RESPONSES TO COMMENTS ON: Recommendations for Use of Point-of-Care (POC) Troponin Assays in Assessment of Acute Coronary Syndrome

RESPONSES TO COMMENTS ON: Recommendations for Use of Point-of-Care (POC) Troponin Assays in Assessment of Acute Coronary Syndrome Page 1

Responses were received from: 1. Chemical Pathology Clinical Stream, NSW Health Pathology 2. The Royal College of Pathologists of Australasia (RCPA) Dr Graham Jones 3. Dr Grahame Caldwell, Private Pathology 4. Pathology Queensland, Public Pathology 5. The Australasian College for Emergency Medicine (ACEM) Council of Advocacy, Practice and Partnerships Dr Yusef Nagree and Prof Anthony Lawler. ACEM is happy to disseminate the Recommendations to its members. 6. The NSW Cardiac Network - a comment was received from Dr Matthew Bragg, the Director of ED at the Bowral and District Hospital. 7. The Cardiac Society of Australia and New Zealand (CSANZ) and Derek Chew. CSANZ does not endorse documents where it has not been formally involved during development. The CSANZ Quality Standards Committee did not have any further comments to add. CSANZ is happy to provide a link to the recommendations document on their website. The AACB thanks all organisations and individuals for their comprehensive comments and support of the recommendations. Page 2

1. Chemical Pathology Clinical Stream, NSW Health Pathology No. Comment Response 1 As a general comment, `dot points are not useful when referencing sections of the paper. 2 Page 2: Index of Contents Suggest change to Table of Contents 3 Page 5 (first dot point) and reiterated on page 7, (first dot point): The results of various assays, available both for POC and laboratory-based troponin testing are NOT directly comparable and values CANNOT be transferred between assays. This advice is at odds with the `Analytical recommendation made on page 6 (first dot point) and reiterated on page 9 (fourth dot point) that: POC troponin assays should be validated against laboratory-based sensitive or high sensitive troponin assays. 4 Page 5 (fifth dot point) and reiterated on page 7 (third dot point): Re-baseline the troponin values for individual patients if transported. The retest interval for these patients is unclear. 5 Page 5 (seventh dot point) and reiterated on page 7 Further information needed. Suggestion that the document present information in the form of numbered paragraphs for ease of reference. More detailed information on the type of validation may be needed. Response: We agree and have changed the text as follows: POC troponin methods should be validated for clinical concordance, or at least compared against the local laboratory-based sensitive or highly sensitive troponin assay where patients may be referred. In this way POC results can be assessed against laboratory results, but not used for monitoring unless the differences are larger than the variation between assays. Suggest a uniform test and retest interval for these patients to simplify understanding by clinical staff Response: see page 6 and page 9 which read as follows: At the second location collect a new baseline sample and a second sample at 2-3 hours (if testing with a highly sensitive troponin assay) and 6-8 hours (if testing with a less analytically sensitive assay) unless the patient has been symptom-free for >12 hours, when a single sample is appropriate. Page 3

(fifth dot point): Quantitative troponin delta change has not been determined for use with POC assays due to assay inaccuracy at low troponin concentration; see local algorithms for recommendation. However, a quantitative delta change is suggested later in the document (page 21 algorithm from Pathology Queensland for Abbott i-stat). 6 Page 6 (fifth dot point) and reiterated on page 10 (first dot point): A general approach to reducing micro-clots is to mix blood tubes immediately after collection to avoid formation of micro-clots that may not be visible on testing but which can cause falsely low troponin values. Micro-clots can also cause falsely raised troponin values. It is misleading to specify that only falsely low values may occur. 7 Page 7 (fourth dot point): Serial measurements help to improve the reliability of the detection of low elevated troponin although POC assays are less accurate than laboratory-based troponin assays. Modelling shows that there is not much difference in misclassification rates for an assay with a 10% CV at the 99th percentile vs. one with 25% CV provided serial measurements are used in the model. Response: Pathology Queensland recommends a delta for i-stat POC assay and this has been substantiated by local data. ICCnet and NSW Health Pathology do not recommend a delta. The sentence on page 6 and on page 10 has been modified slightly to read: In general quantitative troponin delta change has not been determined for use with POC assays as POC assays are less precise at low troponin concentration (see local algorithms for examples). Change wording to: may cause incorrect troponin values. Response: We have changed the wording as suggested. Re-wording is needed Response: This section has been removed according to the College s comment (# 24). The expression, not much difference, seems a bit Page 4

