Kemp, Jennifer

Abstract Background We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. Methods Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR–positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR–positive, antigen-positive samples for further analysis. Results All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50 000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50 000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). Conclusions Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500 000 copies/mL.

Abstract Introduction: Immunoassay technology is subject to matrix interferences that can produce inaccurate results and incorrect conclusions when using samples not previously validated. While many commercially available research grade (RG) immunoassay kits are available, caution should be applied when using RG kits for thyroid health assessments, particularly on samples from pregnant individuals whose blood chemistry is unique to non-pregnant individuals. Question: Do RG immunoassay kits reliably provide precise and accurate measurements of thyroid health biomarkers in serum Standard Reference Materials (SRMs) from pregnant and non-pregnant donors? Methods: T4, T3, rT3, Tg and TSH measurements were conducted on SRMs 971, Hormones in Frozen Human Serum, and SRM 1949, Frozen Prenatal Serum, using RG immunoassay kits. When available, performance was assessed against validated FDA approved immunoassays or mass spectrometric (MS) methods. Results: RG kits were variable, inaccurate, or imprecise for four of the six biomarkers assessed. RG kit total thyroid hormone measurements overall performed comparably to MS methods, except rT3 measurements, which were twofold greater than mass spectrometric measurements (971M RG mean = 0.51 ng/mL, MS mean = 0.20 ng/mL, p < 0.0001; 971F RG = 0.48 ng/mL, MS = 0.18 ng/mL, p < 0.0001) and had CVs over 30 %. RG kit Tg measurements varied sometimes by as much as tenfold (971M means of 6.50 ng/mL up to 63.3 ng/mL, p < 0.0001; 971F means of 0.350 ng/mL up to 14.5 mg/mL, p < 0.0001). TSH values differed by RG kit manufacturer (971M means of 1.27 µIU/mL up to 1.82 µIU/mL, p < 0.0001; 971F means of 1.36 µIU/mL up to 2.27 µIU/mL, p < 0.0001) and by dilution scheme using the same manufacturer with one case indicating a diagnosis of hypothyroid versus normal TSH levels (1949 non-pregnant undiluted mean = 2325 pg/mL, half dilution mean = 1631 pg/mL, p < 0.0001). Conclusions: RG immunoassays are often used for research projects because they do not require expensive equipment and are simple to conducted. However, we demonstrate here that not all kits are accurate for all patient samples. By utilizing a matrix matched SRM with well-defined quantities of thyroid health biomarkers, one can assess method accuracy, making measurements from different methods comparable. Thereby, data can be harmonized to contribute reliable data on thyroid biomarkers to advance the field of thyroid health.

AbstractObjectivesMatrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy.MethodsSerum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only).ResultsCertified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003).TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS.ConclusionsThe analyte concentrations’ changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.