York (197 8)
Received July 31, 1978 . Accepted in revised form August 17, 1978
Key words: Immunofluorescence - Kidney biopsies - Glomerulonephritis Privatdozent Dr. Fred Gudat, Institut fur Pathologie der Universirat, Schonbeinstr. 40, CH-4056 Basel, Schweiz
Dept. of Pathology, Un iversity of Basel, Basel, Switzerland
Applied Immunology in Clinico-Pathologic Diagnostics F.GUDAT
Immunopathology tries to yield a morphological basis for clinical immune phenomena within the framework of general and special pathology. Immune reactions take place between antigens and specific immunoglobulins or immune lymphocytes and lead to the secondary and tertiary activation of mediators of inflammation such as the complement or clotting system. Immunological methods are necessary to identify the antigens and specific molecules involved in these interactions. The major morphological tool of immunopathology is immune histology which is immunolog y applied to tissue sections allowing the demonstration of immunopathologically relevant components. But any high molecular substance which possesses antigenic sites to which specific antibodies can be attached can also be visualized. The diagram in Fig. 1 shows the basic technic of immune histology. In its simplest form, the direct method, the section containing the antigen in question is incubated directly with a labeled antigen-specific antibody which binds irre versibly to its specific antigen . Immunofluorescent technics use fluorescein or rhodamine as a marker which in the fluorescence
Direct method
Indirect method Species -specific anti globulin
n
~~
~
Antigen -speeitrc gommoglobulin
Fig. I. The principle of direct and ind irect immune histologic al incubation. The marker ("") may be fluorescein, rhodamine or an enzyme such as perox idase. 2 Path. Res. Pract. Vol. 164
18 . F. Gudat
microscope allows localization of the site of the specific antigen/antibody reaction. Alternatively, peroxidase or other enzymes may be used as a marker in immunohistochemistry (see article of Dr. Heitz in this series) or ferritin and peroxidase for immune electron microscopy. For detailed technical procedures the reader is referred to appropriate textbooks. It is the purpose of this contribution to illustrate the limitations and possibilities of immune histology in the particular field of kidney biopsy. Its diagnostic value is exemplified in the differential diagnosis of acute kidney failure since immune histology of unfixed biopsy material may yield the first evaluation after two to three hours, before light or electron microscopy is available. As indicated in Fig. 2, immune histology will allow a basic distinction between a glomerulonephritic or nonglomerulonephritic process. If there is no deposition of immunoglobulins, complement components, and fibrin Immune histological patterns in the differential diagnosis of acute kidney failure Immunofluorescence localization of immunoglobulins, complement and fibrin (-ogen): GN Interstitium Glomerula
+
~ granular
linear
Anti-BMantibody nephritis -+ Goodpasture?
GN:
glomerulonephritis
BM:
basement membrane
Fig.
2.
NON-GN
-+ extraglomerular? -+ extrarenal?
Immune complex glomeruloparhy: a) intrarenal: diffuse or focal Epimembraneous GN b) intraglomerular: global or segmental c) intralobular: peripheral BM, mesangial or mixed Endothelio-mesangial GN Membrano-proliferative GN Segmental-focally sclerosing GN IgA nephropathy Kidney transplants: - Glomerulopathy - Vasculopathy - Recurrent GN
Flow sheat for the evaluation of immune histological findings.
Applied Immunology· 19
(-ogen) within the glomeruli, an acute glomerulonephritis is highly unlikely and the clinician has to consider other extraglomerular or extrarenal causes for the acute failure. If such mediators of inflammation are present within the glomeruli, a second basic distinction between linear and granular depositions can be met. The linear pattern which indicates a diffuse, homogenous localization of immunoglobulins and complement along the glomerular basement membrane is diagnostic of anti-basement membrane glomerulonephritis which represents the analogue of the experimental Masugi type nephritis (Fig. 3). Since this glomerulonephritis may be part of Goodpasture's syndrome the clinician has to look for pulmonary symptoms. In the case of granular glomerulopathy, a more detailed analysis of the deposition pattern is necessary. When considering the overall involvement of the glomeruli we speak of diffuse and focal involvement, respectively. With respect to the individual glomerulum, the terms "global" or "segmental" are employed. Finally, at the same level of the single glomerulum the deposits may be localized in the peripheral basement membrane, within the mesangium, or in both locations. Based on these different deposition patterns and also considering the nature of the depositions (immune globulin classes, complement, etc.) the following tentative diagnoses may be recognized from immune histology: I.
