- Email: [email protected]
Update on anti-NMO IgG antibodies
ABSTRACTS
QAP), Helen Martin (AACB), Richard Steele (RCPA), Louise Wienholt (ASCIA)] Department of Immunology, Pathology Queensland, Royal Brisbane and Women’s Hospital, Brisbane, Qld, Australia Background: Electrophoresis is a long established technique in both pathology laboratories and is a principal test in the diagnosis and follow up of plasma cell dyscracias. In spite of this there have been few projects looking into standardisation. Clinical issues: A Working Party was established last year from a number of interested groups and has developed recommendations in reporting of serum (SPEP) and urine (UPEP) specimens. Clinical aspects of these recommendations will be discussed together with implications for laboratories. Clinical aspects which will be discussed include general interpretative comments, comment on samples with a paraprotein, comments on paraproteins in the non-gamma regions, small abnormal bands on SPEP in patients with a known paraprotein, e.g., post-stem cell transplantation, and comments on first presentation of a small abnormal band on SPEP in patients without a known paraprotein. It is expected that standardised protein electrophoresis work practices will reduce variation in reporting of SPEP and UPEP between laboratories. ADVANCES IN FLOW CYTOMETRY – HOW MANY COLOURS DO YOU NEED? William A. Sewell1,2, Sandy Smith1 SydPath, St Vincent’s Hospital Sydney and 2Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
1
Technological advances in flow cytometry include increasingly sophisticated instruments and an expanding range of fluorochromes. These advances are making it possible to detect an increasing number of markers simultaneously on the one cell. In clinical cytometry, the use of multiple markers has several advantages. For example, populations can be analysed more comprehensively and efficiently. There is improved sensitivity in the assessment of small populations, which is critical in the evaluation of minimal residual disease. When few cells are available in samples such as cerebrospinal fluid (CSF), more information can be obtained from a single tube. Disadvantages of the use of multiple markers include greater challenges with instrument compensation, and higher levels of training required for pathologists and laboratory staff. Developments in data analysis have lagged behind advances in instrumentation and fluorochromes. Implementation depends on a highly skilled laboratory scientist. Eightcolour systems are becoming increasingly widely used in clinical flow cytometry. For example, the EuroFlow Consortium is developing standardised antibody panels with suitable fluorochromes for the diagnosis, classification and monitoring of treatment of haematological malignancies. These protocols are based on the use of eight-colour flow cytometry. FLOW CYTOMETRY FOR THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE MYELOID LEUKAEMIA Ken Bradstock Haematology Department, Westmead Hospital, Sydney, NSW, Australia The recognition of specific aberrant patterns of leucocyte differentiation antigens on leukemic cells now allows flow cytometry to
S43
be used as a tool for assessing treatment response in acute myeloid leukaemia (AML), based on detection of small numbers of abnormal cells surviving therapy, therefore allowing individualisation of patient management. The availability of flow cytometers with eight or more fluorescence detectors, together with a wide range of antibodies conjugated with appropriate fluorochromes, has greatly improved the efficiency of identifying leukaemia aberrant phenotypes (LAP), which can be found in more than 90% of AML cases at diagnosis. Knowledge of the LAP profile of a case can then be used to detect abnormal cells in the marrow after chemotherapy, at sensitivities of 104 and lower. Recent data1 have shown that detection of minimal residual disease (MRD) in AML provides strong independent prognostic information, and can be used to identify patients with good and poor outcomes within standard and intermediate cytogenetic risk groups. A national consortium sponsored by the Australasian Leukaemia and Lymphoma Group (ALLG) is establishing a network to standardise methods, analysis and reporting of flow MRD in AML in Australia. Reference 1. Buccisano F, Maurillo L, Spagnoli A, et al. Cytogenetic and molecular diagnostic characterization combined to postconsolidation minimal residual disease assessment by flow cytometry improves risk stratification in adult acute myeloid leukemia. Blood 2010; 116: 2295-303.
