- Email: [email protected]
Levels of holo-transcobalamin are more closely associated with pernicious anaemia than total B12 levels
ABSTRACTS
Annular pancreas is a rare congenital anomaly consisting of a partial or complete ring of pancreatic tissue encircling the second part of the duodenum. The encircling tissue is continuous with the head of the pancreas and may include a pancreatic duct. The true incidence is not well established and is estimated to be 3 in 20 000. Annular pancreas is diagnosed equally in children and adults. The diagnosis in children is often suggested prenatally by neonatal ultrasound and confirmed within the first 1–2 days of life. Adults present with pain and gastrointestinal symptoms including nausea, vomiting and abdominal bloating. Childhood cases are frequently associated with congenital anomalies whereas adults show an increased risk of pancreatobiliary neoplasia. We present a Whipple’s resection of an annular pancreas associated with duodenal adenocarcinoma in a 63-year-old male, a rarely reported combination. The second part of the duodenum was thickened by not only the circumferential tumour mass but also a variably thick rim of pancreatic tissue containing an aberrant pancreatic duct. The macroscopic evaluation of this specimen was challenging and in the absence of a cancer, the duodenum thickened by an annular pancreas could potentially be mistaken for a tumour mass.
S69
methods. However, a new anti-dsDNA ELISA in which the dsDNA is attached to the plate by nucleosomes (dsDNA-NcX) has recently been reported to be equivalent. This study is a case-control validation comparing the two assays. Methods: Consecutive samples sent for anti-dsDNA testing were measured by the Farr radioimmunoassay (Amerlex) and the antidsDNA-NcX ELISA (Euroimmun). Performance characteristics and correlation with disease activity were assessed. Results: There were 26 patients with SLE and 26 controls with no connective tissue disease. The sensitivity and specificity for the Farr was 85% and 85% versus 77% and 96%, respectively, for the ELISA. There was no statistical difference between these results. The ELISA showed a better correlation with disease activity. Conclusion: Our preliminary data also suggest that the performance of the Anti-dsDNA-NcX ELISA is comparable to the Farr radioimmunoassay, with lower sensitivity but better specificity at the manufacturer cut-offs. Correlation with disease activity appears to be better by ELISA. Further evaluations on a larger sample size are now to be performed. PITFALLS IN FNA DIAGNOSIS OF WARTHIN’S TUMOUR
ACCURACY OF SEROLOGICAL TESTING IN THE CITY, COUNTRY AND BEYOND! S. Badman1, D. Byers1, R. Widjaja1, S. Ali1, Y. S. Lim1, R. Devarajo1, W. Rawlinson1,2 1 RCPA Serology QAP, and 2Virology Division, SEALS Microbiology, Prince of Wales Hospital, NSW, Australia Aims: External quality assurance programs provide samples on a regular basis, for which the expected or target result is unknown to the participants enrolled in the scheme. This enables participants to evaluate their performance compared to their peers in the preanalytical, analytical and post-analytical phases of sample analysis. The Royal College of Pathologist of Australasia (RCPA) Serology Quality Assurance Program (QAP) evaluated how a laboratory can assess their accuracy simply by reviewing their individualised Participant Assessment Report. Methods: The RCPA Serology QAP uses the consensus of qualitative results, as the assigned value to which participant performance is evaluated, whilst robust statistics are used to evaluate (not score) quantitative serological results returned by participants. Results: An analysis of 2011 survey results highlighted issues with participant accuracy when compared with their peers. Conclusion: Quality assurance is a means of evaluating overall laboratory performance. A laboratory can record reproducible daily quality control, indicating that their assays are precise, but QA gives an indicator of accuracy. The RCPA Serology QAP individualised Participant Assessment Reports provide a ‘score’ for the qualitative data but apply robust statistics to evaluate quantitative data to provide participants with an indication of a bias in their operating system and an overall assessment of their accuracy compared with their peers. PHAR LAP AND THE FILLY: VALIDATING THE ANTI-DSDNA-NCX IgG ELISA Russell Barker, Patricia Lehmann, Sean O’Neill, Catherine Toong Liverpool Hospital, Liverpool, NSW, Australia Aims: Anti-dsDNA antibody measurement by radioimmunoassay has traditionally had better performance characteristics for the diagnosis of systemic lupus erythematosus (SLE) than ELISA
