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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2019  |  Volume : 30  |  Issue : 2  |  Page : 350-358
A study of detection and comparison of immunofluorescence on formalin-fixed paraffin-embedded tissue with fresh frozen renal biopsy specimen

Department of Pathology, SMS Medical College, Jaipur, Rajasthan, India

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Date of Submission02-Jul-2018
Date of Acceptance18-Mar-2018
Date of Web Publication23-Apr-2019


Direct immunofluorescence on the fresh frozen tissue is established way of demonstrating of immunoglobulins and complement deposition in renal biopsies. IF studies can be done on paraffin-fixed tissue (IF-P) and give comparable results to those obtained on frozen tissue for most pathogenic immunoglobulins and immunoglobulin fragments; although, the detection of C3c may be more problematic. In our study, we used proteinase-K method for antigen retrieval. We aimed to detect immunoglobulins and complements in formalin-fixed paraffin-embedded (FFPE) tissue sections from renal biopsies and have comparison of IF staining intensity on FFPE sections with conventional IF on fresh frozen tissue. Based on our results, we conclude that IF-P can serve as salvage technique and has significant diagnostic utility.

How to cite this article:
Solanki R, Solanki MK, Hemrajani D, Saha J. A study of detection and comparison of immunofluorescence on formalin-fixed paraffin-embedded tissue with fresh frozen renal biopsy specimen. Saudi J Kidney Dis Transpl 2019;30:350-8

How to cite this URL:
Solanki R, Solanki MK, Hemrajani D, Saha J. A study of detection and comparison of immunofluorescence on formalin-fixed paraffin-embedded tissue with fresh frozen renal biopsy specimen. Saudi J Kidney Dis Transpl [serial online] 2019 [cited 2022 Dec 4];30:350-8. Available from: https://www.sjkdt.org/text.asp?2019/30/2/350/256842

   Introduction Top

The practice of renal pathology interpretation requires the integration of light microscopy, immunofluorescence (IF) microscopy, and electron microscopy (EM) with a correlation of clinical history and biochemical parameters to arrive at a correct diagnosis. IF and EM are essential components, and these should be employed in the pathologic evaluation of renal biopsies. Direct IF (DIF) on the fresh frozen tissue (IF-F) is the established method for the demonstration of immunoglobulins and complement deposition in kidney tissue. This is critical for the accurate diagnosis of many immunologically mediated glomerular diseases.

DIF is simple, fast, and very sensitive technique in which fluorescent tagged monoclonal antibodies [fluorescein isothiocyanate (FITC)] are used for staining on frozen kidney biopsies sections. The major limitation of currently used IF-F method is the need for the fresh frozen tissue, which may not be available in all cases, as well as frozen tissue at times may not contain glomeruli due to medullary sampling.

To overcome this problem, various methods to detect the immune reactants on formalin-fixed paraffin-embedded (FFPE) tissue sections have been tried. The major problem with FFPE is that tissue antigen is denaturated and effective antigen analysis using IF becomes difficult. Various antigen retrieval methods have been developed to salvage fixed tissue when inadequate frozen tissue is available for routine IF examination.

In this study, we used Proteinase-K method for antigen retrieval. Enzymatic digestion using Proteinase-K helps breaks down the protein cross-linkages formed during formalin fixation, exposing the antigenic immune complexes to staining with FITC labeled antibodies.

   Materials and Methods Top

A total of 50 consecutive renal biopsies were taken under ultrasound guidance after appropriate anesthesia with detailed clinical history and investigations. Two cores of biopsy were obtained in each case, one in 10% formalin and another in normal saline. The specimen so obtained was appropriately processed and sections were studied with Hematoxylin and Eosin (H&E), Periodic acid–Schiff stain (PAS), Jones silver stain, and Masson’s trichrome stain (MT) for light microscopic evaluation. IF examination was done using FITC conjugated antibodies against IgG, IgA, IgM, complements C3c, C1q, and kappa and lambda light chains by routine protocol on fresh frozen tissue and after Proteinase-k digestion on FFPE renal biopsies.

