- Email: [email protected]
Incidence of very high international normalised ratio (INR) and factors contributing to discrepant results
ABSTRACTS
Methods: We undertook an online voluntary survey of senior transfusion scientists across Australia regarding current pre-transfusion practice with regards to MSBOS through a confidential online form. Results: 72 laboratories participated: 49% serviced public hospitals, 11% private hospitals and 40% both. The majority (68%) had transfusion activity of >200 episodes of red-cell e-issue/crossmatch monthly. E-issue was performed in 63%. Most serviced blood fridges within their institution (67%) and/or remote sites (61%). An MSBOS was used in only 36% with no significant difference in utilisation of an MSBOS based upon transfusion episodes, sector serviced or E-issue. Seventy-three percent used an MSBOS for all patients, 27% using it only for remote allocations and 14% for those with allo-antibodies. MSBOS were derived from the ANZSBT guideline (70%) and local data (52%). Sixty-three percent reported a high frequency of receiving clinical information on transfusion requests and 68% reported requested units complied with MSBOS. Thirty-one percent of laboratories felt a revised MSBOS would be useful. Conclusion: A MSBOS is used in a minority of laboratories. Where used, good referrer compliance and significant interest in updated guidelines is reported.
VALIDATION OF NANOSTRING MICRORNA ANALYSIS IN LEUKAEMIC BLOOD Jane E. A. Gordon1, Chuck G. Bailey1, William Ritchie1, Dale Watkins4,5,6, Tamara Leclercq4,5, Deborah L. White4,5,6,7, Timothy P. Hughes4,5,6,7 and John E. J. Rasko1,2,3 1Centenary Institute, Sydney, 2Cell and Molecular Therapies, Royal Prince Alfred Hospital, Sydney, 3University of Sydney, Sydney, NSW, 4Centre for Personalised Cancer Medicine, University of Adelaide, 5Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), 6Department of Medicine, University of Adelaide, and 7Centre for Cancer Biology, Adelaide, SA, Australia Aim: To assess the technical performance of the NanoString nCounter platform by profiling microRNA (miRNA) in CML patients. Methods: RNA (100 ng) from 75 peripheral blood mononuclear cell samples was analysed on the nCounter miRNA panel (n ¼ 800 probes) in seven batches. Samples were hybridised 12–19 h. We used nSolver, R, GraphPad Prism and Normfinder software for normalisation and analysis. Results: Ninety-eight miRNAs were expressed at >100 copies (invariant normalised, linear counts). These exhibited an intersample fold change >10, and maximum fold change 12,315. QC values were: positive control linearity R2 ¼ 0.99–1, binding density ¼ 0.27–1.03 (acceptable 0.05–2.25) and imaging ¼ 0.88–1 (acceptable >0.75). Replicate sample raw miRNA counts exhibited a high Spearman’s rank correlation coefficient ¼ 0.8678, p < 0.0001. Raw data batch effects resolved after invariant miRNA or cyclic loess normalisation. Hybridisation time affected neither background nor reaction efficiency. Discussion: Sample processing is more complex for miRNA analysis than mRNA analysis on the NanoString nCounter platform. Nevertheless, normalisation abrogated inter-run differences. The applications of mRNA based diagnostics using NanoString have recently expanded to diverse cancers, but ours is a first attempt at establishing utility for miRNAs. We conclude that NanoString performance is sufficiently robust to explore this
S93
advanced RNA counting technology for diverse applications in pathology.
