Metrological traceability for monkeypox virus analysis: development of an inter-laboratory validated digital PCR reference measurement procedure

The analysis of heterogeneous protein analytes is very complicated to standardize, but these measurements must be viewed as fundamental to the practice of clinical chemistry. The clinical importance of selected protein measurements such as hemoglobin A1c (Hb A1c),5 thyroid-stimulating hormone, and cardiac troponin I (cTnI) is underscored by their routine use in the diagnosis, prognosis, monitoring, and management of disease, and their incorporation into professional guidelines. Standardization is difficult for proteins because there are few reference measurement procedures for these analytes, few primary (pure substance) reference materials (RMs) have been developed, and some of the RMs that are available can be used only for assay calibration with restrictions. In addition, few secondary (matrix-based) RMs with assigned values are available. In fact, the reference measurement system for the majority of clinically relevant proteins fall into the relatively weak Category 4, according to International Organization for Standardization (ISO) document 17511 (1). Furthermore, protein reference measurements sometimes appear to belong in a higher category but are in fact subject to analytical artifacts and prone to bias. By the criteria of the recently published “roadmap for harmonization” (2), standardization and harmonization of measurement would be regarded as high priority and mission critical for many proteins in laboratory medicine. cTnI is one such protein because its measurement represents the cornerstone for the diagnosis, prognosis, and management of patients with suspected and confirmed acute coronary syndromes (3, 4). Standardization is based on the concept of metrological traceability, as described, for instance, in the ISO 17511 document (1). According to ISO 17511, standardization-of-measurement results for a substance require the metrological traceability chain outlined in Fig. 1A. This chain begins with a primary reference measurement procedure, which assigns quantity values to a primary RM. Primary RMs are used to assign values …

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) published the reference measurement procedure (RMP) for ALP measurement in 2011. However, the RMP is of high requirements for laboratories, complicated, time-consuming and high cost of reagents. Many manufacturers do not trace results to the higher procedure. And there is currently no designated comparison method (DCM) for ALP measurement. Thus, the standardization of ALP measurement is hindered. Automatic biochemical analyzers are easy to operate and widely used in clinical laboratories. Therefore, according to the RMP, establishing a DCM based on an automatic biochemical analyzer will be a practical way to establish traceability to the accuracy base and promote the standardization of ALP measurement. On the basis of conforming to the RMP recommended by IFCC as far as possible, the DCM was established based on a Thermo Indiko automatic biochemical analyzer. Performances of the method were validated. The DCM repeatability and within laboratory imprecision was <1% and <2.5%, respectively. For evaluation of trueness, the biases were within the equivalent limits. Measurement procedure comparisons and biases estimation were carried out between the DCM, the RMP, and the six routine methods using a panel of 40 individual human serum samples. The comparisons between the DCM and the RMP gave satisfying results. Compared with the DCM, the relative biases of some routine methods failed to meet the bias limit derived from biological variation.

Measurement accuracy has been assessed for many different blood glucose monitoring systems (BGMS) over the years by different study groups. However, the choice of the comparison measurement procedure may impact the apparent level of accuracy found in such studies. Measurement accuracy of 18 different BGMS was assessed in a setting based on ISO 15197 using two different comparison methods in parallel: a glucose oxidase (GOD)-based and a hexokinase (HK)-based method. Accuracy limits of ISO 15197 were applied, and additional analyses were performed, including bias, linear regression, and mean absolute relative difference (MARD) to assess the impact of possible differences between comparison methods on the apparent level of accuracy. While ≈80% of BGMS met the accuracy criteria of ISO 15197 when compared with the respective manufacturers' reference measurement procedure, only two-thirds did so against both comparison methods. The mean relative bias ranged from -6.6% to +5.7% for the analysis against the GOD-based method and from -11.1% to +1.3% for the analysis against the HK-based method, whereas MARD results ranged from 3.7% to 9.8% and from 2.3% to 10.5%, respectively. Results of regression analysis showed slopes between 0.85 and 1.08 (GOD-based method) and between 0.81 and 1.01 (HK-based method). The results of this study indicate that there are systematic differences between the reference measurement procedures used for BGMS calibration as well as for system accuracy assessment. Because of the potential impact on therapy of patients with diabetes resulting from these differences, further steps toward harmonization of the measurement procedures' results are important.