Simone Birch, Jenny Grew Queensland Medical Laboratory, Buderim, QLD, Australia Aim: To illustrate an important potential diagnostic pitfall in salivary gland cytology. In particular, to demonstrate that acinic cell carcinoma can have an appearance indistinguishable from Warthin’s tumour, and should be considered in the differential diagnosis. Methods: Corresponding cytology and histology findings are presented for a case of acinic cell carcinoma. Results: A 78-year-old male presented with a right parotid swelling. Fine needle aspiration (FNA) showed features typical of a Warthin’s tumour with clusters of oncocytes in a background of small lymphocytes and granular debris. However, histological examination of the excision specimen showed an acinic cell carcinoma invading into surrounding fat and normal parotid. It had very prominent lymphoid stroma with germinal centres. Conclusions: Acinic cell carcinomas often have very bland appearing nuclei and may have abundant granular or foamy cytoplasm. These features combined with the prominent lymphoid stroma that may be present in acinic cell carcinomas can easily be misdiagnosed as Warthin’s tumour on FNA and should therefore be considered in the differential diagnosis. It should be remembered that there are numerous neoplastic and non-neoplastic lesions that can give a similar cytological appearance to Warthin’s tumour. FNA results for salivary gland neoplasms should always be interpreted in conjunction with clinical and radiological features. LEVELS OF HOLO-TRANSCOBALAMIN ARE MORE CLOSELY ASSOCIATED WITH PERNICIOUS ANAEMIA THAN TOTAL B12 LEVELS Mona Botros, Abdulhadi Bima, Zhong Lu, Craig Redden, Ken Sikaris Melbourne Pathology, Collingwood, Vic, Australia Aims: Vitamin B12 deficiency is usually due to dietary factors, however clinicians always consider the possibility of autoimmune pernicious anaemia. The Schillings test is no longer routinely available and therefore diagnosis often includes the specific
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. Copyright
S70
PATHOLOGY 2012 ABSTRACT SUPPLEMENT
intrinsic factor auto-antibodies (IFAb) þ/– parietal cell autoantibodies (PCAb). Holo-transcobalamin (HoloTC), also known as active B12, has proved to be a more reliable indicator of B12 deficiency than measuring total B12 (TB12). We therefore reviewed our experience with this test to see if HoloTC was more closely associated with pernicious anaemia testing. Methods: We reviewed 1359 patients where HoloTC þ/– TB12 was measured as well as one or more of IFAb and PCAb. We used each patient’s first B12 result(s) for the analysis. HoloTC was measured by Abbott AxSym and total B12 was measured by Abbott Architect. PCAb antibodies are detected by immunofluorescence whereas IFAb are measured by Alegria automated ELISA. Results: When HoloTC was very low (<10 pmol/L), 21% of patients had positive IFAb and 42% had positive PCAb. When HoloTC was mildly low (10–22 pmol/L), 8% had positive IFAb and 18% had positive PCAb. For very low TB12 (<100 pmol/L), 8% had positive IFAb and 24% had positive PCAb. When TB12 was mildly low (100–149 pmol/L), 4% had positive IFAb and 11% had positive PCAb. Conclusions: PCAb were more likely to be positive at any B12 level consistent with its known poor specificity. The more specific IFAb positivity is more likely to be associated with low HoloTC levels than low TB12 levels. A STANDARDISED REPORTING PROGRAM: REPORTING IN THE 21ST CENTURY Travis Brown Royal Melbourne Hospital, Melbourne, Vic, Australia Ever since the division of surgery from pathology in the mid to late 19th century, the anatomical pathology report has been the primary link between pathologists and clinicians. In earlier times, the pathology report was significantly shorter and rarely longer than a few sentences in total. Nowadays, the pathology report includes five sections (patient data and history, macroscopic description, microscopic description, diagnosis and comments), ranges from paragraphs to pages and contains so much jargon that few, if any, outside the pathology field fully understand it. To add to the confusion, a single clinician can receive a multitude of reporting formats and styles for identical diagnoses simply because they were reported by different pathologists. In an effort to address these issues, the Royal College of Pathologists of Australasia have developed a series of structured reporting protocols that provides guidance as to the content and presentation for cancer reports. Using these protocols, I have written a computer program that generates standardised pathology reports for pathologists to use in everyday practice. This application greatly reduces the time spent on reporting while providing important and consistent information to clinicians in accordance these recommendations.