vague and unscientific. 8 Throughout document the word bradyarrhythmia is misspelt. 9 Several abbreviations are used throughout the document to describe point-of-care testing i.e. POC, POCT and PoCT. Correct spelling needed. Response: Thank you for detecting this error. Hopefully every misspelling has been corrected. A recommendation on the most appropriate abbreviation to use would be useful. Response: We agree and have used POC throughout the document. 10 End-stage renal disease A comment on the impact of ESRD on Troponin values may be useful. Response: Apart from listing in Table 1 that Dialysis dependent renal failure is one cause of elevated cardiac troponin. We don t believe that a comment on the impact of ESRD should be included in the scope of this document. 11 Delayed presentation A comment on the impact of delayed presentation on interpretation of troponin values may be useful. 12 The document provides conflicting algorithms making recommendations for harmonised units and protocols across the country difficult. Response: Delayed presentation and the timing of sample collection are sticking points with some. Many assume that since you have symptoms for many hours, you can do a single test. We note in the Clinical Recommendations sections under Point V: Less sensitive troponin assays may require a longer time period to show a clinically significant elevation in troponin. A recommendation on harmonised units and algorithm for use with POC devices would be useful. Response: As noted the algorithms are not harmonised across state borders. Currently i- STAT reports to 2 decimal places in µg/l and hence a change to ng/l would report all values in deciles. Other POC platforms can report to 3 decimal places hence reporting in ng/l is possible for these assays. Not until i-stat can produce an assay that reports in ng/l, it is not feasible to use ng/l uniformly (unfortunately!) and local algo rithms are recommended (as shown in Appendix 2). Please note that NSW Health Pathology has now decided to use ng/l for the Radiometer AQT90 TnT assay. Page 5

In a new section in the document (Postanalytical reporting units), we advocate for the future use of harmonised reporting units in whole numbers in ng/l across Australia. 13 2 sets of units for reporting Troponin are in use. Suggest uniform units of measurement. States/Services should then justify any variation for a national guideline on units to be used. 14 The transfer of patient between sites presents a challenge in terms of Troponin testing. Response: We agree. In the Postanalytical section under reporting units we state the following: To improve patient safety we recommend that the clinical and laboratory communities standardise on troponin units of measurement in Australia namely ng/l and report values in whole numbers. This will allow for harmonised reporting of troponin in the electronic medical record. Suggest that additional guidance is provided regarding protocols to be considered when there is a transfer of testing/patient from sites with Troponin I to sites with Troponin T (or vice versa), or from sites with low sensitivity to high sensitivity assays (and vice versa). 15 Shorter time-interval testing for troponins (after 2-3 hours) is currently under investigation. Response: We agree in that additional guidance would be helpful. However, although the recent NHF/CSANZ 2016 Guideline for the assessment of ACS recommends that an initial sample is taken with the patient to the second location for retesting of troponin (usually by a more analytically sensitive assay), the various logistical issues of safe transport of patient samples and the associated paperwork cannot be overcome at this time. Hence, we recommend to re-baseline the troponin value for individual patients if transported to a different location where a different troponin assay is used. Consider adding a future review of short time interval testing for troponins to the position statement. Response: Louise Cullen has commented as follows in the Clinical recommendations sections on page 5 and page 9 under point VI: Recommended timing of samples using accelerated diagnostic pathways, using laboratory-based highly sensitive assays, is obtaining the first sample at admission to ED and the second sample at 2-3 hours later. In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. Page 6

(Page 9) Few POC troponin assays have been studied and reported as part of an accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. 16 Please include this comment. Please note that should the patient or sample be referred to a hospital/lab with a different assay then it is recommended to perform another troponin test at the time of arrival of the patient to the hospital or sample to the laboratory. Otherwise it is recommended that repeat testing, if warranted, should occur in 6 hours Response: We have put the following (page 6 and page 10): At the second location collect a new baseline sample and a second sample at 2-3 hours (if testing with a highly sensitive troponin assay) and 6-8 hours (if testing with a less analytically sensitive assay) unless the patient has been symptom-free for >12 hours, when a single sample is appropriate. Page 7

