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Eidan AJ. Direct immunofluorescence on formalin-fixed, paraffin-embedded versus fresh frozen human renal biopsies: a comparative study. J Immunoassay Immunochem 2024; 45:292-305. [PMID: 38263688 DOI: 10.1080/15321819.2024.2306324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2024]
Abstract
BACKGROUND The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is controversial. The aim of this study was to investigate whether renal biopsies evaluated by routine immunofluorescence on frozen tissue (IF-Frozen) would yield adequate findings to confirm diagnoses when the IF-Paraffin technique was applied. METHODS To show immunoglobulins, complement components, and light chains, 55 native renal biopsies were subjected to IF-Paraffin and IF-Frozen staining techniques. The intensity of the staining was compared, and the sensitivity and specificity were calculated. RESULTS The IF-Paraffin technique showed a sensitivity of 89%, 81%, 86%, 30%, 71%, 60%, and 77% for IgG, IgM, IgA, C1q, C3, κ, and λ, respectively, whereas specificity was 91%, 100%, 100%, 96%, 94%, 98%, and 100%. It showed diagnostic findings in 87% of cases. Compared to cases that had both IF-Paraffin and IF-Frozen staining techniques, 43 of 55 showed either equal intensity for the diagnostic immunoglobulin/complement or a little difference. CONCLUSIONS Direct immunofluorescence on formalin-fixed, paraffin-embedded sections cannot replace immunofluorescence on frozen sections in the assessment of renal biopsies, but may be a "salvage technique" when frozen tissue is insufficient or unavailable and must be interpreted with great caution.
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Affiliation(s)
- Ali J Eidan
- Immunology, Department of Basic Science, Faculty of Nursing, University of Kufa, Al-Najaf, Iraq
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Diagnostic Accuracy of Direct Immunofluorescence Test on Paraffin-Embedded Blocks in Comparison with Frozen Section Blocks in Renal Biopsies. Int J Nephrol 2022; 2022:4974031. [PMID: 36226196 PMCID: PMC9550505 DOI: 10.1155/2022/4974031] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 08/07/2022] [Accepted: 09/12/2022] [Indexed: 01/10/2023] Open
Abstract
Background In several published research, the evaluation of renal disorders using immunofluorescence on formalin-fixed, paraffin-embedded (FFPE) tissue sections versus immunofluorescence on frozen sections was compared. Each technique's accuracy varies greatly. This study's objective was to assess IF-P as a potential replacement for IF-F in the diagnosis of renal biopsy specimens. Materials and Methods To show immunoglobulin IgA, IgG, IgM, and C3 immune deposits, proteinase K digestion of paraffin-embedded renal biopsy was standardized and used in 51 renal biopsies. Sensitivity, specificity, false-positive, and false-negative values were calculated. Results IF-P showed a sensitivity of 93.1%, 76.9%, 63.6%, and 33.3%, and a specificity of 100%, 97.3%, 95%, and 100% for IgG, IgA, IgM, and C3, respectively. Compared to cases that had both routine IF and IF-P, 50 of 51 showed either the same amount of staining for the diagnostic immunoglobulin/complement or a small amount of difference. In most of the cases (49 of 51), diagnostic findings were found. Conclusion IF-P is a sensitive and precise approach for assessing immune deposits in renal tissue biopsies. We come to the conclusion that IF-P serves as a beneficial salvage immunohistochemistry method for renal biopsies that do not contain enough cortical tissue for IF-F.
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Abstract
Immunofluorescence is an important immunochemical technique that utilizes fluorescence-labeled antibodies to detect specific target antigens. It is used widely in both scientific research and clinical laboratories. Immunofluorescence allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry. However, analysis of samples labeled with fluorescence-labeled antibodies has to be performed using a fluorescence microscope or other type of fluorescence imaging. There are two methods available: direct (primary) and indirect (secondary) immunofluorescence. Here, we describe the principle of immunofluorescence methods as well as the preparation of fresh-frozen and formalin-fixed, paraffin embedded tissues for both direct and indirect immunofluorescence labeling.
