Bangladesh Academy of Pathology

Cytogenetic Pattern in Adult Patients with de novo Acute Myeloid Leukaemia: a Single Center Study in Bangladesh

 

*Baqi SA,¹ Munmun UK,² Khan MR,³ Shah MS,⁴ Islam S,⁵ Rahman F,⁶ Aziz MA,⁷ Begum M⁸

 

 

1. Saqi Md. Abdul Baqi, Resident, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka. saqi.dmc@gmail.com
2. Umme Kulsum Munmun, Lecturer, Department of Pathology, Dhaka Medical College, Dhaka.
3. Md. Rafiquzzaman Khan, Associate Professor, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,
4. Md. Salahuddin Shah, Associate Professor, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,
5. Shafiqul Islam, Medical Officer, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,
6. Farzana Rahman, Assistant Professor, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,
7. Md. Abdul Aziz, Professor, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,
8. Masuda Begum, Professor, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka,

 

*For correspondence

 

 

Abstract

Background: Cytogenetic analysis performed at diagnosis is considered to be the most important prognostic factor in AML.

Objective: The purpose of this study was to observe the pattern of cytogenetic abnormalities in adult patients with de novo AML.

Method: Total fifty two newly diagnosed de novo AML patients were selected for the study. Six cytogenetic abnormalities including t(8;21), t(15;17), inv(16), BCR-ABL1, FLT3-ITD & NPM1 mutations were detected by Real-Time PCR.

Results: In this study, 36 (69.2%) patients showed different cytogenetic abnormalities. The t(15;17) was found to be the most common. t(15;17), t(8;21) and inv(16) were found only in M3, M2 and M4 subtypes, respectively. Significant association was found with increasing age and cytogenetic risk groups. BCR-ABL1 mutation showed significant relation with increased age. FLT3-ITD mutation showed significant association with increased WBC count and inv16 was significantly associated with relatively less bone marrow blast percentage.

Conclusion: So, Cytogenetic study should be performed routinely in all cases of AML for proper diagnosis, prediction of prognosis and implementation of effective therapeutic measures.

 

 

[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):86-92]

 

Key words: Acute myeloid leukaemia, cytogenetics

 

Introduction

Acute myeloid leukaemia (AML) is a clonal, malignant disease of haematopoietic tissue which is characterized by accumulation of abnormal (leukaemic) blast cells, principally in the bone marrow, and impaired production of normal blood cells.¹The incidence of AML is higher in the white population (3.8 per 100,000 person) than that of the Asian population (3.2 per 100,000 person).² AML is the most common acute leukaemia in adult and it is more common in males. Majority of the AML patients are older than 60 years.³ Cytogenetic abnormalities were first described in AML in the 1960s.⁴ In 1988, the Morphologic, Immunologic and Cytogenetic (MIC) classification was published. Since then, cytogenetic investigations have become more important in AML classification.⁵ AML is heterogeneous in terms of morphology, immunophenotype, cytogenetics and molecular genetics.⁶ Using the WHO criteria, the diagnosis of AML is established by the presence of 20% or more of leukaemic myeloblasts in the peripheral blood or bone marrow. However, in a subgroup of AML, the presence of recurrent genetic abnormalities alone is sufficient for the diagnosis of AML regardless of the blast percentage.⁷ Geographical, ethnic, and environmental influences should be considered in determining the cytogenetic and morphological features of this disease.⁸ Cytogenetic abnormalities are identified in 50-60% of adult newly diagnosed AML patients.⁹ Age and cytogenetic abnormalities are the most important prognostic factors in AML.¹⁰There is an increase in the proportion of patients with unfavourable risk cytogenetics and a decrease in favourable risk cytogenetics with advancing age.¹¹ Besides the prognostic importance, cytogenetic analysis is also recommended to monitor minimal residual disease (MRD) in case of AML patients with abnormal cytogenetics.¹²

 

From these points of view, the current study was designed to observe the pattern of cytogenetic abnormalities in adult patients with de novo AML, to categorize the patients into three risk groups and to observe the association of cytogenetic findings with FAB subtypes, age, sex & other laboratory findings.