Background Quantitative results based on reference measurement procedures (RMP) require the calculation of the uncertainty of the result. In order to take the uncertainty of the full traceability chain into account a modularisation of the different execution steps is needed, starting from the primary reference material until the final concentration value. Different strategies have to be applied and combined to obtain the final uncertainty.We combine different ways for calculating the uncertainty of an isotope dilution -liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure for Cortisol, and explain the differences between Type A and Type B uncertainties and best strategies to choose from them. In addition, combinations of results from subsequent steps to a final overall uncertainty are presented. Methods An ID-LC-MS/MS based reference measurement procedure requires as a starting point a primary reference material with known purity of the analyte under consideration. Nowadays this purity assessment is often done with quantitative nuclear magnetic resonance (qNMR) spectroscopy. The qNMR uncertainty calculation is a Type A approach, based on replicate sample preparations and measurements. With this primary material a first set of primary calibrators is produced, often through multiple steps of weighings, preparation of stock solutions and dilutions. For these steps balances, volumetric flasks, or pipettes are used, all of them coming with an error budget which has to enter the uncertainty budget of the primary calibrator preparation. We will show how this can be done through Type B uncertainty calculation. Based on the primary calibrators the LC/MS instrument is calibrated and measurements of individual samples are carried out. We will introduce a hierarchical variability experiment, which takes into account the most important error sources and results in a Type A uncertainty for the LC/MS measurements.Finally, the errors of the LC/MS measurements have to be combined with those of the primary calibrator preparation to obtain the overall uncertainty of an LC/MS reference measurement procedure. Results We show the approaches and calculation strategies based on the Cortisol candidate reference measurement procedure, developed within the Roche LC/MS Reference Measurement Procedure project. The purity assessment by qNMR carries an uncertainty of 0.18%. This is incorporated into the uncertainty calculations of the primary calibrators, where in addition uncertainties from weighing and dilutions are incorporated resulting in uncertainties of the primary calibrator values between 0.6% and 1.1%. Combining these uncertainties with the variabilities of the LC/MS measurement procedure results in final uncertainties between 1.6% and 2.8%. Conclusion We have developed a sound framework for uncertainty calculation of reference measurement procedures, taking into account the different procedure steps, and included this into a process for the management of the different input variables up to comprehensive reports of the calculations.

We undertook this study to evaluate method differences for 5 components analyzed by immunoassays, to explore whether the use of method-dependent reference intervals may compensate for method differences, and to investigate commutability of external quality assessment (EQA) materials. Twenty fresh native single serum samples, a fresh native serum pool, Nordic Federation of Clinical Chemistry Reference Serum X (serum X) (serum pool), and 2 EQA materials were sent to 38 laboratories for measurement of cobalamin, folate, ferritin, free T4, and thyroid-stimulating hormone (TSH) by 5 different measurement procedures [Roche Cobas (n = 15), Roche Modular (n = 4), Abbott Architect (n = 8), Beckman Coulter Unicel (n = 2), and Siemens ADVIA Centaur (n = 9)]. The target value for each component was calculated based on the mean of method means or measured by a reference measurement procedure (free T4). Quality specifications were based on biological variation. Local reference intervals were reported from all laboratories. Method differences that exceeded acceptable bias were found for all components except folate. Free T4 differences from the uncommonly used reference measurement procedure were large. Reference intervals differed between measurement procedures but also within 1 measurement procedure. The serum X material was commutable for all components and measurement procedures, whereas the EQA materials were noncommutable in 13 of 50 occasions (5 components, 5 methods, 2 EQA materials). The bias between the measurement procedures was unacceptably large in 4/5 tested components. Traceability to reference materials as claimed by the manufacturers did not lead to acceptable harmonization. Adjustment of reference intervals in accordance with method differences and use of commutable EQA samples are not implemented commonly.

