Identification of urinary proteins potentially associated with diabetic kidney disease.

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets.

Expanding the antibody-mediated component of plasma cell-rich acute rejection: A case series.

Renal allograft rejection is mediated by T-cells (T-cell mediated rejection) or by donor-specific antibodies (DSAs) (antibody mediated rejection, ABMR). Plasma cell-rich acute rejection (PCAR) is a unique entity due to its peculiar morphology and poor prognostic behavior. All allograft biopsies done at our center from January 2013 to October 2014 were reviewed, and seven were identified with a diagnosis of PCAR with antibody mediated rejection (ABMR). The allograft biopsies were classified as per the Banff 2007 schema. Immunohistochemistry with C4d, SV 40, CD3, CD20, CD138, kappa and lambda light chain was performed. Total 210 allograft biopsies were performed in the study period of which seven biopsies (3.3%) were diagnosed as PCAR with ABMR. All these were late ABMRs (more than 6 months) with median posttransplant duration of 17 months. The allograft biopsy showed features of PCAR along with glomerulitis, peritubular capillaritis, and positive C4d. DSA was positive in six patients. All the patients were treated with standard therapeutic measures of acute cellular rejection (ACR) and ABMR including steroids, plasma exchange, rituximab and intravenous immunoglobulins. All the patients had persistent graft dysfunction or graft loss on follow-up.

A clinicopathologic study of malignant melanoma based on cytomorphology.

CONTEXT: Melanoma accounts for 1-3% of all malignancies. It is usually diagnosed as metastasis in the draining lymph nodes, that is inguinal lymph node. Due to paucity of cytological studies on melanoma in India, the present study was undertaken. AIM: To study the cytomorphology of malignant melanoma and to correlate the cytological diagnosis with histopathology and clinical details. SETTINGS AND DESIGN: Sixteen cytomorphological features were studied and analysed from the cases diagnosed as melanoma on cytology with histopathological correlation. MATERIALS AND METHODS: Thirty patients diagnosed with malignant melanoma on fine-needle aspiration cytology (FNAC) were studied over a period of 10 years and compared by histopathology. Papanicolaou (Pap)- and May Grunwald Giemsa (MGG)-stained smears were studied and analysed for the cytomorphological spectrum of melanoma. RESULTS: Among the 30 patients studied, males were 19 and females were 11 (M:F:1.7:1) with a mean age of 49.3 years. The commonest primary site was foot followed by maxilla and thigh. The most common site for metastasis was inguinal lymph node. All 30 cases had histopathological correlation. Pigmented melanophages (83%) was the predominant feature followed by presence of melanin pigment (77%) and pseudo-inclusions (73%). Condensed chromatin was observed in the majority of the patients (53%). Smooth and irregular nuclear contours were seen in combination in 37% of the cases. A combination of nuclear placement, that is eccentric and central was observed as a predominant feature (53%). Spindle cell pattern was the predominant shape. Among cytoplasmic features, melanin granules and vacuoles were equally distributed (50%). Mixed pattern, that is singly scattered plasmacytoid cell pattern and cell clusters was the predominant cell architecture. CONCLUSION: A spectrum of cytomorphological features in correlation with clinical details leads to greater precision in diagnosis and helps to avoid pitfalls in diagnosing melanoma.

Spectrum of malignancies in human immunodeficiency virus - positive patients at a Tertiary Care Centre in South India.

CONTEXT: India has a very large number of patients living with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome. Opportunistic infections in these patients are commonly encountered. However, malignancies in such patients also do occur. AIM: The aim was to study the spectrum of malignancies in HIV-positive patients at a tertiary health care center. SETTINGS AND DESIGN: Retrospective study. MATERIALS AND METHODS: The cases were retrieved from pathology record files at our Institute from January 2003 to December 2008. The follow-up was obtained from Medical oncology records. The morphology of each case was reviewed along with immunohistochemistry wherever done. RESULTS: There were 61 such cases (51 males, 10 females). The age range was 7-78 years with a median of 35 years. The clinical presentation varied according to the malignancy. The largest group was non-Hodgkin lymphoma (18 nodal, 23 extra-nodal). The others included carcinoma breast (4), chronic myeloid leukemia (3), Burkitt Leukemia (2), squamous cell carcinoma anal region (2), multiple myeloma (2) and one each of miscellaneous malignancies (7). CONCLUSION: Malignancies in HIV positive individual occurred in younger individuals. Non-Hodgkin lymphomas, especially extra-nodal lymphomas, were the most common malignancy. There were no cases of proven Kaposi's sarcoma or invasive cervical carcinomas. There were two cases of multiple myeloma which are infrequently reported.