2. The Royal College of Pathologists of Australasia (RCPA) No. Comment Response 17 Page 5, Clinical recommendations The basis for some of these comments is not apparent until the laboratory recommendations are seen, i.e. that current POC devices are less analytically sensitive than most laboratory assays. This should be stated with the clinical recommendations so they make sense. The College s preference would be to link the use of an assay to its analytical performance rather than its location. This could future proof the document against new high sensitive POC assays should they arrive. 18 Page 5, dot point 1 The results of various assays, available both for POC and laboratory-based troponin testing are NOT directly comparable and values CANNOT be transferred between assays. 19 Page 5, dot point 3 Troponin results must always be interpreted in the clinical context. Biomarkers are only one component of clinical information to be used with ECG(s) and clinical history for risk stratification according to Response: Agreed See pages 5 and 9 (point II): The following clinical recommendations regarding use of POC troponin assays are based on laboratory data showing that current POC assays are less analytically sensitive than most laboratory assays for troponin. As currently written, this suggests it refers to assays available in both POC and laboratory formats. There are none of these. The key point is that different, troponin assays are not directly comparable, irrespective of whether they are POC or laboratory based. The College suggests adding a rider, unless there is evidence of such comparability to allow for such an eventuality. Response: Agreed. Page 5 (point III); also see page 9 (point III): The results of various troponin assays, available both for POC and laboratory-based troponin testing whether they are available in POC or laboratory formats, are NOT directly comparable and values CANNOT be transferred between assays unless there is evidence of such comparability. The examples here seem overly restricting. The College suggests the 1st sub-point be rewritten as Elevated troponins alone do not rule in the diagnosis of ACS. It still remains unclear whether the authors are indicating the need for the right clinical setting or for a rise and/or fall from this dot point. The second sub-point here again seems difficult. Do the authors mean we should keep all patients for more than 12 hours? Is a >99% safety rule out not sufficient? Page 8

clinical guidelines. Elevated troponin results do not rule in the diagnosis of ACS Normal troponin results (at 0 and 3 hours post presentation) do not rule out ACS; slowly evolving AMIs may not elevate troponin until >12 hours post presentation and unstable angina (UA) will not be detected. 20 In the time of older laboratory assays 8-12 hours was considered sufficient for rule out, why is a single POC troponin in this time period not satisfactory? Response: The intent was the need for the right clinical setting so as not to miss any MI cases using POC troponin assays, i.e. the safe rule-out of ACS/MI. We have elaborated more fully on this. On page 5 and page 9 under point V we have written: Troponin results must always be interpreted in the clinical context. Biomarkers are only one component of clinical information to be used with ECG(s) and clinical history for risk stratification according to clinical guidelines. - Elevated troponin results alone do not rule in the diagnosis of ACS as elevated troponin can occur in non-acs conditions (causes of non -ACS troponin elevations are shown in Table 1). - Normal troponin results (at 0 and 3 hours after admission of patient to the Emergency Department [ED]) do not rule out ACS where there is a high clinical suspicion; in such cases further troponin testing may be indicated at 6 hours or at 12 hours post admission to ED (slowly evolving AMIs may not elevate troponin until >12 hours after admission and unstable angina (UA) will not be detected). - Less sensitive troponin assays may require a longer time period to show a clinically significant elevation in troponin. In the time of older laboratory assays 8-12 hours was considered sufficient for rule out, why is a single POC troponin in this time period not satisfactory? Response: from Dr Louise Cullen Serial troponin measurements should be performed for all patients presenting to an ED with symptoms suggestive of ACS, unless the patient has been reliably ACS symptom-free for 12 hours when a single sample is appropriate. The time of symptom onset, even if reliable, does not define the time point of coronary occlusion. This is in keeping with the 2016 NHF/CSANZ guidelines. In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw (vs. 2-3 hours using highly sensitive assays and an accelerated diagnostic pathway). Few POC troponin assays have been studied and reported as part of an Page 9

21 Dot point 8 When a central laboratory is used, results should be available as soon as possible, with a goal of within 60 minutes after the sample is received in the laboratory. Otherwise POC testing should be considered. 22 Page 7, dot point 1, dash point 3 There are many different assays available both for POC and laboratory-based troponin testing. Importantly, the results of various assays are NOT directly comparable and values CANNOT be transferred between assays. Users of POC and laboratory services need to be aware of the limitations of the troponin assay they are using in the management of their patients. Lack of awareness of these differences may impact adversely on patient management and patient safety. Normal troponin results (at 0 and 3 hours post presentation) do not rule out ACS; slowly evolving AMIs may not elevate troponin until >12 hours post presentation and unstable angina (UA) will not be detected. 23 Page 7, dot point 2, dash point 3 Serial troponin measurements should be performed for all patients presenting with symptoms suggestive of ACS, unless the patient has been reliably ACS symptom-free for 24 hours Recommended timing of samples when using POC assays is obtaining the first sample at presentation accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. What is a central laboratory? Does this mean a laboratory? How is the timing within the laboratory relevant if the sample takes 3 hours to get there? Response: Yes, we mean any laboratory. We have removed the word central. It is unclear whether this statement refers to all troponin assays or only POC assays (which are less analytically sensitive at this time). Response: We have modified this section and added the following sentences prior to the section you refer to: Point II. The following recommendations take into account the impact of the analytical performance of currently available Tn assays on their clinical use and interpretation. The recommendations regarding use of POC troponin assays are based on laboratory data showing that current POC assays are less analytically sensitive than most laboratory assays (labelled as high sensitivity or sensitive assays) for troponin. At the end of point V. we have also added: Less sensitive troponin assays may require a longer time period to show a clinically significant elevation in troponin. The implications of this that every chest pain patient at an ED with a POC device will be in the ED for at least 6 hours. Is this what is intended? Response: We have modified the section to put the emphasis on safe rule-out of ACS/MI as follows. Recommended timing of samples using accelerated diagnostic pathways, using laboratory-based highly sensitive assays, is obtaining the first sample at admission to ED Page 10

