RCPA Quality Assurance Program Research Articles

A-106 Sigma Metric Performance of Chemistries on the Abbott Alinity c Analyzer

Abstract Background The analytical performance of the Abbott Alinity c (Abbott Park, IL) was compared to our current Abbott Architect analyzers using sigma metrics. Methods Sigma metrics of 28 chemistry analytes on our six Abbott Alinity analyzers was compared to three Abbott Architect 16 000 analyzers and one Architect 8000 analyzer using Bio-Rad Multiqual quality control (QC) (Hercules, CA). Method imprecision was estimated for each assay using the cumulative coefficient of variation (CV) of 12 months data for each analyzer at three QC concentrations and calculating the average sigma. Method bias was estimated by comparing each analyzer QC mean to peer group QC means. Sigma values were calculated for each method as (TEa—Bias%)/CV% using allowable total error (TEa) from three sources: the CLIA proficiency testing evaluation limits, Australasian Association of Clinical Biochemistry allowable limits of performance (RCPA quality assurance programs Pty limited, Adelaide, Australia) and desirable biological variation (Ricos C et al, Scand J Clin Lab Invest. 1999, 59(7):491–500). Average sigma values were generated for each analyte and graded as optimal >6 sigma; good 5–6 sigma; marginal 3–5 sigma; or poor <3 sigma. Results Assay sigmas varied across QC concentrations and between analyzers of the same platform. Na, Cl, and CO2 had the lowest sigma performance. Since the sigma calculations are dependent on TEa, tighter TEa limits (Ricos and RCPA) generated lower sigmas compared to the wider CLIA TEa limits especially for Ca, Cl, total bilirubin, CO2, Na, total protein, and HDL. Conclusion None of the chemistries met optimal six sigma performance for all analytes, concentrations of QC and sources of TEa. However, the assay performance between analyzer platforms was comparable.

Challenging assay robustness with RCPAQAP external quality assurance programs

Background: External quality assurance (EQA) programs commonly test for precision and accuracy of quantitative assays, but less frequently for common interferences that could potentially impact patient results. RCPA Quality Assurance Programs (RCPAQAP) in conjunction with the Australasian Association of Clinical Biochemists (AACB) now includes challenge samples in Chemical Pathology programs.

RCPA quality assurance programs pilot – online haematology morphology course

RCPA quality assurance programs pilot – online haematology morphology course

Interesting QA cases

Sexually transmitted infections (STI) are a significant cause of morbidity and some mortality globally. Identification of STIs is important for prevention, treatment and public health. ‘Increases in STI transmissions are a concern in their own right, but also because they can increase the possibility of HIV transmission. Condoms are the best way to reduce the risk of acquiring STIs, however regular comprehensive sexual health testing is also vital, as this is the only way that cases can be diagnosed and the cycle of transmission can be broken.' 1 Seventy percent of medical decisions rely on laboratory results. 2 The RCPA Quality Assurance Programs Pty Limited (RCPAQAP) offered 45 surveys in 12 modules in 2014 for potentially sexually transmitted infections, and 536 participants enrolled in these modules. RCPAQAP reports enable participants to review the validity of their assay results and identify procedural and technical problems through the comparison of data from other participants performing the same test. Participants receive recommendations with regard to possible reasons for results outside the consensus as well as suggested testing algorithms where appropriate. Multiple surveys throughout the year facilitate trend analysis for ongoing monitoring of performance in testing for STIs. 3

Vitamin B1 and B6 method harmonization: Comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program

ObjectivesThe RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, β-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6. Design and methodsA questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). ResultsAll nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. ConclusionsThe immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

RCPA quality assurance program: review of the urology module

In the course of the presentation a review of the urology module of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP) will be made, including an assessment of the results pertaining to straightforward urogenital cases, and those that presented more of a challenge to respondents. One aspect that will be examined in more depth is the range of responses given in regard to Gleason scoring of prostate adenocarcinoma, an area that has not been formally assessed to date but will be assessed going forward. Discussion will also be made concerning the performance monitoring project and the framework to be introduced in regard to anatomical pathology.

