- Email: [email protected]
Immunophenotyping of plasma cell and the utility of flow cytometry in plasma cell dyscrasias
S94
Pathology (2015), 47(S1)
PATHOLOGY 2015 ABSTRACT SUPPLEMENT
nuclear dots (MND) and nuclear pore complex (NPC) respectively. They have not previously been reported in subjects with normal liver function tests (LFTs). We review the incidental incidence of these autoantibodies in our test population and their association with normal and cholestatic LFTs. Method: Retrospective analysis of lineblots performed based on ANA IIF pattern of MND and NPC. LFTs were recorded from date of ANA testing and at one and two years of follow up. Results: 187 lineblots were positive for autoantibody to sp100 and/or gp210 detected on the basis of ANA pattern to MND and NPC. Twenty-nine patients with anti-gp210 antibodies and 51 patients with anti-sp100 antibodies detected incidentally on the basis of ANA pattern remained biochemically and symptomatically stable during a follow up period of one year. Discussion: The population identified here could serve as a basis for long term studies, contributing to our knowledge as the predictive value of these specificities detected incidentally is currently unknown.
AUTOANTIBODIES DIRECTED TO CENTROMERE PROTEIN F IN A PATIENT WITH BRCA1 GENE MUTATION Fiona Moghaddas1, Fredrick Joshua2, Bobbie Taylor1, Marv Fritzler3 and Ban Hock Toh1,4 1Healthscope Pathology, Melbourne, Vic, 2Prince of Wales Hospital, Sydney, NSW, Australia, 3University of Calgary, Calgary, Alberta, Canada, and 4Monash University, Melbourne, Vic, Australia Aim: Autoantibodies directed to centromere protein F (CENP-F) were first reported in 1993 and their association with malignancy has been well documented. We present a case of this autoantibody detected in a 47-year-old female with BRCA1 gene mutation associated with bilateral breast cancer and ovarian cancer. Method and results: Antinuclear autoantibody immunofluorescence carried out for possible inflammatory arthropathy showed high titre nuclear speckled-II (NSII) pattern consistent with CENP-F that was confirmed by addressable laser bead immunoassay (ALBIA) for the C-terminal p-F4, an immunodominant CENP-F peptide. Discussion: We review the current literature on CENP-F, its association with breast cancer and present the first documented case of this antibody being identified in a person with BRCA1 gene mutation.
TM
RETROSPECTIVE AUDIT OF THE FREELITE SERUM FREE LIGHT CHAIN (SFLC) ASSAY: TESTING PATTERNS, CONCORDANCE WITH SERUM AND URINE ELECTROPHORESIS/IMMUNOFIXATION AND CORRELATION WITH THE N LATEX FLC ASSAY S. C. Sasson1,2, K. McGill1, L. Wienholt3, A. Carr1,4, D. Brown1,4, A. D. Kelleher1,2, S. N. Breit1,4 and W. A. Sewell1,5,6 1Immunology Laboratory, SydPath, St Vincent’s Hospital, Sydney, 2The Kirby Institute, UNSW, 3Immunology Laboratory, Royal Prince Alfred Hospital, Sydney, 4St Vincent’s Centre for Applied Medical Research, Sydney, 5The Garvan Institute, Sydney, and 6St Vincent’s Clinical School, UNSW, Sydney, NSW, Australia
Aims: A retrospective audit of the FreeliteTM SFLC assay was conducted to compare concordance with electrophoresis (EPG)/immunofixation (IFX) and correlation with the N latex FLC assay. Methods: 244 samples collected over 3.5 months were studied by nephelometry using the FreeliteTM and N Latex FLC assays. Results were compared with serum (S) and/or urine (UEPG)/ IFX. The precision and linearity of the N latex FLC assay were examined. Results: 94% of samples with kappa restriction, and 100% with lambda restriction had detectable paraprotein by SEPG/IFX or UEPG/IFX. The correlation between the two assays was good for kappa (R2 ¼ 0.935) but not lambda (R2 ¼ 0.775) especially when lambda was above the ULN (R2 ¼ 0.406). Agreement in the categorical diagnosis of restricted SFLC was good (Cohen’s kappa ¼ 0.701). In discordant samples the FreeliteTM assay displayed higher agreement with IFX (53% vs 47%). The N latex FLC assay displayed good precision and linearity. Discussion: SFLC testing is popular despite traditional methods detecting paraproteins in most cases. Correlation between the FreeliteTM and N latex FLC assays is better for kappa than lambda FLC. The two assays are not equivalent and the FreeliteTM assay displays better agreement with IFX. Care should be taken by interpreting physicians and laboratories considering switching assays.