and the second sample at 6-8 hours post presentation (N.B. this is different to laboratory-based troponin assays). 24 Page 7, dot point 4 Serial measurements help to improve the reliability of the detection of low elevated troponin although POC assays are less accurate than laboratory-based troponin assays. Modelling shows that there is not much difference in misclassification rates for an assay with a 10% CV at the 99th percentile vs. one with 25% CV provided serial measurements are used in the model. 25 Page 9, dot point 1 dash point 1 It is important that clinicians recognise the limitations of POC troponin vs. laboratory-based troponin assays in terms of analytical performance which impacts their clinical performance. All current troponin POC assays are analytically less sensitive for the detection of the troponin molecule and cannot measure troponin in the healthy population (example of i -STAT ctni POC vs. AccuTnI and Architect ctni lab-based assays is shown in Figure 1)" and the second sample at 2-3 hours later. In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. Few POC troponin assays have been studied and reported as part of an accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. This classification of assays according to CV is interesting, but the College suspects it has been misinterpreted. The study is based on an exponential distribution where there are not many normal results near the 99th centile (and thus not many false positives). As we now know, the distribution is not this shape and the conclusions may need to be revised. If this is taken as read, it should be clarified that any one of multiple POC troponins above the 99th centile should indicate a possible AMI. Similarly, if we really wanted this extra clinical sensitivity we could take more samples in a shorter time frame. Response: Thank you for this comment. We have removed this dot point from the document. The graphs in Figure 1 do not support this statement as the istat assay can measure TnI in some patients with laboratory assay results below the 99th centile. If taken as written, this point means that all measurable troponins on all current POC devices are pathological, which does not seem to be true. Response: We agree and have modified the section to read as follows: All current troponin POC assays are analytically less sensitive for the detection of the troponin molecule and troponin is measurable above the limit of detection in <10% of the healthy population (Apple 2012). Studies using the i-stat POC assay have shown that by lowering the decision cut-off to half of that of the 99th percentile troponin concentration of the reference population distribution that more AMIs are detected (Schneider 2013) (example of analytical sensitivity i-stat ctni POC vs. AccuTnI and Architect ctni lab-based assays is shown in Figure 1). Page 11

26 Page 9, dot point 1, dash point 2 It is important that clinicians recognise the limitations of POC troponin vs. laboratory-based troponin assays in terms of analytical performance which impacts their clinical performance. POC assays are less diagnostically accurate at earlier time points (<6 hours after the onset of symptoms) compared with high sensitive and sensitive assays used by laboratories (see Figures 2-5). 27 Page 9, dot points 2 and 3 Laboratories should confirm imprecision around the decision cut-off by doing an imprecision profile. Table 2 shows the analytical characteristics of commercial POC troponin assays declared by the manufacturer. Generally POC troponin assays are clinically usable based on a scorecard performance approach.8 This means that imprecision at the 99th percentile troponin concentration is >10 % CV and 20 % CV. I-STAT POC Troponin I assay is clinically usable at a cut-off 0.04 μg/l with an imprecision of 16.5 % CV reported (Pathology Queensland local data) AQT90 Troponin T assay is also clinically usable at a cut-off of 0.02 μg/l ctnt with an imprecision of 11% CV (NSW Health Pathology local data). POC troponin assays should be assessed for analytical imprecision using the manufacturer s QC and ideally, also, a low concentration sample at close to the 99th percentile ctn concentration. Figures 6A-E show the analytical imprecision that can be obtained close to the 99th percentile ctn concentration for high The figures show the POC assays are less diagnostically accurate. They are clearly less clinically sensitive, but also less clinically specific. The College suggests using less diagnostically sensitive than less diagnostically accurate in this sentence. Response: We agree and have used the term less diagnostically sensitive. Are these the same thing or is one assay verification and one routine QC? Also, there is a problem trying to measure SDs or CVs when the data is highly granular. For example, with the i-stat TnI near 0.04 ug/l, the results are measured to the nearest 0.01 ug/l and the measured CV depends whether the average is on a number, or between two numbers. Response: The first dot point II gives the manufacturers precision and examples of local imprecision data. The second dot point III describes verification of imprecision using precision profiles. The sections have been modified as follows: II. Table 2 shows the analytical characteristics of commercial POC troponin assays declared by the manufacturer. Generally POC troponin assays are clinically usable based on a scorecard performance approach.8 This means that imprecision at the 99th percentile troponin concentration is >10 % CV and 20 % CV. For example: - I-STAT POC Troponin I assay is clinically usable at a cut-off 0.04 µg/l with an imprecision of approx. 20% CV reported (Pathology Queensland local data collated over several months) - AQT90 Troponin T assay is also clinically usable at a cut-off of 0.02 µg/l ctnt with an imprecision of 11% CV (NSW Health Pathology local data). III. All troponin assays should be verified for analytical imprecision using the manufacturer s QC and ideally, also, a low concentration QC sample or patient pool at close to the 99th percentile ctn concentration. Figures 6A-E show the analytical Page 12

