|Year : 2015 | Volume
| Issue : 4 | Page : 452-457
Role of direct immunofluorescence in the diagnosis of glomerulonephritis
Archana C Buch, Sonam K Sood, Sunita A Bamanikar, Shirish S Chandanwale, Harsh Kumar, Karnik Swapnil
Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, India
|Date of Web Publication||14-Jul-2015|
Archana C Buch
B-603 Gold Coast, Ivory Estates, Someshwarwadi, Pune - 411 008, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Immunofluorescence microscopy is a vital tool for the diagnosis of glomerular diseases. This study was carried out to study patterns of glomerulonephritis (GN) and to record the sensitivity of direct immunofluorescence (DIF) in renal lesions. The DIF findings were correlated with clinical and histopathology findings and discrepancies were analyzed. Materials and Methods: The cross-sectional analytical study was conducted during the period July 2011 to July 2013 at a tertiary care Hospital, Department of Pathology. A total of 75 renal biopsies were received for routine and immunofluorescence studies in which histopathology and clinical data were reviewed and analyzed. Results: The sensitivity of DIF was 87.9% and specificity was 70.5%. The maximum number of cases were seen in the age group 41-50 years. The pattern of GN by DIF was minimal change disease (MCD) in 24%, IgA nephropathy in 13%, focal segmental glomerulosclerosis in 9% and membranoproliferative glomerulonephritis in 8% of the cases. Twelve histopathologically proven cases of GN were negative on DIF. One case of MCD on histopathology was diagnosed as IgM nephropathy based on DIF. Conclusion: Direct immunofluorescence forms an important diagnostic tool in reaching the exact diagnosis in various types of GN presenting with overlapping clinical and histomorphological features.
Keywords: Direct immunofluorescence, glomerulonephritis, renal biopsy
|How to cite this article:|
Buch AC, Sood SK, Bamanikar SA, Chandanwale SS, Kumar H, Swapnil K. Role of direct immunofluorescence in the diagnosis of glomerulonephritis. Med J DY Patil Univ 2015;8:452-7
|How to cite this URL:|
Buch AC, Sood SK, Bamanikar SA, Chandanwale SS, Kumar H, Swapnil K. Role of direct immunofluorescence in the diagnosis of glomerulonephritis. Med J DY Patil Univ [serial online] 2015 [cited 2021 May 14];8:452-7. Available from: https://www.mjdrdypu.org/text.asp?2015/8/4/452/160784
| Introduction|| |
Glomerular diseases are an important cause of morbidity and mortality. The introduction of renal biopsy in clinical practice has represented one of the most important advances in the field of clinical nephrology. Renal biopsy has contributed greatly to a rational classification of intrinsic renal diseases and therefore, to a better knowledge of the pathogenetic mechanisms involved. In spite of the availability of new and less invasive tests, renal biopsy is still an irreplaceable tool in assessing diagnosis and prognosis and guiding the treatment of many renal diseases. 
Immune mechanisms are responsible for glomerular injury in most cases of primary glomerulonephritis (GN) and many of the secondary GN. 
Correct diagnosis of GN requires renal biopsy histopathological examination by light microscopy, immunofluorescence microscopy and electron microscopic examination, along with correlation with clinical features and biochemical parameters. Facilities for electron microscopic study however are not readily available in many institutions.
In the diagnosis of renal diseases, the direct immunofluorescence (DIF) has become a necessary morphological tool for proving immunological mechanisms involved. 
The utility of this technique is limited by cost, site and time of the biopsy, technical and tissue processing factors, history of treatment and the nature of the disease. 
This study was carried out to evaluate the patterns of GN in a tertiary care hospital in Pune and to correlate the DIF and light microscopic (LM) findings with clinical data and serological markers.
| Materials and Methods|| |
A cross-sectional study was undertaken at the Department of Pathology, of a tertiary care hospital and research center during the period July 2011 to July 2013. A total of 75 cases were selected. A detailed clinical history as submitted by the nephrologists was recorded in a proforma. Various investigations like levels of serum creatinine, urea, total proteins and albumin were done along with complete urine analysis. All patients who presented with the clinical diagnosis of nephrotic and nephritic syndrome were included in the study. Patients with renal neoplasms, uncontrolled severe hypertension, hemorrhagic diathesis, solitary kidney, small and shrunken kidneys, urinary tract obstruction were excluded. After taking written informed consent of the patient and excluding contraindications, two renal biopsies were performed in all cases.