Background Monkeypox virus (MPXV) is a member of Poxviridae family and belongs to the genus of orthopoxvirus. The Monkeypox virus has a double stranded DNA genome of approximately 197 kb with almost 190 nonoverlapping ORFs. Monkeypox virus encodes all the enzymes needed for transcription and replication process of the viral genome. It has been hypothesized that progressive gene loss, primarily at the terminal end of the genome, has been the justification behind evolution of a new adaptive virus capable of attacking humans and causing serious illness. Monkeypox virus infection in human can lead to a smallpox-like illness with almost 11% fatality rate in individuals who are non-vaccinated. Monkeypox virus infects a wide range of mammalian cells without the need for specific host receptors, cell entry molecules and replication machinery. No specific approved treatment exists for Monkeypox. The symptoms could be managed by treating secondary bacterial infections, reducing symptoms, and providing supportive care. ST-246 (tecovirimat), FDA approved drug against Smallpox virus, has shown some efficacy against Monkeypox, and therefore, accurate diagnosis, usually through PCR-based testing is critical. LGC Clinical Diagnostics has developed AccuPlex Monkeypox Reference Material, which is a non-infectious, stable, and reproducible standard to aid nucleic acid testing for Monkeypox virus. Methods AccuPlex™ Monkeypox Reference Material consists of recombinant Adeno virus bearing the complete Monkeypox genes: J2L (TNF receptor gene), F8L (DNA polymerase gene), F3L (Double-stranded RNA-binding protein, inhibitor of IFN signaling), and N3R (orthopoxvirus MHC class I-like protein -OMCP). The design of the recombinant virus is based on the sequence of ON585038.1 USA strain from the recent outbreak. A digital PCR assay was developed using the E9L Non-variola (NVAR) Orthopox Generic Assay design described in Y. Li et al. /Journal of Clinical Virology 36 (2006) 194–203. Digital PCR testing using the BioRad QX-200 droplet digital PCR system was used to quantitate the viral copies/mL and guide formulation. The final product is targeted as a low positive control, approximately 2–3 times the detection limit of commercial PCR testing methods and is formulated in defibrinated human plasma. Results AccuPlex™ Monkeypox Reference Material gave an average value of 5.33E+03 copies/mL ± 6.34E+02 copies/mL by digital PCR. Along with the undiluted sample, 1:2 and 1:4 dilutions were made which were then tested on Quest Diagnostics Qualitative RT-PCR platform to verify performance and compatibility with the assay detection limits. All three samples were positively detected. Conclusion LGC Clinical Diagnostics has developed a stable, non-infectious reference material using recombinant Adenovirus technology for assays that detect Monkeypox DNA. Our study helps ensure that the control will be low positive for Monkeypox virus and could be used for monitoring the sensitivity of the assay. Data presented indicates the compatibility of the reference material with commercially available real time PCR assays. These materials may be used by labs for development, validation, training, and ongoing QC of Monkeypox detection assays and workflows.

AT THE DESK OR ONLINE: REFERENCE TRAINING, MEASUREMENTS, AND GUIDELINES RUSA Professional Tools: Reference Guidelines http://www.ala.org/ala/rusa/rusaprotools/referenceguide/referenceguidelines.htm. Reviewed by Penny Scott RUSA Professional Tools: Guidelines for Implementing and Maintaining Virtual Reference Services http://www.ala.org/ala/rusa/rusaprotools/referenceguide/virtrefguidelines.htm. Reviewed by Susanne Markgren. Program http://www.arl.org/stats/ by Dawn Lowe-Wincentsen. Digital Reference Services Bibliography http://www.lis.uiuc.edu/∼b-sloan/digiref.html. Reviewed by Susanne Markgren. Digital Reference Education Initiative http://drei.syr.edu/index.cfm. Reviewed by Lydia Eato Harris. Ohio Reference Excellence (ORE on the Web) http://www.olc.org/Ore/index.html. Reviewed by Dawn Eckenrode. Library Staff Competencies http://www.librarysupportstaff.com/4competency.html. Reviewed by Beth Thomsett-Scott Association of Research Libraries Statistics and Measurement Research Methods Knowledge Base http://www.socialresearchmethods.net/kb/. Reviewed by Barbara Burd.

Standardization of insulin and C-peptide – A status report

Traceability as a unique tool to improve standardization in laboratory medicine

Proficiency testing (PT), or external quality assessment (EQA), is intended to verify on a recurring basis that laboratory results conform to expectations for the quality required for patient care. Key factors for interpreting PT/EQA results are knowledge of the commutability of the samples used and the process used for target value assignment. A commutable PT/EQA sample demonstrates the same numeric relationship between different measurement procedures as that expected for patients' samples. Noncommutable PT/EQA samples frequently have a matrix-related bias of unknown magnitude that limits interpretation of results. PT/EQA results for commutable samples can be used to assess accuracy against a reference measurement procedure or a designated comparison method. In addition, the agreement of the results between different measurement procedures for commutable samples reflects that which would be seen for patients' samples. PT/EQA results for noncommutable samples must be compared to a peer group mean/median of results from participants who use measurement procedures that are expected to have the same or very similar matrix-related bias. Peer group evaluation is used to asses whether a laboratory is using a measurement procedure in conformance to the manufacturer's specifications and/or in conformance to other laboratories using the same technology. A noncommutable PT/EQA sample does not give meaningful information about the relationship of results for patients' samples between different measurement procedures. PT/EQA provides substantial value to the practice of laboratory medicine by assessing the performance of individual laboratories and, when commutable samples are used, the status of standardization or harmonization among different measurement procedures.

Commutability assessment of NIST SRM 3666 albumin and creatinine in frozen human urine among routine clinical laboratory measurement procedures.