Staining method of DIF on Paraffin-embedded formalin-fixed (FFPE) renal biopsy (IF-P)

  1. Cut FFPE renal biopsy at 3-μ thickness on poly L lysine coated slides
  2. Then place the slides in oven at 60°C for overnight, next day raise the temperature of oven about 80°C for 15 min
  3. Then give two change in xylene each for 5 min, one change in acetone for 5 min, one change in alcohol for 5 min then in running tap water for 2 min
  4. Immerge slides in Tris EDTA pH 9 for 30 min at room temperature.
  5. Circle the tissue by PAP pen
  6. Perform enzymatic digestion with Proteinase-K (1.25 mg/mL) for 25–30 min Preparation of stock solution- Add 10 mg proteinase-K in 1 mL of PBS buffer Preparation of working solution – Add 125 μL of stock solution in 875 μL of PBS solution
  7. Stop enzyme digestion by immerging in Tris EDTA at 4°C and leave into it for 40 min
  8. Ringe the slides in PBS for 10 min
  9. Apply diluted FITC antibodies for 1 h in a moist chamber in dark room at room temperature. (IgG, IgA, IgM, kappa, lambda -1:40, C3c-1:10, C1q-1:20 DAKO)
  10. Wash the excess antibodies with PBS-5 minutes, normal saline-5 min, distilled water -5 min
  11. Mount in glycerol (1:10)-10 part PBS and 1 part glycerol.

The intensity of IF was graded as a score of 0–3 in both IF-F and IF-P method. The final diagnosis was based on biopsy findings on LM, IF, and their correlation with clinical data and serological results wherever necessary.

   Results Top

Fifty native renal biopsies with various glomerulopathies were included in the study. Fifty-two percent of cases were male whereas 48% were female patients. Mean age of patients was 20.99 years with standard deviation of ±14.61 years (range: 2–70 years), while the median age was 16 years. The disease spectrum is shown in [Table 1]. Maximum cases were of immune complex-mediated GN constituting 38% of total cases followed by lupus nephritis 18% and membranous nephropathy 16%.
Table 1: Renal glomerular diseases included in the study (n = 50).

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[Table 2] shows 39 cases (78%) were positive for IgG by IF-F method, while on IF-P method, IgG was detected in 36 cases (72%). C3c was detected in 32 cases (64%) by IF-F method, while on IF-P, it was positive in 24 cases (48%) and only one case showed 3+ intensity on IF-P method as compared to 12 cases in IF-F method. Kappa light chain IF was detected in 31 (73.8%) cases on IF-F, while on IF-P, it was detected in 25 (59.5%) cases. Lambda light chain was detected in 33 (78.5%) cases on IF-F while on IF-P, it was detected in 27 (64.2%) cases.
Table 2: Overall positivity and intensity of various immune-reactants according to different methods of immunofluorescence.

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[Table 3] shows that seven out nine (78%) cases of LN, 15 out of 18 (83.33%) cases of ICGN showed no difference of intensity for IgG in both methods [Figure 1] and [Figure 2]. Two out of four (50%) cases of HSP nephritis showed equal intensity for IgA and one case of IgM nephropathy also showed no difference in intensity for IgM with both methods. However, in C3 dominant GN on IF-P method only three cases showed positivity for C3c complement with difference of 1+ intensity as compared to IF-F method. The two cases of IF-P method failed to detect C3c complement component.
Table 3: Comparison of immunofluorescence intensityofdiagnostic immunoglobulins/complement on IF-F and IF-P methods.

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Figure 1: Photomicrographs showing comparison of immunofluorescence by IF-F and IF-P method in a case of Lupus nephritis class-III. FITC tagged antibodies to IgG, C3c and C1q-×200.

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Figure 2: Photomicrographs showing comparison of immunofluorescence by IF –F and IF-P method in a case of Membranous nephropathy. FITC tagged antibodies to IgG, C3c, kappa andlambda-×200.

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IF-P method was diagnostic in 88.8% cases of LN, 100% of ICGN cases, 87.5% of membranous nephropathy cases, 100% of HSP nephritis and IgM nephropathy cases; however, it contributed only in 60% cases of C3GN [Table 4].
Table 4: Percentage of cases in which diagnostic IF-P findingswere obtained.

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In most of the cases, there was no difference or +1 difference for IgG and IgM with two methods. Mean intensity for IgG was 2.2+ on IF-P as compared to 2.7+ on IF-F method in LN cases, and in 19 cases of ICGN mean intensity for IgG was 2+ on IF-F as compared to 1.73+ on IF-P. However, detection of IgA was found difficult in LN cases which showed in only 3 cases on IF-P as compared to 7 cases on IF-F. In C3GN cases the intensity of C3c was 1+ lesser on IF-P as compared to IF-F method with a mean intensity of 2.8+ on IF-F as compared to 1+ on IF-P. IF-P method was also able to detect kappa and lambda light chain with equal or difference of 1+ intensity in most of the cases [Table 5].
Table 5: Comparison of mean intensity between IF-F and IF-P method (n = 50).