 

Methods

This study was conducted in the department of Haematology, BSMMU from February 2018 to January 2019. Total fifty two newly diagnosed adult patients with de novo AML had been enrolled for the study. Complete blood count was done by automated cell counter machine and checked manually. Morphological diagnosis of AML had been done by bone marrow study. For cytogenetic study, 3-5 ml of peripheral blood or bone marrow aspirate was collected in EDTA tube. Six cytogenetic abnormalities including t(8;21), t(15;17), inv(16), BCR-ABL1, FLT3-ITD & NPM1 mutations were detected on an internationally standard laboratory by Real-Time PCR technique.The ABI 7500 real time PCR system (using the Taqman chemistry) was used for doing the cytogenetic analysis. A pre-designed semi-structured data collection sheet was used for data collection. The statistical analysis was carried out using the SPSS version 24.0 for Windows. For all statistical tests, p-value less than 0.05 was considered as statistically significant.

 

Results

The mean age of the patients was 36.87 (+13.29) years. Maximum (30.8%) patients belonged to 18-27 years age group. Female was slightly predominant with a male: female ratio was 1: 1.08. Maximum patients belonged to FAB M2 (32.7%), followed by M3 (26.9%) and M1 (17.3%). Among the 52 cases, 36 (69.2%) cases showed different cytogenetic abnormalities and 16 (30.8%) cases had normal cytogenetics. The t(15;17) was found to be the most common detected in 13(25%) patients. NPM1 mutation was found in 10 (19.23%), t(8;21) in 8 (15.38%) and FLT3-ITD mutation in 8 (15.38%) cases (Table I).

 

M0 and M6 subtypes did not show any cytogenetic abnormality. Most of the M1 cases also had normal cytogenetic pattern. The t(15;17), t(8;21) & inv16 were found only in M3, M2 & M4 subtypes respectively. Only one case showed BCR-ABL1 mutation which belongs to M1 subtype. A combination of NPM1 & FLT3-ITD mutation was found in M4, M1, M3 and M2 subtypes (Table II).

Significant association was found between the mean age of 78 years and BCR-ABL1 mutation (p value 0.038). Favourable cytogenetics were more frequent in younger age groups. Intermediate and unfavourable cytogenetics were common in relatively older age groups. These findings were statistically significant (p value 0.004).

FLT3-ITD mutation was significantly associated with increased WBC count (p value 0.042). inv(16) was significantly associated with relatively less bone marrow blast percentage (p value 0.029). Patients with normal cytogenetics showed significant association with relatively increased bone marrow blast percentage (p value 0.033). No other parameter (sex, haemoglobin level, platelet count) showed any significant association with cytogenetic pattern.

 Discussion

AML is characterized by a spectrum of clinical, morphological, immunophenotypic and associated cytogenetic abnormalities. In this study the mean age at presentation of AML was 36.87 years. Studies in most of the other countries showed much higher mean age.^6,8,13,14,15 The reason for this difference may be due to inclusion of only de novo cases of AML in this study or may be geographic/ethnic influence.

Cytogenetic abnormalities were detected in 69.2% of patients with an increased frequency of t(15;17) in this study. These findings are consistent with the results of several other studies conducted by Ayesh et al. (2012)¹⁶ and Enjeti et al. (2004).¹³ Here, favourable cytogenetic risk group belonged to young patients and frequency of unfavourable cytogenetics were higher in older age groups. Similarly, Meng et al. (2013) found 75% favourable cytogenetics in young patients and mostly complex karyotype in elderly patients.⁶

Male: female ratio in this study was 1:1.08. This finding differs from most of the studies in other countries where male predominance was found.^6,13,17This difference is probably due to greater proportion of female bed in our department. Geographic or ethnic influence may also be responsible. An increased frequency of t(15;17) (28%) was found among the male patients in this study. Enjeti et al. (2004) also found increased frequency of t(15;17) among male patients in a study from Singapore.¹³ No other significant difference was found in this study among male and female patients in relation to cytogenetics.

