Impact of Immunofluorescence on the Histological Pattern of Pediatric Kidney Biopsies from Northern Pakistan

Abstract

Kidney biopsy is an investigation for diagnosis and prognosis of a variety of nephritides. It also influences therapeutic options. Immunofluorescence (IMF) greatly adds in identifying the pathologies which may not be obvious on light microscopy (L/M), such as IgM, IgA nephropathy, pauci-immune glomerulonephritis, and anti-glomerular basement membrane disease. We present here data of 170 pediatric kidney biopsies from July 2005 to December 2009 from Department of Nephrology and Hypertension, Lady Reading Hospital, Peshawar, Pakistan. The study was undertaken to see whether IMF would alter the histological pattern of pediatric kidney biopsies and to compare these data with an earlier data from our department of 415 pediatric kidney biopsies done over 7-year period from 1998 to 2005, which were analyzed with L/M alone. Out of 170 kidney biopsies using L/M and IMF, IgM turns out to be most common pattern (20%), followed by minimal change disease (MCD) (17.05%), focal and segmental glomerulosclerosis (FSGS) (15.88%), chronic sclerosing glomerulonephritis (Chr. sclerosing GN) (12.35%), mesangio proliferative glomerulonephritis (MPGN) (7.65%), mesangio capillary glomerulonephritis (MCGN) (6.47%), membranous glomerulonephritis (Mem. GN) (5.29%), IgA nephropathy (5.29%), cresentic glomerulonephritis (Cres. GN) (3.53%), lupus nephritis (2.96%), and others (3.53%). Comparing these results of 170 cases with 415 renal biopsies without IMF, IgM dominated the histological pattern in IMF group whereas MCD followed by FSGS and MPGN were prominent in group without IMF. Therefore, variation in the overall histological pattern with IMF technique proved statistically significant (p < 0.0001). Addition of IMF has altered the frequency of MCD, a change from 24% (100/415) to 17% (29/170), FSGS from 18.3% (76/415) to 15.88% (27/170), and MPGN from 17.35% (72/415) to 7.65% (13/170).

Keywords:

• Renal biopsy
• light microscopy
• immunofluorescence
• IgM nephropathy
• MCD
• FSGS
• MPGN

INTRODUCTION

Renal biopsy is a basic diagnostic tool for diagnosis, prognosis, and staging of renal diseases. It also helps in diagnosing a variety of nephritides. Standard kidney biopsy involves at least five stains on light microscopy (L/M), that is, eosin and hematoxylin, periodic acid skiff, silver nitrate, and Congo red.

The morphological pattern seen with these stains is good enough to diagnose most of the nephritides. However, the cases of IgA nephropathy, IgM nephropathy, pauci-immune glomerulonephritis, and anti-glomerular basement membrane disease do need special staining via immunofluorescence (IMF). This also adds greatly in staging systemic lupus erythematosus. The technique however involves more money and additional piece of tissue for the laboratory and adds to the patients’ pocket.

IMF is an important diagnostic tool that enhances diagnostic accuracy. Nowadays kidney biopsies are almost invariably examined both by L/M and IMF. We have already published pediatric kidney biopsy data of 415 biopsies from northern part of Pakistan which analyzed patients without IMF. However, histological pattern was comparable to the biopsy data of pediatric population from south of the country which was with IMF.

The importance of IMF in analysis of renal biopsies cannot be undermined. In this regard, our department has data of 170 pediatric kidney biopsies from 2005 to 2009. This study is undertaken to see the difference in the disease pattern of pediatric nephrotic syndrome after the availability of this diagnostic technique (IMF) and to analyze its impact on the frequency of different variety of nephritides.

MATERIAL AND METHODS

This study was carried out in the Department of Nephrology and Hypertension, PGMI, Lady Reading Hospital, Peshawar, from July 2005 to December 2009. All the pediatric kidney biopsies during this period were included in this study. Patients from the age of 3 to 15 years of either sex were involved. All those with acute renal failure of varied etiology, renal stone disease, diabetic nephropathy, renal transplant, and obstructive nephropathy were excluded from this study. Detailed history and clinical examination was performed prior to biopsy. Informed consent was taken from the guardian and was documented. All the patients had complete hematological, immunological, and coagulation profile checked prior to biopsy.

These biopsies were done under ultrasound guidance using 2% lignocaine as local anesthesia. Lower and outer pole of kidney was localized at real-time ultrasound and two core of tissues taken using automated spring-loaded monotony gun. No basal sedation or general anesthesia was used. The sample was preserved in N/10 formal saline and sent to the reference lab. All the biopsies were analyzed at Ziauddin University Hospital, Karachi. All patients were kept under observation for 24 h and their vitals were monitored. Syrup paracetamol was used as analgesic.