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Affiliation(s)
| | | | - Sergio Piña-Oviedo
- Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
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Paraffin Immunofluorescence: A Valuable Ancillary Technique in Renal Pathology. Kidney Int Rep 2018; 3:1260-1266. [PMID: 30450452 PMCID: PMC6224795 DOI: 10.1016/j.ekir.2018.07.008] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Revised: 06/28/2018] [Accepted: 07/02/2018] [Indexed: 01/10/2023] Open
Abstract
Immunofluorescence on frozen tissue is the gold standard immunohistochemical technique for evaluation of immune deposits in the kidney. When frozen tissue is not available or lacks glomeruli, immunofluorescence can be performed on paraffin tissue after antigen retrieval (paraffin immunofluorescence). Excellent results can be obtained by paraffin immunofluorescence in most immune complex-mediated glomerulonephritides and dysproteinemia-associated kidney lesions, and thus this technique has become a valuable salvage technique in renal pathology. Furthermore, new data have emerged suggesting that paraffin immunofluorescence can be used as an unmasking technique, as it is more sensitive than frozen tissue immunofluorescence in some kidney lesions, such as crystalline light chain proximal tubulopathy and is needed to establish the diagnosis of certain unique lesions, such as membranous-like glomerulopathy with masked IgG kappa deposits and membranoproliferative glomerulonephritis with masked monotypic Ig deposits. However, it is important to recognize and be aware of the limitations and pitfalls associated with paraffin immunofluorescence. These include poor sensitivity for detection of C3 deposits and for the diagnosis of primary membranous nephropathy. Here, we summarize the available techniques of paraffin immunofluorescence, review its role and performance as a salvage and unmasking technique in renal pathology, address its limitations and pitfalls, and highlight unusual forms of glomerulopathy that require paraffin immunofluorescence for diagnosis.
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The Value of Direct Immunofluorescence on Proteinase-Digested Formalin-Fixed Paraffin-Embedded Skin Biopsies. Am J Dermatopathol 2018; 40:111-117. [DOI: 10.1097/dad.0000000000000934] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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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; 5:461-470. [PMID: 27648410 PMCID: PMC5011253 DOI: 10.5527/wjn.v5.i5.461] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2016] [Revised: 05/11/2016] [Accepted: 08/18/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature.
METHODS Immunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed.
RESULTS Over the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue.
CONCLUSION As a “salvage” technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls.
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Clinicopathologic Features of Membranous-Like Glomerulopathy With Masked IgG Kappa Deposits. Kidney Int Rep 2016; 1:299-305. [PMID: 29142932 PMCID: PMC5678740 DOI: 10.1016/j.ekir.2016.08.012] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Revised: 08/06/2016] [Accepted: 08/10/2016] [Indexed: 12/30/2022] Open
Abstract
Introduction Ig deposits identified on renal biopsy samples by paraffin immunofluorescence that show negative staining by routine immunofluorescence on frozen tissue have become known as “masked” deposits. Membranous-like glomerulopathy with masked IgG kappa (κ) deposits is a recently recognized pattern of immune complex deposition characterized by masked deposits that show IgG κ restriction and are subepithelial and mesangial by electron microscopy. Based on the frequent presence of C3-only staining by routine immunofluorescence microscopy (IF), these cases could be misdiagnosed as C3 glomerulonephritis in the absence of paraffin immunofluorescence evaluation. Methods The clinicopathologic details of all cases of membranous-like glomerulopathy with masked IgG κ deposits diagnosed in our laboratory were included, beginning with the initial recognition of this entity in 2011 through the end of 2015. Inclusion was based on renal biopsy sample morphologic features including glomerular deposits that stain for IgG κ and have a staining intensity that is significantly brighter by paraffin IF than by routine IF on frozen tissue. Results This pattern of immune complex deposition has been seen in 41 patients in our laboratory over a 5-year period. The patients with these biopsy findings are most commonly young female individuals with a mean age of 27.5 years, with 88% being less than 40 years. All patients had proteinuria with a mean 24-hour urine protein of 3.5 g (range 0.5−12.8 years) and 35% with nephrotic-range proteinuria. Hematuria was present in 88% of patients, and 29% had elevated serum creatinine at presentation. Autoimmune serologic tests were positive in 55% of patients, with a weakly positive antinuclear antibody being most common. Despite this, only 1 patient (2%) fulfilled the diagnostic criteria for systemic lupus erythematosus. The outcome data were mixed, as some patients showed spontaneous remission and mild disease whereas others progressed to end-stage renal disease. There was no apparent correlation between the treatment used and outcome in this retrospective analysis. One patient underwent transplantation and developed biopsy-proven recurrence of disease in the graft 42 months posttransplantation. The etiology of this entity remains unknown. Discussion We provide an expanded case series detailing the clinicopathologic findings of patients with membranous-like glomerulopathy with masked IgG κ deposits. Patients are most commonly young female individuals <40 years of age and commonly have positive autoimmune serologic studies such as antinuclear antibody, although few carry a diagnosis of any well-defined autoimmune disease such as lupus. The outcome data were mixed, as some patients showed spontaneous remission and mild disease whereas others progressed to ESRD. There was no apparent correlation between the treatment used and outcome in this retrospective analysis.