IMMUNOPHENOTYPING OF PLASMA CELL AND THE UTILITY OF FLOW CYTOMETRY IN PLASMA CELL DYSCRASIAS Daniel Tran, Sandy Smith, David Brown and William Sewell Sydpath, St Vincent’s Hospital, Sydney, and Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Aims: To prospectively compare the performance of CD27/CD28/ CD81/CD117/CD126/CD147/CD152/CD200/CD229 to current markers (gating- CD38/CD138/CD45; clonality- k-/l-light chain; phenotype- CD19/CD56) to assess utility in identifying neoplastic plasma cells (PC) and distinguishing monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM). To retrospectively assess polychromatic flow cytometry’s (PFC) contribution to diagnosing plasma cell dyscrasias through correlation with other investigations. Methods: Antibody panels including new markers (CD27/CD28/ CD81/CD117/CD126/CD147/CD152/CD200/CD229) were run on BM samples. Database analysis of bone marrow samples at St Vincent’s hospital was conducted to correlate PFC with other investigations. Results: CD19 and CD19/CD147 were the best single marker and 2-marker combination respectively for identifying monoclonals. CD229 was best at differentiating MM and MGUS, and additional markers did not improve differentiation. PFC correlated with electrophoresis, cytogenetics and imaging. PFC detected 29 monoclonal cases that trephine analysis classified as polyclonal. These cases had low PC numbers and polyclonal PC backgrounds. Conclusion: CD19/CD147 could be more useful than CD19/ CD56 in distinguishing monoclonal and polyclonal populations. CD229 may help PFC differentiate MM from MGUS. Despite PFC’s underestimation of PCs, PFC correlates with other investigations and may be particularly useful in minimal residual disease
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
ABSTRACTS
where small PC populations occur or where background reactive plasmacytosis occurs.
USING GENOTYPE TO COMBAT RESISTANCE: DEVELOPMENT OF A POTENTIAL TREATMENT AND SIMPLE PHENOTYPIC TEST DURING A ‘PANRESISTANT’ KLEBSIELLA PNEUMONIAE MINIOUTBREAK Jeremy Brown1, Mitchell Brown1, Andrew Ginn1,4, Alex Agyekum2,3, Alicia Fajardo-Lubian2,4 and Jon Iredell1,2,3,4 1Centre for Infectious Diseases and Microbiology Laboratory Services, Institute for Clinical Pathology and Medical Research, Westmead Hospital, 2NHMRC Centre for Research Excellence in Critical infection, Westmead Millennium Institute, 3Faculty of Medicine, Westmead Clinical School, University of Sydney, and 4Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia Aims: Aztreonam/clavulanic acid combination therapy is a potential treatment for multi-resistant Enterobacteriaceae containing MBLs and ESBLs. We describe an aztreonam/clavulanic acid based phenotypic detection and susceptibility test for MBL and ESBL-containing multi-resistant Enterobacteriaceae derived from initial genotypic characterisation. Methods: Three phenotypically pan-resistant (by Etest and automated broth microdilution) K. pneumoniae isolates from a clinical colonisation mini-outbreak were shown by PCR to contain MBL and ESBL b-lactamases. These isolates and thirteen other Enterobacteriaceae containing MBLs and ESBLs (with three also containing AmpC b-lactamases) were assessed for susceptibility to aztreonam/clavulanic acid by either broth microdilution or Etest, and our phenotypic disc-diffusion assay. b-lactamases were confirmed by multiplex PCR/reverse line blot. Results: Clavulanic acid restored susceptibility to aztreonam in all ‘pan-resistant’ outbreak isolates but not strains also carrying an AmpC b-lactamase. Our disc-diffusion assay predicted 16/16 MBLs, 15/16 ESBLs and 3/3 AmpC b-lactamases and showed susceptibility to aztreonam/clavulanic acid in all non-AmpC isolates. Discussion: Pan-resistant Gram-negative isolates are likely to become increasingly common. We have described a potential treatment for an initially phenotypically pan-resistant organism that was predicted from a genotype and developed a simple combined phenotypic detection and susceptibility test. Both potential treatment and test could be employed in resource poor settings.