Cases with FLT3-ITD mutation were found in this study to have increased WBC count. Penget al. (2008) also demonstrated a correlation between the presence of FLT3 mutation and the increased WBC count.¹⁸ Similarly, Schnittger et al. (2002) and Haferlach et al. (2012) reported NPM1 and FLT3 mutations to be correlated with higher peripheral WBC count.^19,20

The t(15;17) was found to be the most common (25%) cytogenetic abnormality in this study. The frequency of t(15;17) found in this study is much higher than other parts of Asia, like Taiwan (15%), compared to those observed in North America and Europe (3-10%).^21,22 The t(15;17) was found in 85.7% of M3 subtype patients in the current study, which is very close to the findings in Singapore (82.5% of M3 patients)¹³, compared to Japan (75.4%)⁸ and Europe (72.5%).²²

On the other hand, t(8;21) was found in 41.2% of AML M2 patients in this study. This percentage is closer to the incidence found in Japanese and Taiwanese reports (33.1% and 34% of their AML M2 patients).^8,21 The t(8;21) is less frequent among  the AML M2 patients in Singapore (14.5%), Australia (15.3%) and North America (22%).^13,23,24

In this study, significant difference was found in case of inv(16) pattern between M4 and non-M4 subtypes. Enjeti et al. (2004) also found the highest frequency of inv16 among their M4 cases.¹³

FLT3-ITD mutation was found more frequently in M4 subtype in this study. Similarly, another study carried out by Koczkodaj et al. (2016) in Southeastern Poland found the highest frequency of FLT3-ITD mutation in AML M4 cases.²⁵ In an Iranian study, Rezaei et al (2017) divided the patients into FAB M3 and non-M3 groups and analyzed the FLT3-ITD and NPM1 mutational status among these patients. They also found that these mutations were more frequent in non-M3 patients.²⁶ On the other hand, Smith et al. (2011) found a higher frequency of FLT3-ITD mutations among M3 patients.²⁷

 

Falini et al. (2005) observed that FLT3-ITD mutations occur twice as often in the cases with simultaneous NPM1 mutations as in the cases without this mutation.²⁸ The current study also shows that co-existent FLT3-ITD & NPM1 mutations are more frequent than FLT3-ITD mutation alone.

These differences in the frequency of cytogenetic/molecular genetic abnormalities might be due to geographic & ethnic heterogenecity, variation in the sample size or inclusion of paediatric patients in some studies. Paediatric patients were excluded in our study.

 Conclusion

Significant number of patients showed different cytogenetic abnormalities. Therefore, cytogenetic study should be performed routinely in all cases of AML for proper diagnosis, prediction of prognosis and implementation of effective therapeutic measures.