sensitive hs-tnt and hs-tni assays vs. POC ctn assays. Note that the laboratory-based high sensitive Roche hs-tnt (Fig. 6A) and Abbott Architect hs -TnI (Fig. 6B) assays have lower imprecision than the three PoC ctn assays, AQT90 Troponin I (Fig. 6C), AQT90 Troponin T (Fig. 6D) and i-stat ctni (Fig.6E). imprecision that can be obtained close to the 99th percentile ctn concentration for highly sensitive hs-tnt and hs-tni assays vs. POC troponin assays. - Note that the laboratory-based highly sensitive Roche hs-tnt (Fig. 6A) and Abbott Architect hs-tni (Fig. 6B) assays have lower imprecision than the three PoC troponin assays, AQT90 Troponin I (Fig. 6C), AQT90 Troponin T (Fig. 6D) and i-stat ctni (Fig. 6E). 28 Figure 6A-E Also, expressing this can be difficult, e.g. page 15, figure e. Repeats of TnI of 0.04 do not fall between 0.03 and 0.05 but rather fall from 0.03 to 0.05 (inclusive). 29 Precision studies are also difficult for laboratories to perform as they tend to be with one lot number of reagents which may not reflect performance over time. With regard to method validation/verification, it would also be good to offer advice regarding what is required on a representative number of analysers and what is recommended for all POC analysers of the same type. 30 Page 9, dot point 4 POC troponin assays should be validated against laboratory-based sensitive or high sensitive troponin assays.9 Response: We have modified the text in the figure legends on page 15 accordingly. Precision studies are also difficult for laboratories to perform as they tend to be with one lot number of reagents which may not reflect performance over time. With regard to method validation/verification, it would also be good to offer advice regarding what is required on a representative number of analysers and what is recommended for all POC analysers of the same type. Response: We have included an extra table 3 with long-term imprecision shown with the data from various local and published studies. This should be stated as validated, or at least compared with the laboratory assay where patients may be referred, so that the POC results can be assessed against the laboratory results (but not used for monitoring unless the differences are larger than the variation). Response: We have used your sentence here. Please refer to page 6, point IV and page 11, point V. POC troponin methods should be validated for clinical concordance, or at least compared against the local laboratory-based sensitive or highly sensitive troponin assay where patients may be referred. In this way POC results can be assessed against laboratory results, but not used for monitoring unless the differences are larger than the variation between assays. Page 13

31 Missing sections Postanalytical The College suggests a section on post analytical factors. This is a consideration of the test name, the units, other supporting information (e.g. indicating the method), the need to provide education to users about the assay (and comparison with other assays in use), the advisability of placing results into the electronic record. Response: We agree and thank you for these suggestions. Postanalytical Test name: I. The naming of generations of troponin assays has been inconsistent and largely marketdriven. Highly sensitive assays measure the same protein as the sensitive assays, and even more sensitive cardiac troponin assays will certainly be developed, thus making any high sensitivity designation obsolete. II. For the purpose of reporting of results, if the analytical performance of a new assay is significantly different from a previous troponin assay the test name should indicate this, e.g. Troponin T Highly sensitive. III. We recommend there is a general consensus in Australia regarding troponin test names for the same manufacturer s assay to allow for harmonised reporting of troponin in the electronic medical record. Reporting units: I. The international consensus is that troponin should be reported in ng/l unitage and expressed as whole numbers (REFS). Highly sensitive troponin assays generally use units of ng/l, whereas results from less sensitive assays have usually been reported in mg/l to two or more decimal points. As a consequence, the reporting of these more sensitive assays produces results that may be a thousand-fold different compared to results by other less sensitive assays, and thus has the potential for confusion and medical error, e.g. 4000 ng/l is 4.000 mg/l and 4 ng/l is 0.004 mg/l. II. To improve patient safety we recommend that the clinical and laboratory communities standardise on troponin units of measurement in Australia, namely ng/l and report values in whole numbers. This will allow for harmonised reporting of troponin in the electronic medical record. Page 14

