Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

IgM and C3 Deposition in Primary Focal Segmental Glomerulosclerosis (FSGS): A Clinical and Histopathological Spectrum.

Focal segmental glomerulosclerosis (FSGS) is a common renal disorder, characterized by progressive segmental sclerosis of renal glomeruli and clinical symptoms corresponding to proteinuria. Classically, it is not considered to be an antibody-mediated disease, however, IgM and C3 deposition may be seen in a subset of cases of FSGS. The impact of this immune deposition on histopathological features in renal core biopsies, on the urinary biochemical parameters, and the clinical outcomes, has not been previously investigated in our population. The aim of this study is to analyze the aforementioned parameters in patients with primary FSGS having antibody deposition as compared to those who do not have any antibody deposition. Some 155 patients diagnosed with FSGS were retrospectively enrolled in our study. The renal biopsies were reviewed for histopathological features and immunofluorescence (IF) findings of IgM and C3 glomerular deposition. These histological features were then compared with the biochemical parameters as well as the clinical outcomes of patients. The patients were assigned to Groups 1 and 2 based on the IF findings. The IgM and/or C3 glomerular deposition had a low incidence in patients with primary FSGS in our study (28.3%). Patients having IgM and C3 co-deposition had a significantly longer time duration since the onset of their clinical symptoms; active disease duration (42 months vs 22 months, p=0.049). The mean pre-treatment serum creatinine of patients with IgM and C3 co-deposition was 6.00 mg/dL as compared to 3.29 mg/dL in patients with no immune deposition (p=0.037). The immune deposition was associated with higher rates of segmental and global glomerulosclerosis, but this finding along with other evaluated histological parameters did not show statistical significance. The number of patients having IgM and/or C3 deposition and with active steroid use/renal dialysis was similar to patients having no IgM and/or C3 deposition. The IgM and/or C3 deposition in FSGS has a low incidence within and is not associated with any significant differences in histological parameters on renal core biopsies of patients from the Pakistani population. IgM and/or C3 deposition is also associated with a significantly longer duration of active disease and these patients may present with higher pre-treatment serum creatinine. Other biochemical parameters and clinical outcomes appear comparable between the groups based on the available clinical data.

Blind Spot in the Radar of MEST-C Score: Type and Severity of Tubulointerstitial Nephritis in IgA Nephropathy.

Background: The updated version of predictive classification for immunoglobulin A nephropathy (IgAN) prognosis "The Oxford Classification" identifies five histopathological features including mesangial hypercellularity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S), tubular atrophy/interstitial fibrosis (T) and crescents (C), the MEST-C. However, few studies suggest that tubulointerstitial inflammation, which is not included in the MEST-C, is also linked to disease progression and is, consequently, a neglected determinant of prognosis among others. Therefore, there is a need to evaluate this histopathological parameter in patients with IgA nephropathy. Materials and Methods: This cross-sectional descriptive study was conducted at Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan. Data of histopathological and immunofluorescence proven renal biopsies (300) of IgA nephropathy patients from January 2016 through May 2022 were extracted using a convenient sampling technique. Biopsies were histologically reviewed for type and severity of tubulointerstitial inflammation, in addition to the MEST-C score. Renal biopsies of patients who had a history of transplant, autolyzed tissue, no glomeruli on histological examination, and/or a tubular atrophy/interstitial fibrosis score of 2 (T2) in MEST-C scoring were excluded. Data were analyzed using SPSS 20. An association between the variables was analyzed using the chi-square and Fischer exact tests. A p value less than 0.05 was considered statistically significant. Results: A total of 247/300 biopsies were eligible for inclusion. The mean age at the time of biopsy was 31.90 ± 12.48 with 63.6% in the age group between 21 and 40 years, and 69.6% were male. Tubulointerstitial inflammation was observed in 90.2% cases with 49.4% showing moderate while 4.5% showing severe degree of inflammation. A strong association of both the type and severity of tubulointerstitial inflammation was found with M, E, T, and C scores (p value < 0.05). Conclusion: The high-frequency and strong statistical association of tubulointerstitial inflammation with the M, E, T, and C scores in our study elucidate its prognostic role in the progression and management of IgA nephropathy.