In laboratory medicine, the terms “standardization” and “harmonization” are frequently used interchangeably as the final goal is the same: the equivalence of measurement results among different routine measurement procedures over time and space according to defined analytical and clinical quality specifications. However, the terms define two distinct, albeit closely linked, concepts based on traceability principles. The word “standardization” is used when results for a measurement are equivalent and traceable to the International System of Units (SI) through a high-order primary reference material and/or a reference measurement procedure (RMP). “Harmonization” is generally used when results are equivalent, but neither a high-order primary reference material nor a reference measurement procedure is available. Harmonization is a fundamental aspect of quality in laboratory medicine as its ultimate goal is to improve patient outcomes through the provision of accurate and actionable laboratory information. Patients, clinicians and other healthcare professionals assume that clinical laboratory tests performed by different laboratories at different times on the same sample and specimen can be compared, and that results can be reliably and consistently interpreted. Unfortunately, this is not necessarily the case, because many laboratory test results are still highly variable and poorly standardized and harmonized. Although the initial focus was mainly on harmonizing and standardizing analytical processes and methods, the scope of harmonization now also includes all other aspects of the total testing process (TTP), such as terminology and units, report formats, reference intervals and decision limits as well as tests and test profiles, requests and criteria for interpretation. Several projects and initiatives aiming to improve standardization and harmonization in the testing process are now underway. Laboratory professionals should therefore step up their efforts to provide interchangeable and comparable laboratory information in order to ultimately assure better diagnosis and treatment in patient care.

A high allele burden of the KIT D816V mutation in peripheral blood or bone marrow aspirates indicates multi-lineage hematopoietic involvement and has been associated with an aggressive clinical course of systemic mastocytosis. Since mast cells are substantially underrepresented in these liquid specimens, their mutation burden likely underestimates the tumor burden of the disease. We used a novel previously validated digital polymerase chain reaction (PCR) method for KIT D816V analysis to systematically analyze the mutation burden in formalin-fixed, paraffin-embedded bone marrow tissue sections of 116 mastocytosis patients (91 with indolent and 25 with advanced systemic mastocytosis), and to evaluate for the first time the clinical value of the tissue mutation burden as a novel biomarker. The KIT D816V mutation burden in the tissue was significantly higher and correlated better with bone marrow mast cell infiltration (r=0.68 vs. 0.48) and serum tryptase levels (r=0.68 vs. 0.58) compared to that in liquid specimens. Furthermore, the KIT D816V tissue mutation burden was: (i) significantly higher in advanced than in indolent systemic mastocytosis (P=0.001); (ii) predicted survival of patients in multivariate analyses independently; and (iii) was significantly reduced after response to cytoreductive therapy. Finally, digital PCR was more sensitive in detecting KIT D816V in bone marrow sections of indolent systemic mastocytosis patients than melting curve analysis after peptide nucleic acid-mediated PCR clamping (97% vs. 89%; P<0.05). In summary, digital PCR-based measurement of KIT D816V mutation burden in the tissue represents a novel biomarker with independent prognostic significance that can also be employed for monitoring disease progression and treatment response in systemic mastocytosis.

The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.

Background Laboratory professionals should independently verify the correct implementation of metrological traceability of commercial measuring systems and determine if their performance is fit for purpose. We evaluated the trueness, uncertainty of measurements, and transferability of six clinically important enzyme measurements (alanine aminotransferase [ALT], alkaline phosphatase [ALP], aspartate aminotransferase [AST], creatine kinase [CK], γ-glutamyltransferase [γGT], and lactate dehydrogenase [LDH]) performed on the Abbott Alinity c analytical system. Methods Target values and associated uncertainties were assigned to three pools for each enzyme by using the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference measurement procedures (RMPs) and the pools were then measured on the Alinity system. Bias estimation and regression studies were performed, and the uncertainty associated with Alinity measurements was also estimated, using analytical performance specifications (APS) derived from biological variability of measurands as goals. Finally, to validate the transferability of the obtained results, a comparison study between two Alinity systems located in Milan, Italy, and Bydgoszcz, Poland, was carried out. Results Correct implementation of traceability to the IFCC RMPs and acceptable measurement uncertainty fulfilling desirable (ALP, AST, LDH) or optimal APS (ALT, CK, γGT) was verified for all evaluated enzymes. An optimal alignment between the two Alinity systems located in Milan and Bydgoszcz was also found for all enzyme measurements. Conclusions We confirmed that measurements of ALT, ALP, AST, CK, γGT, and LDH performed on the Alinity c analytical system are correctly standardized to the IFCC reference measurement systems and the system alignment is consistent between different platforms.