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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. [PMID: 27194832 PMCID: PMC4862263 DOI: 10.4103/0971-4065.159558] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.
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Affiliation(s)
- R Nada
- Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - A Kumar
- Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - V G Kumar
- Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - K L Gupta
- Department of Nephrology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - K Joshi
- Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
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Shi S, Cheng Q, Zhang P, Wang N, Zheng Y, Bai XY, Chen X. Immunofluorescence with dual microwave retrieval of paraffin-embedded sections in the assessment of human renal biopsy specimens. Am J Clin Pathol 2013; 139:71-8. [PMID: 23270901 DOI: 10.1309/ajcprzg8exn7baid] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathologic examination. However, it has certain disadvantages, such as diffuse antigen distribution and few or even no glomeruli in the section. We developed a new technique of immunofluorescence staining using dual microwave retrieval in paraffin-embedded renal tissue sections (IF-DMP) and compared IF-DMP with IF-F in 406 renal biopsy samples. IF-DMP detected significantly more glomeruli than did IF-F (P< .001). There was no significant difference for the specificity and sensitivity in the detection of immunoglobulins, complements, κ, and λ between IF-F and IF-DMP. Concordant observations were 98% for all immunofluorescence, complements, κ, and λ staining and 100% for immunoglobulin staining. Both techniques were completely accurate in confirming diagnoses of various glomerular diseases. IF-DMP provided clearer images of tissue structure and more precise localization of antigens, and it is a suitable alternative for traditional IF-F in clinical renal immunopathologic diagnosis.
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Affiliation(s)
- Suozhu Shi
- Department of Nephrology, State Key Lab of Kidney Diseases, Chinese PLA General Hospital, Beijing, China
| | - Qingli Cheng
- Department of Geriatric Nephrology, Chinese PLA General Hospital, Beijing, China
| | - Ping Zhang
- Department of Pathology, Wang Jing Hospital, Chinese Academy of Chinese Medical Sciences, Beijing, China
| | - Nan Wang
- Department of Nephrology, State Key Lab of Kidney Diseases, Chinese PLA General Hospital, Beijing, China
| | - Ying Zheng
- Department of Nephrology, State Key Lab of Kidney Diseases, Chinese PLA General Hospital, Beijing, China
| | - Xue-Yuan Bai
- Department of Nephrology, State Key Lab of Kidney Diseases, Chinese PLA General Hospital, Beijing, China
| | - Xiangmei Chen
- Department of Nephrology, State Key Lab of Kidney Diseases, Chinese PLA General Hospital, Beijing, China
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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. [PMID: 24475396 DOI: 10.5812/nephropathol.7518] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Accepted: 06/05/2012] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND The technique of direct immunoflourescence (IF) is essential in the accurate diagnosis of renal glomerular diseases. The optimal results are obtained when the procedure is done on fresh frozen tissue (IF-F). However, techniques are available for IF study on formalin fixed and paraffin embedded (FFPE) renal biopsy specimens with variable reported success rates. OBJECTIVES We evaluated three such techniques on FFPE tissue and compared the results with those obtained by IF-F from the same patients. MATERIALS AND METHODS Heat treatment with Tris buffer and citrate buffer, and pronase treatment of the FFPE material was carried out. Direct IF was done for renal panel immunoglobulins and complement components on all biopsies and the results were compared with the historical IF-F study. RESULTS When compared to the IF-F, the immunoflourescence staining on the paraffin sections was less sensitive and less intense in all immune complex-mediated renal diseases, but the diagnostic findings were detected in majority of the cases. CONCLUSIONS In conclusion, it is possible to establish the diagnosis in most cases of immune complex-mediated glomerular diseases with IF on paraffin embedded tissue specimens.
