Total lab automation: sample stability of clinical chemistry parameters in an automated storage and retrieval module

Objectives Automated storage and retrieval modules (SRM), as part of total lab automation (TLA) systems, offer tremendous practical and economic benefits. In contrast to manual storage systems, SRMs indicate continuous motion of samples and may leave samples prone to temperature fluctuations. This study investigates analyte stability in serum and heparin plasma within an automated storage module. Methods The stability of 28 common biochemistry analytes was investigated using 57 freshly obtained routine serum samples and 42 lithium-heparin plasma samples. Following baseline measurement, samples were stored at 2–8 °C in the automated SRM of the Accelerator a3600 TLA and reanalyzed at fixed time points (2, 4, 8, 12, 24, 48 and 72 h) on the Abbott Architect c16000 chemistry analyzer. The concentration at each time point was expressed as %-difference to the baseline value and mean results were compared to the criteria for desirable bias derived from the biological variation database. Results Nine of the analytes exceeded the bias criterion within 72 h after initial measurement in either serum samples, plasma samples or both. Lithium-heparin plasma samples showed increasing values for phosphor, potassium and lactate dehydrogenase (LDH), which were only considered stable for respectively 24, 12 and 4 h, glucose was considered stable for 8 h. Electrolyte concentrations and LDH activity significantly increased in serum samples beyond 48 h. Bicarbonate should not be performed as add-on test at all. Conclusions The presented data indicate that the conditions within an SRM have no clinical impact on sample stability and allow stable measurement of routine analytes within 72 h, comparable to manual storage facilities.

A Low Aldosterone/Renin Ratio and High Soluble ACE2 Associate with COVID-19 Severity

Background The severity of COVID-19 after SARS-CoV-2 infection is unpredictable. Angiotensin-converting enzyme-2 (ACE2) is the receptor responsible for coronavirus binding, while subsequent cell entry relies on priming by the serine protease TMPRSS2 (transmembrane protease, serine 2). Although renin-angiotensin-aldosterone-system (RAAS) blockers have been suggested to upregulate ACE2, their use in COVID-19 patients is now considered safe. The aim of our study was to investigate parameters that determine COVID-19 severity, focusing on RAAS-components and variation in the genes encoding for ACE2 and TMPRSS2.MethodsAdult patients hospitalized due to SARS-CoV-2 infection between May 2020 and October 2020 in the Haga Teaching Hospital were included, and soluble ACE2 (sACE2), renin, aldosterone (in heparin plasma), and polymorphisms in the ACE2 and TMPRSS2 genes (in DNA obtained from EDTA blood) were determined. Measurements and main resultsOf the 188 patients that were included, 60 were defined as severe COVID-19 (ICU and/or death). These patients more often used antidiabetic drugs, were older, had higher renin and sACE2 levels, lower aldosterone levels, and a lower aldosterone/renin ratio. In addition, they displayed the TMPRSS2-rs2070788 AA genotype less frequently. No ACE2 polymorphism-related differences were observed. Multivariate regression analysis revealed independent significance for age, sACE2, the aldosterone/renin ratio, and the TMPRSS2 rs2070788 non-AA genotype as predictors of COVID-19 severity, together yielding a C-index of 0.79. Findings were independent of the use of RAAS blockers. Conclusion High sACE2, a low aldosterone/renin ration, and having the TMPRSS2 rs2070788 non-AA genotype are novel independent determinants that may help to predict COVID-19 disease severity. Trial registration: retrospectively registered

EXPRESS: Repeat measurements on patient samples identifies unpredictable and poorly reproducible cardiac troponin results with a high-sensitivity cardiac troponin assay

There is much current interest in the use of low-normal high-sensitivity cardiac troponin (hsTn) concentrations, with or without minimal change, to rule out myocardial infarction (MI). Clifford-Mobley’s observations demonstrate that this a challenge even for platforms measuring hsTnT.1 Analytical imprecision may also affect algorithms that use hsTn change alone to rule in MI. For example, the imprecision observed with the Ortho hsTnI assay (Ortho Clinical Diagnostics, New Jersey, United States), using quality control or patient pools, has been found to exceed the European Society of Cardiology 0/1h algorithm criterion.2 The Ortho hsTnI assay has also been shown to yield high and non-reproducible results (i.e., outliers), in addition to the problems with imprecision.3 Outliers are often identified by repeat centrifugation and repeat testing. However, Ortho hsTnI results above the 99th percentile cutoffs may be discordant with respect to other cardiac troponin assays and the clinical diagnosis, even when imprecision from duplicate analysis is acceptable.4 As part of a stability study assessing Ortho hsTnI concentrations in both EDTA plasma and lithium heparin plasma over 24h, we have observed that this interference is random and not related to time on cells.