Nephrotic syndrome remained the main indication for renal biopsies in 100% of the cases. However, steroid resistance (defined as no response to steroid after 8 weeks of treatment), frequently relapsing nephrotic syndrome (defined as >2 relapses in 6 months or >3 relapses in a year) and nephrotic syndrome with renal insufficiency remained indications for renal biopsy in some cases.

The biopsy sample was subjected to L/M using standard five stains, that is, eosin and hematoxylin, silver nitrate, periodic acid skiff, and Congo red. In addition, each sample was subjected to IMF using standard antibodies against IgA, IgM, IgG, C3, C4, C1q, and other components of complement cascade. In a given case, with relevant history and clinical suspicion, linear pattern of IMF was also used. However, routine staining for kappa (κ) and lambda (λ) light chains were not done as amyloidosis and myelo-proliferative disorders were not suspected in pediatric population of this age group. Moreover, Congo red staining was used as part of L/M staining process to pick up amyloidosis.

Table 1. Histological patterns of renal disease.

Minimal change disease (MCD)

Focal and segmental glomerulosclerosis (FSGS)

Mesangio proliferative glomerulonephritis (MPGN)

Mesangio capillary glomerulonephritis (MCGN)

Proliferative glomerulonephritis (Pro. GN)

Membranous glomerulonephritis (Mem. GN)

Hypertensive Nephropathy (H. Neph)

Cresentic glomerulonephritis (Cres. GN)

Chronic sclerosing glomerulonephritis (Chr. sclerosing GN)

Chronic tubular interstitial necrosis (Chr. TIN)

IgM nephropathy

IgA nephropathy

Alport syndrome

Acute tubular necrosis (ATN)

Lupus nephritis III

Renal amyloidosis

WHO classification for renal disease was employed to classify nephrotic syndrome (Table 1). Histological pattern of these biopsies was compared with a separate data of 415 pediatric renal biopsies that were performed in our department from 1998 to 2005, without using IMF.

STATISTICAL ANALYSIS

Chi-square (χ²) test of independence was employed as means of statistical analysis. All results were considered significant at α = 5%, when p-value was <0.05.

Figure 1. Male to female ratio across different patterns of renal disease.

RESULTS

Total of 170 kidney biopsies were analyzed using IMF and involved 95 males and 75 females (Figure 1). It was observed that male to female ratio across different histological patterns did not vary significantly with an overall male to female ratio being 1.27:1 (p = 0.554). Patients from 3 to 15 years of age were included with mean age of 11.78 years. Overall IgM nephropathy turned out to be most common histological pattern seen in 34 patients (20%), followed by minimal change disease (MCD), 29 patients (17.05%); focal and segmental glomerulosclerosis (FSGS), 27 patients (15.88%); chronic sclerosing glomerulonephritis (Chr. sclerosing GN), 21 patients (12.35%); mesangio proliferative glomerulonephritis (MPGN), 13 patients (7.65%); mesangio capillary glomerulonephritis (MCGN), 11 patients (6.47%); membranous glomerulonephritis (Mem. GN), 9 patients (5.29%); IgA nephropathy, 9 patients (5.29%); cresentic glomerulonephritis (Cres. GN), 6 patients (3.53%); lupus nephritis, 5 patients (2.96%); others, 6 patients (3.53%).

Among 95 male patients, IgM nephropathy followed by MCD and FSGS were the dominant histological pattern seen in 21.1%, 17.9%, and 13.7% of cases, respectively. In female population of 75 patients, histological spectrum showed FSGS and IgM nephropathy sharing common position in 18.67% of cases followed by MCD seen in 16% of cases.

Majority of the patients were between 11 and 15 years of age comprising 118 patients (69.40%). Forty-nine patients (28.82%) were between 6 and 10 years of age while only three patients (1.78%) were under 6 years of age.

Out of 118 patients from 11 to 15 years of age, FSGS was the most common pattern seen in 16.9% followed by IgM nephropathy 15.25%; Chr. sclerosing GN, 15.25%; MCD, 13.56%.

However, in 49 patients between 6 and 10 years of age, IgM nephropathy was dominant seen in 32.65% followed by MCD, 20.4%; FSGS, 14.29%; MPGN, 10.20%.

MCD was the only pattern seen in three patients of less than 6 years of age. Thus, variation across the histological pattern between ages 6 to 10 and 11 to 15 years proved statistically significant (p = 0.046).