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Affiliation(s)
- Muhammed Mubarak
- Histopathology Department, Sindh Institute of Urology and Transplantation, Karachi, Pakistan
| | - I Kazi Javed
- Histopathology Department, Sindh Institute of Urology and Transplantation, Karachi, Pakistan
| | - Umme Kulsoom
- Histopathology Department, Sindh Institute of Urology and Transplantation, Karachi, Pakistan
| | - Muhammed Ishaque
- Histopathology Department, Sindh Institute of Urology and Transplantation, Karachi, Pakistan
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11
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Jiang YS, Ehara T. Trypsin digestion on paraffin sections is a useful tool for diagnosis of alport syndrome. Ultrastruct Pathol 2010; 34:199-206. [PMID: 20594039 DOI: 10.3109/01913121003725739] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Trypsin digestion for 90 min revealed the alpha(5) chain of type IV collagen along the glomerular basement membrane and Bowman's capsule in paraffin-embedded renal sections of controls. In the 9 patients with the ultrastructures suggestive of Alport syndrome (AS), 8 patients were classified as X-linked dominant type due to the lack or mosaic pattern of alpha(5) chain in paraffin sections of renal biopsies by trypsin digestion, and 1 patient was classified as autosomal recessive type due to the lack of alpha(5) chain in the glomerular basement membrane only. Trypsin digestion is useful for the diagnosis of AS in paraffin-embedded renal tissue.
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Affiliation(s)
- Ying Song Jiang
- Department of Histopathology, Shinshu University School of Medicine, Matsumoto, Japan
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12
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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. [PMID: 17063172 DOI: 10.1038/sj.ki.5001990] [Citation(s) in RCA: 126] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Direct immunofluorescence (IF) on frozen tissue is the method of choice for the study of medical renal diseases. When no glomeruli are available, IF can be performed on the formalin-fixed paraffin-embedded tissue allocated for light microscopy after antigen retrieval with proteases. In this study, the results of IF on frozen tissue (IF-F) and on deparaffinized, pronase-treated tissue (IF-P) were compared in 71 renal biopsies representing 12 major renal diseases. Using IF-P, diagnostic findings were obtained in 100% of cases of lupus nephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, fibrillary glomerulonephritis, primary amyloidosis, myeloma cast nephropathy, and light-chain Fanconi syndrome (LCFS), 88% of cases of immunoglobulin (Ig)A nephropathy, 80% of cases of light-chain deposition disease, 60% of cases of membranoproliferative glomerulonephritis type 1, 50% of cases of idiopathic membranous glomerulopathy (MGN) and 20% of cases of anti-glomerular basement membrane (GBM) disease. IF-P was less sensitive than IF-F for the detection of C3 in all disease categories and for the detection of IgG in cases of MGN and anti-GBM disease. The diagnostic kappa light-chain staining was demonstrated in 100% of cases of LCFS by IF-P versus 40% by IF-F. We conclude that IF-P is a valuable salvage immunohistochemical technique for renal biopsies lacking adequate cortical sampling for IF-F, and is superior to IF-F for the diagnosis of LCFS.
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Affiliation(s)
- S H Nasr
- Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
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Inoue M, Akikusa B, Masuda Y, Kondo Y. Demonstration of microaneurysms at the interlobular arteries of the kidneys in microscopic polyangiitis: a three-dimensional study. Hum Pathol 1998; 29:223-7. [PMID: 9496823 DOI: 10.1016/s0046-8177(98)90039-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Aneurysms, mostly saccular, of the medium-sized muscular arteries are frequently encountered in classic polyarteritis nodosa, whereas their occurrence in smaller arteries is unclear. The objective of this study is to clarify the three-dimensional morphology of the small-sized muscular arteries involved in microscopic polyangiitis (MPA). Six autopsy cases of MPA of the acute inflammatory stage were chosen. Using serial paraffin-embedded sections of the kidney, vasculitic lesions observed in the interlobular arteries were three-dimensionally reconstructed. All of the 19 lesions showed microaneurysms, of which 18 were sausage-shaped and the other was saccular. In the former type, average outer diameter at the most expanded point was 235.4 +/- 83.8 microm (mean +/- SD), which was 2.78 +/- 0.73 times that of an uninvolved adjacent arterial diameter. The major axis-minor axis ratio in a cross-aneurysmal section was 1.17 +/- 0.16, indicating rather regular centrifugal expansion of the aneurysm. The aneurysmal length was 742.7 +/- 254.8 microm. These sausage-type aneurysms showed whole circumferential vasculitic involvement, whereas the saccular-type aneurysm contained an uninvolved arterial portion. In both types, the luminal spaces showed similar three-dimensional features to the outer surface of microaneurysms It was concluded that the interlobular arteries of the kidneys in MPA were characterized by formation of microaneurysms, most of which were sausage-shaped