Pathology (2012), 44(S1)
improved response to alkylating agent chemotherapy and patient survival. Hence MGMT promoter methylation is often assessed as a prognostic marker for determining the course of treatment. Cytosine hydroxymethylation is another DNA modification whose significance is only now becoming clear, and in mammals it is known to occur at high levels in the brain and pluripotent stem cells. Importantly, 5’hydroxymethylcytosine is indistinguishable from 5’methylcytosine by bisulphite-based techniques. We hypothesised that hydroxymethylation may confound MGMT methylation assessment in a subset of glioblastomas, and may account in part for those glioblastomas that are classified as having MGMT hypermethylation but who fail to respond to chemotherapy. In this study the levels of both hydroxymethylcytosine and methylcytosine were assessed in a cohort of gliomas. Hydroxymethylation was found to be present in most samples, and ranged from 0.7% to 80% of the total ‘methylation’ signature of this region. These results demonstrate that hydroxymethylation can comprise a substantial amount of the ‘methylation’ levels detected at the MGMT promoter in glioblastomas, and may potentially confound the classification of patients for treatment with alkylating agents. DIGITAL IMAGE ANALYSIS SHOWS HIGH REPRODUCIBILITY AND AGREEMENT WITH HUMAN INTERPRETATION ON HEP-2 CELLS Carol E. Buchner, Cassandra C. Bryant, Pieter A. Baker, Rachel A. Rosenblum, Gabriella Lakos, Rufus W. Burlingame INOVA Diagnostics Inc., San Diego, CA, USA Objectives: Evaluate the precision of the NOVA View1 automated digital image analysis system for anti-nuclear antibody (ANA) testing on HEp-2 cells, and compare the NOVA View interpretation of positive/negative to human interpretation. Methods: For assessing intra-assay precision, four specimens were assayed in 12 replicates. For inter-assay precision, five serum samples were tested in 25 runs. Endpoint titres of four sera were established in 25 separate runs. To evaluate positive/negative agreement, 204 clinically defined sera were processed on HEp-2 cells and analysed manually and on the NOVA View. Results: The intra-assay and inter-assay variability were <20%. The same endpoint was found by titration on average 15 out of 25 runs, and was one dilution lower or higher in the rest of the cases. The total agreement for positive and negative interpretation between the automated system and the human was 92%. Conclusion: The high precision of the NOVA View, and the good agreement of results on 204 clinically defined sera demonstrate the capability of the NOVA View to reliably discriminate between positive and negative specimens. In addition, the archived images can be stored, which provides a basis for objective ANA interpretation, and may facilitate the establishment of ANA testing standardisation.
MGMT HYDROXYMETHYLATION IN GLIOBLASTOMA C. Li1,3, M. Lee1,2, S. Flanagan1,2, R. Gupta1,2, C. M. Suter3, M. E. Buckland1,2 1 Discipline of Pathology, Sydney Medical School, University of Sydney, 2Department of Neuropathology, Royal Prince Alfred Hospital, Sydney, and 3Victor Chang Cardiac Research Institute, Sydney, NSW, Australia O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme which is commonly hypermethylated in glioblastomas. Methylation of the MGMT promoter is associated with an
PERFORMANCE OF A NOVEL CHEMILUMINESCENCE ASSAY FOR THE DETECTION OF ANTI-PR3, ANTI-MPO AND ANTI-GBM AUTOANTIBODIES Michael Mahler1, Andrea Seaman1, Elena Csernok2, Jan Damoiseaux3, Renato A. Sinico4, Antonella Radice5, Zhao Cui6, Alenka Vizjak7, Rufus W. Burlingame1 1 INOVA Diagnostics, Inc, San Diego, CA, USA, 2Department of Rheumatology, University of Lu¨beck and Klinikum Bad Bramstedt, Germany, 3Laboratory of Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands, 4Nephrology
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. Copyright
Annular pancreas is a rare congenital anomaly consisting of a partial or complete ring of pancreatic tissue encircling the second part of the duodenum. The encircling tissue is continuous with the head of the pancreas and may include a pancreatic duct. The true incidence is not well established and is estimated to be 3 in 20 000. Annular pancreas is diagnosed equally in children and adults. The diagnosis in children is often suggested prenatally by neonatal ultrasound and confirmed within the first 1–2 days of life. Adults present with pain and gastrointestinal symptoms including nausea, vomiting and abdominal bloating. Childhood cases are frequently associated with congenital anomalies whereas adults show an increased risk of pancreatobiliary neoplasia. We present a Whipple’s resection of an annular pancreas associated with duodenal adenocarcinoma in a 63-year-old male, a rarely reported combination. The second part of the duodenum was thickened by not only the circumferential tumour mass but also a variably thick rim of pancreatic tissue containing an aberrant pancreatic duct. The macroscopic evaluation of this specimen was challenging and in the absence of a cancer, the duodenum thickened by an annular pancreas could potentially be mistaken for a tumour mass.