References

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14. Mauritzson N, Johansson B, Albin M, Billström R, Ahlgren T, Mikoczy Z, Nilsson PG, Hagmar L, Mitelman F. A single‐center population‐based consecutive series of 1500 cytogenetically investigated adult hematological malignancies: karyotypic features in relation to morphology, age and gender. European journal of haematology. 1999 Feb;62(2):95-102.
15. Preiss BS, Kerndrup GB, Schmidt KG, Sørensen AG, Clausen NA, Gadeberg OV, Mourits‐Andersen T, Pedersen NT, AML Study Group Region of Southern Denmark. Cytogenetic findings in adult de novo acute myeloid leukaemia. A population‐based study of 303/337 patients. British journal of haematology. 2003 Oct;123(2):219-34.
16. Ayesh M, Khassawneh B, Matalkah I, Alawneh K, Jaradat S. Cytogenetic and morphological analysis of de novo acute myeloid leukemia in adults: a single center study in jordan. Balkan Journal of Medical Genetics. 2012 Jan 1;15(1):5-10.
17. Amare PK, Jain H, Kabre S, Deshpande Y, Pawar P, Banavali S, Menon H, Sengar M, Arora B, Khattry N, Narula G. Cytogenetic profile in 7209 Indian patients with de novo acute leukemia: a single centre study from India. J Cancer Ther. 2016 Jun 27;7:530-44.
18. Peng HL, Zhang GS, Gong FJ, Shen JK, Zhang Y, Xu YX, Zheng WL, Dai CW, Pei MF, Yang JJ. Fms-like tyrosine kinase (FLT) 3 and FLT3 internal tandem duplication in different types of adult leukemia: analysis of 147 patients. Croatian medical journal. 2008 Oct 15;49(5):650-9.
19. Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C, Löffler H, Sauerland CM, Serve H, Büchner T, Haferlach T. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood. 2002 Jul 1;100(1):59-66.
20. Haferlach T, Bacher U, Alpermann T, Haferlach C, Kern W, Schnittger S. Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype. Leukemia research. 2012 Jan 1;36(1):51-8.
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24. Grimwade D, Walker H, Harrison G, Oliver F, Chatters S, Harrison CJ, Wheatley K, Burnett AK, Goldstone AH. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood. 2001 Sep 1;98(5):1312-20.
25. Koczkodaj D, Zmorzyński S, Michalak-Wojnowska M, Wąsik-Szczepanek E, Filip AA. Examination of the FLT3 and NPM1 mutational status in patients with acute myeloid leukemia from southeastern Poland. Archives of medical science: AMS. 2016 Feb 1;12(1):120.
26. Rezaei N, Arandi N, Valibeigi B, Haghpanah S, Khansalar M, Ramzi M. FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin 1 (NPM1) in Iranian adult acute myeloid leukemia patients with normal karyotypes: mutation status and clinical and laboratory characteristics. Turkish Journal of Hematology. 2017 Dec;34(4):300.
27. Smith ML, Hills RK, Grimwade D. Independent prognostic variables in acute myeloid leukaemia. Blood reviews. 2011 Jan 1;25(1):39-51.
28. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. New England Journal of Medicine. 2005 Jan 20;352(3):254-66.

Classification of Membranoproliferative Glomerulonephritis According to New Evolving Classification System by Using Immunofluorescence Technique

*Rahman DA,¹ Asaduzzaman,² Saleheen S,³ Sultana S,⁴ Banu SG ⁵

Abstract

Background: Membranoproliferative glomerulonephritis (MPGN) are progressive diseases with poor prognoses. Recently, a newly proposed classification of these diseases separated them into immune complex – and complement- mediated diseases. We investigated the frequency of C3 glomerulopathy among  histologically diagnosed MPGN patients by using immunofluorescence study.
Methods: We conducted a cross sectional study was carried out at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka during the period from June 2015 to December 2016. The histopathological, immunofluorescence studies were carried out at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.
Results: We found 8 cases of complement mediated MPGN (C3 glomerulopathy) out of 67 cases of MPGN, accounting for 12% of the total cases of MPGN. Histomorphological and other clinical parameters in immune complex mediated MPGN and complement mediated MPGN show no significant difference.
Conclusion: Complement-mediated glomerulonepathy was present in 12% of patients previously diagnosed with MPGN.

[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):76-85]

 Keywords: Membranoproliferative glomerulonephritis, C3 glomerulopathy, Immunofluorescence

 

 

 

1. *Dr. DM Arifur Rahman, Assistant Professor, Pathology, TMSS Medical College, Bogura. arifurrahmandm@gmail.com
2. Asaduzzaman, Lecturer, Pathology, Sheikh Sayera Khatun Medical College, Gopalgonj
3. Saied Saleheen, Assistant Professor (Current Charge), Pathology, Sheikh Hasina Medical College, Tangail.
4. Sabera Sultana, Lecturer, Virology, Rajshahi Medical College
5. Sultana Gulshana Banu, Associate Professor, Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.