RCPA Quality Assurance Programs Pty Ltd Presentations

RCPA Quality Assurance Programs Pty Ltd Presentations

RCPA Quality Assurance Programs Pty Ltd Presentations

RCPA Quality Assurance Programs Pty Ltd Presentations

RCPA biosecurity QAP introduction

The Royal College of Pathologists of Australasia, Quality Assurance Programs Pty Ltd (RCPA QAP) has been utilising a grant from the Department of Health and Ageing to establish a proficiency testing program for Security Sensitive Biological Agents (SSBAs). The BioSecurity Quality Assurance Program (QAP) was introduced in June 2009 with an initial focus on preparing surveys for the proficiency testing of Bacillus anthracis. The surveys are available to laboratories within Australia that are registered to handle SSBAs and will soon be available to laboratories that may receive a suspected SSBA. Participants enrol to take part in the surveys which are offered four/five times per annum and are sent the survey containing a clinical history and specimens for testing. The BioSecurity QAP receives the results which are then collated and written into a report to be reviewed by program collaborators. All participant information is de-identified after the collation process. The surveys will be free of charge in 2010 and the reports available electronically to all participants and other interested organisations on request. The reports are aimed at educating the participants in the detection, characterisation and confirmation or the ‘ruling out’ of B. anthracis. Two BioSecurity QAP surveys were completed in 2009 with reports sent to ten participating laboratories. The Royal College of Pathologists of Australasia, Quality Assurance Programs Pty Ltd (RCPA QAP) has been utilising a grant from the Department of Health and Ageing to establish a proficiency testing program for Security Sensitive Biological Agents (SSBAs). The BioSecurity Quality Assurance Program (QAP) was introduced in June 2009 with an initial focus on preparing surveys for the proficiency testing of Bacillus anthracis. The surveys are available to laboratories within Australia that are registered to handle SSBAs and will soon be available to laboratories that may receive a suspected SSBA. Participants enrol to take part in the surveys which are offered four/five times per annum and are sent the survey containing a clinical history and specimens for testing. The BioSecurity QAP receives the results which are then collated and written into a report to be reviewed by program collaborators. All participant information is de-identified after the collation process. The surveys will be free of charge in 2010 and the reports available electronically to all participants and other interested organisations on request. The reports are aimed at educating the participants in the detection, characterisation and confirmation or the ‘ruling out’ of B. anthracis. Two BioSecurity QAP surveys were completed in 2009 with reports sent to ten participating laboratories.

Quality assurance in immunohistochemistry – an update

Introduction Diagnosis of tumours and treatment decisions for patients including breast cancer patients are based on determination of immunohistochemistry biomarkers. The accurate assessment of hormone receptor status in breast cancer and the correct classification of lymphomas is essential for appropriate patient care. The immunohistochemistry (IHC) Quality Assurance Program (QAP) includes a number of modules: diagnostic, lymphoma markers, breast markers, bright-field HER2 ISH and technical. Other countries have similar quality assurance programs. 1,2 Methods The IHC modules consist of a number of technical exercises per year with assessment of different biomarkers. The technical module evaluates different biomarkers across a range of antibodies that would be important for diagnosis of tumours. The diagnostic module allows participants to select a panel of antibodies to obtain a correct diagnosis mimicking real patient samples. The breast markers module evaluates oestrogen receptor, progesterone receptor and HER2 with a separate component for bright-field HER2 ISH testing. An audit of patient results is also performed for the breast biomarkers. The lymphoma module is used to assess markers used for classification and diagnosis of both Hodgkin’s disease and non-Hodgkin’s lymphoma. Slides are sent to participants comprising either tissue microarray blocks or composite blocks from different tissues. Homogeneity and stability testing are performed on the sections and every 20th section is stained to ensure representative tissue. Antigenic site stability is also evaluated. Slides are stained by the participating laboratory and returned for evaluation by a committee of scientists and a pathologist. Each slide is evaluated independently and an average score is returned. Each slide is scored from 0 to 5. The assessment criteria used are: • intensity of true positivity is of reasonable strength • absence of background staining (good signal to noise ratio) • sensitivity – all target tissues labelled • localisation – only target antigenic sites labelled • chromogen character – crisp and distinct • counterstain quality – complementary not obscuring • absence of artifacts • adequacy of controls – sufficiently sensitive. A score A control slide is also assessed as satisfactory, borderline or unsatisfactory. Results Participants’ results vary depending on the module being assessed and there has generally been an improvement of results over time, however deficiencies are still noticed for some biomarkers. Results of some of the modules will be presented. Discussion Immunohistochemistry is used to diagnose patients’ tumours and to determine patient treatment. Poor quality IHC will therefore have a significant impact on patient outcome. It is therefore essential that the results generated by the laboratory are accurate. External QAP forms an essential part of laboratory assessment. Participants frequently comment that the supplied material from the QAP is different to their in-house samples and that because the in-house controls worked the results are correct. This statement does not recognise the difference between Quality Control and Quality Assurance. The in-house controls, even if placed on the same slide, are Quality Controls and indicate that the antibody and the detection system are working. They do not indicate whether the result obtained is in fact correct and accurate. This is well recognised in clinical pathology where a method is validated against a reference method to determine accuracy and then QC is run internally to ensure that the reagents and instruments are working. In many cases for immunohistochemistry the controls selected are inappropriate, excessively large, show fixation variation or only reflect one level of staining. Conclusion High quality IHC results are essential to enable treating clinicians to optimise therapy for patients. The introduction and expansion of a specific quality assurance module for immunohistochemistry has enabled an ongoing assessment of the performance of biomarkers in Australia, New Zealand and South-East Asia. Despite the extraction of large amounts of information in regards to the methodology used for staining, it is usually not possible to identify one specific method that results in optimal staining although sample methods with high marks are reported. Optimisation of retrieval for a wide range of sensitivities is considered to be the important factor in achieving satisfactory results. The development of the program has resulted in an improvement in the staining quality for a number of biomarkers. Comments in this report were prepared for and on behalf of the RCPA QAP.