Epimembraneous glomerulonephritis
In this condition, there is a diffuse and global deposition of immune complexes which are found exclusively along the peripheral basement membrane in a beaded fashion (Fig. 4). 2. Endothelio-mesangial glomerulonephritis This is the glomerulonephritis of the post-streptococcal type, characterized by diffusely distributed granular depositions within the mesangium as well as in the basement membrane (Fig. 5). 3. Membrano-proliferative glomerulonephritis
In typical forms, there is a garland-like staining of the peripheral basement membrane with sparing of the distended mesangium. 4. Segmentally, focally sclerosing glomerulonephritis
In this type, there is a segmental deposition of complexes within the mesangium and bordering the basement membrane. 5. Kidney transplant
As a rule, acute cellular rejection does not give impressively positive immune histological findings. Depending on the duration of the transplant,
20 •
F. Gud at
Fig. 3. Linear staimng of the glomerular basement membrane for immunoglobulin (and complement) in anti-basement membrane glomerulonephritis.
Fig. 4. Diffuse (all glomeruli affected), global, and granular deposition of immunoglobulin (and complement) along the glomerular basement membrane in epimembraneous glomerulonephritis.
Applied Immunology .
21
Fig. 5. Granular deposition of immun e comple xes in mesangium and basement membrane as seen in ("poststreptococcal ") endorhelio-rnesangial glomeru lonephritis.
however, there might be a transplant glomerulopathy which is characterized by mesangial deposit s of IgM and complement. In addition, there might be a vasculopathy of the Vasa afferentia. These findings, however, are not the morphological substrate of acute, but rather of chronic rejecnon. In the rare event of recurrent glomerulonephritis in a transplant, the deposition of IgG in one of the above mentioned forms would be highly diagnostic. In some cases of apparently antibody-mediated vascular rejection, deposition of immunoglobulins, fibrinogen and complement may be apparent in arterial vessels larger than Vasa afferentia. Apart from these tentati ve immune histological diagnoses which should always be confirmed by light and electron microscopy there are some absolutely diagnostic immune histologic patterns which are listed in the following: 1. Linear fluorescence of the peripheral basement membrane in antibasement antibody glomerulonephritis (see above and Fig. 3). 2. Berger's glomerulonephritis: Preferential deposition of IgA within the mesangium (Fig. 6).
22 •
F. Gudat
Fig. 6. Mesangial deposition of IgA in glomerulonephritis of Berger.
3. Immune histology is the method of choice for the demonstration of the antigenic part of immune complexes which is possible in the minority of biopsies. Examples are the demonstration of nuclear antigens in systemic lupus erythematodes or of viral antigens as in hepatitis B-associated glomerulonephritis. It should be added that in many instances immune histology is less clear in cases which we prefer to call nonspecific glomerulopathy which is characterized by mesangial deposits of IgM and/or C3 but lack of IgG or IgA without light microscopic inflammatory reactions and clinical signs of glomerular damage. This is seen in hypertension, chronic pyelonephritis or in kidneys resected for urologic reasons. Identical findings, however, might be found in minimal-change nephropathy classifying it as secondary, i.e., glomerulonephritic minimal change. For the sake of completeness it might be mentioned that primary minimal-change nephropathy (i.e, "lipoid nephrosis") and eclampsia are immune histologically negative by definition. The examples mentioned briefly in this review illustrate that immune histology may yield additional insights into the immune pathogenesis of glomerulonephritis in addition to mere deposition patterns of biologically active components. The following aspects are worthy of consideration:
Applied Immunology· 23 I. The overwhelming majority of glomerulonephritic kidney diseases is due to immune complexes.
2.
Immune complex deposition may occur in diagnostic patterns.
3. The diversity in these patterns suggests different pathogenic properties of immune complexes: complexes of high molecular components, especially those of IgM and IgA are preferentially deposited in the subendothelial and mesangial area which behaves as a functional entity in this respect. Small complexes are localized to the peripheral basement membrane. It should be mentioned in parenthesis that primary local damage of the basement membrane or of the mesangium predisposes for the deposition of high molecular complexes leading to focal nonspecific depositions of IgM and/or complement in a segmental pattern in nonglomerulonephritic diseases such as Alpert's syndrome and the above mentioned conditions of nonspecific glomerulopathy. This brief review has been an attempt to delineate characteristic immune histological patterns which are useful in diagnostic kidney biopsy. In daily routine, however, the interpretation is of course more difficult. The following listing is meant to emphasize that besides methodological problems additional diagnostic procedures and cooperation of the clinician are essential for an efficient diagnostic approach. Essentials for diagnostic immune histology are: I •
Availability of purified specific antigen and antibody prepara-
tions. 2. Availability of a reasonable number of biopsies from normal and pathological tissue.
3. Availability of additional serological, light and/or electron microscopic methods for additional confirmation.
4. Cooperation of the clinician.
References For detailed literature on methodology and individual diseases see H. U. Zollinger and M. J. Mihatsch: Renal Pathology in Biopsy. Springer, Berlin-Heidelberg-New York (197 8)
Received July 31, 1978 . Accepted in revised form August 17, 1978
Key words: Immunofluorescence - Kidney biopsies - Glomerulonephritis Privatdozent Dr. Fred Gudat, Institut fur Pathologie der Universirat, Schonbeinstr. 40, CH-4056 Basel, Schweiz