Expression of NKX2.2 in Non-Ewing Tumors With Round Cell Morphology.

Background Round cell sarcomas pose diagnostic challenges due to overlapping histopathological features, necessitating precise immunohistochemical markers for accurate categorization. NKX2.2 has emerged as a sensitive diagnostic tool, particularly in Ewing sarcoma. This study extends this understanding to various round-cell sarcomas, shedding light on the potential diagnostic utility of NKX2.2 beyond its established role. The nuanced exploration of NKX2.2 expression aims to enhance diagnostic strategies, prognostic assessments, and therapeutic developments in the landscape of sarcoma research. Methodology Cases were retrieved from the surgical pathology and consultation files of Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan. Representative hematoxylin and eosin-stained slides of six different types of already confirmed tumors, including lymphoblastic lymphoma, neuroblastoma, rhabdomyosarcoma, synovial sarcoma, Wilms tumor, and Ewing sarcoma, were reviewed by a panel of pathologists. Immunohistochemistry, utilizing a rabbit anti-NKX2.2 monoclonal antibody, was performed on formalin-fixed paraffin-embedded tissue sections. The presence of NKX2.2 was defined as moderate or high nuclear immunoreactivity in at least 5% of cells. Results The histopathological examination revealed characteristic features in each sarcoma subtype, aligning with established diagnostic criteria. In Lymphoblastic lymphoma, T-cell lineage was confirmed through TdT expression, while the atypical finding of focal NKX 2.2 expression hinted at genetic diversity. Neuroblastoma exhibited the expected salt and pepper chromatin pattern, with NKX 2.2 expression raising questions about its prognostic significance. Rhabdomyosarcoma presented primitive cells expressing desmin, and NKX 2.2 focal expression echoed previous subtype-associated studies. Synovial sarcoma displayed both monophasic and biphasic growth patterns and TLE1 expression, with NKX 2.2 variation suggesting tumor heterogeneity. In Wilms tumor, the characteristic WT1 expression was observed, while NKX2.2's absence reaffirmed its irrelevance in this context. Ewing sarcoma displayed the anticipated homogenous cell population, strong NKX2.2 expression, and CD99 positivity across various sites. Furthermore, age and gender impact on this range of sarcomas found no significant relation with an expression of NKX2.2. Conclusion In conclusion, the diverse expression profiles of diagnostic markers discovered in this study, particularly the atypical expression of NKX2.2 beyond its established role in Ewing sarcoma, signify a significant advancement. This unique finding accentuates the potential diagnostic importance of NKX2.2 in various sarcomas, presenting a novel dimension to our understanding of these malignancies.

Angiomatoid Fibrous Histiocytoma: A Tumor With Uncertain Behavior and Various Clinicopathological Presentations.

Background Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of uncertain differentiation, which has various clinical and morphological presentations. Although it behaves in a benign manner, it has malignant potential. Aim To share various histological patterns and survival data in our population of this rare entity. Materials and methods We studied 25 patients who reported AFH from January 2011 to December 2021. Clinical information, gross and histological features, immunohistochemical results, and survival data were compiled and analyzed. Results Among 25 cases reported as AFH, the majority (68%) were males with a mean age of 31.8 years at the time of diagnosis. The most common location was the lower extremity, especially the thigh (56%), and the mean size of the lesion was 55 mm. Most of the lesions were superficial (84%). Grossly, the majority of lesions (76%) had a solid appearance. Microscopically, classic spindle cell morphology was the most common (76%) with a lymphoid cuff and intralesional hemorrhage. Mild cellular atypia was seen in most (92%) of the cases, while some biopsies (8%) had a high-grade morphology. The majority of patients were alive, while one patient died of the disease. Conclusion AFH is an under-recognized entity with various clinical and histological presentations and a low malignant potential.