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Affiliation(s)
- M Inoue
- Second Department of Pathology, School of Medicine, Chiba University, Japan
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14
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Akikusa B, Kondo Y, Irabu N, Yamamoto S, Saiki S. Six cases of microscopic polyarteritis exhibiting acute interstitial pneumonia. Pathol Int 1995; 45:580-8. [PMID: 7496503 DOI: 10.1111/j.1440-1827.1995.tb03506.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Out of 21 autopsy cases of microscopic polyarteritis (MPA), six cases that died of acute respiratory failure are described. All of these six cases exhibited pauci-immune necrotizing glomerulonephritis. Of these six cases, systemic vasculitis and respiratory failure occurred almost simultaneously in two, whereas respiratory failure developed when vasculitis recurred in the other four. Pathologically, pulmonary changes were diffuse and almost uniform in each case. Interstitial inflammatory cells (alveolitis), hyaline membrane, restructuring, and fibrosis were observed. The degrees of these changes differed from case to case; four cases showed predominantly exudative changes and two cases exhibited proliferative or organizing ones. Clinical and pathological features were consistent with acute interstitial pneumonia (AIP). Immunofluorescent and ultrastructural studies did not suggest deposition of immune complexes at the lesions of alveolitis. An etiologic agent could not be identified in these cases. Some lung lesions including pulmonary hemorrhage have been described in association with MPA; however, cases such as those presented here have rarely been reported. It was considered that AIP could be one of the pulmonary disorders in MPA. Furthermore, the possible pathogenetic role of antineutrophil cytoplasmic antibodies (ANCA) in the evolution of present lung lesion is discussed. Although ANCA were not examined in the present cases, these antibodies should be investigated in future cases.
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Affiliation(s)
- B Akikusa
- Department of Pathology, School of Medicine, Chiba University, Japan
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15
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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. [PMID: 2678034 DOI: 10.1016/s0344-0338(89)80256-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Two types of tissue preparation for immunofluorescent staining were compared with sections of the same kidney. In one, formalin-fixed paraffin-embedded kidney sections were incubated with pronase (IF-PRON). The optimal pronase exposure for bright, specific IF and a mild background IF, suitable for the whole set of antisera was determined (pronase 0.75 g/l of Tris buffer for 60 min) and this was used for the subsequent steps of the study. Snap-frozen sections were also stained (IF-FROZ). Positive and negative cases, IF intensity, distribution and location in IgA nephropathy, membranous nephropathy, proliferative lupus nephritis were compared by the 2 methods. The main antigens for each disease were adequately revealed by IF-PRON, so that a correct diagnosis was possible in all cases. IF-PRON was also applied to sections before and after prolonged storage of blocks in the files, to see whether or not retrospective analysis is possible. Only minor differences were found between the 2 series of sections. Finally, the exposure of the sections to the continuous fluorescent light showed that IF fading was less in fixed sections. We conclude that IF-PRON is a reliable method for renal pathology with some advantages over the IF-FROZ.
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Affiliation(s)
- G B Fogazzi
- Division of Nephrology and Dialysis, Maggiore Hospital, Milan, Italy
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16
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Bresser J, Evinger-Hodges MJ. Comparison and optimization of in situ hybridization procedures yielding rapid, sensitive mRNA detections. GENE ANALYSIS TECHNIQUES 1987; 4:89-104. [PMID: 3333762 DOI: 10.1016/0735-0651(87)90002-1] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
This paper describes methods that are commonly used for performing mRNA in situ hybridizations. Each stage of the procedure has been analyzed to identify the parameters that most significantly affect the final cell morphology and sensitivity of the system. We have identified key elements of the procedure as the fixation employed, the type of polynucleotide probe and label chosen, and the detection system used. By optimizing these critical components, we have developed a procedure for performing mRNA in situ hybridizations that takes 2-4 hours and has a sensitivity of 1-10 molecules of mRNA per cell. This system has been used to detect levels of oncogene expression in normal bone marrow and peripheral blood. It is possible to detect the expression of three oncogenes (c-myc, c-sis, and c-abl) simultaneously in a small population of cells from the peripheral blood of leukemic patients.