32 Missing sections Governance Education to users about assay (JONES 2012): - Various healthcare professionals request and measure troponin. It is essential that there is a comprehensive education process communicated to all users. - Supporting information detailing troponin assay utility and comparing the local assay to others in use should be provided (REF JONES) - General educational materials including a slide presentation of the recommendations and case studies are available on the AACB website at: http://www.aacb.asn.au/professionaldevelopment/harmonisation We also suggest a section on governance, e.g. that the method in use and the way it is used should have input from relevant clinicians (ED, cardiology, general medicine as needed) as well as laboratory and users (eg nursing staff). There should be a review process to ensure ongoing performance and correct use. (e.g. Jones et al, Introducing high sensitivity troponin into routine use - A worked example. AACB Monograph 2012). Response: We agree and have included the following: Governance Input from stakeholders (JONES 2012): I. Introduction of a more sensitive troponin assay requires close communication between the laboratory, Emergency Department, cardiologists, and other relevant clinicians. Ideally decisions regarding assay performance, clinical cut-off, and turnaround time of results should be decided on by all the stakeholders. II. Discussion regarding the likelihood of the impact of a more sensitive assay on the increase in patients with elevated troponin and patient classifications is essential prior to assay implementation. Audit and review of assay performance and clinical use (JONES 2012): I. There should be a review process to ensure ongoing performance and correct laboratory use of troponin assays. II. Review by the site doing the assay is required to determine its performance, e.g. imprecision close to the troponin cut-off concentration. III. Review and audit by the clinical team of clinical use of the assay is required. Page 15

3. Dr Grahame Caldwell, Private Pathology No. Comment Response 33 Page 3 under Clinical recommendations will highlight the importance of understanding dot point 2 34 Page 5 under dot point 2 (and page 7 under dot point 1) Clinical Recommendations 35 Page 5 under dot point 8 (and page 7 under dot point 6) Clinical Recommendations 36 Page 5 under dot point 3 (and page 7 under dot point 1) Clinical Recommendations: Suggest replace that there are no missed acute myocardial infarctions (AMI) by that acute myocardial infarction (AMI) is not missed Suggest replace Users of POC and lab services by Users of POC devices and laboratory analysers Suggest replace When a central laboratory is used by When a laboratory is used Suggest that you reference the clinical guideline: Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD: the Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third Universal Definition of Myocardial Infarction. Circulation 2012;126:2020-35. 37 Page 7 under dot point 1 Clinical Recommendations Suggest say (causes of non-acs troponin elevations are shown in Table 1) 38 Page 5 under dot point 4 (and page 7 under dot point 2) Clinical Recommendations In both the executive summary and the comprehensive document the reason for the difference in the timing of the collection of the second sample when using POC and labbased troponin assays should be explained and the safety issue stressed for rule out of ACS. Response: Louise Cullen has commented as follows in the Clinical recommendations sections on page 5 and page 9 under point VI: Recommended timing of samples using accelerated diagnostic pathways, using laboratory-based highly sensitive assays, is obtaining the first sample at admission to ED and the second sample at 2-3 hours later. In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. Page 16

39 Page 5 under dot point 1 (and page 9 under dot point 1) Analytical Laboratory Recommendations 40 Page 5 under dot point 1 (and page 9 under dot point 1) Analytical Laboratory Recommendations 41 Page 5 under dot point 1 (and page 9 under dot point 1) Analytical Laboratory Recommendations 42 Page 6 under dot point 2 (and page 9 under dot point 5) Analytical Laboratory Recommendations: 43 Page 9 subheadings under Laboratory Recommendations 44 Page 9 under Analytical suggest including a dot point about analytical interferences 45 Under Table 2 which shows the analytical characteristics of POC assays according to the manufacturers data, suggest including a paragraph about the international recommendation for the use (Page 9) Few POC troponin assays have been studied and reported as part of an accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. Suggest replace clinical performance by either clinical interpretation or clinical utility Suggest replace compared with high sensitive and sensitive assays used laboratories by compared to high sensitive and sensitive assays Suggest that as these are recommendations the first sentence should read Clinicians should recognise the limitations Suggest the end of the sentence reads related to the management of patients at risk of ACS Suggest use Analytical and Preanalytical without repeating the word recommendations Suggest including a dot point about analytical interferences. Response: We agree and have added the following: Both laboratory-based and POC troponin assays may be affected by analytical interferences including heterophilic antibodies, immunoglobulin complexes, fibrin clots, etc. These may interfere with troponin measurement in some assays and cause falsepositive or false-negative values. Suggest including a paragraph about the international recommendation for the use of the units, ng/l (or pg/ml) and that results should be reported in whole numbers. Response: The College has also suggested including a section on Postanalytical by Page 17

