| Abstract|| |
Background: Immunofluorescence (IF) on frozen sections has been considered to be the gold standard for evaluation of kidney biopsy specimens. Immunohistochemistry (IHC) method can also be used for this purpose with advantages of being applicable on paraffin embedded tissue, providing permanent sections, and not requiring a specialized microscope for interpretation. Our aim was to evaluate IHC as an alternative to IF in the diagnostic assessment of kidney biopsy specimens. Methods: One hundred kidney biopsy specimens were subjected to both IF and IHC staining for immunoglobulins (Ig), IgG, IgA, IgM and complement components c3 and c1q. IF staining was done on frozen sections. IHC staining was performed on paraffin-embedded tissue following proteolytic antigen retrieval. The sections were evaluated, and the results of IHC were compared with IF. Results: Concordant observations were 98%, 87%, 89%, 83%, and 89% for IgA, IgM, IgG, C3 and C1q, respectively. The sensitivity of IHC method for Igs was found to be high (92%, 86.5%, and 95.1%, respectively for IgA, IgM, and IgG). 91% cases showed concordance of the intensity of the deposits while 100% cases showed a concordance of the pattern. Statistically, there was no significant difference in outcomes between IF and IHC for IgA, IgM, and IgG. However, statistically significant difference was found in the results for complement proteins. Conclusion: In this study, it is documented that IHC is, with few exceptions, equal to IF for the detection of Igs. Standardized immunoperoxidase method on the paraffin embedded, formalin fixed needle kidney biopsies could successfully replace the IF method in the diagnosis of glomerulonephritis.
Keywords: Immunofluorescence, immunoperoxidase, kidney biopsy
|How to cite this article:|
Shubham S, Bhardwaj M, Mahapatra HS. Comparative evaluation of immunoperoxidase versus immunofluorescent techniques in interpretation of kidney biopsies. Indian J Pathol Microbiol 2016;59:305-9
|How to cite this URL:|
Shubham S, Bhardwaj M, Mahapatra HS. Comparative evaluation of immunoperoxidase versus immunofluorescent techniques in interpretation of kidney biopsies. Indian J Pathol Microbiol [serial online] 2016 [cited 2020 Jan 24];59:305-9. Available from: http://www.ijpmonline.org/text.asp?2016/59/3/305/188105
| Introduction|| |
Kidney biopsy is the gold standard in diagnosis of various glomerular diseases. In common practice, the kidney biopsy is examined by
- Light microscopy for morphologic changes using various stains such as hematoxylin and eosin (H and E), periodic acid-Schiff (PAS), silver methenamine (SM), and Masson's Trichrome (MT),
- Direct immunofluorescence (IF) for evaluation of immune deposits and in some cases
- Electron microscopy for ultrastructural evaluation.
IF examination provides vital information regarding the pathogenesis of glomerular diseases. The method for IF labeling was developed by Coons and Kaplan in 1950s. Soon afterward, IF became the gold standard investigation in the evaluation of kidney biopsies. The IF method is relatively simple, highly sensitive, and very specific. However, it has certain drawbacks including the requirement of a cryostat and a fluorescent microscope, fading and impermanence of sections, need of an experienced observer and requirement of a sample that is different from H and E sample leading to a less satisfactory correlation. On the other hand, immunohistochemistry (IHC) has rapidly become indispensable for histopathological routine diagnoses and is available widely in small and large centers. It does not have the disadvantages of IF method. In addition, since paraffin embedded tissue is used for IHC, the kidney biopsy sample processed for H and E can be used omitting the need for a second biopsy core for IF. Several studies have already indicated high sensitivity and specificity of C4d antibody stain using IHC method in biopsies from transplanted kidneys in recognition of humoral graft rejection.,,
In a modern pathology laboratory, use of IHC for evaluation of immune deposits in kidney biopsies will be easier, will omit the need for specialized equipment and reagents for IF and will provide stable sections which can be stored and re-evaluated. The IHC method requires retrieval of antigen in the tissue sections before immunolabeling. This is done to expose reactive site of the antigens that were masked during formalin fixation due to cross-linking. Several method is used depending on the type of tissue and reagents used including pressure cooking, microwaving, and enzymatic digestion or various combinations of these. The enzymatic digestion method was found to be effective in the study of immunoglobulin (Ig) deposits in formalin fixed paraffin embedded tissue in studies by Curran and Gregory and Huang et al., Curran and Gregory also found that paraffin sections demonstrated intracellular Ig more effectively than cryostat sections while IF and IHC methods were equally effective.