RCPA IMMUNOLOGY QAP REPORT – DATA FROM RECENT PILOT PROGRAMS Sue Jovanovich RCPA Immunology QAP, Flinders Medical Centre, SA, Australia Several pilot programs were provided in 2010, including ganglioside antibodies, CCP using patient sera, and monitoring amyloidosis with serum free light chains. Results from these will be discussed, along with updates to the 2011 program. DSDNA ASSAYS: TRIALS AND TRIBULATIONS Daman Langguth Sullivan Nicolaides Pathology, Brisbane, Qld, Australia Over the last 20 years, there have been many attempts to replace the traditional radioimmunoassay with modern automatable techniques. There is reasonable evidence that relying on one assay for all systemic lupus erythematosus (SLE) patients may not be the best approach. This presentation will review the available methods and present some in-house data on varying methods used to detect dsDNA antibodies. UPDATE ON ANTI-NMO IGG ANTIBODIES R. C. W. Wong1, D. Gillis1, K. Prain1, S. Broadley2, R. J. Wilson2 1 Division of Immunology, Pathology Queensland and 2School of Medicine, Griffith University, Gold Coast, Qld, Australia Neuromyelitis optica (NMO) or Devic’s syndrome is a severe idiopathic and relapsing, demyelinating disease characterised by optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM).1 Neuropathologically, NMO is characterised by extensive demyelination with partial necrosis of the spinal cord white and grey matter, acute axonal injury, neutrophil and
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
S44
PATHOLOGY 2011 ABSTRACT SUPPLEMENT
eosinophil infiltrates, IgG and IgM deposition and perivascular complement activation. NMO is distinguished from classical multiple sclerosis (MS) by clinical, neuroimaging, CSF and serological criteria.1 The identification of NMO-IgG by indirect immunofluorescence (IIF) was initially described as a highly specific marker with the titre paralleling the disease activity. However, the sensitivity of IIF is variable (58–75%) for clinical NMO. Consequently, numerous methods using aquaporin 4 (AQP4) as the target antigen have been developed in an attempt to improve sensitivity. Apart from cellbased assays, these have failed to improve on the sensitivity of IIF. Recently, it has been demonstrated that NMO-IgG targets mainly the M-23 isoform of AQP4 that structurally organises itself into orthogonol arrays of particles in cell membranes. As AQP4-Ab recognises conformational epitopes, the lower sensitivity of all but the cell-based assays may be due to changes resulting during purification, fixation or other causes of NMO, i.e., parainfectious NMO. As indirect immunofluorescence is the most widely available method for detecting NMO-IgG, the Pathology Queensland experience has raised issues of the ‘true’ sensitivity of NMO-IgG testing for NMO as well as identifying other autoantibodies (including anti-reticulin and anti-nuclear antibodies) that cause interference and make IIF interpretation difficult. These issues may contribute to both false positive and false negative IIF results. Some authors have suggested2 that the published sensitivities of NMO-IgG may be artificially high and this has prompted the proposed Australian and New Zealand NMO study designed to evaluate the sensitivity and specificity of all currently available methods for NMO-IgG. References 1. Waters P, Vincent A. Detection of anti-aquaporin-4 antibodies in neuromyelitis optica: current status of the assays. Int MS J 2008; 15: 99-105. 2. Qiu W, Wu JS, Zhang MN, et al. Longitudinally extensive myelopathy in Caucasians: a West Australian study of 26 cases from the Perth Demyelinating Diseases Database. J Neurol Neurosurg Psychiatry 2010; 81: 209-12.