The role of external quality assurance in progressing quality in haemostasis: initiatives from the RCPA QAP

The Haematology Quality Assurance Program (QAP) has been conducting proficiency testing in haemostasis for over 40 years. Regular multilaboratory surveys, with peer group assessments, are sent out 2–8 times a year, depending on the specialty. The face of haemostasis testing has changed considerably over the past decade and this has presented many challenges for external quality assurance in haemostasis. One area that has seen considerable expansion is international normalised ratio (INR) point of care (POC) instruments for monitoring warfarin therapy, and this has resulted in the development of the POC-INR program in recent years. The past 7 years has also seen the expansion of the Special Haemostasis program to include testing for lupus anticoagulant, protein C and S, activated protein C resistance and von Willebrand factor. Many other new initiatives have been introduced including the factor inhibitor study exercises, PFA-100 testing trials, and D-Dimer special exercises. The evaluation of results from a broad range of laboratories, expert and non-expert alike, have helped identify potential issues within the haemostasis testing. With our expert advisory committee, the RCPA QAP has been able to provide suggestions to help minimise potential diagnostic errors in this field. 1

Developments in quality assurance in anatomical pathology (RCPA quality assurance programs P/L)

Quality assurance programs in anatomical pathology consist of technical and diagnostic modules. The technical modules evaluate proficiency in both standard histopathology and immunohistochemistry. The diagnostic modules evaluate skills of pathologists;these provide annually one general module (30 cases) and eight specialist modules (total 105 cases), e.g., dermatopathology (10 cases). Cases are a ‘structured mix': most are of conditions seen regularly in an average histopathology practice interspersed with uncommon or emerging entities and those with practical problems of diagnosis, e.g., dual pathology, presence of microbial pathogens. The use of virtual microscope images allows surveys to use small biopsy specimens or cases with only a small focus of abnormal tissue. Target diagnoses are set for each case but fundamentally performance is assessed by peer review comparison using a novel non-parametric system. This provides an aggregate performance profile for each module for all participants against which each compares their individual performance. It also permits comparison of different types of case for performance evaluation, e.g., in validating the use of virtual microscopy. Studies are also undertaken of data reported by participants but not currently assessed, e.g., tumour grading. The outcomes of some diagnostic surveys for 2008 will be presented and plans for the future outlined.

RCPA QAP immunopathology update: how can the immunology QAP help your laboratory?

The Immunology Quality Assurance Program (QAP) provides an accredited external quality assurance program to over 560 laboratories in Australia, New Zealand and overseas. Laboratories enrolled in the program have access to educational services such as case studies, participation in pilot programs, and online data analysis using the QAP database of results and methodologies. Other services provided include personalised consultation on your laboratory’s reports, available on request.

Challenges in haematology morphology external quality assurance

Morphology remains a key skill for haematology staff despite the introduction of new non-morphological techniques. The Haematology Morphology program of the RCPA Quality Assurance Program (QAP) began over 30 years ago and now has >600 participating laboratories who are sent 12 survey cases annually. With this increase, challenges have arisen to ensure: • Sufficient diagnostic material sometimes including bone marrow for all participants. • A balance of basic cases for proficiency testing and more unusual cases for education. • A balance between scenarios requiring morphological skills alone and those providing more information which may better resemble clinical practice but allow bypassing of morphological skills. • The most effective means for submitting and recording participant responses. • A graded assessment of participants’ ability to recognise morphological features and to make a diagnosis which is relevant for the full spectrum of participants and provides some credit for partially correct responses. A virtual microscopy system is being developed to enhance the program in relation to the first three issues. An online data entry option and a numerical scoring system for assessment have been introduced to address the last two issues. Ongoing modifications continue in response to feedback and to participants’ needs.