THE CHANGING EPIDEMIOLOGY OF KLEBSIELLA PNEUMONIAE CARBAPENEMASE (KPC) RESISTANCE IN AUSTRALIA K. Cronin and M. J. Waters Department of Microbiology, St Vincent’s Hospital Melbourne, Vic, Australia Aims: The aims of this study were to firstly describe the epidemiology, risk factors and outcomes of patients with KPC producing organisms at our institution, and secondly, to describe an outbreak involving seven patients admitted to a geriatric evaluation and management (GEM) ward.
S95
Methods: A retrospective review was conducted to assess 29 patients with KPC producing organisms in a tertiary referral centre in Australia from 2012 to 2014. In addition, a prospective assessment of an outbreak was conducted including a molecular evaluation of the isolates and patient outcomes. Results: Of the 29 patients, 55% were male with a median age of 69 years. The greatest risk factors for acquiring a KPC producing organism were length of hospital stay and recent exposure to broad spectrum antibiotics. The 30 day mortality rate was 17.2%, which increased to 37.9% at 60 days. The seven patients identified in the outbreak were exposed to an index case where a KPC was isolated from urine. The whole genome sequence analysis revealed all seven isolates were highly clonally related. Two of the patients died of causes not directly attributable to KPC colonisation. Discussion: This study highlights the increasing threat of KPC producing organisms in Australia.
AN UNUSUAL SKIN LESION CAUSED BY LASIODIPLODIA THEOBROMAE Lindsey J. Papacostas1,4, Andrew Henderson2, Keat Choong3 and David Sowden4 1Coastal Pathology, Buderim, 2Pathology Queensland, Townsville Hospital, 3Infectious Diseases, Nambour Hospital, and 4Pathology Queensland, Nambour Hospital, Qld, Australia Lasiodoplodia theobromae is a common plant pathogen of tea and fruiting plants from the Coelomycetes family. It is a rare plant pathogen in both immunocompetent and immunocompromised hosts with infections reported including sinusitis, keratitis, pneumonia and cutaneous lesions. We describe a case of subcutaneous infection as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated in multiple swabs from the foot of an active healthy male following a penetrating sea water injury. Following the trauma, the patient used wild aloe vera plant leaf to promote wound healing and this was thought to be a potential source of inoculation. Identification was performed by traditional mycology culture methods with sporulation occurring only with cornmeal agar at 28 days. Identification was confirmed by DNA sequencing. The patient was successfully treated with voriconizole.