S69
methods. However, a new anti-dsDNA ELISA in which the dsDNA is attached to the plate by nucleosomes (dsDNA-NcX) has recently been reported to be equivalent. This study is a case-control validation comparing the two assays. Methods: Consecutive samples sent for anti-dsDNA testing were measured by the Farr radioimmunoassay (Amerlex) and the antidsDNA-NcX ELISA (Euroimmun). Performance characteristics and correlation with disease activity were assessed. Results: There were 26 patients with SLE and 26 controls with no connective tissue disease. The sensitivity and specificity for the Farr was 85% and 85% versus 77% and 96%, respectively, for the ELISA. There was no statistical difference between these results. The ELISA showed a better correlation with disease activity. Conclusion: Our preliminary data also suggest that the performance of the Anti-dsDNA-NcX ELISA is comparable to the Farr radioimmunoassay, with lower sensitivity but better specificity at the manufacturer cut-offs. Correlation with disease activity appears to be better by ELISA. Further evaluations on a larger sample size are now to be performed. PITFALLS IN FNA DIAGNOSIS OF WARTHIN’S TUMOUR
ACCURACY OF SEROLOGICAL TESTING IN THE CITY, COUNTRY AND BEYOND! S. Badman1, D. Byers1, R. Widjaja1, S. Ali1, Y. S. Lim1, R. Devarajo1, W. Rawlinson1,2 1 RCPA Serology QAP, and 2Virology Division, SEALS Microbiology, Prince of Wales Hospital, NSW, Australia Aims: External quality assurance programs provide samples on a regular basis, for which the expected or target result is unknown to the participants enrolled in the scheme. This enables participants to evaluate their performance compared to their peers in the preanalytical, analytical and post-analytical phases of sample analysis. The Royal College of Pathologist of Australasia (RCPA) Serology Quality Assurance Program (QAP) evaluated how a laboratory can assess their accuracy simply by reviewing their individualised Participant Assessment Report. Methods: The RCPA Serology QAP uses the consensus of qualitative results, as the assigned value to which participant performance is evaluated, whilst robust statistics are used to evaluate (not score) quantitative serological results returned by participants. Results: An analysis of 2011 survey results highlighted issues with participant accuracy when compared with their peers. Conclusion: Quality assurance is a means of evaluating overall laboratory performance. A laboratory can record reproducible daily quality control, indicating that their assays are precise, but QA gives an indicator of accuracy. The RCPA Serology QAP individualised Participant Assessment Reports provide a ‘score’ for the qualitative data but apply robust statistics to evaluate quantitative data to provide participants with an indication of a bias in their operating system and an overall assessment of their accuracy compared with their peers. PHAR LAP AND THE FILLY: VALIDATING THE ANTI-DSDNA-NCX IgG ELISA Russell Barker, Patricia Lehmann, Sean O’Neill, Catherine Toong Liverpool Hospital, Liverpool, NSW, Australia Aims: Anti-dsDNA antibody measurement by radioimmunoassay has traditionally had better performance characteristics for the diagnosis of systemic lupus erythematosus (SLE) than ELISA
Simone Birch, Jenny Grew Queensland Medical Laboratory, Buderim, QLD, Australia Aim: To illustrate an important potential diagnostic pitfall in salivary gland cytology. In particular, to demonstrate that acinic cell carcinoma can have an appearance indistinguishable from Warthin’s tumour, and should be considered in the differential diagnosis. Methods: Corresponding cytology and histology findings are presented for a case of acinic cell carcinoma. Results: A 78-year-old male presented with a right parotid swelling. Fine needle aspiration (FNA) showed features typical of a Warthin’s tumour with clusters of oncocytes in a background of small lymphocytes and granular debris. However, histological examination of the excision specimen showed an acinic cell carcinoma invading