In each case for the first biopsy, multiple thin sections (3 μ) were cut for light microscopy after fixation in 10% formalin followed by special stains like Periodic Acid Schiff and Gomori's methenamine silver (GMS) stain. The second biopsy core placed in normal saline/phosphate buffered saline (PBS, pH 7.2) was immediately taken to laboratory for DIF. The tissue was embedded in the cryostat embedding medium, frozen and cut at 3 μm thickness at −22°C. Sections were taken on Poly L Lysine coated glass slides, fixed with ether-alcohol mixture and air dried for 10 min. Later the slides were rinsed in phosphate buffer saline at pH 7.2 for 15 min. The sections were treated with fluorescein isothiocyanate labeled and optimally diluted (1:40) antisera, that is, IgG, IgA, IgM, C 3 and C1q respectively. Positive and negative controls were run simultaneously. The slides were incubated in wet chamber for 1 h in dark room, washed with PBS and mounted with glycerine jelly. The slides were observed under green filter of fluorescence microscope at 494 nm wavelength. DIF was reported based on nature and distribution of immune deposits, glomerular localization, semi quantitative assessment of intensity of staining and pattern of immune complex deposits. The final diagnosis was rendered after correlating clinical data with LM and IF findings.
Statistical methods used to analyze this study include, percentages, ratio, sensitivity, specificity and positive and negative predictive values (NPVs).
Sensitivity: True positive (TP)/TP+ false negative (FN)
Specificity: True negative (TN)/false positive (FP) +TN
Positive predictive value: TP/TP + FP
| Results|| |
Of the 75 patients, nephrotic syndrome was the most common mode of presentation. The age ranged from 3 to 69 years. The maximum (24%) cases belonged to the age group of 41-50 years with 43 (57.3%) males and 32 (42.6%) females.
Severe proteinuria (3+) was recorded in 33 (44%) cases, moderate (2+) in 14 (18.6%) and mild (1+) proteinuria in 28 (37.3%) cases. Hematuria was present in 32 (42.6%) cases.
Creatinine levels <3 mg/dl were seen in 44 (58.6%) cases, 3-6 mg/dl in 24 (32%) cases and 7 (9.3%) cases had levels >6 mg/dl.
Thirty-two (42.6%) cases showed urea levels to be <50 mg/dl, 33 (44%) had 50-150 mg/dl and 10 (13.3%) cases showed levels between 150 and 250 mg/dl.
The details of histopathological findings and DIF correlation of all cases are shown in [Table 1]. DIF eventually helped in diagnosing 9 (12%) cases of IgA nephropathy, 4 (5.3%) of lupus nephritis and 1 of IgM nephropathy (IgMN). The light microscopy and DIF of different types of GN are shown in [Figure 1] [Figure 2] [Figure 3] [Figure 4].
|Figure 1: (a) Minimal change disease showing normal glomeruli (H and E, ×100). (b) Direct immunofl uorescence (DIF) of IgM nephropathy showing diffuse, granular IgM (3+) deposits in the mesangium (×400). (c) Focal segmental glomerulosclerosis (FSGS) showing segmental glomerular sclerosis (H and E, ×400). (d) DIF of FSGS showing IgM (2+) deposits in sclerotic segments (×400)|
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|Figure 2: (a) Membranoproliferative glomerulonephritis (MPGN) type-I showing mesangial matrix expansion along with lobular accentuation (H and E, ×400). (b) Direct immunofluorescence (DIF) showing C3 (2+) along capillary walls in MPGN type-I (×400). (c) IgA nephropathy showing mesangial proliferation/expansion (PAS, ×400). (d) DIF showing IgA (2+) deposits in the mesangium in IgA nephropathy (×400)|
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|Figure 3: (a) Membranous glomerulonephritis (GN) showing glomerular basement membrane thickening; capillary loops appear round and rigid (H and E, ×400). (b) Direct immunofluorescence (DIF) of membranous GN showing granular IgG (2+) deposits along capillary walls (×400). (c) PIGN showing diffuse endocapillary proliferation along with neutrophilic infiltration (H and E, ×400). (d) DIF of PIGN showing granular C3 (2+) deposits in the mesangium (×400)|
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|Figure 4: (a) Rapidly progressive glomerulonephritis showing a fibrous crescent within the glomeruli (H and E, ×100). (b) Direct immunofluorescence (DIF) showing linear IgG (2+) along glomerular capillary walls in anti-glomerular basement membrane disease (×400). (c) Systemic lupus erythematosus showing diffuse proliferative lupus nephritis (WHO Class-IV) (H and E, ×100). (d) DIF showing IgM (3+) in diffuse lupus nephritis (×400)|
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|Table 1: Association between histopathological diagnosis and DIF in glomerulonephritis|
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The study found that IgA nephropathy can exhibit a number of morphological patterns, including mesangial proliferation/expansion (n = 6), chronic sclerosing/diffuse glomerulosclerosis (n = 2) and minimal change (n = 1).