Our aim was to evaluate IHC as an alternative to IF in the diagnostic assessment of kidney biopsy specimens. This study was undertaken to compare the IHC technique for Igs (IgA, IgM, IgG) and complement proteins C3 and C1q in native kidney biopsies with the IF technique.
| Methods|| |
All native kidney biopsies performed in the Department of Nephrology, from December 2010 to November 2011 were included in the study. The biopsies which were found to be inadequate for diagnosis on examination were excluded. Biopsies were considered inadequate if <4 glomeruli were seen in H and E sections and <2 glomeruli in IF sample. However, if the H and E sections showed typical diagnostic features in a biopsy sample, it was considered adequate even in the presence of <4 glomeruli. Two cores of kidney biopsies were obtained using Bards biopsy gun (16-gauge needle) from each patient, one for paraffin section and IHC and the other core for IF. Thus, kidney biopsies from each patient were stained using both IF and IHC methods. One core was fixed in 10% neutral buffered formalin while the other core was kept in Michel's medium. Both the cores were immediately transported to the Department of Pathology.
Formalin-fixed core was paraffin processed and 4 μm thick tissue sections were routinely stained with H and E, PAS, SM, and MT. Additional special stains were applied as required. Paraffin blocks of the same tissue cylinders which were used for light microscopy were treated with IHC method for IgG, IgA, IgM, C3, C1q. The second core was immediately washed, snap frozen, and sections taken with a cryostat upon arrival. These sections were then incubated with antibodies labeled with fluorescein isothiocyanate against human Igs (IgA, IgG, IgM) and complement components (C3 and C1q).
Paraffin blocks (of the same tissue cylinders which were used for light microscopy) were used to obtain 3 μm thick tissue sections. Retrieval of antigens by proteolytic enzyme digestion was performed by placing the slides in Proteinase K (Dako, Denmark) working solution (20 μg/ml in Tris-EDTA buffer, pH 8.0) for 10 min at 37°C. They were treated with IHC method using antibodies against IgG, IgA, and IgM (Diagnostic Biosystems, Pleasanton, CA, USA), C3 (Assay Biotech, Sunnyvale, CA, USA) and C1q (Novus Biologics, Littleton, CO, USA). Positive controls included sections from a lymph node.
IF stained sections were examined using Nikon fluorescent microscope while IHC stained sections were evaluated with Nikon bright field microscope. In both IF and IHC stained sections, the pattern and distribution of immune deposits were analyzed qualitatively. The intensity was determined semi-quantitatively using a scale from 0 to 4+, in a manner similar to guidelines established by the Centers for Disease Control and Prevention, Atlanta, Georgia for indirect IF: 0, 1+, 2+, 3+, and 4+ for nil, mild, moderate, moderately severe, and severe staining. Final diagnosis was made considering the results of H and E, PAS, MT, SM, other special stains wherever applied and IF. IHC sections were assessed separately, without knowing IF results. The staining patterns of antibodies by IHC were compared with the staining pattern of the same antibodies achieved by IF, which was taken as a gold standard. The variation of intensity between IF and IHC was also compared.
All analyzed samples were included in 2 × 2 tables of paired samples in which IF method was regarded as the reference test. The positive results of IHC concordant with the gold standard were taken as true positive, and the negative concordant results were taken as true negative. Similarly, discordant positive results of IHC were taken as false positive and negative discordant results were taken as false negative. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated. Statistical analysis was done with the continuity correction included and referred to the Chi-square distribution for probability with 1 df (McNemar's test).
| Results|| |
A total of 116 biopsies were received out of which 16 biopsies were excluded from the study as either the H and E or IF samples were inadequate. After excluding the inadequate samples, 100 eligible cases were assessed. These included various glomerular and nonglomerular diseases; focal segmental glomerulosclerosis, n = 28; lupus nephritis, n = 15; membranoproliferative glomerulonephritis, n = 9; crescentic Glomerulonephritis, n = 8; diffuse proliferative glomerulonephritis, n = 8; membranous glomerulopathy, n = 5; renal amyloidosis, n = 5; tubulointerstitial disease n = 5; acute tubular necrosis, n = 5; IgA nephropathy, n = 3; chronic glomerulonephritis n = 3; minimal change disease, n = 2; diabetic glomerulosclerosis, n = 2; mesangioproliferative glomerulonephritis, n = 1; and hemolytic uremic syndrome, n = 1.