Reference Intervals for Selected Hematology and Clinical Chemistry Measurands in Temminck's Pangolin (Smutsia temminckii)

Pangolins are the world's most trafficked non-human mammals. A significant number of Temminck's pangolins (Smutsia temminckii) are presented for veterinary care and rehabilitation in southern Africa. Little is known about the physiology and normal health of this species, making diagnosis and medical management difficult. This study aimed to establish reference intervals (RIs) for hematology and plasma clinical chemistry in the Temminck's pangolin. RIs were generated according to international guidelines using samples from 27 healthy free-living (n = 18) and rehabilitated (n = 9) pangolins. Hematology was performed using the Abaxis VetScan HM5 analyzer with manual differentials; clinical chemistry was performed using heparin plasma on the Abaxis VetScan VS2 and Cobas Integra 400 Plus analyzers. Hematology RIs were: RBC 3.88–8.31 × 1012/L, HGB 73–150 g/L, HCT 26–51%, MCV 59–72 fL, MCH 15.6–21.4 pg, MCHC 257–325 g/L, RDW 14.3–19.1%, WBC 1.80–10.71 × 109/L. Vetscan VS2 clinical chemistry RIs were: albumin 27–41 g/L, ALP 26–100 U/L, ALT 25–307 U/L, amylase 267–826 U/L, bilirubin 4–10 μmol/L, calcium 2.1–2.2 mmol/L, globulin 21–55 g/L, glucose 3.8–10.0 mmol/L, phosphate 1.3–2.6 mmol/L, potassium 3.6–5.9 mmol/L, sodium 132–140 mmol/L total protein 52–84 g/L, and urea 5.3–11.4 mmol/L. RIs for creatinine were not calculated as analytical imprecision exceeded analytical performance goals. Cobas Integra clinical chemistry RIs were: albumin 22–33 g/L, ALP 20–104 U/L, ALT 17–291 U/L, amylase 466–1,533 U/L, bilirubin 1–14 μmol/L, calcium 2.0–2.4 mmol/L, creatinine <58 μmol/L, globulin 23–49 g/L, glucose 3.6–10.1 mmol/L, phosphate 1.0–2.2 mmol/L, potassium 3.1–5.8 mmol/L, sodium 137–150 mmol/L, total protein 47–72 g/L, and urea 6.0–12.5 mmol/L. There was significant bias between the two chemistry analyzers for several measurands. Differences were found for some analytes between free-living and rehabilitated animals, probably reflecting differences in nutrition and hydration. These are the first RIs generated for Temminck's pangolin. These results will allow veterinarians to better determine pangolin health status, formulate optimal treatment plans and increase patient survival rates in this endangered species.

A Comparison of Serum and Plasma Blood Collection Tubes for the Integration of Epidemiological and Metabolomics Data

Blood is a rich biological sample routinely collected in clinical and epidemiological studies. With advancements in high throughput -omics technology, such as metabolomics, epidemiology can now delve more deeply and comprehensively into biological mechanisms involved in the etiology of diseases. However, the impact of the blood collection tube matrix of samples collected needs to be carefully considered to obtain meaningful biological interpretations and understand how the metabolite signatures are affected by different tube types. In the present study, we investigated whether the metabolic profile of blood collected as serum differed from samples collected as ACD plasma, citrate plasma, EDTA plasma, fluoride plasma, or heparin plasma. We identified and quantified 50 metabolites present in all samples utilizing nuclear magnetic resonance (NMR) spectroscopy. The heparin plasma tubes performed the closest to serum, with only three metabolites showing significant differences, followed by EDTA which significantly differed for five metabolites, and fluoride tubes which differed in eleven of the fifty metabolites. Most of these metabolite differences were due to higher levels of amino acids in serum compared to heparin plasma, EDTA plasma, and fluoride plasma. In contrast, metabolite measurements from ACD and citrate plasma differed significantly for approximately half of the metabolites assessed. These metabolite differences in ACD and citrate plasma were largely due to significant interfering peaks from the anticoagulants themselves. Blood is one of the most banked samples and thus mining and comparing samples between studies requires understanding how the metabolite signature is affected by the different media and different tube types.