A CASE OF AMOEBIC APPENDICITIS IN NORTH QUEENSLAND Gemma Robertson, Andrew Henderson, David Williams and Robert Norton Pathology Queensland, The Townsville Hospital, Townsville, Australia A 13-year-old female from Mornington Island presented with diarrhoea, vomiting, and abdominal pain. Laparoscopic investigation was performed and a gangrenous appendix removed. Histological examination was initially unremarkable, but a later review of the periodic acid-Schiff (PAS) stain demonstrated several areas of PAS-positive bodies consistent with invasive Entamoeba histolytica. Further testing was performed to confirm the diagnosis of E. histolytica, including serology and PCR from paraffinised tissue. The patient was treated with metronidazole and paromomycin to ensure treatment of both tissue and luminal phases of the parasite.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Pathology (2015), 47(S1)
PATHOLOGY 2015 ABSTRACT SUPPLEMENT
nuclear dots (MND) and nuclear pore complex (NPC) respectively. They have not previously been reported in subjects with normal liver function tests (LFTs). We review the incidental incidence of these autoantibodies in our test population and their association with normal and cholestatic LFTs. Method: Retrospective analysis of lineblots performed based on ANA IIF pattern of MND and NPC. LFTs were recorded from date of ANA testing and at one and two years of follow up. Results: 187 lineblots were positive for autoantibody to sp100 and/or gp210 detected on the basis of ANA pattern to MND and NPC. Twenty-nine patients with anti-gp210 antibodies and 51 patients with anti-sp100 antibodies detected incidentally on the basis of ANA pattern remained biochemically and symptomatically stable during a follow up period of one year. Discussion: The population identified here could serve as a basis for long term studies, contributing to our knowledge as the predictive value of these specificities detected incidentally is currently unknown.
AUTOANTIBODIES DIRECTED TO CENTROMERE PROTEIN F IN A PATIENT WITH BRCA1 GENE MUTATION Fiona Moghaddas1, Fredrick Joshua2, Bobbie Taylor1, Marv Fritzler3 and Ban Hock Toh1,4 1Healthscope Pathology, Melbourne, Vic, 2Prince of Wales Hospital, Sydney, NSW, Australia, 3University of Calgary, Calgary, Alberta, Canada, and 4Monash University, Melbourne, Vic, Australia Aim: Autoantibodies directed to centromere protein F (CENP-F) were first reported in 1993 and their association with malignancy has been well documented. We present a case of this autoantibody detected in a 47-year-old female with BRCA1 gene mutation associated with bilateral breast cancer and ovarian cancer. Method and results: Antinuclear autoantibody immunofluorescence carried out for possible inflammatory arthropathy showed high titre nuclear speckled-II (NSII) pattern consistent with CENP-F that was confirmed by addressable laser bead immunoassay (ALBIA) for the C-terminal p-F4, an immunodominant CENP-F peptide. Discussion: We review the current literature on CENP-F, its association with breast cancer and present the first documented case of this antibody being identified in a person with BRCA1 gene mutation.
TM
RETROSPECTIVE AUDIT OF THE FREELITE SERUM FREE LIGHT CHAIN (SFLC) ASSAY: TESTING PATTERNS, CONCORDANCE WITH SERUM AND URINE ELECTROPHORESIS/IMMUNOFIXATION AND CORRELATION WITH THE N LATEX FLC ASSAY S. C. Sasson1,2, K. McGill1, L. Wienholt3, A. Carr1,4, D. Brown1,4, A. D. Kelleher1,2, S. N. Breit1,4 and W. A. Sewell1,5,6 1Immunology Laboratory, SydPath, St Vincent’s Hospital, Sydney, 2The Kirby Institute, UNSW, 3Immunology Laboratory, Royal Prince Alfred Hospital, Sydney, 4St Vincent’s Centre for Applied Medical Research, Sydney, 5The Garvan Institute, Sydney, and 6St Vincent’s Clinical School, UNSW, Sydney, NSW, Australia
Aims: A retrospective audit of the FreeliteTM SFLC assay was conducted to compare concordance with electrophoresis (EPG)/immunofixation (IFX) and correlation with the N latex FLC assay. Methods: 244 samples collected over 3.5 months were studied by nephelometry using the FreeliteTM and N Latex FLC assays. Results were compared with serum (S) and/or urine (UEPG)/ IFX. The precision and linearity of the N latex FLC assay were examined. Results: 94% of samples with kappa restriction, and 100% with lambda restriction had detectable paraprotein by SEPG/IFX or UEPG/IFX. The correlation between the two assays was good for kappa (R2 ¼ 0.935) but not lambda (R2 ¼ 0.775) especially when lambda was above the ULN (R2 ¼ 0.406). Agreement in the categorical diagnosis of restricted SFLC was good (Cohen’s kappa ¼ 0.701). In discordant samples the FreeliteTM assay displayed higher agreement with IFX (53% vs 47%). The N latex FLC assay displayed good precision and linearity. Discussion: SFLC testing is popular despite traditional methods detecting paraproteins in most cases. Correlation between the FreeliteTM and N latex FLC assays is better for kappa than lambda FLC. The two assays are not equivalent and the FreeliteTM assay displays better agreement with IFX. Care should be taken by interpreting physicians and laboratories considering switching assays.