of the units, ng/l (or pg/ml) and that results should be reported in whole numbers. This issue requires further harmonisation in Australia. recommendations including reporting units. Please see this section under Analytical recommendations. Under Table 2 the following has been added: NB: The international consensus is that troponin should be reported in ng/l (or pg/ml) unitage and expressed as whole numbers. This issue requires further harmonisation in Australasia. 46 Page 18 and the local algorithms Suggest there is a preamble explaining that the intent of the local algorithms is to provide information and they are only meant as a guide. As a disclaimer, the Working Party notes that there are differences between algorithms at present, for example reporting units, decision limits, etc. Response: We have added your suggested sentences. 47 To add a glossary Recommend including a glossary to explain terminology such as highly sensitive troponin assays, sensitive (contemporary) assays; POC device; 99th percentile as an upper reference limit; reference population; limit of blank; limit of detection. Response: a glossary of terms has been added (Appendix 5). Under Postanalytical recommendations there is also a description of highly sensitive troponin assays. Page 18

4. Pathology Queensland, Public Pathology No. Comment Response 48 Page 5 (also page 7) under serial troponin sampling sub-point 3 and the timing of the second sample Recommendation does not allow for a more rapid rule-in of AMI where the clinical situation indicates a likely AMI. A shorter time interval for collection of the second sample may be useful to rule in an MI. The 3rd Universal Definition of Myocardial Infarction recommends that repeat testing be at 3-6 hours provided troponin assays have an imprecision of 20% CV at the 99th percentile concentration. This would include most POC troponin assays as indicated in Table 2 in your position paper. 49 Page 5 (also page 7) under clinical recommendations final dot point 50 Page 5 (also page 7) under clinical recommendations final dot point Response: This section has been revised. However, the emphasis remains on safely ruling out patients from the ED. The section reads as follows: Recommended timing of samples when using accelerated diagnostic pathways is obtaining the first sample at admission to ED and the second sample at 2-3 hours later. In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. Few POC troponin assays have been studied and reported as part of an accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. The Pathology Queensland algorithm recommends that repeat testing is done at 3-6 hours. Not only central laboratories but all laboratories whether they are central or not measure troponin. Response: The word central has been removed. It would be worthwhile emphasising that the laboratory-based troponin assay is the preferred testing option. Response: We have modified the point as follows: When a laboratory is used, results should be available as soon as possible, with a goal of within 60 minutes after the sample is received in the laboratory. Otherwise POC testing Page 19

51 Page 6 (also page 9 & 10) under preanalytical recommendations 52 Page 5 (also page 9) under analytical laboratory recommendations first dot point, second sub-point could be considered although the laboratory-based troponin assay is the preferred testing option when available. Regarding false negative and positive troponin results due to micro-clots, it might be worthwhile softening this sentence and to state that false negative and false positive troponin results are relatively common in ED samples due to preanalytical and analytical causes. Response: We have modified the sentence in the preanalytical section and added a sentence regarding analytical interferences in the Analytical section. It is not proven that POC assays are less diagnostically accurate at earlier time points. The assays are less analytically sensitive at low troponin levels and reference change value will be wider because of the higher imprecision at these low levels. We suggest you replace the words less diagnostically accurate by less analytically sensitive. Response: the change has been made. 53 Page 5, point 6 and page 7, point 4 Regarding POC assays being less accurate, reference change values are based on serial measurements usually within a period of 2-12 hours and hence are dependent on precision only. As POC assays are less precise at low troponin levels, a larger change value is required to give a statistically significant rise and/or fall of troponin. We suggest you replace the words less accurate by less precise. Response: the change has been made. 54 Page 9 second dot point Regarding istat imprecision, the between-run precision is approx. 20 % CV. Response: The sentence now reads: I-STAT POC Troponin I assay is clinically usable at a cut-off 0.04 µg/l with an imprecision of approx. 20% CV reported (Pathology Queensland local data collated over several months). Page 20