Out of 170 patients, serum creatinine on admission was found between 0 and 1.2 mg/dL in 116 patients (68.24%). The dominant pattern in this age group was found to be MCD, seen in 25% of the cases, followed by IgM nephropathy, 22.4%; FSGS, 18.1%; MPGN, 12.1%.

Twenty-six patients (15.3%) had their serum creatinine between 1.3 and 3 mg/dL. Chr. sclerosing GN was found to overshadow most of the histological pattern being present in 38.5% of the cases, followed by IgM nephropathy 19.2%; Cres. GN, 11.5%; FSGS, 11.5%.

Serum creatinine between 3.1 and 14 mg/dL on admission was present in 28 patients (16.46%). Chr. sclerosing GN was the dominant pattern in this group being present in 39.3% of the cases, followed by MCGN 10.7%; Cres. GN, 10.7%; FSGS, 10.7%; IgA nephropathy, 10.7% (Table 2). Therefore, the histological pattern was found to vary significantly across patients with different serum creatinine on admission (p < 0.0001).

Table 2. Histological patterns of renal disease across varying creatinine levels.

	Creatinine (0–1.2 mg/dL)	Creatinine (1.3–3 mg/dL)	Creatinine (3.1–14 mg/dL)	Total
Chr. Sclerosing GN	0	10	11	21
Cresentic GN	0	3	3	6
MCD	29	0	0	29
IgM nephropathy	26	5	3	34
FSGS	21	3	3	27
MPGN with no deposits on immunoflorescence	14	0	0	14
IgA nephropathy	6	0	3	9
MCGN	6	2	3	11
Others	14	3	2	19
Total	116	26	28	170

Results of 170 renal biopsies performed using IMF were compared with the results of 415 pediatric renal biopsies from our department, performed without employing IMF technique (Figure 2). It was observed that IMF technique had a significant impact on the overall histological pattern. IgM nephropathy was found to be the dominating pattern in presence of IMF whereas MCD, followed by FSGS and MPGN, was more prominent in the absence of this technique. Therefore, variation in the overall histological pattern with employment of IMF technique proved statistically significant (p < 0.0001).

Figure 2. Impact of immunoflourescence on different histological patterns of renal disease.

DISCUSSION

Our department has been doing renal biopsy since its inception, that is, 1990. Initially, the biopsies were referred to Armed Forces Institute of Pathology, Rawalpindi, for analysis and subsequently to Aga Khan University Hospital, Karachi. IMF was not available in either of these hospitals till late 2000.

Since 2005, we have been sending our renal biopsies to Ziauddin Teaching Hospital, Karachi, both for IMF and L/M. These are analyzed by a single experienced renal histopathologist.

The main question addressed in this study has been whether IMF has altered our histological pattern of pediatric renal biopsies; however, renal outcome based on IMF was not evaluated in current study. An earlier study of 841 consecutive biopsies excluding transplant patients showed that overall diagnostic inadequacy that would occur without IMF was 8.9%.¹ Anand Date while analyzing the kidney biopsies of 39 patients without IMF have noted no significant effect on the patient management and found that clinical, biochemical, and light microscopic findings were enough to make a genuine clinical judgment.²

This issue is partly addressed in a study by Khawar Abbas et al.³ where the authors have seen the impact of IMF and serology on the morphology of renal biopsy in nephrotic patients, both adult and children. Total percentage change in L/M diagnosis after IMF study was 23.5% and combined change after serological and IMF study was 35% in L/M diagnosis.

Earlier data from our department consisting of 415 patients suffering from nephritic syndrome over 7-year period from 1998 to 2005 have also been analyzed. The histological patterns of renal disease in this pediatric population involved only L/M as a diagnostic modality. The data showed MCD, FSGS, MPGN, MCGN, post-infectious proliferative GN, membranous GN, and crescentic GN as common histological pattern (Figure 2). This was comparable to the data from Sindh Institute of Urology and Transplantation (SIUT)⁴ where 560 pediatric kidney biopsies with IMF were analyzed showing MCD and allied (including MCD, IgM in the same category). Comparing these two large studies the histological pattern of renal disease from south and north of Pakistan does not differ significantly. To some extent, this comparison appears to undermine the importance of IMF as a diagnostic technique in addition to L/M!

Would the study from SIUT⁴ have considered IgM nephropathy as a separate entity, the impact of IMF on the histological pattern would have become more obvious!

In the current study, our data have clearly shown that IMF significantly altered the histological pattern of renal biopsies when compared with L/M alone.