FLOW CYTOMETRY FOR SYSTEMIC MASTOCYTOSIS Gary Unglik1,3, Surender Juneja2, Peter Chapel2, George Varigos4, Karl Bleasel3 1 Clinical Immunology Laboratory, Department of Haematology, 2 Department of Haematology, 3Department of Clinical Immunology and Allergy, and 4Department of Dermatology, Royal Melbourne Hospital, Parkville, Vic, Australia Mastocytosis is a heterogeneous group of disorders characterised by abnormal proliferation of mast cells. In most cases of adult mastocytosis it is possible to demonstrate evidence of clonal proliferation. As is the case with other clonal disorders of haematopoietic lineage, flow cytometry has become a major diagnostic modality. Flow cytometry is particularly useful in the diagnosis of systemic mastocytosis, where the mast cells may still occur at a very low frequency in the bone marrow, despite neoplastic growth. Numerous studies have compared immunophenotype of mast cells in mastocytosis and those in normal and diseased controls. Some aberrantly expressed antigens on mast cells are of such high specificity for systemic mastocytosis that their expression on mast cells has become a minor World Health Organisation (WHO) criterion for diagnosis (CD2, CD25). This presentation will discuss the phenotype of normal mast cell and compare with abnormal immunophenotype – both in the context of the WHO diagnostic criteria as well as other diagnostically useful antigens that are not part of the criteria.
Pathology (2011), 43(S1)
MUTATION ANALYSIS FOR SYSTEMIC MASTOCYTOSIS Chelsee Hewitt1, Michelle McBean1, Angela Tan1, Alexander Dobrovic1, Grant McArthur2, David Westerman1 1 Department of Pathology and 2Haematology and Medical Oncology, The Peter MacCallum Cancer Centre, Melbourne, Vic, Australia Mast cells from over 80% of adult systemic mastocytosis (SM) patients carry an activating mutation at codon 816 of the KIT gene. In fact, a KIT mutation constitutes one of the minor WHO 2008 classification criteria for SM. The most common is the c.2447A>T: p.Arg816Val (D816 V) mutation. The KIT gene encodes a type III transmembrane receptor tyrosine kinase whose ligand is stem cell factor. Activation of KIT mediates proliferation and maturation signalling. D816 V causes constitutive activation of the KIT tyrosine kinase activity, which also results in resistance to the tyrosine kinase inhibitor imatinib mesylate. Therefore knowing the mutational status of KIT codon 816 is important for diagnosis and treatment strategy. The population of mutant mast cells is often low in relation to normal cells and so a sensitive molecular detection technique is required. Several have been reported in the literature, the most sensitive include RT-PCR þ RFLP, PNA-mediated PCR and allele-specific PCR. Within the diagnostic setting the Peter MacCallum uses the rapid and highly sensitive allelespecific competitive blocker PCR (ACB-PCR) assay with a sensitivity level of between 1 and 0.1%. Between Jan 2008 and Oct 2010, 338 samples were tested, of which 58% were positive. MATURATION OF THE HUMORAL IMMUNE RESPONSE TO HIV-1 INFECTION Kim M. Wilson National Serology Reference Laboratory, St Vincent’s Institute of Medical Research, Melbourne, Vic, Australia We have examined the maturation of the humoral immune response to human immunodeficiency virus (HIV-1) infection, by correlating the individual antibody isotype profiles (titre, affinity and epitope specificity) with time elapsed following initial infection. The methodology we have used includes isotype specific Western blots, antibody isotype and antigen specific enzyme immunoassays and the analysis of direct antibody-antigen interaction using surface plasmon resonance. We have analysed sequential samples obtained from individuals undergoing seroconversion, individuals placed on highly active anti-retroviral treatment (HAART) early following infection and individuals who have undertaken structured treatment interruptions (STI) following early HAART intervention. As the humoral immune response to HIV develops we have observed different antibody isotypes, directed to discrete HIV antigens, occurring at different time points following infection, and often these interactions only occur transiently. The administration of HAART decreases the viral load to undetectable levels. This loss of antigenic stimulation delays maturation of the humoral immune response. Re-exposure to viral antigens during STI stimulates continuing maturation of the immune response. The humoral immune profiles generated in response to HIV-1 infection may assist in providing insight into the best strategies to employ for effective vaccines and potential correlates of disease progression.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