On individual assessment, all of the deposits showed an intensity of 1+ to 3+ on both IF and IHC. We did not find an intensity of 4+ in any of the deposits. Of 178 results which were positive by both IF and IHC methods, 162 (91%) cases showed equal intensity of the immune deposits and only 16 (9%) showed variation in the intensity of the immune deposits. Some of the variations between the staining intensities could be explained by variable degrees of involvement among glomeruli in nephritis. Even in the same section, some variation was often noted in the intensity of the deposits by both IF and IHC methods. The pattern of staining, for example, segmental or global, linear or granular and localization of deposits, for example, capillary or mesangial was also well appreciated in IHC sections. The morphological details of the glomeruli in these sections were discernible [Figure 1].
|Figure 1: A panel of representative cases of glomerular diseases (light microscopy with corresponding immunofluorescence and immunohistochemistry findings, respectively). Membranous nephropathy: (1a) H and E; (1b) immunoglobulin G IF; (1c) immunoglobulin G IHC. Acute proliferative glomerulonephritis: (2a) H and E; (2b) immunoglobulin G, IF; (2c) immunoglobulin G, IHC. Immunoglobulin A nephropathy: (3a) H and E; (3b) immunoglobulin A, IF; (3c) immunoglobulin A, IHC. Lupus nephritis: (4a) H and E; (4b) C3, IF; (4c) C3, IHC . Note that the pattern of deposits in immunohistochemistry sections is similar to that in immunofluorescence in each case (×200)|
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A concordance of 89.2% (446 out of 500 observations) was noted between IF and IHC staining. A comparison of results is summarized in [Table 1]. For IgA, IgM, and IgG, sensitivity of IHC staining was high (92%, 86.5%, and 95.1%, respectively). A higher specificity was seen for IgA, C3 and C1q (100% each) as compared to IgG and IgM (79.5% and 87.3%, respectively). No statistically significant difference was found between IHC staining and IF staining (P > 0.1) for Igs. However, for C3 and C1q, this difference was found to be significant.
|Table 1: Results of comparison of immunohistochemistry and immunofluorescence technique for IgA, IgM, IgG, C3, and C1q|
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Staining of serum proteins present within the capillary lumina was sometimes seen in sections with or without immune deposits. However, they were easily identified and distinguished from capillary immune deposits and did not interfere with the interpretation [Figure 2].
|Figure 2: Nonspecific staining: Staining of serum proteins present within capillary lumina and a tubular cast showing positivity. The glomerulus is clearly negative for immune deposits, immunoglobulin A immunohistochemistry (×400)|
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| Discussion|| |
IF is a very sensitive and reliable technique for evaluation of glomerular diseases but it can only be applied on fresh unfixed frozen tissue. Therefore, a sample different from the one used for routine histopathological examination is required. This also leads to a poor correlation between light microscopy and IF. However, as the utility of direct IF is practically limited in evaluation of kidney biopsies and skin biopsies, most centers do not have the facility. If IHC proves to be a viable alternative to IF, any center with IHC facility can evaluate kidney biopsies adequately.
The concordance rate in this study (89%) was higher than the previous studies (71–88%).,,,, Overall, the sensitivity of IHC method was 82% while in the previous studies it ranged from 73% to 98%. The specificity of IHC was 94% which was comparable to that found by Howie et al. However, other studies showed lower specificity ranging from 55% to 79%. The positive and negative predictive values in the present study were 91% and 87%, respectively.
The use of IHC in formalin fixed paraffin embedded kidney biopsies has been evaluated in several studies.,,,, A comparison of the present study with previous studies is shown in [Table 2]. Out of these, Mölne et al. and Karasalihovic et al. analyzed their data in paired proportions and calculated sensitivity, specificity, positive predictive value, negative predictive value, and P value., The overall concordance of IHC and IF was 71% and 78% in the studies by Mölne et al. and Karasalihovic et al. respectively as compared to 89% in this study. According to Mölne et al., sensitivities of IHC for IgG, IgA, IgM, and C3 were 0.72, 0.8, 0.98, and 0.84 respectively which was comparable to our results. However, sensitivity of C1q as assessed by Mölne et al. was 1.0 while in our study, it was 0.6. Furthermore, in their study, the specificity for IgM, C1q and C3 was 0.24, 0.15, and 0.63, respectively which are much lower than our results. However, they used Protease type XXIV for antigen retrieval which could have contributed toward these differences. Karasalihovic et al. found a sensitivity of 1 and a specificity of 0.64 for C1q, which is in contrast to our results. However, they considered identical only those findings in which deposits had the same intensity.
Statistically significant difference between IHC and IF was found in the case of complement proteins in our study (P < 0.1). This may in part be due to higher sensitivity of complement proteins to enzymatic digestion and the difference in the enzyme used for antigen retrieval compared to other studies. MacIver et al. compared 0.1% and 0.05% trypsin digestion for antigen retrieval and concluded that the latter was more effective for demonstration of immune deposits. They also noted that increasing the time of digestion led to the loss of complement from the tissue section.