 

*For correspondence

 

Introduction

Membranoproliferative glomerulonephritis (MPGN) is the fourth most common cause of primary glomerulonephritis in Bangladeshi patients and seventh most common cause of primary glomerulonephritis in Indian patients.^1,2 Because the proliferation is predominantly in the mesangium but may also involve the capillary loops, membranoproliferative glomerulonephritis is also known as mesangiocapillary glomerulonephritis.³ MPGN accounts for 10% to 20% of cases of nephrotic syndrome in children and young adults.⁴

Traditionally, primary MPGN has been classified based on the findings of electron microscopy (EM) as MPGN type I (MPGN I), MPGN II and MPGN III. This classification does not reflect the disease pathogenesis. It is proposed that MPGN be classified into two major groups on the basis of Immunofluorescence (IF) findings: immunoglobulin (Ig)-mediated and complement-mediated (C3G) membranoproliferative glomerulonephritis(MPGN). If the IF studies show predominantly C3 and are negative or show no significant staining for Igs, this is called complement mediated MPGN, also known as C3 glomerulopathy. If immunoglobulins are present on IF studies, it is considered as Ig mediated MPGN.^5-8

C3 glomerulopathy again classified as dense deposit disease (DDD) (formerly membranoproliferative GN type II and C3 glomerulonephritis (C3GN) based on electron microscopic findings. C3 glomerulopathies in which the deposits do not fulfill the criteria for dense deposit disease have been designated as ‘C3 glomerulonephritis’.⁹

The term C3 glomerulonephritis was coined to describe glomerular lesions in which there is glomerular accumulation of C3 with little or no immunoglobulin in the absence of the characteristic highly electron-dense transformation seen in dense deposit disease. Practically according to the concensus meeting towards the definition of C3 glomerulopathy stated that C3 glomerulopathy should be used as a morphological termfor those cases with dominant staining for C3. Dominant is defined as C3 intensity ≥2 orders of magnitude more than any other immune reactant on a scale of 0 to 3 (including 0, trace, 1+, 2+, 3+).⁷

The new classification system based on immunofluorescence findings has been proposed to replace the traditional classification system in order to better identify the underlying causes of MPGN and to provide guidance for more individualized treatment. Eculizumab, the first available anticomplement therapy, blocks at the level of complement (C5) and has revolutionized the treatment of complement-mediated diseases as well as C3 glomerulopathy.⁵

Objectives

The main objective of the present study is to find out the distribution of complement mediated membranoproliferative glomerulopathy in Bangladesh. A second objective is to study the various histopathological changes and immunofluorescence pattern in renal biopsy specimens with MPGN as well as to compare between various clinical presentation and biochemical parameters between immune complex mediated MPGN and complement mediated MPGN.

Methods

This cross sectional study enrolled 67 cases of membranoproliferative glomerulonephritis during the period from June 2015 to December 2016. Histopathological and  immunofluorescence studies were carried out at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka. Cases diagnosed as membranoproliferative glomerulonephritis, based on renal biopsy findings; irrespective of age and sex were included in this study. Cases diagnosed as lupus nephritis, post streptococcal glomerulonephritis, IgA nephropathy and C1q nephropathy were excluded from the study.

All clinical information were recorded in a pre-designed proforma  before doing histopathology and direct immunofluorescence microscopy including patient’s age, sex, clinical presentation and results of laboratory investigation like urinary total protein (UTP), urinary RBCs, serum creatinine, blood urea, anti-nuclear antibody (ANA), anti-double stranded DNA (Anti-ds DNA) antibody etc.

Two samples of renal tissue were obtained from each case. Specimen for light microscopy was preserved in 10% formalin and that for immunofluorescence in normal saline. After arrival at the pathology department, tissue in 10% formalin was embedded in paraffin block and processed routinely. Then sections were cut thin (3-4 µm) with microtome and stained with haematoxyllin and eosin, periodic acid Schiff (PAS) and methenamine silver stains. The tissue in normal saline was stored at -20^oC temperature in deep freeze for subsequent cryostat sectioning. The sections were stained by fluorescein isothiocyanate (FITC) tagged conjugated rabbit polyclonal anti human IgG, IgA, IgM, complement component 3 (C3), fibrinogen and complement component 1q (C1q).