IMMUNOPHENOTYPING OF PLASMA CELL AND THE UTILITY OF FLOW CYTOMETRY IN PLASMA CELL DYSCRASIAS Daniel Tran, Sandy Smith, David Brown and William Sewell Sydpath, St Vincent’s Hospital, Sydney, and Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Aims: To prospectively compare the performance of CD27/CD28/ CD81/CD117/CD126/CD147/CD152/CD200/CD229 to current markers (gating- CD38/CD138/CD45; clonality- k-/l-light chain; phenotype- CD19/CD56) to assess utility in identifying neoplastic plasma cells (PC) and distinguishing monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM). To retrospectively assess polychromatic flow cytometry’s (PFC) contribution to diagnosing plasma cell dyscrasias through correlation with other investigations. Methods: Antibody panels including new markers (CD27/CD28/ CD81/CD117/CD126/CD147/CD152/CD200/CD229) were run on BM samples. Database analysis of bone marrow samples at St Vincent’s hospital was conducted to correlate PFC with other investigations. Results: CD19 and CD19/CD147 were the best single marker and 2-marker combination respectively for identifying monoclonals. CD229 was best at differentiating MM and MGUS, and additional markers did not improve differentiation. PFC correlated with electrophoresis, cytogenetics and imaging. PFC detected 29 monoclonal cases that trephine analysis classified as polyclonal. These cases had low PC numbers and polyclonal PC backgrounds. Conclusion: CD19/CD147 could be more useful than CD19/ CD56 in distinguishing monoclonal and polyclonal populations. CD229 may help PFC differentiate MM from MGUS. Despite PFC’s underestimation of PCs, PFC correlates with other investigations and may be particularly useful in minimal residual disease
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
ABSTRACTS
where small PC populations occur or where background reactive plasmacytosis occurs.
USING GENOTYPE TO COMBAT RESISTANCE: DEVELOPMENT OF A POTENTIAL TREATMENT AND SIMPLE PHENOTYPIC TEST DURING A ‘PANRESISTANT’ KLEBSIELLA PNEUMONIAE MINIOUTBREAK Jeremy Brown1, Mitchell Brown1, Andrew Ginn1,4, Alex Agyekum2,3, Alicia Fajardo-Lubian2,4 and Jon Iredell1,2,3,4 1Centre for Infectious Diseases and Microbiology Laboratory Services, Institute for Clinical Pathology and Medical Research, Westmead Hospital, 2NHMRC Centre for Research Excellence in Critical infection, Westmead Millennium Institute, 3Faculty of Medicine, Westmead Clinical School, University of Sydney, and 4Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia Aims: Aztreonam/clavulanic acid combination therapy is a potential treatment for multi-resistant Enterobacteriaceae containing MBLs and ESBLs. We describe an aztreonam/clavulanic acid based phenotypic detection and susceptibility test for MBL and ESBL-containing multi-resistant Enterobacteriaceae derived from initial genotypic characterisation. Methods: Three phenotypically pan-resistant (by Etest and automated broth microdilution) K. pneumoniae isolates from a clinical colonisation mini-outbreak were shown by PCR to contain MBL and ESBL b-lactamases. These isolates and thirteen other Enterobacteriaceae containing MBLs and ESBLs (with three also containing AmpC b-lactamases) were assessed for susceptibility to aztreonam/clavulanic acid by either broth microdilution or Etest, and our phenotypic disc-diffusion assay. b-lactamases were confirmed by multiplex PCR/reverse line blot. Results: Clavulanic acid restored susceptibility to aztreonam in all ‘pan-resistant’ outbreak isolates but not strains also carrying an AmpC b-lactamase. Our disc-diffusion assay predicted 16/16 MBLs, 15/16 ESBLs and 3/3 AmpC b-lactamases and showed susceptibility to aztreonam/clavulanic acid in all non-AmpC isolates. Discussion: Pan-resistant Gram-negative isolates are likely to become increasingly common. We have described a potential treatment for an initially phenotypically pan-resistant organism that was predicted from a genotype and developed a simple combined phenotypic detection and susceptibility test. Both potential treatment and test could be employed in resource poor settings.