ASPIRIN RESISTANCE: ANALYSIS OF THE INCIDENCE AMONGST STABLE PATIENTS ON PROPHYLACTIC THERAPY VERSUS UNSTABLE PATIENTS EXPERIENCING ACUTE ISCHAEMIC EVENTS UTILISING THE MULTIPLATE ANALYSER IN A TERTIARY INSTITUTION Yasmin Harvey, Connie Solano, Robert Bird, Andrew McCann and John Quinn Princess Alexandra Hospital, Brisbane, Qld, Australia Aim: To determine the incidence of aspirin resistance amongst stable patients treated with aspirin as primary prophylaxis for cardiovascular disease (control group) versus unstable patients experiencing acute arterial ischaemic events including acute coronary syndrome, transient ischaemic attack, cerebrovascular accident and critical limb ischaemia (event group). Methods: Patients on aspirin therapy, presenting to cardiology outpatient departments, vascular surgical wards and coronary care units in a tertiary institution were identified. Compliance with aspirin therapy was confirmed directly by verbal interview. Patient consent was obtained prior to the collection of peripheral blood samples into citrate and hirudin tubes. The samples were analysed to determine the platelet count and platelet aggregation response to arachidonic acid (ASPItest) and thrombin receptor activating peptide (TRAPtest) using impedence aggregometry on the Multiplate analyser. Results: Results of statistical analysis to follow. Discussion: Correlation between the incidence of aspirin resistance amongst stable versus unstable patients can be used to assess the role of the Multiplate analyser in the laboratory and the implications of these results in guiding patient therapy.
INCIDENCE OF VERY HIGH INTERNATIONAL NORMALISED RATIO (INR) AND FACTORS CONTRIBUTING TO DISCREPANT RESULTS S. Htet and P. Turner Haematology Department, Healthscope Pathology Background: Healthscope Pathology offers a warfarin dosing service in Victoria and Queensland with a large number of patients. Very high INR results will inevitably occur and we routinely recommend hospital presentation for patients with INR>9. Discrepant results compared to the subsequent hospital INR test are sometimes seen. Aims: To identify the incidence of INR>9 in Healthscope Pathology across Victoria and Queensland, the factors contributing to discrepant results, the incidence of bleeding in patients with INR>9 and the incidence of repeat INRs before therapeutic intervention. Methods: Retrospective follow-up phone calls to patients and health care providers for all episodes of INR>9 from 01/07/ 2013 to 09/10/2013. Results: INR>9 was detected in 130 episodes out of 100,321 INRs done (0.13%). Among 130 episodes, there was a discrepant result in 10 cases, a difficult venepuncture was documented in three cases, five cases were collected in paediatric tubes implying a difficult venepuncture, transfer of underfilled adult citrate tube to paediatric tube occurred in one case and the reason was unknown in
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
S94
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
one case. Fourteen out of 130 had a bleeding episode. In 41 episodes, vitamin K was given without rechecking INR first, among them only two were actively bleeding. Discussion: In a busy dosing service, our rates of very high INR are very low. Even in very high INR, bleeding complications were uncommon. Problems at the point of collection are the major cause for discrepant INR results. Education of phlebotomists, doctors and nurses about the importance of proper collection technique will improve quality of INR result. Repeating INR is also crucial before instituting therapeutic measures.
EFFICACY OF A POINT OF CARE INTERNATIONAL NORMALISED RATIO DEVICE FOR MONITORING OF PATIENTS ON VITAMIN K ANTAGONISTS IN THE INPATIENT AND AMBULATORY SETTING Akash Kalro, Carol Mackrow, Pamela Clarke, Nicholas Douglas and Catherine Marshall Royal Darwin Hospital, Tiwi, NT, Australia Aim: To compare the international normalised ratio (INR) values of warfarinised patients using a Point of care (POC) (Coaguchek) device against a standard laboratory assay. Methods: Patients on therapeutic warfarin with or without concomitant heparin therapy between December 2012 and October 2013 were consented to enrol in the study. Patients with hyperbilirubinaemia and antiphospholipid antibody syndrome were excluded. All patients gave a simultaneous venous and capillary sample for INR. The POC device was transported in a temperature controlled esky and underwent monthly quality control as per manufacturer’s instructions. The laboratory INR was measured using a Sysmex CA 500 analyser. Both methods were compared by Bland-Altman analysis and by means of the correlation coefficient. Results: 72 patients with median age of 60.4 years (range 12–90 years) consented. Results from 10 of these patients could not be analysed due to inability to obtain paired samples. Eightysix percent of the samples were collected at patients’ homes. Of the 90 INR values available, 47 values were therapeutic (INR 2.0–3.5), 38 were below and 5 were above. There was good correlation between the two methods (r ¼ 0.971) and the mean difference in INR was 0.09 (reference range –0.3 to 0.53). The proportion of INR values that differed from laboratory values by more than 0.3 was 9/90 (10%). Discussion: This study demonstrates that the Coaguchek device accurately measures INR in the ambulatory setting, considering the hot and humid conditions encountered in the Northern Territory. We believe it would be safe to base warfarin dosing decisions on the results of this device provided supratherapeutic values were crosschecked using the standard laboratory assay.