into surrounding fat and normal parotid. It had very prominent lymphoid stroma with germinal centres. Conclusions: Acinic cell carcinomas often have very bland appearing nuclei and may have abundant granular or foamy cytoplasm. These features combined with the prominent lymphoid stroma that may be present in acinic cell carcinomas can easily be misdiagnosed as Warthin’s tumour on FNA and should therefore be considered in the differential diagnosis. It should be remembered that there are numerous neoplastic and non-neoplastic lesions that can give a similar cytological appearance to Warthin’s tumour. FNA results for salivary gland neoplasms should always be interpreted in conjunction with clinical and radiological features. LEVELS OF HOLO-TRANSCOBALAMIN ARE MORE CLOSELY ASSOCIATED WITH PERNICIOUS ANAEMIA THAN TOTAL B12 LEVELS Mona Botros, Abdulhadi Bima, Zhong Lu, Craig Redden, Ken Sikaris Melbourne Pathology, Collingwood, Vic, Australia Aims: Vitamin B12 deficiency is usually due to dietary factors, however clinicians always consider the possibility of autoimmune pernicious anaemia. The Schillings test is no longer routinely available and therefore diagnosis often includes the specific
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. Copyright
S70
PATHOLOGY 2012 ABSTRACT SUPPLEMENT
intrinsic factor auto-antibodies (IFAb) þ/– parietal cell autoantibodies (PCAb). Holo-transcobalamin (HoloTC), also known as active B12, has proved to be a more reliable indicator of B12 deficiency than measuring total B12 (TB12). We therefore reviewed our experience with this test to see if HoloTC was more closely associated with pernicious anaemia testing. Methods: We reviewed 1359 patients where HoloTC þ/– TB12 was measured as well as one or more of IFAb and PCAb. We used each patient’s first B12 result(s) for the analysis. HoloTC was measured by Abbott AxSym and total B12 was measured by Abbott Architect. PCAb antibodies are detected by immunofluorescence whereas IFAb are measured by Alegria automated ELISA. Results: When HoloTC was very low (<10 pmol/L), 21% of patients had positive IFAb and 42% had positive PCAb. When HoloTC was mildly low (10–22 pmol/L), 8% had positive IFAb and 18% had positive PCAb. For very low TB12 (<100 pmol/L), 8% had positive IFAb and 24% had positive PCAb. When TB12 was mildly low (100–149 pmol/L), 4% had positive IFAb and 11% had positive PCAb. Conclusions: PCAb were more likely to be positive at any B12 level consistent with its known poor specificity. The more specific IFAb positivity is more likely to be associated with low HoloTC levels than low TB12 levels. A STANDARDISED REPORTING PROGRAM: REPORTING IN THE 21ST CENTURY Travis Brown Royal Melbourne Hospital, Melbourne, Vic, Australia Ever since the division of surgery from pathology in the mid to late 19th century, the anatomical pathology report has been the primary link between pathologists and clinicians. In earlier times, the pathology report was significantly shorter and rarely longer than a few sentences in total. Nowadays, the pathology report includes five sections (patient data and history, macroscopic description, microscopic description, diagnosis and comments), ranges from paragraphs to pages and contains so much jargon that few, if any, outside the pathology field fully understand it. To add to the confusion, a single clinician can receive a multitude of reporting formats and styles for identical diagnoses simply because they were reported by different pathologists. In an effort to address these issues, the Royal College of Pathologists of Australasia have developed a series of structured reporting protocols that provides guidance as to the content and presentation for cancer reports. Using these protocols, I have written a computer program that generates standardised pathology reports for pathologists to use in everyday practice. This application greatly reduces the time spent on reporting while providing important and consistent information to clinicians in accordance these recommendations.
