Point-of-care testing (POCT) is becoming an increasingly popular way to perform laboratory tests closer to the patient. This option has several recognized advantages, such as accessibility, portability, speed, convenience, ease of use, ever-growing test panels, lower cumulative healthcare costs when used within appropriate clinical pathways, better patient empowerment and engagement, and reduction of certain pre-analytical errors, especially those related to specimen transportation. On the other hand, POCT also poses some limitations and risks, namely the risk of lower accuracy and reliability compared to traditional laboratory tests, quality control and connectivity issues, high dependence on operators (with varying levels of expertise or training), challenges related to patient data management, higher costs per individual test, regulatory and compliance issues such as the need for appropriate validation prior to clinical use (especially for rapid diagnostic tests; RDTs), as well as additional preanalytical sources of error that may remain undetected in this type of testing, which is usually based on whole blood samples (i.e., presence of interfering substances, clotting, hemolysis, etc.). There is no doubt that POCT is a breakthrough innovation in laboratory medicine, but the discussion on its appropriate use requires further debate and initiatives. This collective opinion paper, composed of abstracts of the lectures presented at the two-day expert meeting "Point-Of-Care-Testing: State of the Art and Perspective" (Venice, April 4-5, 2024), aims to provide a thoughtful overview of the state-of-the-art in POCT, its current applications, advantages and potential limitations, as well as some interesting reflections on the future perspectives of this particular field of laboratory medicine.
Neurofilament light chain (NfL) is an early nonspecific biomarker in neurodegenerative diseases and traumatic brain injury, indicating axonal damage. This work describes the detailed structural characterization of a selected primary calibrator with the potential to be used in future reference measurement procedure (RMP) development for the accurate quantification of NfL. As a part of the described workflow, the sequence, higher-order structure as well as solvent accessibility, and hydrogen-bonding profile were assessed under three different conditions in KPBS, artificial cerebrospinal fluid, and artificial cerebrospinal fluid in the presence of human serum albumin. The results revealed that NfL is a structurally heterogeneous protein, eliciting a large conformational flexibility. Its structural ensemble changed when it was diluted with an aqueous buffer versus a surrogate matrix, artificial cerebrospinal fluid (aCSF), and/or aCSF with human serum albumin. Various regions of protection and deprotection in the protein head, central helical, and tail domains that experienced altered solvent accessibility and conformational changes caused by different solvent conditions were identified. Moreover, interfacial residues, which may play a role in a potential direct interaction between NfL and human serum albumin, emerged from hydrogen-deuterium exchange mass spectrometry (HDX-MS). These data pinpointed distinct regions of the protein that may participate in such an interaction. Overall, critical quality attributes of a potential primary calibrator for NfL measurements are provided. These findings will ultimately inform ongoing biochemical and clinical assay development procedures and manufacturing practices, giving careful consideration during sample handling and method development.
OBJECTIVES:Management of phenylketonuria (PKU) relies upon life-long monitoring of phenylalanine (Phe) in dried blood spots (DBS), thus comparability of measurements is important. The lack of harmonisation and standardisation between laboratories, combined with the variable quality of patient-collected DBS specimens, are currently preventing this from being achieved. A traceable, matrix-matched Phe certified reference material, common methodology and means to ensure patient collected DBS specimens are of consistent quality would improve comparability between laboratories. METHODS:Baseline inter-laboratory (n=15) variation of DBS Phe was determined by triplicate measurement of four DBS materials, on three days. Laboratories prepared and analysed these samples using their routine method of analysis. A sub-set of laboratories (n=5) repeated the process using a common sample preparation and instrument methodology (LC-MS/MS), and three different calibration approaches. Samples prepared on dried blood spot microsampling cards (DBS-MCs) from whole blood, value assigned for Phe concentration by National Measurement Laboratories (NML), were then analysed using the harmonised methodology. RESULTS:Inter-laboratory co-efficient of variation (CV) differed with calibration approach; internal calibration 27.7 %; in-house aqueous calibration 4.7 %; centrally distributed aqueous calibration, 2.1 %. Inter-laboratory CV was reduced from 8.7 to 2.1 % by using common sample preparation and LC-MS/MS methodology. No significant difference was observed between consensus and assigned values for Phe in the four materials (p>0.05). CONCLUSIONS:This study demonstrates a simple approach to harmonising and standardising DBS Phe measurements, traceable to value assigned materials. Combined with the introduction of DBS-MCs to ensure specimen quality, clinical laboratories can achieve comparability of patient results over time.