Four cases of lupus nephritis presented morphologically as diffuse proliferative GN.
One case of IgMN showed minimal change disease (MCD) morphologically.
Direct immunofluorescence was helpful in confirming the diagnoses of 16 cases of MCD, 2 cases of focal segmental glomerulosclerosis (FSGS), 3 cases of membranous GN, 3 of membranoproliferative glomerulonephritis (MPGN).
The statistical analysis is shown in [Table 2].
| Discussion|| |
Glomerulonephritis constitutes a major health issue on the already overburdened health services in the developing countries like India. Early diagnosis and treatment can prevent long-term complications.
Renal biopsy has contributed greatly to a rational classification of intrinsic renal diseases. Developed countries have all the facilities needed for the correct diagnosis of renal biopsy, which include LM, IF and EM examination. 
Light microscopy provides only the morphologic pattern of GN, but to arrive at a specific diagnosis of any glomerular disease, it requires the correlation between clinical data, LM, IF and EM if available. 
In our institution, IF when combined with LM, gave accurate results in most cases despite the lack of availability of EM.
In our study, it was noted that most common GN diagnosed on renal biopsies was MCD followed by IgA nephropathy and FSGS. Nasir et al.  study reported the most common GN to be membranous GN followed by FSGS and MCD.
The other study, however reported the highest prevalence to be that of FSGS. 
All the cases of MCD were diagnosed correctly on LM, which was negative on IF except 2 cases. 1 case of MCD showed positivity for IgG which can be secondary to proteinuria and not of primary pathogenic importance. The other case showed IgM positivity on IF and hence after clinical, LM and IF correlation, the case was diagnosed as IgMN.
Out of 7 cases diagnosed as FSGS on LM; 2 cases showed positivity for IgM and C3 in sclerotic segments (28.5%). This finding is similar to the studies by Nasir et al.  in which FSGS showed non-immune trapping of IgM in 7 (40%) cases and C3 in 3 (20%) cases and Abbas et al.  which showed focal positivity of IgM in 87.3% and C3 in 83.63% of cases.
In our study, 5 cases of FSGS were negative on IF. This is because if the segmental lesion is present in a small proportion of the glomeruli within the whole kidney (<10% of the glomeruli), there is a heightened probability of not finding a diagnostic lesion within the biopsy sample. The sampling problem and the juxtamedullary distribution of the involved glomeruli suggest that missing an involved glomerulus is a distinct possibility in a small and superficial biopsy. 
Membranous GN showed glomerular basement membrane (GBM) thickening and spikes on GMS staining. All these cases showed strong granular GBM deposition of IgG and C3. This finding is similar to other studies. ,,,
Detection of early stage of membranous GN is important, as the majority of these cases will be diagnosed as MCD on LM alone. Positive DIF will help to differentiate MCD from membranous GN. This is particularly important as the treatment and correct diagnosis changes the prognosis of the disease. 
In the present study, 3/6 cases of MPGN showed IgG and C3 deposition along the glomerular capillary wall in a fine to coarse granular pattern, other 3 cases showed IgM and IgA along the capillary wall in the granular pattern. This finding is similar to study by Zucchelli et al. 
A predominance of IgM is typically observed in MPGN type I arising from chronic bacterial infection, such as osteomyelitis or infected ventriculoatrial shunt.  However, no such correlation was noted in our cases. Since IgA nephropathy has varied morphological patterns on LM, it is important to ascertain on IF whether IgA is the predominant or codominant immunoreactant to diagnose a membranoproliferative form of IgA nephropathy, which is rare.
An improved understanding of the role of complement in the pathogenesis of MPGN has led to a proposed reclassification into immunoglobulin-mediated disease (driven by the classical complement pathway) and nonimmunoglobulin-mediated disease (driven by the alternative complement pathway). This reclassification has led to improved diagnostic clinical algorithms and the emergence of a new grouping of diseases known as the C3 glomerulopathies, best represented by dense deposit disease and C3 GN. 
