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Assessing basophil function
ABSTRACTS
Background: Paraneoplastic pemphigus (PNP) is a rare autoimmune mucocutaneous blistering condition which has a universal association with malignancy and a poor prognosis. PNP, which is characterised by the presence of autoantibodies against plakins in the skin epithelial layer, can be a challenging diagnosis to make, particularly in those without an existing history of malignancy, as tissue biopsy findings may resemble other disorders that have similar clinical features. Thus, serology may be helpful in the diagnosis. Historically, indirect immunoflourescence (IIF) has been the most widely used assay in diagnostic laboratories with reasonable, but not ideal, specificity. Western blotting for various anti-plakin antibodies is more specific but not routinely available. Recently many antiplakin antibody specific assays have been developed commercially. Aim: The aim of our study was to compare the utility of three traditional assays, (IIF on rat bladder, monkey bladder and rat cardiac tissue) versus two newer plakin-based assays (envoplakin ELISA and HEK-transfected cell IIF) in patients presenting initially with oral ulceration alone. Method: Fifty-nine samples [PNP n ¼ 5, healthy controls n ¼ 10, and disease controls n ¼ 44, including pemphigus vulgaris (PV), mucous membrane pemphigoid (MMP), lichen planus (LP) and miscellaneous oral ulcers] were analysed on the five different assays and results were compared. Results: The current assay employed by our laboratory, rat bladder IIF, is the most sensitive assay for our three confirmed PNP cases (3/3, 100%), and has high specificity among all controls (53/54, 98.1%). The Envoplakin ELISA was even more specific (54/54, 100%), although less sensitive (1/3, 33.3%). Monkey bladder IIF had equal sensitivity to rat bladder IIF, although poorer specificity (51/54, 94.4%). Rat cardiac and HEK-transfected cell IIF performed the most poorly with 78–88% specificity, with the latter also having 0% sensitivity. Conclusion: Our findings show that the newer envoplakin ELISA compares favourably with more traditional IIF in its high negative predictive value for PNP in both disease and healthy controls. However, traditional IIF seems to have superior sensitivity in our limited cohort of PNP patients.
IMPLAMENTING A SEROLOGICAL TEST FOR PRIMARY MEMBRANOUS GN: THE PLA2R AB ELISA Lawrence Ong and Ming-Wei Lin Department of Immunopathology, ICPMR, Westmead Hospital, Sydney, NSW, Australia Background: The discovery of the phospholipase-A2 receptor antibody (PLA2R Ab) has attracted interest for its ability to discriminate primary membranous glomerulonephritis (GN) from secondary forms. The development of commercially available indirect immunofluorescence (IIF) and ELISA assays has purportedly increased test sensitivity to up to 97.5%. We sought to evaluate this in a cohort of patients at Westmead Hospital. Methods: Subjects had biopsy proven membranous GN. A cross sectional design was employed to evaluate the performance characteristics of the Euroimmun PLA2R Ab ELISA. Patients with positive ELISA results were followed prospectively to determine whether PLA2R Ab levels correlated with disease activity. Results: Preliminary findings show that the PLA2R Ab ELISA is highly specific, regardless of time since initial diagnosis. However, sensitivity was much lower than claimed by the manufacturers amongst all patients with primary membranous GN. Sensitivity
S39
improved, however, when the PLA2R Ab was assayed within 100 days of diagnosis. There appeared to be a relationship between antibody levels and measures of disease activity (n ¼ 30). Conclusion: Positivity by PLA2R Ab ELISA allows a clinician to rule-in diagnosis of primary membranous GN with confidence. Despite higher sensitivity nearing diagnosis, the test cannot ruleout disease or verify the diagnosis of primary membranous GN during quiescence. Antibody levels appear to correlate with disease activity.