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   Discussion Top

IF is indispensable tool in the diagnosis of renal biopsy. DIF is an essential tool to delineate the type of medical renal diseases.[1] DIF on fresh frozen tissue is the gold standard method for detecting various immunoglobulins and complement components. However, it requires a separate core of renal biopsy in normal saline as well cryostat equipment which may not be available in every institution. At times, only a single core of biopsy is received in formalin or there may be a lack of glomeruli due to medullary sampling which hinders the interpretation of renal pathology. Therefore, a need for an alternative method to perform DIF on formalin-fixed paraffin-embedded (FFPE) renal biopsy as “salvage technique” is always felt by renal pathologists.

Masking effect of formalin fixation is well known and is due to extensive cross-linking of environmental proteins which results in tight network that block the access of FITC conjugated antibody to interact with antigen. The cross-linking, however, has the advantage of preserving the tissue morphology.[2]

To unmask the antigen various methods have been tried such as enzyme digestion and heat method. The enzymes, namely trypsin, pepsin, protease VII, pronase, protease XXIV, and proteinase-K have been used for various time duration, temperature, as well as at different concentration for the purpose of antigen retrieval.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16]

Most crucial step in performing DIF on formalin-fixed renal biopsies by enzyme method is to achieve optimal digestion to unmask the antigen sites. Singh et al.[3] have described that loss of outline of epithelial cell of proximal tubules correlates well with optimal digestion. We were able to achieve optimal digestion by following this check point. Apart from enzyme digestion disease process as such and time of exposure in formalin also seems to play important role in masking the antigens.

Mubarak et al[17] unmasked the antigen on a total of 40 formalin-fixed renal biopsy by three different methods, namely heat-induced antigen retrieval (HIAT) using tris buffer (TBS), HIAT using citrate buffer, and pronase (at 37°C for 1 h) method, then compared the IF results with IF on fresh tissue (IF-F). Nasr et al[4] used pronase 0.75 mg/mL of tris buffer for 30 min. at 37°C. Nada et al[5] used proteinase-K 0.25 mg/mL was at 37°C for 5 min. Singh et al[3] used proteinase-K (Sigma-Aldrich, United States) at 1.25 mg/ mL at room temperature for 15–20 min. We used proteinase-K (Sigma-Aldrich, United States catalog no. P2308) at a concentration of 1.25 mg/mL of PBS for 25–30 min at room temperature for optimal digestion.

We performed IF-P in our samples and compared the results with IF-F and were able to make a diagnosis in 46 (92%) cases. The study was comparable with the results of Singh et al,[3] Nasr et al[4] and Nada et al[5] who diagnosed 214 out of 246 (87%) cases, 59 out of 71 (83%) cases and 66 out of 75 (88%) cases, respectively.

In this study, we detected immunoglobulins in 83% of cases and complements (C3c and C1q) in 75% of cases. This was compatible with the earlier studies by Qualman and Keren[6] and Fogazzi et al.[7]

Mubarak et al[17] was able to detect IgG, IgA, IgM, C3, and C1q by IF-P method in 15 (55.55%), 17 (85%), 30 (93%), 18 (58%), and 10 (45.45%) cases as compared to 27, 20, 32, 31, and 22 cases respectively by IF-F method in a total of 40 renal biopsies. In this study, IgG was detected in 36 (92.3%) cases by IF-P as compared to 39 cases by IF-F. IgA was detected in 12 (70.6%) cases by IF-P as compared to 17 cases by IF-F. IgM was detected in 15 (78.9%) cases by IF-P as compared to 19 cases by IF-F. C3c was detected in 23 (71.9%) cases by IF-P as compared to 32 cases by IF-F. C1q was detected in nine (81.8%) cases by IF-P as compared to 11 cases by IF-F. Kappa light chain was detected in 25 (80.6%) cases by IF-P as compared to 31 cases by IF-F. Lambda light was detected in 27 (81.8%) cases by IF-P as compared to 33 cases by IF-F.

We found equal intensity or difference of 1+ for IgG, IgM, C1q, and Kappa and lambda light chain in most of the cases. In three cases, one from each LN, ICGN and MGN IgG showed a difference of 2+ intensity on IF-P in comparison to IF-F. IgA showed 1+ less intensity on IF-P as compared to IF-F for most cases. This method of IF-P was able to diagnose all four cases of HSPN with equal intensity in two cases and 1+ less intensity in two cases. Similar to the previous studies[4],[6],[7],[8],[17] we also observed that C3c showed 1+ less intensity as compared to IF-F and was even negative in two cases of C3GN which showed 3+ intensity on IF-F method. This may be possibly due to technical issue during enzyme digestion. The difference in staining intensities was observed after the standardization of the technique. In one case of ICGN, no glomerulus was present on IF-F which showed 1+ positivity for IgG and C3 on IF-P method. Hence, it helped in making the diagnosis in this case.