THE CHANGING EPIDEMIOLOGY OF KLEBSIELLA PNEUMONIAE CARBAPENEMASE (KPC) RESISTANCE IN AUSTRALIA K. Cronin and M. J. Waters Department of Microbiology, St Vincent’s Hospital Melbourne, Vic, Australia Aims: The aims of this study were to firstly describe the epidemiology, risk factors and outcomes of patients with KPC producing organisms at our institution, and secondly, to describe an outbreak involving seven patients admitted to a geriatric evaluation and management (GEM) ward.
S95
Methods: A retrospective review was conducted to assess 29 patients with KPC producing organisms in a tertiary referral centre in Australia from 2012 to 2014. In addition, a prospective assessment of an outbreak was conducted including a molecular evaluation of the isolates and patient outcomes. Results: Of the 29 patients, 55% were male with a median age of 69 years. The greatest risk factors for acquiring a KPC producing organism were length of hospital stay and recent exposure to broad spectrum antibiotics. The 30 day mortality rate was 17.2%, which increased to 37.9% at 60 days. The seven patients identified in the outbreak were exposed to an index case where a KPC was isolated from urine. The whole genome sequence analysis revealed all seven isolates were highly clonally related. Two of the patients died of causes not directly attributable to KPC colonisation. Discussion: This study highlights the increasing threat of KPC producing organisms in Australia.
AN UNUSUAL SKIN LESION CAUSED BY LASIODIPLODIA THEOBROMAE Lindsey J. Papacostas1,4, Andrew Henderson2, Keat Choong3 and David Sowden4 1Coastal Pathology, Buderim, 2Pathology Queensland, Townsville Hospital, 3Infectious Diseases, Nambour Hospital, and 4Pathology Queensland, Nambour Hospital, Qld, Australia Lasiodoplodia theobromae is a common plant pathogen of tea and fruiting plants from the Coelomycetes family. It is a rare plant pathogen in both immunocompetent and immunocompromised hosts with infections reported including sinusitis, keratitis, pneumonia and cutaneous lesions. We describe a case of subcutaneous infection as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated in multiple swabs from the foot of an active healthy male following a penetrating sea water injury. Following the trauma, the patient used wild aloe vera plant leaf to promote wound healing and this was thought to be a potential source of inoculation. Identification was performed by traditional mycology culture methods with sporulation occurring only with cornmeal agar at 28 days. Identification was confirmed by DNA sequencing. The patient was successfully treated with voriconizole.
A CASE OF AMOEBIC APPENDICITIS IN NORTH QUEENSLAND Gemma Robertson, Andrew Henderson, David Williams and Robert Norton Pathology Queensland, The Townsville Hospital, Townsville, Australia A 13-year-old female from Mornington Island presented with diarrhoea, vomiting, and abdominal pain. Laparoscopic investigation was performed and a gangrenous appendix removed. Histological examination was initially unremarkable, but a later review of the periodic acid-Schiff (PAS) stain demonstrated several areas of PAS-positive bodies consistent with invasive Entamoeba histolytica. Further testing was performed to confirm the diagnosis of E. histolytica, including serology and PCR from paraffinised tissue. The patient was treated with metronidazole and paromomycin to ensure treatment of both tissue and luminal phases of the parasite.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.