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Implementation of an ex vivo TH17 assay
Pathology (2017) 49(S1), pp. S43–S46
Immunopathology
QUANTITY NOT QUALITY – SHOULD ENAs BE REPORTED DIFFERENTLY? Gerard Chu1,2, Stephen Adelstein1,2 1 Department of Clinical Immunology and the Central Sydney Immunology Laboratory, Royal Prince Alfred Hospital, Camperdown, and 2Sydney Medical School, University of Sydney, NSW, Australia The diagnosis of limited cutaneous systemic sclerosis (lcSS) is aided by the detection in serum of antibodies to nuclear (ANA) and extractable nuclear antigens (ENA). ENA antigens were initially measured by counterimmunoelectrophoresis and reported as either present or absent. Newer techniques, such as enzyme linked immunosorbant assays and line immunoassays to CENP-B, permit quantitative or semi-quantitative reporting of antibody levels. Therefore, we undertook a study to see such reporting aids in the diagnosis or classification of lcSS. A retrospective analysis was performed on 100 recent centromere-positive samples referred to the Central Sydney Immunology Laboratory. The results of the ANA and ENA signal intensity on line immunoassay were correlated with information obtained from review of the subjects’ clinical records. 87% of patients with lcSS have an ANA anticentromere titre of 2560, compared to only 44% of patients without lcSS. Patients with lcSS also have a higher anticentromere signal intensity on line immunoassay with an average intensity of 101.7 compared to 50.1 for patients without lcSS (p < 0.01). Consistent with these findings, patients without lcSS were more likely to have a low semi-quantitative score of + (33%), whereas all patients with lcSS had a score of ++ and above. Therefore, in this highly selective population, a signal intensity for anticentromere antibodies >30 gives a sensitivity of 100% (95%CI 89–100%) and specificity of 37% (95%CI 24–51%) for the diagnosis of lcSS. A signal intensity of >50 gives a sensitivity of 94% (95%CI 79–99%) and a specificity of 58% (95%CI 43–71%). Hence, the likelihood of having lcSS increases with increasing signal intensity, information that is not available without quantitative reporting. IMPLEMENTATION OF AN EX VIVO TH17 ASSAY Laine Hosking1,2, Christine Czakjo1, Sharon Choo1,2 1 Immunology Laboratory, Laboratory Services, Royal Children’s Hospital, Melbourne, and 2Department of Allergy and Immunology, Royal Children’s Hospital, Melbourne, Vic, Australia Background: IL-17 secreting T-helper type 17 cells (Th17) have been shown to play a crucial role in mucosal immunity. Deficiency of Th17 cells is seen in primary immunodeficiency disorders (PID) such as autosomal dominant hyper IgE syndrome [STAT3 loss of function (LOF)], autosomal dominant chronic
Print ISSN 0031-3025/Online ISSN 1465-3931
mucocutaneous candidiasis [STAT1 gain of function (GOF)] and IL12 and IL12RB1 deficiencies. Rapid and cost effective screening is required to identify these patients. Aim: To validate and implement a whole blood one-tube flow cytometric assay (modified from Dhalla et al.1) for the identification of Th17 deficient patients. Method: Peripheral blood from 16 adult healthy controls, one STAT3 LOF patient and one STAT1 GOF patient underwent surface staining to identify Th17 cells, defined as CD45+CD3+CD4+CD45RA–CCR6+CXCR3–. Results: 4.4–10.8% of CD4+ T cells and 9.7–20.3% of CD4+CD45RA– T cells (5th–95th centiles) in the adult healthy controls were Th17 cells. Th17 cells comprised 0.3% of CD4+ T cells and 1.9% of CD4+CD45RA– T cells in the STAT3 LOF patient, and 0.7% and 1.3% respectively in the STAT1 GOF patient. Conclusion: This Th17 assay can be performed with minimal blood volume and provides rapid identification of PID patients with reduced Th17 cells. Reference 1. Dhalla F, Fox H, Davenport EE, et al. Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility. Clin Exp Immunol 2016; 184: 216–27.