Results

Among 67 biopsies, 59 cases (88%) were diagnosed as immune complex mediated MPGN. Eight (12%) cases were found to be conforming to complement mediated MPGN (C3 glomerulopathy), as defined by the diagnostic criteria mentioned earlier.

The mean age of patients with immune complex mediated MPGN was 35.71±11.76 and with  complement mediated MPGN was 35.87±14.98. The mean age difference is not statistically significant (p>0.05) between two groups. In cases of immune complex mediated MPGN, number of male patients were 31 (53%) and female patients were 28 (47%). In cases of complement mediated MPGN, number of male patient was 5(62%) and female patient was 3(38%).

Nephritic syndrome was the most common presentation in cases of complement mediated MPGN (50%) compared to immune complex mediated MPGN (27%). On the other hand nephrotic syndrome was the most common presentation (58%) in cases of immune complex mediated MPGN compared to complement mediated MPGN (37.5%).

The mean creatinine value of immune complex mediated glomerulonephritis was 1.56 mg/dl and of  complement mediated glomerulonephritis was 1.33 mg/dl. Serum C3 level decreased in 62% and 57% cases of immune complex mediated and complement mediated glomerulonephritis respectively. This differences are not statistically significant between two groups (p>0.05).

Crescent formation is observed 2 cases (3%) of immune complex mediated MPGN and one case of complement mediated MPGN (12.5%). Haematuria was present in 69% cases of immune complex mediated MPGN. On the other hand haematuria was present in 75% cases of complement mediated MPGN. These differences are not statistically significant (P value is >0.05)

Table I shows immunofluorescence deposits in complement mediated MPGN. It describes sites of deposition of antibody and staining intensity. Staining intensity is expressed by a scale of 0 to 3 including 0, trace, 1+, 2+, 3+ (Pickering et al., 2013). Table II shows biochemical parameters compared to immunofluorescence deposits in complement mediated MPGN cases.

 Discussion

The traditional classification of membranoproliferative glomerulonephritis is confusing. It is not based on disease pathogenesis and the types are overlapping. A new classification system of membranoproliferative glomerulonephritis based on immunofluorescence finding has been proposed. It helps to identify the underlying causes of MPGN and to provide guidance for more individualized treatment. This is a cross sectional study carried out to find out the distribution of immunoglobulin mediated and complement mediated membranoproliferative glomerulonephritis as well as to detect immunofluorescence deposits in renal biopsy specimens with MPGN. Clinical presentations and biochemical parameters in immune complex mediated MPGN and complement mediated MPGN were also observed.

A total of 67 membranoproliferative glomerulonephritis cases were included in this study. Clinical information and written consent were taken from the patients or their attendants. All clinical information were recorded in a pre-designed proforma. Routine H&E, PAS and methenamine silver stained sections of the renal biopsy samples were examined by two experienced pathologists. Sections were examined for changes in four components: glomeruli, tubules, interstitium and blood vessels. Cryostat sections were examined under fluorescence microscope for antibody deposition in the renal tissue. We categorized 59 cases (88%) as immune complex mediated MPGN and 8 cases (12%) as complement mediated MPGN out of 67 cases. Similar studies were conducted by Lu et al., in 2012, in China, Woo et al., in 2014, in South Korea, Pavinic and Miglinas, in 2015, in Lithuania and Mathur et al., in 2015, in India. They showed the percentage of C3 glomerulopathyin a series of studies on previously diagnosed MPGN cases as 20%, 4.3%, 8.6% and 1.16%, respectively.^10-13

The mean age of immune complex mediated MPGN cases was 35.71 years and of complement mediated MPGN (C3 glomerulopathy) cases was 35.87 years. Study conducted by Servais et al had showed the mean age of presentation of their C3 glomerulonephritis cases 30.3 years (SD±19.3).⁹ We had 5 cases of pediatricpatients (7.5% of total) out of which 4 were immune complex mediated MPGN (80%) and 1 was C3 glomerulopathy (20%).⁹ This finding is comparable with the study conducted by Giedraite et al on pediatric patients. They diagnosed 8 out of 23 pediatric MPGN cases as C3 glomerulopathy which was about 34% of total MPGN cases.¹⁴