A PROSPECTIVE ASSESSMENT OF METHOXYFLURANE AND OTHER ANALGESIC AGENTS DURING BONE MARROW BIOPSY David Kliman1, Lynn Rugg1, Sue Bailey1, Ann Cargill1 and Poomahal Kumar1,2,3 1Department of Haematology, Royal North Shore Hospital, St Leonards, 2Pacific Laboratory Medicine Services (PaLMS)/ Pathology North, Sydney, and 3University of Sydney, Sydney, NSW, Australia
Pathology (2014), 46(S1)
Aims: Analgesic agents for use during bone marrow biopsy include lignocaine, nitrous oxide, intravenous midazolam and methoxyflurane. There are no published data regarding the use of methoxyflurane during bone marrow biopsy and thus no guidelines as to whether this agent should be provided to every patient. Methods: A prospective audit was conducted via the use of questionnaires given to patients post-biopsy. Patients assessed the pain experienced on a 100 mm visual analogue scale. If applicable, the pain from previous biopsies was rated. Information was collected regarding side effects. The change in the number of biopsies requiring midazolam sedation was determined. Results: 163 questionnaires were returned from 1034 biopsies performed during the collection period (16% response rate). There was no significant difference in pain score between the lignocaine (31 mm), nitrous oxide (34 mm) and methoxyflurane (37 mm) groups, however the patients in the methoxyflurane group were younger (53 years versus 68 years with lignocaine, p < 0.01). Side effects were more common in the methoxyflurane group (62%), predominantly dizziness. Since the introduction of methoxyflurane, midazolam use declined (9% to 2%, p < 0.01). Discussion: Methoxyflurane is a potent adjuvant agent, with the drawbacks of cost and side effects. An approach of patient selection rather than universal usage does not result in increased pain perception.
CLINICAL AND HAEMATOLOGICAL FEATURES OF NON-DELETIONAL ALPHA THALASSAEMIA MUTATIONS IN SINGAPORE J. C. M. Lam1, S. Y. Soh1 and H. Y. Law2 1Paediatric Haematology/Oncology Service, Department of Paediatric Subspecialties, and 2National Thalassaemia Registry, KK Women’s and Children’s Hospital, Singapore Aims: Non-deletional mutations of the a-globin gene are rare causes of alpha thalassaemia. Our study aims to characterise the frequency, haematological and clinical features of non-deletional forms of alpha thalassemia in patients who presented to the Singapore National Thalassaemia Registry (NTR) from 1997 to 2012. Methods: A list of patients with non-deletional a-globin mutations were generated from laboratory records at the NTR. The relevant patient casenotes were retrieved for clinical details. Results: There were 83 patients identified. The three most common a-globin chain mutations were Hb Constant-Spring (CS) (47%), Hb Quong Sze (QS) (14.5%) and Hb Adana (CD59) (12%). There were distinct ethnic differences noted in the distribution of the mutations, as the majority of patients with HbCS (46%) and all patients with HbQS were of Chinese ethnicity whereas most patients with Hb Adana (93%) were of Malay ethnicity. The majority of patients were asymptomatic carriers. Most patients with a thalassaemia intermedia phenotype had HbH disease, co-inheriting deletional a-globin mutations with nondeletional mutations, most commonly - - SEA / aCSa in 80% of cases. Coinheritance of Hb Adana / Hb CS (aCSa / aCD59a) in particular was associated with considerable clinical heterogeneity, ranging from asymptomatic carriers to transfusion dependence. Discussion: Non-deletional a-globin chain mutations are associated with diverse clinical and haematological features, depending on the specific mutation and interaction with other a and b-globin chain mutations.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Methods: We undertook an online voluntary survey of senior transfusion scientists across Australia regarding current pre-transfusion practice with regards to MSBOS through a confidential online form. Results: 72 laboratories participated: 49% serviced public hospitals, 11% private hospitals and 40% both. The majority (68%) had transfusion activity of >200 episodes of red-cell e-issue/crossmatch monthly. E-issue was performed in 63%. Most serviced blood fridges within their institution (67%) and/or remote sites (61%). An MSBOS was used in only 36% with no significant difference in utilisation of an MSBOS based upon transfusion episodes, sector serviced or E-issue. Seventy-three percent used an MSBOS for all patients, 27% using it only for remote allocations and 14% for those with allo-antibodies. MSBOS were derived from the ANZSBT guideline (70%) and local data (52%). Sixty-three percent reported a high frequency of receiving clinical information on transfusion requests and 68% reported requested units complied with MSBOS. Thirty-one percent of laboratories felt a revised MSBOS would be useful. Conclusion: A MSBOS is used in a minority of laboratories. Where used, good referrer compliance and significant interest in updated guidelines is reported.