IgG AND THE SUM OF THE SUBCLASSES: WHAT IS IT GOOD FOR? Heather Dunckley, Rachael H. N. S. Fulcher and David A. Fulcher Immunopathology, ICPMR, Pathology West, Sydney, NSW, Australia Determination of IgG subclasses has a role in the diagnosis of IgG4 disease, and possibly IgG subclass deficiency. It is recommended that the total IgG level be measured as a quality check, with the requirement that the sum of the subclasses be within 15%. Although widely adopted, there seems little scientific justification for such an approach. We performed a prospective audit of 297 samples sent for IgG subclass measurement. Fifty of 297 samples failed the 15% rule; subsequently, eight passed on repeating the IgG, and another six on repeating the subclasses. EPG on the 36 remaining samples showed no abnormalities in 22, polyclonal hypergammaglobulinaemia in seven, paraprotein in two, and a questionable band in five; seven of 36 had isolated increase in IgG4. We next examined bias in these assays, and retrospectively examined the specific effects of isolated IgG4 elevation. We performed mathematical simulations of over 3000 combinations of IgG subclass results, each subjected to 10,000 virtual nephelometric assays, to determine how frequently the 15% rule would be violated by random chance alone, as reflected by the co-efficient of variation. Our findings have very significant implications for the use of the IgG check in routine diagnostic IgG subclass estimation, and these will be presented.
ASSESSING BASOPHIL FUNCTION Alexander Headley1, Connie Katelaris2 and Stephen Adelstein1 1Department of Immunology, Royal Prince Alfred Hospital, and 2Department of Immunology, Campbelltown Hospital, Sydney, NSW, Australia Basophils play an important and non-redundant role as inductor and effector cells of the Th2 response, e.g., helminth infection. In addition, they are involved in a broadening range of pathological conditions including systemic lupus erythematosus, eosinophilic oesophagitis and IgG-mediated systemic anaphylaxis. There is a real need to develop robust in vitro techniques to enable study their role in human disease. Basophil histamine release, sulfidoleukotriene synthesis and the flow cytometric basophil activation test are three methods for diagnosing IgE-mediated allergy in vitro. This talk will describe the potential application of these tests in both allergic and nonallergic contexts, as well as outlining challenges in the performance and interpretation of these functional assays.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Background: Paraneoplastic pemphigus (PNP) is a rare autoimmune mucocutaneous blistering condition which has a universal association with malignancy and a poor prognosis. PNP, which is characterised by the presence of autoantibodies against plakins in the skin epithelial layer, can be a challenging diagnosis to make, particularly in those without an existing history of malignancy, as tissue biopsy findings may resemble other disorders that have similar clinical features. Thus, serology may be helpful in the diagnosis. Historically, indirect immunoflourescence (IIF) has been the most widely used assay in diagnostic laboratories with reasonable, but not ideal, specificity. Western blotting for various anti-plakin antibodies is more specific but not routinely available. Recently many antiplakin antibody specific assays have been developed commercially. Aim: The aim of our study was to compare the utility of three traditional assays, (IIF on rat bladder, monkey bladder and rat cardiac tissue) versus two newer plakin-based assays (envoplakin ELISA and HEK-transfected cell IIF) in patients presenting initially with oral ulceration alone. Method: Fifty-nine samples [PNP n ¼ 5, healthy controls n ¼ 10, and disease controls n ¼ 44, including pemphigus vulgaris (PV), mucous membrane pemphigoid (MMP), lichen planus (LP) and miscellaneous oral ulcers] were analysed on the five different assays and results were compared. Results: The current assay employed by our laboratory, rat bladder IIF, is the most sensitive assay for our three confirmed PNP cases (3/3, 100%), and has high specificity among all controls (53/54, 98.1%). The Envoplakin ELISA was even more specific (54/54, 100%), although less sensitive (1/3, 33.3%). Monkey bladder IIF had equal sensitivity to rat bladder IIF, although poorer specificity (51/54, 94.4%). Rat cardiac and HEK-transfected cell IIF performed the most poorly with 78–88% specificity, with the latter also having 0% sensitivity. Conclusion: Our findings show that the newer envoplakin ELISA compares favourably with more traditional IIF in its high negative predictive value for PNP in both disease and healthy controls. However, traditional IIF seems to have superior sensitivity in our limited cohort of PNP patients.