Pathology (2012), 44(S1)
improved response to alkylating agent chemotherapy and patient survival. Hence MGMT promoter methylation is often assessed as a prognostic marker for determining the course of treatment. Cytosine hydroxymethylation is another DNA modification whose significance is only now becoming clear, and in mammals it is known to occur at high levels in the brain and pluripotent stem cells. Importantly, 5’hydroxymethylcytosine is indistinguishable from 5’methylcytosine by bisulphite-based techniques. We hypothesised that hydroxymethylation may confound MGMT methylation assessment in a subset of glioblastomas, and may account in part for those glioblastomas that are classified as having MGMT hypermethylation but who fail to respond to chemotherapy. In this study the levels of both hydroxymethylcytosine and methylcytosine were assessed in a cohort of gliomas. Hydroxymethylation was found to be present in most samples, and ranged from 0.7% to 80% of the total ‘methylation’ signature of this region. These results demonstrate that hydroxymethylation can comprise a substantial amount of the ‘methylation’ levels detected at the MGMT promoter in glioblastomas, and may potentially confound the classification of patients for treatment with alkylating agents. DIGITAL IMAGE ANALYSIS SHOWS HIGH REPRODUCIBILITY AND AGREEMENT WITH HUMAN INTERPRETATION ON HEP-2 CELLS Carol E. Buchner, Cassandra C. Bryant, Pieter A. Baker, Rachel A. Rosenblum, Gabriella Lakos, Rufus W. Burlingame INOVA Diagnostics Inc., San Diego, CA, USA Objectives: Evaluate the precision of the NOVA View1 automated digital image analysis system for anti-nuclear antibody (ANA) testing on HEp-2 cells, and compare the NOVA View interpretation of positive/negative to human interpretation. Methods: For assessing intra-assay precision, four specimens were assayed in 12 replicates. For inter-assay precision, five serum samples were tested in 25 runs. Endpoint titres of four sera were established in 25 separate runs. To evaluate positive/negative agreement, 204 clinically defined sera were processed on HEp-2 cells and analysed manually and on the NOVA View. Results: The intra-assay and inter-assay variability were <20%. The same endpoint was found by titration on average 15 out of 25 runs, and was one dilution lower or higher in the rest of the cases. The total agreement for positive and negative interpretation between the automated system and the human was 92%. Conclusion: The high precision of the NOVA View, and the good agreement of results on 204 clinically defined sera demonstrate the capability of the NOVA View to reliably discriminate between positive and negative specimens. In addition, the archived images can be stored, which provides a basis for objective ANA interpretation, and may facilitate the establishment of ANA testing standardisation.
MGMT HYDROXYMETHYLATION IN GLIOBLASTOMA C. Li1,3, M. Lee1,2, S. Flanagan1,2, R. Gupta1,2, C. M. Suter3, M. E. Buckland1,2 1 Discipline of Pathology, Sydney Medical School, University of Sydney, 2Department of Neuropathology, Royal Prince Alfred Hospital, Sydney, and 3Victor Chang Cardiac Research Institute, Sydney, NSW, Australia O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme which is commonly hypermethylated in glioblastomas. Methylation of the MGMT promoter is associated with an
PERFORMANCE OF A NOVEL CHEMILUMINESCENCE ASSAY FOR THE DETECTION OF ANTI-PR3, ANTI-MPO AND ANTI-GBM AUTOANTIBODIES Michael Mahler1, Andrea Seaman1, Elena Csernok2, Jan Damoiseaux3, Renato A. Sinico4, Antonella Radice5, Zhao Cui6, Alenka Vizjak7, Rufus W. Burlingame1 1 INOVA Diagnostics, Inc, San Diego, CA, USA, 2Department of Rheumatology, University of Lu¨beck and Klinikum Bad Bramstedt, Germany, 3Laboratory of Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands, 4Nephrology
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. Copyright