The Diagnostic Dilemma: Dual Presentations of Clinical Mucosal Bleeding and Venous Thrombosis Associated with the Presence of Thrombophilia Markers and Mild Reduction in von Willebrand Factor

A prothrombotic and hemorrhagic state can separately manifest in one patient and can potentially cause several diagnostic problems. We report an intriguing case as an example of a potential hemostasis-based diagnostic dilemma. A 29-year-old female patient presented with a personal history of menorrhagia and other mucosal bleeding and renal ovarian thrombosis. Previous investigations had uncovered several diagnostic anomalies, including von Willebrand disease (VWD), factor V Leiden (FVL), antiphospholipid syndrome, and thrombocytopaenia. Previous therapy in this patient included heparin and warfarin for the thrombosis and desmopressin acetate (DDAVP) and antifibrinolytic therapy for surgical management. Subsequent laboratory testing with fresh samples consistently confirmed an equivocal (borderline normal/abnormal) level of von Willebrand factor (VWF) and FVL with activated protein C resistance (APCR). A patient sample, differentially labeled according to the tests being performed, was later distributed for blind testing to participants within several modules of the RCPA Quality Assurance Program (QAP). Most participants reported a low level of VWF consistent with possible mild Type 1 VWD, and most (but not all) reported a positive finding for APCR. All participants correctly reported the sample as heterozygous for the FVL mutation, negative for the Prothrombin gene mutation G20210A, and heterozygous for the methylenetetrahydrofolate reductase (MTHFR) mutation C677T. Interestingly, a significant number of laboratories performing Protein S testing using clot-based procedures also identified a false Protein S deficiency. In conclusion, this exercise showed how, either depending on the clinical review and specific laboratory investigation and tests performed, a pro-bleeding diagnosis (of either VWD or thrombocytopenia) or pro-thrombophilia risk (Antiphospholipid Syndrome or FVL/APCR or false Protein S deficiency) could potentially and differentially arise in the one patient.

Reporting units for therapeutic drug monitoring: a correctable source of potential clinical error

Variation between laboratories and reference sources in the units used for reporting pathology results raises the possibility of medical error. Data submitted to the RCPA Quality Assurance Programs demonstrate wide variation in the units used for reporting therapeutic drug concentrations. This potential source of medical error needs to be addressed by all parties involved in communicating drug concentrations and providing support information.

Anti-cardiolipin antibody testing and reporting practices among laboratories participating in a large external Quality Assurance Program

To investigate differences in anti-cardiolipin antibody (aCL) testing and reporting practices among diagnostic laboratories participating in the RCPA Quality Assurance Program (QAP) Immunology Program. A survey was sent to all 58 laboratories enrolled for aCL testing in the RCPA QAP Program requesting the following information: (1). manufacturer/type of assay; (2). isotype tested; (3). use of calibrators and controls; (4). whether calibrators, control and patient specimens were performed in singles or duplicates; (5). whether results were reported semi-quantitatively and/or numerically; (6). values used to define negative/positive and semi-quantitative cut-offs and how they were determined; and (7). whether interpretative comments were provided and their content. Thirty-two surveys (55%) were received. Significant differences were present particularly in the following areas: (1). whether IgM isotype aCL testing was performed routinely or only on specific request; (2). whether controls/calibrators/specimens were tested in singles or duplicates; (3). whether results were reported numerically and/or semi-quantitatively; (4). the values used to define negative/positive and semi-quantitative range cut-offs; and (5). whether interpretative comments were provided and their content. These differences in testing and reporting practices are likely to contribute to the variation in aCL results reported by different laboratories, particularly among those using the same assay.

Laboratory Testing for von Willebrand’s Disease: An Assessment of Current Diagnostic Practice and Efficacy by Means of a Multi-laboratory Survey