Most of the patients of our study with both immune complex mediated and complement mediated MPGN are male. We found 53% and 62% male patients in cases of immune complex mediated MPGN and complement mediated MPGN respectively. Study conducted by Walker et al  reveal no significant sex difference.¹⁵

At presentation, almost all patients had proteinurea and most of the patients had haematuria. Nephrotic syndrome was present in 57% patients of immune complex mediated MPGN and 37% patients of complement mediated MPGN. The study conducted by Servais et al showedthat 38% of patients with dense deposit disease (MPGN type II) had nephrotic syndrome at presentation as compared with 65% of patients with MPGN type I.⁹

Nephritic syndrome was present in 27% patients of immune complex mediated MPGN and 50% patients of complement mediated MPGN in the present study. The study conducted by Mathur et al showed 50% patients with complement mediated MPGN of their study cases had nephritic syndrome and 50% had nephrotic syndrome at presentation.¹³

Haematuria was present in 75% cases of complement mediated MPGN and 69% cases of immune complex mediated MPGN. The study conducted by Nasr et al had showed 87% patients of dense deposit disease had haematuria on presentation.¹⁶

Mean serum creatinine value in immune complex mediated MPGN was 1.56 mg/dl. It was slightly lower in complement mediated MPGN (1.33 mg/dl). Mean serum creatinine was 2.2 mg/dl on 32 patients diagnosed as dense deposit disease.¹⁶

Serum C3 level was decreased in 62% cases and normal in 38% cases of immune complex mediated MPGN in our study. On the other side serum C3 level was decreased in 57% cases and normal in 43% cases of complement mediated MPGN. The study conducted by Servais et al showed that 40% of the C3GN patients had low C3 levels in circulation, and 46% patients of MPGN type I showed low C3 level.⁹

According to Concensus Report on C3 Glomerulopathy published in, C3 glomerulopathy is defined as C3 intensity ≥2 orders of magnitude more than any other immune reactant on a scale of 0 to 3 including 0, trace, 1+, 2+, 3+. In the present study immunofluorescence microscopy revealed, in most cases of complement mediated MPGN, a granular mesangial deposition of C3 with 2+ staining intensity. In cases with other immunoreactants, the dominant mesangial staining was C3 with at least two order intensity than others.⁷ The second most common deposited antibody was IgG along the glomerular basement membrane followed by IgM and IgA in the mesangium. We found only one case having mesangial deposition of C3   without any other antibody. Nasr et al and walker et al noted intense staining for C3 along the glomerular basement membrane and in the glomerular mesangial regions in their cases. Nasr et al also showed that immunoglobulins were absent or showed only focal and segmental staining with much less intensity than C3. They found IgM the most common second antibody; IgG and especially IgA were less common. Giedraite et al showed the second most common deposited antibodyamong 8 cases of C3 glomerulopathy was IgG followed by IgM.¹⁶

In the present study the immunofluorescence microscopy of immune complex mediated MPGN showed IgG antibody in 50 (86%) cases which was seen along glomerular basement membrane. 4 cases (6%) showed deposition of IgG in mesangium. Second most common immunoreactant was IgM followed by C3, IgA and C1q. 47 (79%) cases showed positive staining for IgM and 46 (77%) cases showed positive staining for C3. IgA positivity was seen in one third cases. Davis and Cavallo showed that in the immune complex variant of MPGN, the most consistent finding was positive IgG staining in granular pattern along the glomerular basement membrane.¹⁷ IgM was less common than IgG, but it was present in 86% of patients in one series, and 73% in another.^18,19

 Conclusion

In the present study we have classified the membranoproliferative glomerulonephritis cases according to the new classification system which is based on detection of immunoreactants in the renal tissue. The immunoreactants reflect the underlying pathogenesis of the disease process. We think this classification can help to manage the patients in a developing country like ours where modern facilities including electron microscope are lacking.

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