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Affiliation(s)
- J Bresser
- Department of Clinical Immunology, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston 77030
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Akikusa B, Irabu N, Kamei K, Tsuchida H, Kondo Y. Glomerulonephritis in patients with rheumatoid arthritis (RA). Report of five cases and review of the literature. ACTA PATHOLOGICA JAPONICA 1986; 36:235-52. [PMID: 3518336 DOI: 10.1111/j.1440-1827.1986.tb01476.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
We reported five cases of glomerulonephritis developed in the course of rheumatoid arthritis (RA), which had no apparent relation with therapeutic agents. Systemic angiitis was observed in patient 1 and overlapping of systemic lupus erythematosus (SLE) with RA was implied in patient 2, while the other three patients did not show these changes. Renal biopsies were performed in all of the cases and glomerular deposition of immune complexes was suggested by both the immunofluorescent and electron microscopic findings, though the amount was variable. Furthermore, cases of glomerulonephritis in patients with RA were reviewed in the literature and classified into three groups: occurring in association with angiitis, overlapping with SLE or other collagen diseases, and developing without the above two factors. Group 3 was further divided into two subgroups according to immunologic abnormalities. All cases included in the three groups can be regarded as glomerulonephritis of RA. The cases of group 2, however, can more preferentially be considered to be caused by SLE or some other collagen disease that has overlapped with RA. Deposition of immune complexes was suggested in most of the cases examined with immunofluorescence. Various changes were seen in the glomerulonephritis of RA presented here (patients 1, 3, 4, and 5) and reviewed in the literature (groups 1 and 3). Immune complexes might be involved in the pathogenesis of glomerulitis.
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Suzuki Y, Maesawa A, Matsui K, Shimizu F, Oite T, Koda Y, Suzuki S, Ueki K, Fukagawa M, Arakawa M. Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion. ACTA PATHOLOGICA JAPONICA 1984; 34:563-74. [PMID: 6431748 DOI: 10.1111/j.1440-1827.1984.tb07584.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.
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Nagao K, Matsuzaki O, Saiga H, Akikusa B, Sugano I, Shigematsu H, Kaneko T, Katoh T, Kitamura T, Asano Y, Okamoto M. A histopathologic study of benign and malignant lymphoepithelial lesions of the parotid gland. Cancer 1983; 52:1044-52. [PMID: 6883273 DOI: 10.1002/1097-0142(19830915)52:6<1044::aid-cncr2820520620>3.0.co;2-r] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Twenty-three cases of benign and malignant lymphoepithelial lesions without Sjögren's syndrome were examined: 21 were classical benign lymphoepithelial lesions, and were mostly found in males older than 50 years of age. The other two cases were identified as undifferentiated carcinoma arising from benign lymphoepithelial lesion (8.7%; 2/23). Histologically, this carcinoma is characterized by the formation of cell nests of variable size and by marked lymphoplasmacytic infiltrations into the stromal tissue. In immunofluorescence, an increase of plasma cells containing IgA and IgG was noted in the marginal areas of carcinoma cell clusters. Electron microscopy disclosed that the carcinoma was composed of poorly differentiated cells with occasional squamous differentiation. No dysplastic characteristics were found in the infiltrating lymphocytic components. These two cases of carcinoma were found in middle-aged females.
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AKIKTJSA B, KONBO Y, IRABU N, SHIGBMATSU H. RENAL VASCULAR LESIONS IN SEVERE HYPERTENSION. Pathol Int 1983. [DOI: 10.1111/j.1440-1827.1983.tb01420.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Abstract
Formalin-fixed tissue may be used as substrate for immunohistochemistry after enzymatic treatment with proteases. However, whether the antigenicity of C3b is restored is controversial owing to the protease sensitivity of the native molecule. The antigenicity and protease sensitivity of formalin-fixed and non-fixed C3b were tested by using C3b-opsonized yeast particles and the immunofluorescence technique. The results indicate that formalin, probably due to cross-linking, stabilizes C3b molecules and renders them less susceptible to protease treatment. Failures to detect C3b in immune deposits in fixed tissues may be due to factors other than protease treatment.
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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. [PMID: 6167639 DOI: 10.1016/0022-1759(81)90273-8] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Formalin is known to mask the antigenicity of immune deposits in glomeruli but not of surface immunoglobulins of isolated lymphocytes. We have shown in mice with experimental passive anti-GBM glomerulonephritis that formalin masks the antigenicity of GBM-bound immunoglobulins only if the tissue is fixed before sectioning. The presence of a high concentration of normal bovine serum during fixation of cryostat sections masks the antigenicity of immune deposits, whereas formalin alone has no obvious effect. The same results were obtained with human immunoglobulins (IgG, IgM and IgA) bound to tissue sections. Protease treatment with pepsin and trypsin restored the ability of the immunoglobulins to be stained. The masking effect seems to be due to extensive cross-linking of environmental proteins which prevents fluorescent conjugates reaching their antigens. Methods for detecting immunoglobulins in tissues must, therefore, take into consideration the influence of fixatives not only on epitopes but also on the environment in which the antigenic determinants are localised.
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