We report an evaluation of current laboratory practice for the diagnosis of von Willebrand's disease (VWD) by means of a multilaboratory survey. This assessment was undertaken with the RCPA Quality Assurance Program (QAP) in Haematology, which covers a wide geographic area encompassing Australia, New Zealand and Asia. A total of 25 laboratories actively involved in testing for VWD were selected to participate in a sample testing assessment exercise. Samples comprised 10 plasmas: (i) a normal plasma pool (in duplicate), (ii) this pool diluted to 50% (in duplicate), (iii) a normal individual (X1), (iv) severe Type 1 VWD (X1), (v) Type 2B VWD (x2 unrelated donors), (vi) Type 3 VWD (x1), (vii) Type 2A VWD (x1). Laboratories were asked to perform all tests available to them in order to establish a laboratory diagnosis of VWD, and then to comment on the possibility or otherwise of VWD. Overall findings indicated a wide variation in test practice, in the effectiveness of various test procedures in detecting VWD, and in the ability of various composite test panels to identify type 2 VWD subtypes. Firstly, while all laboratories (n = 25) performed tests for FVIII:C activity, von Willebrand factor 'antigen' (VWF:Ag) and a functional VWF assay [using the ristocetin cofactor assay (VWF:RCo; n = 23) and/or the collagen binding assay (VWF:CBA; n = 12)], only three laboratories carried out VWF:Multimer analysis. Secondly, for the three quantitative VWF assays, 10/25 (40%) laboratories performed all three, whereas 15/25 (60%) performed only two [VWF:Ag and VWF:RCo (n = 13); VWF:Ag and VWF:CBA (n = 2)]. Thirdly, a variety of assay methodologies were evident for VWF:Ag [ELISA, electro-immuno diffusion (EID), latex immuno-assay (LIA), and VIDAS assay] and VWF:RCo (platelet agglutination/'aggregometry' and a 'functional VWF:RCo-alternative' ELISA assay). Between method analysis for the quantitative VWF assays showed that the VWF:RCo yielded the greatest degree of inter-laboratory assay variation, and had the poorest overall performance with respect to sensitivity to low levels of VWF. The VWF:CBA also performed better than the VWF:RCo in terms of ability to detect functional VWF 'discordance' (i.e. Type 2 VWD). Within VWF:Ag method analysis showed that the EID assay procedure was associated with the greatest variation in assay results, while the EID and LIA test methods showed poorer sensitivity at low VWF levels compared to the ELISA method. Within the VWF:RCo assay procedure, greatest variation in assay results and poorest sensitivity to low VWF levels was obtained using the agglutination method; however, the agglutination procedure showed better performance than the 'functional VWF:RCo-alternative' ELISA assay in identifying Type 2 VWD plasma samples. Finally, despite identified variations, most laboratories appeared to understand the complexities involved in the VWD-diagnostic process, and made appropriate diagnostic predictions regarding tested samples. From a total possible 246 interpretation events, laboratories in most cases correctly identified normal samples as normal (67/75 events = 89%), and VWD samples as derived from individuals with VWD (117/121 events = 97%). Moreover, when VWD was suggested by laboratory findings, laboratories usually correctly predicted the general subtype of VWD present (96/109 events = 88%). When 'misinterpretations' occurred, these could often be linked to the test panels utilised by laboratories. That is, laboratories using the VWF:Ag and VWF:RCo combination were more likely to incorrectly identify samples derived from Type 2 VWD patients as being Type 1, Type 1 VWD patients as being Type 2, and normal plasma samples as potentially derived from patients with VWD, compared to those using the VWF:Ag and VWF:CBA.

Difficulties with the serologic diagnosis of infectious mononucleosis: A review of the RCPA quality assurance programs

The Royal College of Pathologists of Australasia's Quality Assurance Programs (QAP) on serologic assays for infectious Mononucleosis (IM) have identified a number of important problems in laboratory diagnosis of this condition. A wide range of assays for the diagnosis of acute Epstein-Barr virus (EBV) infection are available, although heterophile antibody tests are still the most frequently performed procedures for the diagnosis of IM. In 1993, eighty-six (54%) of the 159 participating laboratories performed only a heterophile test; of these, 71% did not provide an interpretation of their results and none mentioned the need for confirmatory EBV-specific antibody testing. This revealed a lack of appreciation that heterophile tests should only be used for screening, due to their inferior sensitivity of less than 50% in children and 80-90% in adults and specificity of 95% compared to EBV-specific assays. For these reasons the use of heterophile tests is discouraged. Although EBV-specific serology has traditionally been done by immunofluorescence (IF), the use of reliable ELISA methods using purified EBV-antigens is increasing. False negative EBV VCA IgM ELISA test results were obtained by laboratories using unpurified or unspecified VCA antigens. As only five laboratories used these tests in the 1992 QAP, the tests' true performances could not be properly assessed, suggesting the need for independent studies. Variable results were obtained by laboratories using commercial EBNA IgG assays (in addition to EBV IgM) suggesting that an assessment of these kits would also be worthwhile. The QAP reaffirm that reliable EBV IgM detection is the most useful test for the diagnosis of IM, even where other EBV markers are also used. In the 1992 and 1993 QAP, laboratory performance of both heterophile and EBV-specific tests was better with non-reactive serum specimens (99% (correct) than with reactive serum specimens (90% correct).