VALIDATION OF NANOSTRING MICRORNA ANALYSIS IN LEUKAEMIC BLOOD Jane E. A. Gordon1, Chuck G. Bailey1, William Ritchie1, Dale Watkins4,5,6, Tamara Leclercq4,5, Deborah L. White4,5,6,7, Timothy P. Hughes4,5,6,7 and John E. J. Rasko1,2,3 1Centenary Institute, Sydney, 2Cell and Molecular Therapies, Royal Prince Alfred Hospital, Sydney, 3University of Sydney, Sydney, NSW, 4Centre for Personalised Cancer Medicine, University of Adelaide, 5Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), 6Department of Medicine, University of Adelaide, and 7Centre for Cancer Biology, Adelaide, SA, Australia Aim: To assess the technical performance of the NanoString nCounter platform by profiling microRNA (miRNA) in CML patients. Methods: RNA (100 ng) from 75 peripheral blood mononuclear cell samples was analysed on the nCounter miRNA panel (n ¼ 800 probes) in seven batches. Samples were hybridised 12–19 h. We used nSolver, R, GraphPad Prism and Normfinder software for normalisation and analysis. Results: Ninety-eight miRNAs were expressed at >100 copies (invariant normalised, linear counts). These exhibited an intersample fold change >10, and maximum fold change 12,315. QC values were: positive control linearity R2 ¼ 0.99–1, binding density ¼ 0.27–1.03 (acceptable 0.05–2.25) and imaging ¼ 0.88–1 (acceptable >0.75). Replicate sample raw miRNA counts exhibited a high Spearman’s rank correlation coefficient ¼ 0.8678, p < 0.0001. Raw data batch effects resolved after invariant miRNA or cyclic loess normalisation. Hybridisation time affected neither background nor reaction efficiency. Discussion: Sample processing is more complex for miRNA analysis than mRNA analysis on the NanoString nCounter platform. Nevertheless, normalisation abrogated inter-run differences. The applications of mRNA based diagnostics using NanoString have recently expanded to diverse cancers, but ours is a first attempt at establishing utility for miRNAs. We conclude that NanoString performance is sufficiently robust to explore this
S93
advanced RNA counting technology for diverse applications in pathology.