The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis.

Extrapulmonary tuberculosis often poses a diagnostic challenge. This study aimed to assess the value of histological examination in diagnosing tuberculous lymphadenitis (LNTB) when performed simultaneously with rapid molecular assay (Xpert MTB/RIF) testing. People presumed to have LNTB were prospectively enrolled in a tertiary care hospital. Excision biopsy was performed and tested by histology, Xpert, and culture. Of 390 lymph nodes, 11 (2.8%) were positive by AFB microscopy, 124 (31.8%) by Xpert, 137 (35.1%) by culture, and histopathology was consistent with TB in 208 (53.3%). Altogether, LNTB was diagnosed in 228 and bacteriologically confirmed TB in 178 cases. Against culture, histopathology versus Xpert had higher sensitivity (93 vs. 62%) but lower specificity (68 vs. 83%). In patients with short clinical history, a significantly higher number of Xpert-positive specimens were culture-positive. Among patients with histology suggestive of TB, no difference was seen in response to treatment between bacteriology positive and negative, but a significant slow response was noted in bacteriology confirmed TB with nonspecific histology. In a country like Pakistan, with high TB and low HIV prevalence, diagnosis is possible for more than 95% of LNTB when Xpert and histopathology examination is used in combination, compared to less than 60% by Xpert alone.

Immunochemistry-Based Diagnosis of Extrapulmonary Tuberculosis: A Strategy for Large-Scale Production of MPT64-Antibodies for Use in the MPT64 Antigen Detection Test.

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done.

Immunohistochemical expression of CD-10, BCL-6 and MUM-1 antibodies and immediate clinical response in patients of diffuse large B-cell lymphomas after six cycles of chemotherapy.

OBJECTIVE: To determine the expression of CD-10, BCL-6 and MUM-1 in patients with diffuse large B-cell lymphoma (DLBCL) and its association with immediate clinical response after six cycles of CHOP chemotherapy. STUDY DESIGN: Analytical study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Pathology (AFIP), Rawalpindi in collaboration with Nuclear medicine, Oncology and Radiotherapy Institute (NORI), Islamabad from September 2010 to September 2011. METHODOLOGY: CD-10, BCL-6 and MUM-1 antibodies were applied on cases diagnosed as DLBCL. Immediate clinical response was noted after 6 cycles of chemotherapy with the help of oncologist and divided into complete response, partial response, stable disease and relapse/ progression. Patient's age, results of expression of CD-10, BCL-6 and MUM-1 and results of immediate clinical response to chemotherapy were noted. Regarding analysis of prognostic markers (CD-10, BCL-6 and MUM-1), chi-square test was used for immediate clinical response to chemotherapy in DLBCL. RESULTS: CD-10 was positive in 40% cases, BCL-6 in 58.7% cases and MUM-1 was positive in 46.7% cases. About 41.3% of patients showed complete response, 10.6% partial response, 17.3% stable disease and 30.8% showed relapse/progression. CD-10 expression in DLBCL was associated with better immediate clinical response (p=0.011) whereas MUM-1 expression in DLBCL was associated with poor immediate clinical response (p<0.0001). However, there was no statistically significant association of BCL-6 with immediate clinical response (p=0.22). CONCLUSION: DLBCL shows expression of CD-10, BCL-6 and MUM-1 in nearly fifty percent of the cases. CD-10 is associated with good whereas MUM is associated with poor response. However, there was no association of BCL-6 with immediate clinical response.

Determination of frequency of epstein-barr virus in non- Hodgkin lymphomas Using EBV latent membrane protein 1 (EBV-LMP1) immunohistochemical staining.