In the past, the main drawback of IHC in a demonstration of Igs was ineffective antigen retrieval by standard pressure-cooking or microwaving. The inability of standard IHC methods to demonstrate Igs was attributed to their destruction by fixation or processing, till Curran and Gregory demonstrated Igs in tonsillar tissue using trypsin digestion as the antigen retrieval method., They suggested that trypsin incubation allows antisera to penetrate sections which are impermeable because of fixation. McIver et al. demonstrated Igs and complements in paraffin sections of kidney biopsies using controlled trypsin digestion. Following this, different studies reported effective antigen retrieval using various enzymes.,,, In this study, proteinase K was used for antigen retrieval, and we found satisfactory results. It was found to be a very sensitive procedure and required careful standardization especially with regard to the concentration of the enzyme in solution and the time of antigen retrieval for satisfactory results. We found a concentration of 20 μg/ml for 10 min optimal in this study. On applying a lesser concentration of enzyme or shorter incubation period, the kidney biopsy sections failed to show antibody deposits. Conversely, a higher concentration of the enzyme or longer incubation period led to structural damage to the sections.
In some cases, mild diffuse background staining was seen. It did not interfere with the interpretation in cases with intense deposits of immune complexes. However, in five cases with diffuse background positivity which we interpreted as negative for immune deposits on immunoperoxidase, IF was seen to show deposits of lower intensity (1+). According to Sinclair et al., thicker sections resulted in markedly increased background staining and staining of serum proteins within the glomerular capillary lumina and could be removed to some extent by thorough washing after incubation with antibody. Mölne et al. stated that endogenous enzyme activity present in the tissue, especially the renal tubular epithelium, may give nonspecific background staining. They also observed that if there was a delay of more than a few minutes before fixation, a diffuse positive reaction of plasma proteins was seen which could not be removed by enzyme treatment.
| Conclusion|| |
This study shows that IHC method is a viable alternative for IF for evaluation of immune deposits in kidney biopsy, especially Ig deposit assessment. Therefore, it can be used as a primary method in histopathology laboratories or as an alternative for IF in cases where tissue for IF is not adequate. However, stringent standardization of the reagents and procedure is required.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Nachman PH, Jennette JC, Falk RJ. Primary glomerular disease. In: Taal MW, editor. Brenner and Rector's the Kidney. 9th
ed. Philadelphia: Saunders, an Imprint of Elsevier; 2012.
Walker PD. The renal biopsy. Arch Pathol Lab Med 2009;133:181-8.
Coons AH, Kaplan MH. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 1950;91:1-13.
Kashgarian M. Immune complex. In: True LD, editor. Atlas of Diagnostic Immunohistopathology. Philadelphia: Lippincott; 1990.
MacIver AG, Mepham BL. Immunoperoxidase techniques in human renal biopsy. Histopathology 1982;6:249-67.
Bowdler AL, Griffiths DF, Newman GR. The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method. Histochem J 1989;21:393-402.
Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91.
Santos A, Viana H, Galvão MJ, Carvalho F, Nolasco F. C4d detection in renal allograft biopsies: Immunohistochemistry vs. immunofluorescence. Port J Nephrol Hypertens 2012;26:272-7.
Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 6th
ed. Philadelphia: Churchill Livingstone; 2008.
Curran RC, Gregory J. Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections. J Clin Pathol 1978;31:974-83.
Huang SN, Minassian H, More JD. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 1976;35:383-90.
National Committee for clinical Laboratory Standardization. Quality assurance for the indirect immunofluorescence test for autoantibodies to nuclear antigen (IF-ANA). Approved Guideline. Wayne, PA: NCCLS I/LA2-A, vol. 16 (11); 1996.
MacIver AG, Giddings J, Mepham BL. Demonstration of extracellular immunoproteins in formalin-fixed renal biopsy specimens. Kidney Int 1979;16:632-6.
Sinclair RA, Burns J, Dunnill MS. Immunoperoxidase staining of formalin-fixed, paraffin-embedded, human renal biopsies with a comparison of the peroxidase-antiperoxidase (PAP) and indirect methods. J Clin Pathol 1981;34:859-65.
Howie AJ, Gregory J, Thompson RA, Adkins MA, Niblett AJ. Technical improvements in the immunoperoxidase study of renal biopsy specimens. J Clin Pathol 1990;43:257-9.
Mölne J, Breimer ME, Svalander CT. Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies. Am J Kidney Dis 2005;45:674-83.
Karasalihovic Z, Iljazovic E, Ferluga D, Cickusic E, Mustedanagic Mujanovic J, Stahov J, et al.
Direct immunofluorescence and immunohistochemistry in diagnostics of glomerulonephritis. Bosn J Basic Med Sci 2008;8:12-9.
Curran RC, Gregory J. Effects of fixation and processing on immunohistochemical demonstration of immunoglobulin in paraffin sections of tonsil and bone marrow. J Clin Pathol 1980;33:1047-57.
Dr. Swasti Shubham
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Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]