ASPIRIN RESISTANCE: ANALYSIS OF THE INCIDENCE AMONGST STABLE PATIENTS ON PROPHYLACTIC THERAPY VERSUS UNSTABLE PATIENTS EXPERIENCING ACUTE ISCHAEMIC EVENTS UTILISING THE MULTIPLATE ANALYSER IN A TERTIARY INSTITUTION Yasmin Harvey, Connie Solano, Robert Bird, Andrew McCann and John Quinn Princess Alexandra Hospital, Brisbane, Qld, Australia Aim: To determine the incidence of aspirin resistance amongst stable patients treated with aspirin as primary prophylaxis for cardiovascular disease (control group) versus unstable patients experiencing acute arterial ischaemic events including acute coronary syndrome, transient ischaemic attack, cerebrovascular accident and critical limb ischaemia (event group). Methods: Patients on aspirin therapy, presenting to cardiology outpatient departments, vascular surgical wards and coronary care units in a tertiary institution were identified. Compliance with aspirin therapy was confirmed directly by verbal interview. Patient consent was obtained prior to the collection of peripheral blood samples into citrate and hirudin tubes. The samples were analysed to determine the platelet count and platelet aggregation response to arachidonic acid (ASPItest) and thrombin receptor activating peptide (TRAPtest) using impedence aggregometry on the Multiplate analyser. Results: Results of statistical analysis to follow. Discussion: Correlation between the incidence of aspirin resistance amongst stable versus unstable patients can be used to assess the role of the Multiplate analyser in the laboratory and the implications of these results in guiding patient therapy.
INCIDENCE OF VERY HIGH INTERNATIONAL NORMALISED RATIO (INR) AND FACTORS CONTRIBUTING TO DISCREPANT RESULTS S. Htet and P. Turner Haematology Department, Healthscope Pathology Background: Healthscope Pathology offers a warfarin dosing service in Victoria and Queensland with a large number of patients. Very high INR results will inevitably occur and we routinely recommend hospital presentation for patients with INR>9. Discrepant results compared to the subsequent hospital INR test are sometimes seen. Aims: To identify the incidence of INR>9 in Healthscope Pathology across Victoria and Queensland, the factors contributing to discrepant results, the incidence of bleeding in patients with INR>9 and the incidence of repeat INRs before therapeutic intervention. Methods: Retrospective follow-up phone calls to patients and health care providers for all episodes of INR>9 from 01/07/ 2013 to 09/10/2013. Results: INR>9 was detected in 130 episodes out of 100,321 INRs done (0.13%). Among 130 episodes, there was a discrepant result in 10 cases, a difficult venepuncture was documented in three cases, five cases were collected in paediatric tubes implying a difficult venepuncture, transfer of underfilled adult citrate tube to paediatric tube occurred in one case and the reason was unknown in
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
S94
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
one case. Fourteen out of 130 had a bleeding episode. In 41 episodes, vitamin K was given without rechecking INR first, among them only two were actively bleeding. Discussion: In a busy dosing service, our rates of very high INR are very low. Even in very high INR, bleeding complications were uncommon. Problems at the point of collection are the major cause for discrepant INR results. Education of phlebotomists, doctors and nurses about the importance of proper collection technique will improve quality of INR result. Repeating INR is also crucial before instituting therapeutic measures.
EFFICACY OF A POINT OF CARE INTERNATIONAL NORMALISED RATIO DEVICE FOR MONITORING OF PATIENTS ON VITAMIN K ANTAGONISTS IN THE INPATIENT AND AMBULATORY SETTING Akash Kalro, Carol Mackrow, Pamela Clarke, Nicholas Douglas and Catherine Marshall Royal Darwin Hospital, Tiwi, NT, Australia Aim: To compare the international normalised ratio (INR) values of warfarinised patients using a Point of care (POC) (Coaguchek) device against a standard laboratory assay. Methods: Patients on therapeutic warfarin with or without concomitant heparin therapy between December 2012 and October 2013 were consented to enrol in the study. Patients with hyperbilirubinaemia and antiphospholipid antibody syndrome were excluded. All patients gave a simultaneous venous and capillary sample for INR. The POC device was transported in a temperature controlled esky and underwent monthly quality control as per manufacturer’s instructions. The laboratory INR was measured using a Sysmex CA 500 analyser. Both methods were compared by Bland-Altman analysis and by means of the correlation coefficient. Results: 72 patients with median age of 60.4 years (range 12–90 years) consented. Results from 10 of these patients could not be analysed due to inability to obtain paired samples. Eightysix percent of the samples were collected at patients’ homes. Of the 90 INR values available, 47 values were therapeutic (INR 2.0–3.5), 38 were below and 5 were above. There was good correlation between the two methods (r ¼ 0.971) and the mean difference in INR was 0.09 (reference range –0.3 to 0.53). The proportion of INR values that differed from laboratory values by more than 0.3 was 9/90 (10%). Discussion: This study demonstrates that the Coaguchek device accurately measures INR in the ambulatory setting, considering the hot and humid conditions encountered in the Northern Territory. We believe it would be safe to base warfarin dosing decisions on the results of this device provided supratherapeutic values were crosschecked using the standard laboratory assay.
