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Flow cytometry for the detection of minimal residual disease in acute myeloid leukaemia
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
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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 10 4 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.
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 10 4 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.