Granulomatous interstitial nephritis: Our experience of 14 patients.

Granulomatous interstitial nephritis (GIN) is a rare condition. Drugs, infections, immune processes, and foreign body reaction are the main causes. We identified a total of 14 patients with GIN during a period of 13 years in 2798 renal biopsies. There were 8 males and 6 females in the age range of 20-70 (mean 35 ± 12) years. The serum creatinine at presentation was 6.7 ± 3.8 (range: 2.3-14.7) mg/dl. In nine patients tuberculosis was the causative agent. Drugs (n = 2) and Wegener's granulomatosis (n = 1) were other etiologies. Systemic lupus erythematosis (SLE) and Immunoglobulin A nephropathy (IgAN) were seen in one patient each. Patients with tuberculosis were treated with antituberculous therapy and three of them improved. Four out of six patients who required dialysis at presentation remained dialysis dependent, one of whom underwent renal transplantation. Two patients progressed to end stage renal disease after 7 years and 9 years each. The patients with drug induced GIN had improvement in renal function after prednisolone treatment. Patients with SLE, and Wegener's granulomatosis responded to immunosuppression. Patient with IgAN was on conservative management. Finally, six patients were on conservative management for chronic renal failure.

Nondiabetic kidney disease in type 2 diabetic patients: A single center experience.

Nondiabetic renal disease (NDRD) is seen as a cause of proteinuria and renal failure in type 2 diabetes mellitus (DM). The clinical differences between NDRD and diabetic glomerulosclerosis (DGS) are not clear. This study was done to find the spectrum of NDRD in type 2 DM patients and differences in clinical profile between NDRD and DGS patients. Data of patients with type 2 DM who underwent renal biopsy in this institute from 1990 to 2008 were analyzed retrospectively. Patients were categorized as isolated NDRD, NDRD with DGS, and isolated DGS. A total of 75 patients were included. Mean age was 45 ± 10.2 years, male to female ratio was 3.1 : 1, median duration of DM was 12 months (range, 1 year-15 years), proteinuria was 4.2 ± 3.4 g/day, and serum creatinine was 4.3 ± 3.9 mg/dl. Hypertension was observed in 63 (84%) cases and microscopic hematuria in 24 (32%) cases. Nephrotic syndrome (38.7%) was the commonest clinical presentation. Forty-eight (64%) cases had NDRD and 27 (36%) had DGS. The commonest NDRD was minimal change disease (12.5%). Three (6.3%) patients had lupus nephritis. Tubulointerstitial nephritis has been observed in 10.4% patients. No significant differences between NDRD and DGS patients were found except hypertension which was significantly high in the DGS group. Acute kidney injury and nephritic syndrome were not observed in the DGS group. In conclusion, the incidence of biopsy-proven NDRD in type 2 DM in this study was high. Kidney biopsy aided in the detection of NDRD in clinically suspected patients.

Membranous nephropathy and granulomatous interstitial nephritis due to tuberculosis.

Tuberculous involvement of the genitourinary tract is well reported in the literature. However, reports of glomerular lesions of the kidney due to tuberculosis are rare. Tuberculosis has been identified as the most common infectious cause of granulomatous interstitial nephritis (GIN). We report a 23-year-old female patient with a membranous nephropathy and GIN due to tuberculosis. She presented with renal failure and nephrotic-range proteinuria, both of which resolved with the treatment of tuberculosis. There is only one report, from Japan, of a patient with membranous nephropathy and tuberculous granulomatous nephritis. Our patient is the second with tuberculous GIN and membranous nephropathy. In our patient, the close temporal relationship between the infection and glomerulonephritis, an ulcerated tuberculin skin test, the response to the treatment and the absence of any other systemic disease that might cause the glomerulonephritis suggested an association between tuberculosis and membranous nephropathy. However, a causal association can only be speculation, because membranous nephropathy could remit spontaneously. It is also possible that it might relapse at a later date when the tuberculosis is inactive. Therefore, the association might be either coincidental or causal, and could become clearer as similar patients are reported.