A PROSPECTIVE ASSESSMENT OF METHOXYFLURANE AND OTHER ANALGESIC AGENTS DURING BONE MARROW BIOPSY David Kliman1, Lynn Rugg1, Sue Bailey1, Ann Cargill1 and Poomahal Kumar1,2,3 1Department of Haematology, Royal North Shore Hospital, St Leonards, 2Pacific Laboratory Medicine Services (PaLMS)/ Pathology North, Sydney, and 3University of Sydney, Sydney, NSW, Australia
Pathology (2014), 46(S1)
Aims: Analgesic agents for use during bone marrow biopsy include lignocaine, nitrous oxide, intravenous midazolam and methoxyflurane. There are no published data regarding the use of methoxyflurane during bone marrow biopsy and thus no guidelines as to whether this agent should be provided to every patient. Methods: A prospective audit was conducted via the use of questionnaires given to patients post-biopsy. Patients assessed the pain experienced on a 100 mm visual analogue scale. If applicable, the pain from previous biopsies was rated. Information was collected regarding side effects. The change in the number of biopsies requiring midazolam sedation was determined. Results: 163 questionnaires were returned from 1034 biopsies performed during the collection period (16% response rate). There was no significant difference in pain score between the lignocaine (31 mm), nitrous oxide (34 mm) and methoxyflurane (37 mm) groups, however the patients in the methoxyflurane group were younger (53 years versus 68 years with lignocaine, p < 0.01). Side effects were more common in the methoxyflurane group (62%), predominantly dizziness. Since the introduction of methoxyflurane, midazolam use declined (9% to 2%, p < 0.01). Discussion: Methoxyflurane is a potent adjuvant agent, with the drawbacks of cost and side effects. An approach of patient selection rather than universal usage does not result in increased pain perception.
CLINICAL AND HAEMATOLOGICAL FEATURES OF NON-DELETIONAL ALPHA THALASSAEMIA MUTATIONS IN SINGAPORE J. C. M. Lam1, S. Y. Soh1 and H. Y. Law2 1Paediatric Haematology/Oncology Service, Department of Paediatric Subspecialties, and 2National Thalassaemia Registry, KK Women’s and Children’s Hospital, Singapore Aims: Non-deletional mutations of the a-globin gene are rare causes of alpha thalassaemia. Our study aims to characterise the frequency, haematological and clinical features of non-deletional forms of alpha thalassemia in patients who presented to the Singapore National Thalassaemia Registry (NTR) from 1997 to 2012. Methods: A list of patients with non-deletional a-globin mutations were generated from laboratory records at the NTR. The relevant patient casenotes were retrieved for clinical details. Results: There were 83 patients identified. The three most common a-globin chain mutations were Hb Constant-Spring (CS) (47%), Hb Quong Sze (QS) (14.5%) and Hb Adana (CD59) (12%). There were distinct ethnic differences noted in the distribution of the mutations, as the majority of patients with HbCS (46%) and all patients with HbQS were of Chinese ethnicity whereas most patients with Hb Adana (93%) were of Malay ethnicity. The majority of patients were asymptomatic carriers. Most patients with a thalassaemia intermedia phenotype had HbH disease, co-inheriting deletional a-globin mutations with nondeletional mutations, most commonly - - SEA / aCSa in 80% of cases. Coinheritance of Hb Adana / Hb CS (aCSa / aCD59a) in particular was associated with considerable clinical heterogeneity, ranging from asymptomatic carriers to transfusion dependence. Discussion: Non-deletional a-globin chain mutations are associated with diverse clinical and haematological features, depending on the specific mutation and interaction with other a and b-globin chain mutations.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.