5. The Australasian College for Emergency Medicine (ACEM) No. Comment Response 55 General comments The document is an appropriately conservative and supportive document for practice for those using and/or interpreting POC troponins. 56 Executive summary: key recommendations (page 5) With regard to clinical recommendation Serial troponin measurements should be performed for all patients presenting with symptoms suggestive of ACS, unless the patient has been reliably ACS symptomfree for 24 hours ACEM considers that this does not align with the practice of most emergency department (ED) physicians. Feedback from members indicates that a single troponin measurement would be performed if the patient has been pain free for 8-12 hours prepresentation. ACEM, therefore, recommends that the AACB consider review of this clinical recommendation as it is incongruent with the timings of troponins suggested in the flowcharts. If it is felt that this recommendation needs to stay, we would suggest a major education initiative will be required to inform all Emergency Departments of this new practice. Response: In keeping with your comment and the NHF/CSANZ 2016 guidelines for assessment of ACS, we have amended the section to read as follows: Serial troponin measurements should be performed for all patients presenting with symptoms suggestive of ACS, unless the patient has been reliably ACS symptom-free for 12 hours when a single sample is appropriate. 57 With regard to clinical recommendation Serial testing should be performed using the same troponin assay Regarding the serial timing of troponins as shown in the flowcharts, the recommendations are as follows: - Recommended timing of samples using accelerated diagnostic pathways, using laboratory-based highly sensitive assays, is obtaining the first sample at admission to ED and the second sample at 2-3 hours later. - In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. It is unclear if the implication is, for example, that if a patient has a POC troponin at Page 21

and platform. Re-baseline the troponin values for individual patients if transported to a different hospital location where a different ctn assay is used, ACEM suggests that additional information is provided regarding the term re-baseline. time 0 due to a middle of the night presentation and then the lab commences work, the time 0 troponin is ignored and 2 lab troponins are performed. If this is the implication, what is the time interval between the two? Response: Where the ED troponin POC assay is located at the same site as the lab-based assay, then local arrangements can be in place to analyse the time 0 sample during laboratory working hours. The timeline for the lab analysis of the initial sample could be anything however, ranging from 1 hour (if time 0 = 8 am and lab commences at 9 am) to 11 hours (if time 0 = 10 pm and lab commences at 9 am) post patient admission to ED. These ED and laboratory arrangements are likely to differ between locations. We have added the following which gives a more general approach: At the second location collect a new baseline sample and a second sample at 2-3 hours (if testing with a highly sensitive troponin assay) and 6-8 hours (if testing with a less analytically sensitive assay) unless the patient has been symptom-free for >12 hours, when a single sample is appropriate. Page 22

6. The NSW Cardiac Network comment from Dr Matthew Bragg, Director of ED at the Bowral and District Hospital. No. Comment Response 58 A query about the nominated testing intervals for POC troponins The performance characteristics of these assays are usually not interpreted in isolation but as part of a risk stratification algorithm (including ECG and clinical assessment). Given that, the sensitivity and NPV of the risk stratification package as a whole may be higher, should the testing intervals be reduced to reflect that? For example if the overall ACS prevalence at entry is 10% but after negative ECG and low risk clinical assessment the prevalence drops to 5% this will increase the NPV of the trop assay to a point perhaps where a 3 or 4 hour testing interval is equivalent to or better than the nominated 6 hours. Or has the effect of risk stratification/ecg, etc, already been incorporated in the assay data you have presented? Response from Dr Louise Cullen: Matthew has raised some good points. The trouble is that few POC assays have been studied and reported as part of integrated assessment processes. Clinical assessment (gestalt) has been shown to be limited in the ability to accurately RO/RI AMI (Body R et al. Can emergency physicians rule in and rule out acute myocardial infarction with clinical judgement? Emerg Med J 2014;0:1 5. doi:10.1136/emermed-2014-203832). ECGs do help identify lower risk patients but again, the combination of ECGs plus POC Tn strategies has not been reported as such. There is only one paper (New Alere POC assay from Sally Aldous in NZ: Aldous S, et al. Comparison of new point-of-care troponin assay with high sensitivity troponin in diagnosing myocardial infarction. Int J Cardiol 2014;177:182-6.). This assay is not in use in Australia at this point in time. Accelerated strategies with POC Tns may be an option; however will the sensitivity/npv for RO AMI (in particular) be of a high enough level to safely discharge ED patients? Currently we cannot recommend any accelerated POC strategies due to a lack of evidence about their performance. What we have presented is a pure analysis using biomarkers only for AMI. This document provides clinicians with a clear starting point. Without good data though, we are not able to speculate about the impact of other clinical information can have on reducing POC Tn interval timing. On page 9, section VI the following has been added to clarify this point: - Recommended timing of samples using accelerated diagnostic pathways, using laboratory-based highly sensitive assays, is obtaining the first sample at admission to ED and the second sample at 2-3 hours later. - In order to safely discharge patients from the ED, it is recommended when using less sensitive (e.g. POC) troponin assays that a sample is collected at 6-8 hours after the first blood draw. Few POC troponin assays have been studied and reported as part of an accelerated, integrated assessment process that includes a combination of clinical assessment, ECGs and POC troponin. Page 23