IgM nephropathy not seen in our earlier data was the most common histological pattern seen in 20% of the cases (34/170) in the current series. This diagnosis was based on predominant IGM deposits seen in mesangium along with a morphological pattern of mesangial hypercellularity. No doubt this remains an IMF-dependent diagnosis. IgM nephropathy also showed renal insufficiency in 8/34 cases, frequently relapsing course in 20 cases and steroid resistance in almost all the cases. Although not yet regarded as independent entity⁵ and sometimes considered as a variant of MCD. However, many of us regard it as a distinct histological entity with different response to steroid, requiring immunosuppressant therapy.^6–11 We believe that IgM nephropathy remains a separate histological entity with steroid responsiveness, course, and prognosis far different from MCD. About 23.5% of our cases showed renal insufficiency with a serum creatinine varying from 1.4 to 5 mg%.

MCD (17.05%), FSGS (15.88%), Chr. sclerosing GN (12.35%), and MPGN with negative IMF (7.65%) were other histological patterns in order of frequency. This has been compared with our earlier series of 415 biopsies without IMF (Figure 2).

The impact of IMF was also seen in diagnosis of IgA nephropathy (9/170) cases. However, frequency of crescentic GN, lupus nephritis, and FSGS did not differ much while comparing the two series of 170 with IMF and 415 patients without IMF (Figure 2). Chr. sclerosing GN was seen in 21 patients (12.35%). This was far higher percentage compared to earlier studies (415 without IMF). However, this entity does not require IMF for diagnosis.

MPGN was the fifth commonest entity showing mesangial hypercellularity of varying degree along with no immune deposits on IMF. This differs from MCD, which by definition had normal morphology on L/M. This entity with no immune deposits responded well to steroids and all the 14 cases showed no renal insufficiency, and relapsing course was only seen in 5/14 cases. We feel this histological pattern cannot be considered as a variant of MCD due to distinct morphological pattern though its clinical behavior mimics MCD to some degree.¹²

IMF however has altered the frequency of MCD which has changed from 24% (100/415) to 17% (29/170), a change of 7%, FSGS 18.3% (76/415) to 15.88% (27/170), and a change of 2.42%. MPGN 17.35% (72/415) to 7.65% (13/170), a change of 10%, when comparing the two series, that is, 170 versus 415. This change in MPGN has come simply because both IgA and IgM nephropathy were included in 72 cases as they all share the common feature of mesangioproliferation of varying degree on L/M. After the use of IMF, these two entities were recorded separately in the current series of 170 cases. Hence MPGN has come down on the merit to the fifth place (Figure 1).

The addition of IMF to the standard biopsy with L/M not only guided us to the therapeutic intervention but also added to the prognostic value. For instance, IgM nephropathy would follow a relapsing course in 58.8% of cases (20/34) and renal insufficiency in 23.5% of cases (8/34). Similarly MPGN with negative IMF has been a good prognostic sign as none of them had renal insufficiency (14/170) and all of them reopened to steroids. Therapeutic interventions were also modified in view of IMF-based diagnosis. IgA nephropathy with microscopic hematuria alone was managed conservatively, where as IgA with features of vasculitis received both steroids and cytotoxic drugs. A proportion of cases of IgA with hematuria and proteinuria in non-nephrotic range with normal kidney function were with ACE inhibitors/ARBs, statins, and diuretics.

IMF has proven itself as an important diagnostic technique which adds to diagnostic accuracy. Although IMF may not enhance the diagnosis of MCD, FSGS, crescentic, amyloid, tubular interstitial necrosis, sclerosing GN, it certainly helps in diagnosis of entities such as IgM, IgA, immune-mediated crescentic GN, earlier stage of membranous GN, and lupus. The analysis of 170 cases in the current study has added IgM nephropathy as the top-most diagnosis in our pediatric population from 2005 to 2009. This was not seen in a single case in our larger series of 415 cases. Also frequency of other common entities such as MCD, FSGS, MPGN have changed 7%, 2.4%, and 10%, respectively, as compared to previous data.

CONCLUSION

IMF while analyzing renal biopsies remains an integral part of diagnostic procedure. It has highlighted the entities which cannot be diagnosed without this technique such as IgM and IgA nephropathy. It certainly helps to diagnose early membranous, type II mesangiocapillary GN, anti-glomerular basement membrane antibody, and WHO Class IIA lupus.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Anjum Mahmood for her priceless efforts and valuable guidance during the process of manuscript preparation. Mr. Akhtar’s (computer operator) efforts are also acknowledged.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of paper.