IMPLAMENTING A SEROLOGICAL TEST FOR PRIMARY MEMBRANOUS GN: THE PLA2R AB ELISA Lawrence Ong and Ming-Wei Lin Department of Immunopathology, ICPMR, Westmead Hospital, Sydney, NSW, Australia Background: The discovery of the phospholipase-A2 receptor antibody (PLA2R Ab) has attracted interest for its ability to discriminate primary membranous glomerulonephritis (GN) from secondary forms. The development of commercially available indirect immunofluorescence (IIF) and ELISA assays has purportedly increased test sensitivity to up to 97.5%. We sought to evaluate this in a cohort of patients at Westmead Hospital. Methods: Subjects had biopsy proven membranous GN. A cross sectional design was employed to evaluate the performance characteristics of the Euroimmun PLA2R Ab ELISA. Patients with positive ELISA results were followed prospectively to determine whether PLA2R Ab levels correlated with disease activity. Results: Preliminary findings show that the PLA2R Ab ELISA is highly specific, regardless of time since initial diagnosis. However, sensitivity was much lower than claimed by the manufacturers amongst all patients with primary membranous GN. Sensitivity
S39
improved, however, when the PLA2R Ab was assayed within 100 days of diagnosis. There appeared to be a relationship between antibody levels and measures of disease activity (n ¼ 30). Conclusion: Positivity by PLA2R Ab ELISA allows a clinician to rule-in diagnosis of primary membranous GN with confidence. Despite higher sensitivity nearing diagnosis, the test cannot ruleout disease or verify the diagnosis of primary membranous GN during quiescence. Antibody levels appear to correlate with disease activity.
IgG AND THE SUM OF THE SUBCLASSES: WHAT IS IT GOOD FOR? Heather Dunckley, Rachael H. N. S. Fulcher and David A. Fulcher Immunopathology, ICPMR, Pathology West, Sydney, NSW, Australia Determination of IgG subclasses has a role in the diagnosis of IgG4 disease, and possibly IgG subclass deficiency. It is recommended that the total IgG level be measured as a quality check, with the requirement that the sum of the subclasses be within 15%. Although widely adopted, there seems little scientific justification for such an approach. We performed a prospective audit of 297 samples sent for IgG subclass measurement. Fifty of 297 samples failed the 15% rule; subsequently, eight passed on repeating the IgG, and another six on repeating the subclasses. EPG on the 36 remaining samples showed no abnormalities in 22, polyclonal hypergammaglobulinaemia in seven, paraprotein in two, and a questionable band in five; seven of 36 had isolated increase in IgG4. We next examined bias in these assays, and retrospectively examined the specific effects of isolated IgG4 elevation. We performed mathematical simulations of over 3000 combinations of IgG subclass results, each subjected to 10,000 virtual nephelometric assays, to determine how frequently the 15% rule would be violated by random chance alone, as reflected by the co-efficient of variation. Our findings have very significant implications for the use of the IgG check in routine diagnostic IgG subclass estimation, and these will be presented.
ASSESSING BASOPHIL FUNCTION Alexander Headley1, Connie Katelaris2 and Stephen Adelstein1 1Department of Immunology, Royal Prince Alfred Hospital, and 2Department of Immunology, Campbelltown Hospital, Sydney, NSW, Australia Basophils play an important and non-redundant role as inductor and effector cells of the Th2 response, e.g., helminth infection. In addition, they are involved in a broadening range of pathological conditions including systemic lupus erythematosus, eosinophilic oesophagitis and IgG-mediated systemic anaphylaxis. There is a real need to develop robust in vitro techniques to enable study their role in human disease. Basophil histamine release, sulfidoleukotriene synthesis and the flow cytometric basophil activation test are three methods for diagnosing IgE-mediated allergy in vitro. This talk will describe the potential application of these tests in both allergic and nonallergic contexts, as well as outlining challenges in the performance and interpretation of these functional assays.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.