BACKGROUND: The presence of Epstein-Barr virus (EBV) in Non-Hodgkin's lymphoma can be identified by immunohistochemistry for detection of EBV latent membrane protein (LMP). The role of EBV as an etiologic agent in the development of non-Hodgkin lymphoma has been supported by detection of high levels of latent membrane protein 1 (LMP-1) expression in tumors. However, no study has been conducted in a Pakistani population up till now to determine the frequency of Epstein-Barr virus positivity. The objective of our study was to determine a value for non-Hodgkin lymphoma patients using EBV LMP-1 immunostaining in our institution. MATERIALS AND METHODS: This study was carried out at the Department of Histopathology, Armed Forces Institute of Pathology (AFIP), Pakistan from December 2011 to December 2012. It was a cross sectional study. A total of 71 patients who were diagnosed with various subtypes of NHL after histological and EBV LMP-1 immunohistochemical evaluation were studied. Sampling technique was non-probability purposive. Statistical analysis was achieved using SPSS version 17.0. Mean and SD were calculated for quantitative variables like patient age. Frequencies and percentages were calculated for qualitative variables like subgroup of NHL, results outcome of IHC for EBV and gender distribution. RESULTS: Mean age of the patients was 53.6 ± 16 years (Mean ± SD). A total of 50 (70.4%) were male and 21 (29.6%) were female. Some 9 (12.7%) out of 71 cases were positive for EBV-LMP-1 immunostaining, 2 (22.2%) follicular lymphoma cases, 1 (11.1%) case of T-cell lymphoblastic lymphoma, 4 (44.4%) cases of diffuse large B cell lymphomas, 1 (11.1%) mantle cell lymphoma and 1 (11.1%) angioimmunoblastic T cell lymphoma case. CONCLUSION: In our study, frequency of EBV in NHL is 12.7% and is mostly seen in diffuse large B cell lymphoma. This requires further evaluation to find out whether this positivity is due to co-infection or has a role in pathogenesis.

Prognostic sub-grouping of diffuse large B-cell lymphomas into germinal centre and post germinal centre groups by immunohistochemistry after 6 cycles of chemotherapy.

INTRODUCTION: Diffuse large B-cell lymphomas (DLBCL) can be divided into germinal centre (GC-DLBCL) and post germinal centre (post GC-DLBCL) groups by applying immunohistochemical antibodies. As these subgroups respond differently to chemotherapy, it is possible at diagnosis to select a poor prognostic subgroup for aggressive treatment. OBJECTIVE: To determine the frequencies of GC-DLBCL and post GC-DLBCL in patients by immunohistochemistry (IHC) and the clinical response after six cycles of chemotherapy. SUBJECTS AND METHODS: In this descriptive study conducted in AFIP and CMH, Rawalpindi and NORI, Islamabad, from September 2010 to September 2011, a total of 75 pretreatment cases of DLBCL diagnosed during the study period were included. Cases were segregated in to GC-DLBCL and post GC-DLBCL groups according to results of immunohistochemistry markers CD10, BCL6 and MUM1. Immediate clinical response was assessed after 6 cycles of chemotherapy. Response was divided into complete response, partial response, stable disease or relapse or progression. RESULTS: The mean age was 54.2 ± 15. Males were 53 (70.7%). Forty (53.3%) cases comprised the GC-DLBCL group; 25(62.5%) of them showed a complete response. Most patients of the post GC-DLBCL 19(54%) showed relapse/progression. Results of immediate clinical response in both prognostic subgroups were significant (p<0.05). Results regarding positivity with immunohistochemical antibodies CD10 (p 0.011), BCL6 (p 0.013) and MUM1 (p 0.000) regarding immediate clinical response were also significant. CONCLUSION: GC-DLBCL group shows better response to CHOP chemotherapy regimen. Immunohistochemistry should be used to further classify DLBCL as this can enable us to select aggressive group for aggressive treatment. This manuscript is important because the study is the first to becarried out exclusively in Pakistan or our part of the world.