QAP), Helen Martin (AACB), Richard Steele (RCPA), Louise Wienholt (ASCIA)] Department of Immunology, Pathology Queensland, Royal Brisbane and Women’s Hospital, Brisbane, Qld, Australia Background: Electrophoresis is a long established technique in both pathology laboratories and is a principal test in the diagnosis and follow up of plasma cell dyscracias. In spite of this there have been few projects looking into standardisation. Clinical issues: A Working Party was established last year from a number of interested groups and has developed recommendations in reporting of serum (SPEP) and urine (UPEP) specimens. Clinical aspects of these recommendations will be discussed together with implications for laboratories. Clinical aspects which will be discussed include general interpretative comments, comment on samples with a paraprotein, comments on paraproteins in the non-gamma regions, small abnormal bands on SPEP in patients with a known paraprotein, e.g., post-stem cell transplantation, and comments on first presentation of a small abnormal band on SPEP in patients without a known paraprotein. It is expected that standardised protein electrophoresis work practices will reduce variation in reporting of SPEP and UPEP between laboratories. ADVANCES IN FLOW CYTOMETRY – HOW MANY COLOURS DO YOU NEED? William A. Sewell1,2, Sandy Smith1 SydPath, St Vincent’s Hospital Sydney and 2Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
1
Technological advances in flow cytometry include increasingly sophisticated instruments and an expanding range of fluorochromes. These advances are making it possible to detect an increasing number of markers simultaneously on the one cell. In clinical cytometry, the use of multiple markers has several advantages. For example, populations can be analysed more comprehensively and efficiently. There is improved sensitivity in the assessment of small populations, which is critical in the evaluation of minimal residual disease. When few cells are available in samples such as cerebrospinal fluid (CSF), more information can be obtained from a single tube. Disadvantages of the use of multiple markers include greater challenges with instrument compensation, and higher levels of training required for pathologists and laboratory staff. Developments in data analysis have lagged behind advances in instrumentation and fluorochromes. Implementation depends on a highly skilled laboratory scientist. Eightcolour systems are becoming increasingly widely used in clinical flow cytometry. For example, the EuroFlow Consortium is developing standardised antibody panels with suitable fluorochromes for the diagnosis, classification and monitoring of treatment of haematological malignancies. These protocols are based on the use of eight-colour flow cytometry. FLOW CYTOMETRY FOR THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE MYELOID LEUKAEMIA Ken Bradstock Haematology Department, Westmead Hospital, Sydney, NSW, Australia The recognition of specific aberrant patterns of leucocyte differentiation antigens on leukemic cells now allows flow cytometry to
S43
be used as a tool for assessing treatment response in acute myeloid leukaemia (AML), based on detection of small numbers of abnormal cells surviving therapy, therefore allowing individualisation of patient management. The availability of flow cytometers with eight or more fluorescence detectors, together with a wide range of antibodies conjugated with appropriate fluorochromes, has greatly improved the efficiency of identifying leukaemia aberrant phenotypes (LAP), which can be found in more than 90% of AML cases at diagnosis. Knowledge of the LAP profile of a case can then be used to detect abnormal cells in the marrow after chemotherapy, at sensitivities of 104 and lower. Recent data1 have shown that detection of minimal residual disease (MRD) in AML provides strong independent prognostic information, and can be used to identify patients with good and poor outcomes within standard and intermediate cytogenetic risk groups. A national consortium sponsored by the Australasian Leukaemia and Lymphoma Group (ALLG) is establishing a network to standardise methods, analysis and reporting of flow MRD in AML in Australia. Reference 1. Buccisano F, Maurillo L, Spagnoli A, et al. Cytogenetic and molecular diagnostic characterization combined to postconsolidation minimal residual disease assessment by flow cytometry improves risk stratification in adult acute myeloid leukemia. Blood 2010; 116: 2295-303.