Our study demonstrated that IF-P method was able to detect various immunoglobulins and C1q; however, detection of C3c was little difficult. Nonetheless, in spite of reduced sensitivity, we were able to make diagnosis in more than 90% of cases.

   Conclusion Top

In our study, we were able to make diagnosis in 92% of cases by IF-P method when compared with routine IF-F. We concluded that it can serve as “salvage technique” and has significant diagnostic utility as also observed by various researchers. IF-F still remains the gold standard in the evaluation of renal biopsy. Nonetheless, in spite of its limitations, IF-P technique has its importance in special situations in arriving at a diagnosis.

Conflict of interest: None declared.

   References Top

Schneider W. Value of immunofluorescence in the diagnosis of kidney diseases. Z Gesamte Inn Med 1983;38:115-20.  Back to cited text no. 1
Hed J, Eneström S. Detection of immune deposits in glomeruli: The masking effect on antigenicity of formalin in the presence of proteins. J Immunol Methods 1981;41:57-62.  Back to cited text no. 2
Singh G, Singh L, Ghosh R, Nath D, Dinda AK. Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature. World J Nephrol 2016;5:461-70.  Back to cited text no. 3
Nasr SH, Galgano SJ, Markowitz GS, Stokes MB, D’Agati VD. Immunofluorescence on pronase-digested paraffin sections: A valuable salvage technique for renal biopsies. Kidney Int 2006;70:2148-51.  Back to cited text no. 4
Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian J Nephrol 2016;26:182-7.  Back to cited text no. 5
[PUBMED]  [Full text]  
Qualman SJ, Keren DF. Immunofluorescence of deparaffinized, trypsin-treated renal tissues. Preservation of antigens as an adjunct to diagnosis of disease. Lab Invest 1979;41:483-9.  Back to cited text no. 6
Fogazzi GB, Bajetta M, Banfi G, Mihatsch M. Comparison of immunofluorescent findings in kidney after snap-freezing and formalin fixation. Pathol Res Pract 1989;185:225-30.  Back to cited text no. 7
Wagrowska-Danilewicz M, Danilewicz M. Immunofluorescence on paraffin-embedded sections in evaluation of immune complex deposits in renal biopsy specimens. Pol J Pathol 2009;60:3-9.  Back to cited text no. 8
Huang SN, Minassian H, More JD. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 1976;35:383-90.  Back to cited text no. 9
Curran RC, Gregory J. The unmasking of antigens in paraffin sections of tissue by trypsin. Experientia 1977;33:1400-1.  Back to cited text no. 10
MacIver AG, Giddings J, Mepham BL. Demonstration of extracellular immunoproteins in formalin-fixed renal biopsy specimens. Kidney Int 1979;16:632-6.  Back to cited text no. 11
Eneström S, Hed J, Hultman P. Detection of immune deposits in glomeruli: A comparative study of paraffin-embedded, enzyme-treated sections and cryostat sections as substrates in immunofluorescence. J Immunol Methods 1980;37:343-51.  Back to cited text no. 12
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.  Back to cited text no. 13
Mölne J, Breimer ME, Svalander CT. Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies. Am J Kidney Dis 2005;45:674-83.  Back to cited text no. 14
Inoue W, Tomino Y, Miura M, et al. Immunofluorescence staining in unfixed or fixed renal biopsy specimens from patients with diabetic nephropathy. Acta Pathol Jpn 1985;35:1135-40.  Back to cited text no. 15
van der Ven K, Nguyen TQ, Goldschmeding R. Immunofluorescence on proteinase XXIV-digested paraffin sections. Kidney Int 2007;72: 896.  Back to cited text no. 16
Mubarak M, Kazi Javed I, Kulsoom U, Ishaque M. Detection of immunoglobulins and complement components in formalin fixed and paraffin embedded renal biopsy material by immunoflourescence technique. J Nephropathol 2012;1:91-100.  Back to cited text no. 17

Correspondence Address:
Madan Kumar Solanki
Department of Pathology, SMS Medical College, Jaipur, Rajasthan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1319-2442.256842

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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