A COMPARISON OF METHODS FOR DETECTING ANTIBODIES TO DFS-70 Jessie Lee, Linda Tran, Renfen Chen, Frederick Lee, Stephen Adelstein Department of Clinical Immunology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia Indirect immunofluorescence (IIF) on Hep-2 cells remains the method of choice for screening in the laboratory for systemic autoimmune disorders. This technique results in different nuclear staining patterns that are associated with particular clinical presentations. In the last five years a new pattern has been described that is associated with antibodies to a 70 kDa intranuclear protein identified as the lens epithelium derived growth factor, and giving a pattern called dense-fine speckled (DFS-70). The ability to identify antibodies against DFS-70 is important, as the occurrence of these antibodies in isolation is not associated with clinical disease1 and their presence can also confuse the interpretation of antinuclear antibodies. The optimal method for the detection of DFS-70 autoantibodies has not yet been determined. We have evaluated 100 serum samples from routine patient referrals to the Central Sydney Immunology Laboratory between January and October 2016, and detected the presence of antiDFS-70 antibodies using IIF on HEp-2 cells and line immunoassays. The presence of anti-DFS-70 antibodies was then further assessed using five additional methods: IIF using Hep2 cells deficient in the DFS-70 gene (IMMCO), DFS-70 ELISA (Werfen), IIF on Hep-2 cells with commercial (Inova) or in-
Immunopathology
QUANTITY NOT QUALITY – SHOULD ENAs BE REPORTED DIFFERENTLY? Gerard Chu1,2, Stephen Adelstein1,2 1 Department of Clinical Immunology and the Central Sydney Immunology Laboratory, Royal Prince Alfred Hospital, Camperdown, and 2Sydney Medical School, University of Sydney, NSW, Australia The diagnosis of limited cutaneous systemic sclerosis (lcSS) is aided by the detection in serum of antibodies to nuclear (ANA) and extractable nuclear antigens (ENA). ENA antigens were initially measured by counterimmunoelectrophoresis and reported as either present or absent. Newer techniques, such as enzyme linked immunosorbant assays and line immunoassays to CENP-B, permit quantitative or semi-quantitative reporting of antibody levels. Therefore, we undertook a study to see such reporting aids in the diagnosis or classification of lcSS. A retrospective analysis was performed on 100 recent centromere-positive samples referred to the Central Sydney Immunology Laboratory. The results of the ANA and ENA signal intensity on line immunoassay were correlated with information obtained from review of the subjects’ clinical records. 87% of patients with lcSS have an ANA anticentromere titre of 2560, compared to only 44% of patients without lcSS. Patients with lcSS also have a higher anticentromere signal intensity on line immunoassay with an average intensity of 101.7 compared to 50.1 for patients without lcSS (p < 0.01). Consistent with these findings, patients without lcSS were more likely to have a low semi-quantitative score of + (33%), whereas all patients with lcSS had a score of ++ and above. Therefore, in this highly selective population, a signal intensity for anticentromere antibodies >30 gives a sensitivity of 100% (95%CI 89–100%) and specificity of 37% (95%CI 24–51%) for the diagnosis of lcSS. A signal intensity of >50 gives a sensitivity of 94% (95%CI 79–99%) and a specificity of 58% (95%CI 43–71%). Hence, the likelihood of having lcSS increases with increasing signal intensity, information that is not available without quantitative reporting. IMPLEMENTATION OF AN EX VIVO TH17 ASSAY Laine Hosking1,2, Christine Czakjo1, Sharon Choo1,2 1 Immunology Laboratory, Laboratory Services, Royal Children’s Hospital, Melbourne, and 2Department of Allergy and Immunology, Royal Children’s Hospital, Melbourne, Vic, Australia Background: IL-17 secreting T-helper type 17 cells (Th17) have been shown to play a crucial role in mucosal immunity. Deficiency of Th17 cells is seen in primary immunodeficiency disorders (PID) such as autosomal dominant hyper IgE syndrome [STAT3 loss of function (LOF)], autosomal dominant chronic
Print ISSN 0031-3025/Online ISSN 1465-3931
mucocutaneous candidiasis [STAT1 gain of function (GOF)] and IL12 and IL12RB1 deficiencies. Rapid and cost effective screening is required to identify these patients. Aim: To validate and implement a whole blood one-tube flow cytometric assay (modified from Dhalla et al.1) for the identification of Th17 deficient patients. Method: Peripheral blood from 16 adult healthy controls, one STAT3 LOF patient and one STAT1 GOF patient underwent surface staining to identify Th17 cells, defined as CD45+CD3+CD4+CD45RA–CCR6+CXCR3–. Results: 4.4–10.8% of CD4+ T cells and 9.7–20.3% of CD4+CD45RA– T cells (5th–95th centiles) in the adult healthy controls were Th17 cells. Th17 cells comprised 0.3% of CD4+ T cells and 1.9% of CD4+CD45RA– T cells in the STAT3 LOF patient, and 0.7% and 1.3% respectively in the STAT1 GOF patient. Conclusion: This Th17 assay can be performed with minimal blood volume and provides rapid identification of PID patients with reduced Th17 cells. Reference 1. Dhalla F, Fox H, Davenport EE, et al. Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility. Clin Exp Immunol 2016; 184: 216–27.
A COMPARISON OF METHODS FOR DETECTING ANTIBODIES TO DFS-70 Jessie Lee, Linda Tran, Renfen Chen, Frederick Lee, Stephen Adelstein Department of Clinical Immunology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia Indirect immunofluorescence (IIF) on Hep-2 cells remains the method of choice for screening in the laboratory for systemic autoimmune disorders. This technique results in different nuclear staining patterns that are associated with particular clinical presentations. In the last five years a new pattern has been described that is associated with antibodies to a 70 kDa intranuclear protein identified as the lens epithelium derived growth factor, and giving a pattern called dense-fine speckled (DFS-70). The ability to identify antibodies against DFS-70 is important, as the occurrence of these antibodies in isolation is not associated with clinical disease1 and their presence can also confuse the interpretation of antinuclear antibodies. The optimal method for the detection of DFS-70 autoantibodies has not yet been determined. We have evaluated 100 serum samples from routine patient referrals to the Central Sydney Immunology Laboratory between January and October 2016, and detected the presence of antiDFS-70 antibodies using IIF on HEp-2 cells and line immunoassays. The presence of anti-DFS-70 antibodies was then further assessed using five additional methods: IIF using Hep2 cells deficient in the DFS-70 gene (IMMCO), DFS-70 ELISA (Werfen), IIF on Hep-2 cells with commercial (Inova) or in-