RCPA IMMUNOLOGY QAP REPORT – DATA FROM RECENT PILOT PROGRAMS Sue Jovanovich RCPA Immunology QAP, Flinders Medical Centre, SA, Australia Several pilot programs were provided in 2010, including ganglioside antibodies, CCP using patient sera, and monitoring amyloidosis with serum free light chains. Results from these will be discussed, along with updates to the 2011 program. DSDNA ASSAYS: TRIALS AND TRIBULATIONS Daman Langguth Sullivan Nicolaides Pathology, Brisbane, Qld, Australia Over the last 20 years, there have been many attempts to replace the traditional radioimmunoassay with modern automatable techniques. There is reasonable evidence that relying on one assay for all systemic lupus erythematosus (SLE) patients may not be the best approach. This presentation will review the available methods and present some in-house data on varying methods used to detect dsDNA antibodies. UPDATE ON ANTI-NMO IGG ANTIBODIES R. C. W. Wong1, D. Gillis1, K. Prain1, S. Broadley2, R. J. Wilson2 1 Division of Immunology, Pathology Queensland and 2School of Medicine, Griffith University, Gold Coast, Qld, Australia Neuromyelitis optica (NMO) or Devic’s syndrome is a severe idiopathic and relapsing, demyelinating disease characterised by optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM).1 Neuropathologically, NMO is characterised by extensive demyelination with partial necrosis of the spinal cord white and grey matter, acute axonal injury, neutrophil and
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
S44
PATHOLOGY 2011 ABSTRACT SUPPLEMENT
eosinophil infiltrates, IgG and IgM deposition and perivascular complement activation. NMO is distinguished from classical multiple sclerosis (MS) by clinical, neuroimaging, CSF and serological criteria.1 The identification of NMO-IgG by indirect immunofluorescence (IIF) was initially described as a highly specific marker with the titre paralleling the disease activity. However, the sensitivity of IIF is variable (58–75%) for clinical NMO. Consequently, numerous methods using aquaporin 4 (AQP4) as the target antigen have been developed in an attempt to improve sensitivity. Apart from cellbased assays, these have failed to improve on the sensitivity of IIF. Recently, it has been demonstrated that NMO-IgG targets mainly the M-23 isoform of AQP4 that structurally organises itself into orthogonol arrays of particles in cell membranes. As AQP4-Ab recognises conformational epitopes, the lower sensitivity of all but the cell-based assays may be due to changes resulting during purification, fixation or other causes of NMO, i.e., parainfectious NMO. As indirect immunofluorescence is the most widely available method for detecting NMO-IgG, the Pathology Queensland experience has raised issues of the ‘true’ sensitivity of NMO-IgG testing for NMO as well as identifying other autoantibodies (including anti-reticulin and anti-nuclear antibodies) that cause interference and make IIF interpretation difficult. These issues may contribute to both false positive and false negative IIF results. Some authors have suggested2 that the published sensitivities of NMO-IgG may be artificially high and this has prompted the proposed Australian and New Zealand NMO study designed to evaluate the sensitivity and specificity of all currently available methods for NMO-IgG. References 1. Waters P, Vincent A. Detection of anti-aquaporin-4 antibodies in neuromyelitis optica: current status of the assays. Int MS J 2008; 15: 99-105. 2. Qiu W, Wu JS, Zhang MN, et al. Longitudinally extensive myelopathy in Caucasians: a West Australian study of 26 cases from the Perth Demyelinating Diseases Database. J Neurol Neurosurg Psychiatry 2010; 81: 209-12.