Renal allograft pathology with C4d immunostaining in patients with graft dysfunction.

Renal allograft biopsy is the gold standard for diagnosis of rejection. Incorporation of C4d as a marker for humoral rejection is a major addition for Banff Schema, 2005. We evaluated the pattern of C4d staining in indicated renal allograft biopsies from January 2005 to December 2009. Of the 67 biopsies analyzed, 21 were C4d-positive. They were 11 cases of acute rejection, seven chronic rejection and one biopsy each of acute tubular necrosis, BK virus nephropathy and normal biopsy. Morphologic features like peritubular capillary dilatation, tubulitis and interstitial inflammation were seen more frequently in C4d-positive biopsies and this was statistically significant. C4d positivity was noted in 50% of the chronic rejection cases indicating a humoral component in the pathogenesis of chronic rejection. There was no significant difference in the serum creatinine levels of C4d-positive and -negative patients, either at the time of biopsy or during the follow-up. This study supports the role of C4d immunostaining in confirming histologically diagnosed acute and chronic humoral rejections and in detecting histologically unsuspected cases.

Pattern of biopsy-proven renal disease in a single center of south India: 19 years experience.

The prevalence of biopsy-proven glomerulonephritis varies according to the geographic area, socioeconomic condition, race, age, demography and indication of renal biopsy. This study analyzed the distribution of biopsy-proven renal disease (BPRD) and its changing pattern over a period of 19 years from a tertiary care hospital in south India. All the renal biopsies performed from 1990 to 2008 were reviewed retrospectively. Biopsies were evaluated by light microscopy and immunofluorescence microscopy and also special stains when warranted. A total of 1849 biopsies were analyzed. The mean patient age was 32.27 ± 18.38 (range 10-80) years. The male:female ratio was 1.4:1. The most common indications of renal biopsy were nephrotic syndrome (49%), followed by chronic renal failure (13.6%) and rapidly progressive renal failure (12%). Primary glomerulonephritis (PGN) comprised 1278 (69.1%) of the total patients. Among the PGN cases, the most common one was minimal change disease (21.8%), followed by focal segmental glomerulosclerosis [FSGS (15.3%)], membranous glomerulonephritis (10%), chronic glomerulonephritis (9.7%), postinfectious glomerulonephritis (8.1%), mesengioproliferative glomerulonephritis (7.5%), diffuse proliferative glomerulonephritis (6.7%), crescentic glomerulonephritis (6.5%), IgA nephropathy [IgAN (6.3%)], membranoproliferative glomerulonephritis (5.7%), focal proliferative glomerulonephritis (1.6%) and IgM nephropathy (0.5). Secondary glomerular disease (SGN) accounted for 337 (18.2%) of the cases. The most common SGN was lupus nephritis (80.1%), followed by amyloidosis (8%) and diabetic nephropathy (6.5%). Tubulointerstitial disease [124 (6.7%)] and vascular disease [60 (3.2%)] were less common. End-stage changes and miscellaneous disease were found in 37 (2%) and 13 (0.7%) cases, respectively. The incidence of FSGS and IgAN has been increasing since 1999. This study provides descriptive biopsy data and highlights the changing incidence of renal disease which is probably contributed by an increase referral due to increased awareness together with increased manpower and infrastructure.

The outcome of proliferative lupus nephritis with pulse cyclophosphamide therapy.