Plasma amino acid analysis plays a vital role in monitoring inherited metabolic diseases (IMDs), such as phenylketonuria (PKU), where dietary therapy is the cornerstone of management and patients are regularly monitored using blood or plasma phenylalanine throughout life. Recent findings from external quality assessment (EQA) schemes demonstrate large variability between results from different clinical laboratories. EQA schemes are hampered by factors including suboptimal sample type and frequency of distributions. Aiming to harmonise and standardise EQA schemes, this paper describes value assignment of an SI traceable matrix-matched amino acid material, and its use in an interlaboratory comparison (ERNDIM QAA-2106 study) that included 89 clinical laboratories from throughout Europe. The outcomes of the interlaboratory comparison are valuable in enabling clinical laboratories to refine their methodologies and, in a longer term, will lead to enhanced accuracy of results in clinical measurements and, ultimately, improved patient care in IMD management. A frozen, pooled human plasma sample was distributed to participating laboratories (n = 88). The sample was value assigned for phenylalanine by the National Measurement Laboratory (NML) traceable to the international system of units (SI), at approximately the target value for dietary monitoring of phenylketonuria (360 µmol/L). Participants used their routine method of analysis to measure 24 amino acids in triplicate and results were compared against desirable analytical performance. Mean intra- and inter-laboratory variability were acceptable for 20/20 and 19/20 amino acids respectively. Mean bias relative to the consensus values was acceptable for 17/20 amino acids. Overall performance of phenylalanine was acceptable; however, 15/87 laboratories failed to meet acceptable imprecision and 8/87 had unacceptable bias; additionally, an overall negative bias for phenylalanine measurement was identified. This study demonstrates the importance of ensuring a matrix material with an assigned value of the analyte(s) of interest (traceable to the SI) is available for regular EQA. The significant bias in the routine measurement of phenylalanine identified has the potential to impact disease management, which aims to control blood phenylalanine concentrations for preventing adverse neurological outcomes.
In the beginning of 2020, the outbreak of the COVID-19 pandemic led to a crisis in which diagnostic methods for the genome detection of SARS-CoV-2 were urgently needed. Based on the very early publication of the basic principles for a diagnostic test for the genome detection of SARS-CoV-2, the first noncommercial laboratory-developed tests (LDTs) and commercial tests were introduced. As there was considerable uncertainty about the reliability and performance of different tests and different laboratories, INSTAND established external quality assessment (EQA) schemes for the detection of SARS-CoV-2 starting in April 2020. In close partnership in a scientific network, the EQA schemes were enhanced, especially the April, June and November 2020 terms. The enhancement included: (i) immediate provision of suitable virus including variants of concern at the beginning of the pandemic outbreak, (ii) short frequency of EQA schemes, (iii) concentration dependency of the testing and sensitivity check, achieved by using SARS-CoV-2-positive samples from a 10-fold dilution series of the same starting material, (iv) specificity check of the testing, achieved by using SARS-CoV-2-negative samples containing human coronaviruses or MERS CoV, (v) revealed samples for orientation on test performance during an ongoing or at the start of an EQA scheme using a pre-quantified SARS-CoV-2-positive EQA sample with a low viral RNA load of only 1 570 copies/mL assigned by digital PCR (dPCR) in June 2020 and (vi) quantified reference materials based on the experiences of the first two EQA schemes with dPCR-assigned values in copies/mL beginning in November 2020 for self-evaluation of the applied test system. This manuscript summarizes the results of a total of 13 EQA schemes for the detection of SARS-CoV-2 between April 2020 and June 2023 in which a total of 1 413 laboratories from 49 countries participated. The qualitative results for the detection of SARS-CoV-2-positive samples were between 95.8 % and 99.7 % correct positive, excluding extremely low concentration samples. For all SARS-CoV-2-negative EQA samples, the qualitative success rates ranged from 95.1 % to 99.4 % correct negative results. The widely varying values for the cycle threshold (Ct)/crossing point (Cq) reported for the different target genes and test systems were striking. A few laboratories reported quantitative results in copies/mL for several VOCs with an acceptable rate of over 93 % correct positive results in the majority of cases. The description of the enhanced EQA schemes for SARS-CoV-2 detection in terms of timing and scope can serve as a blueprint for the rapid development of a quality assessment of diagnostics for an emerging pathogen.