FLOW CYTOMETRY FOR SYSTEMIC MASTOCYTOSIS Gary Unglik1,3, Surender Juneja2, Peter Chapel2, George Varigos4, Karl Bleasel3 1 Clinical Immunology Laboratory, Department of Haematology, 2 Department of Haematology, 3Department of Clinical Immunology and Allergy, and 4Department of Dermatology, Royal Melbourne Hospital, Parkville, Vic, Australia Mastocytosis is a heterogeneous group of disorders characterised by abnormal proliferation of mast cells. In most cases of adult mastocytosis it is possible to demonstrate evidence of clonal proliferation. As is the case with other clonal disorders of haematopoietic lineage, flow cytometry has become a major diagnostic modality. Flow cytometry is particularly useful in the diagnosis of systemic mastocytosis, where the mast cells may still occur at a very low frequency in the bone marrow, despite neoplastic growth. Numerous studies have compared immunophenotype of mast cells in mastocytosis and those in normal and diseased controls. Some aberrantly expressed antigens on mast cells are of such high specificity for systemic mastocytosis that their expression on mast cells has become a minor World Health Organisation (WHO) criterion for diagnosis (CD2, CD25). This presentation will discuss the phenotype of normal mast cell and compare with abnormal immunophenotype – both in the context of the WHO diagnostic criteria as well as other diagnostically useful antigens that are not part of the criteria.
Pathology (2011), 43(S1)
MUTATION ANALYSIS FOR SYSTEMIC MASTOCYTOSIS Chelsee Hewitt1, Michelle McBean1, Angela Tan1, Alexander Dobrovic1, Grant McArthur2, David Westerman1 1 Department of Pathology and 2Haematology and Medical Oncology, The Peter MacCallum Cancer Centre, Melbourne, Vic, Australia Mast cells from over 80% of adult systemic mastocytosis (SM) patients carry an activating mutation at codon 816 of the KIT gene. In fact, a KIT mutation constitutes one of the minor WHO 2008 classification criteria for SM. The most common is the c.2447A>T: p.Arg816Val (D816 V) mutation. The KIT gene encodes a type III transmembrane receptor tyrosine kinase whose ligand is stem cell factor. Activation of KIT mediates proliferation and maturation signalling. D816 V causes constitutive activation of the KIT tyrosine kinase activity, which also results in resistance to the tyrosine kinase inhibitor imatinib mesylate. Therefore knowing the mutational status of KIT codon 816 is important for diagnosis and treatment strategy. The population of mutant mast cells is often low in relation to normal cells and so a sensitive molecular detection technique is required. Several have been reported in the literature, the most sensitive include RT-PCR þ RFLP, PNA-mediated PCR and allele-specific PCR. Within the diagnostic setting the Peter MacCallum uses the rapid and highly sensitive allelespecific competitive blocker PCR (ACB-PCR) assay with a sensitivity level of between 1 and 0.1%. Between Jan 2008 and Oct 2010, 338 samples were tested, of which 58% were positive. MATURATION OF THE HUMORAL IMMUNE RESPONSE TO HIV-1 INFECTION Kim M. Wilson National Serology Reference Laboratory, St Vincent’s Institute of Medical Research, Melbourne, Vic, Australia We have examined the maturation of the humoral immune response to human immunodeficiency virus (HIV-1) infection, by correlating the individual antibody isotype profiles (titre, affinity and epitope specificity) with time elapsed following initial infection. The methodology we have used includes isotype specific Western blots, antibody isotype and antigen specific enzyme immunoassays and the analysis of direct antibody-antigen interaction using surface plasmon resonance. We have analysed sequential samples obtained from individuals undergoing seroconversion, individuals placed on highly active anti-retroviral treatment (HAART) early following infection and individuals who have undertaken structured treatment interruptions (STI) following early HAART intervention. As the humoral immune response to HIV develops we have observed different antibody isotypes, directed to discrete HIV antigens, occurring at different time points following infection, and often these interactions only occur transiently. The administration of HAART decreases the viral load to undetectable levels. This loss of antigenic stimulation delays maturation of the humoral immune response. Re-exposure to viral antigens during STI stimulates continuing maturation of the immune response. The humoral immune profiles generated in response to HIV-1 infection may assist in providing insight into the best strategies to employ for effective vaccines and potential correlates of disease progression.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.