A C3 glomerulopathy is a proliferative GN, usually (but not exclusively) with an MPGN pattern on light microscopy, with C3 staining alone on immunofluorescence, implicating hyperactivity of the alternative complement pathway. 
In the present study, we had 2 cases of post infectious GN which on morphology shows expansion of the lobules, hypercellularity of the tuft and localized thickening of the glomerular capillary walls producing a picture almost identical to that of MPGN. IF along with EM plays an important role in distinguishing these lesions.
Sorger et al.  have described different categories of immunofluorescence patterns in post infectious GN. They noted three arrangements, called the garland pattern, the starry sky pattern and the mesangial pattern. Our cases showed positivity for IgG and C3 in a mesangial pattern thus, correlating with the findings of previous studies. ,
In the present study, 1 case of rapidly progressive glomerulonephritis (RPGN) showed granular IgG and C3 deposits; 2 cases showed negativity on IF which was due to the pauci-immune (Type III) RPGN. This finding is similar to a study done by Naseri et al. 
Our data shows that IF was crucial in establishing the diagnosis of 14 cases. This finding is similar to the studies by Nasir et al.  and Abbas et al.  These cases included 9 cases of IgA Nephropathy, four of lupus nephritis and one of IgMN.
Nine cases of IgA nephropathy had different morphological patterns, including mesangial proliferation/expansion in six, chronic sclerosing/diffuse glomerulosclerosis in two and minimal change in 1 case. These cases could have been misdiagnosed if only morphologic pattern were taken into account. This finding is similar to other studies. ,
IgA Nephropathy is the most common GN worldwide.
Recently, the prevalence of IgA nephropathy is shown as a tip of the iceberg. It is important to diagnose this entity correctly as it progresses to end-stage renal disease in up to 30% cases within 10 years. 
Four cases of lupus nephritis presented morphologically as diffuse proliferative GN. IF helped in arriving at a diagnosis in these cases, which revealed "full house" pattern.
IgM nephropathy is a subset of MCD that may evolve into FSGS.  IgMN is an idiopathic GN characterized by primary mesangioproliferative GN on light microscopy with sole or dominant diffuse, granular IgM deposits within the mesangium on IF and clinically presenting with steroid-resistant/dependent proteinuria, having no associated systemic disease. 
Detection of IgMN can be missed if only light microscopy is performed. In our study, IgMN was diagnosed on IF in 1 case, the initial morphological diagnosis in this case being MCD. The prevalence of IgMN however, was very low in our study, when compared with other studies. 
In the present study, we had 1 case of diabetic nephropathy, which showed linear IgG positivity and granular positivity for IgM and C3 along glomerular capillary wall.
One case of amyloidosis was also seen, which showed nonspecific deposits of IgM and C3 in sclerotic tufts. IgA and IgG were negative.
In the present study, we had 12 cases of Acute Interstitial Nephritis in which IF findings came out to be negative for immunoglobulins and complement. Hence, these cases were included under TNs.
There was 1 case of Hemolytic Uremic Syndrome in our study which showed positivity for IgM and C3. IgG and IgA were negative. Thus this case was included in the TPs as the findings correlated with the findings in the previous studies.
Our study also had 2 cases of Light Chain Cast Nephropathy for which immunofluorescence studies showed that tubular casts predominantly stain with kappa. Lambda was faintly stained. IgG, IgM, IgA and C3 were negative. These 2 cases were also included under TPs.
In the present study, we had 2 cases of Malignant Hypertension. On Immunofluorescence the findings were IgM and C3 deposits along glomerular capillary wall. IgG positivity was reported in 1 case, and IgA was negative. These cases were considered as TPs.
There were 4 cases of chronic glomerulonephritis in which immunofluorescence findings were negative. Hence, we included these cases under TPs.
The study had limitations in terms of its sample size calculation. Since GN is not a very common disease, it was difficult to follow routine sample size methodology, but the number-75 cases - was found worthy enough for our study.
Direct immunofluorescence, apart from renal biopsies, is also very useful in the differential diagnosis of immunobullous lesions of the skin. 
| Conclusion|| |
Immunofluorescence along with light microscopy is required for accurately diagnosing glomerulonephritis. This is especially important in cases of lupus nephritis, IgA nephropathy and IgMN as these entities cannot be diagnosed with light microscopy alone. Treatment strategy for these cases is aggressive, affecting the progression of the disease.
Direct immunofluorescence microscopy and correlation with LM, clinical, biochemical and serological markers should be done on a regular basis for the correct diagnosis of glomerular diseases.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]