Proliferative lupus nephritis deserves aggressive therapy and cyclophosphamide plays a pivotal role. Thirty nine patients with proliferative lupus nephritis (Class III-7 patients and Class IV- 32 patients) with a median follow up of 38 months were considered for this observational study. All the patients received induction therapy with intravenous methylprednisolone. Cyclophosphamide was given intravenously initially in monthly pulses for six months and later quarterly pulses until remission was achieved or until the target dose (200 mg/kg) was reached. The treatment with intravenous methylprednisolone was repeated in the event of a nephritic flare. Later the corticosteroid was reduced to a minimum effective dose and cyclophosphamide was changed to either azathioprine or mycophenolate mofetil. At the time of the last follow up, 82.05% of the patients were in remission (complete remission 51.28% and partial remission 30.77%). The median interval to achieve remission in responders was 15 months. Early diagnosis (P=0.04), a higher creatinine clearance at presentation (P=0.02), and concurrent use of an ACEI or an ARB (P=007) significantly favored attaining remission. Five patients experienced a doubling of serum creatinine and one of them became dialysis dependent. Risk of doubling of serum creatinine correlated with a low Ccr (P=0.03) at presentation, occurrence of renal flares (P=0.034) and failure to achieve remission (P=0.0001). The parameters like serum creatinine, serum C3, serum C4, activity and chronicity indices on renal biopsy, hypertension were not statistically significant. Therapy with cyclophosphamide, if initiated early, helps in inducing remission and hence can retard the progression to CKD.

Estrogen and progesterone hormone receptor status in breast carcinoma: comparison of immunocytochemistry and immunohistochemistry.

CONTEXT: Estrogen receptors (ER) and progesterone receptors (PR) play a significant role in the prognosis of breast cancer. For preoperative chemotherapy in locally advanced lesions, trucut biopsy is used to localize the ER and PR receptors by immunohistochemistry. Immunocytochemistry can be a better alternative to immunohistochemistry as it better fixes cells. AIMS: To evaluate the degree of correlation between immunocytochemical (ICC) and immunohistochemical (IHC) determination of ER and PR in breast cancer. SETTINGS AND DESIGN: Fine needle aspiration cytology (FNAC) was performed on 100 primary breast cancers immunostained for ER and PR during a period of 1 year 7 months, i.e., from January 2006 to July 2007. MATERIALS AND METHODS: Papanicolaou-stained slides were destained, fixed in cold acetone and submitted for immunocytochemistry. In the prospective analysis, FNAC smears were straightaway fixed in cold acetone and submitted for ER and PR. Peroxidase, antiperoxidase technique was used for immunocytochemistry. STATISTICAL ANALYSIS: Spearman Rank correlation test was used. RESULTS: Differences between groups were analysed and correlations were studied. Concordance for ER was 50% and for PR was 29%. Both ER and PR were positive in four cases: ER only in three and PR in one, and both were negative in nine cases. Use of the least best buffer and technical errors contributed to the lower ICC rate. CONCLUSION: Although Immunocytochemistry removes the derogatory step of antigen deterioration, technical errors can cause hindrance in achieving the best of the results.

Intraoperative cytology (squash smear) in neurosurgical practice - pitfalls in diagnosis experience based on 3057 samples from a single institution.

OBJECTIVES: The smear technique is challenging for a neuropathologist where rapid and accurate diagnosis is to be given on small biopsies. The present study, a large retrospective analysis of squash smears in neurosurgical practice, was conducted to assess the usefulness, accuracy and the diagnostic pitfalls of smear diagnosis. METHODS: The authors analysed 3057 central nervous system (CNS) lesions sent for intraoperative cytology (IC) during the years 1988-2005. The stain used was 1% alcoholic toluidine blue. The smear diagnosis was compared with the histological diagnosis to evaluate the diagnostic accuracy. RESULTS: Diagnostic accuracy irrespective of lesion and site ranged from 83.0% to 86.0% per year (mean=85%). The highest rate of correlation among common brain tumours was noted in schwannoma (96.6%) and pituitary adenoma (92.2%), followed by meningiomas (88.9%), astrocytomas (88.4%), chordomas (86.4%) and neurocytomas (86.9%). Infections as a whole contributed 380 cases. The most common infection was tuberculosis. CONCLUSION: This is the largest series reported from India to the best of our knowledge. Squash smear technique is a very reliable and rapid method of intraoperative diagnosis. Knowledge of clinical and neuroimaging details helps the experienced neuropathologist to improve the diagnostic accuracy.