Evidence for the utility of cfDNA plasma concentrations to predict disease severity in COVID-19

COVID-19 is a pandemic caused by the highly infective SARS-CoV-2. There is a need for biomarkers not only for overall prognosis but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Circulating cell-free DNA (cfDNA) has emerged as a promising biomarker in the assessment of various disease conditions. The aim of this retrospective and observational pilot study was to examine the potential value of cfDNA plasma concentrations as a correlative biomarker in hospitalized COVID-19 patients. Lithium-Heparin plasma samples were obtained from twenty-one COVID-19 patients during hospitalization in the University Medical Center of Mainz, Germany, and the cfDNA concentrations were determined by quantitative PCR yielding amplicons of long interspersed nuclear elements (LINE-1). cfDNA plasma concentrations of COVID-19 patients ranged between 247.5 and 6346.25 ng/ml and the mean concentrations were 1831 ± 1388 ng/ml (± standard deviation). Correlations were found between cfDNA levels and the occurrence of acute respiratory distress symptom (ARDS), acute kidney injury (AKI), myositis, neurological complications, bacterial superinfection and disease severity as defined by sepsis-related organ failure assessment score (SOFA) score. D-Dimer and C-reactive-protein (CRP), determined by clinical laboratory analysis, showed the highest correlations with cfDNA levels. The results of this observational study suggest that cfDNA plasma concentrations may serve as a predictive biomarker of disease severity in COVID-19. Prospective studies enrolling larger patient cohorts are ongoing to test this hypothesis.

The use of whole blood capillary samples to measure 15 analytes for a home-collect biochemistry service during the SARS-CoV-2 pandemic: A proposed model from North West London Pathology

Background The COVID-19 pandemic has drastically changed the delivery of secondary care services. Self-collection of capillary blood at home can facilitate the monitoring of patients with chronic disease to support virtual clinics while mitigating the risk of SARS-CoV-2 infection and transmission. Objective To investigate the comparability of whole blood capillary and plasma venous samples for 15 routinely used biochemical analytes and to develop and pilot a user-friendly home-collection kit to support virtual outpatient clinical services. Methods To investigate the comparability of whole blood capillary and plasma venous samples for 15 routinely requested biochemical analytes, simultaneous samples of venous and capillary blood were collected in EDTA and lithium-heparin plasma separation tubes that were of 4–6 mL and 400–600 µL draw volume, respectively. Venous samples were analysed within 4 h of collection while capillary samples were kept at ambient temperature for three days until centrifugation and analysis. Analyte results that were comparable between the matrices were then piloted in a feasibility study in three outpatient clinical services. Results HbA1c, lipid profile and liver function tests were considered comparable and piloted in the patient feasibility study. The home-collect kit demonstrated good patient usability. Conclusion Home collection of capillary blood could be a clinically-useful tool to deliver virtual care to patients with chronic disease.

Comparative study of Chemical Pathology sample collection tubes at the largest hospital in South Africa

Background: BarricorTM lithium heparin plasma tubes are new blood tubes that have been introduced to overcome the effects of gel in serum separator tubes (SST) and the shortcomings of standard lithium heparin plasma. We aimed to evaluate BarricorTM tubes as an alternative to serum separator tubes and compare the stability between the tubes. Methods: Forty-four paired samples were collected using both BarricorTM and SST. We compared five analytes at baseline (<6h) and after every 24h using the Passing-Bablok and Bland-Altman plots. Aspartate aminotransferase (AST), potassium (K), phosphate (PO4), lactate dehydrogenase (LDH), and creatinine were analysed in both tubes. We calculated the percentage difference for each analyte between the baseline and time intervals to assess analyte stability. The percentage difference was compared to the desirable specification for bias and reference change value (RCV). Results: All analytes were comparable at baseline. Statistical differences (p<0.001) became evident after 24h. PO4, K, and creatinine had a mean difference that exceeded the desirable specification for bias (-9.59%, -9.35%, and -4.59%, respectively). Potassium was stable up to 24h in both tubes. LDH showed better stability in SST (144h vs. 96h). PO4 concentrations were more stable in both tubes with the SST (96h vs. 72h). Creatinine and AST had the longest stability in both tubes compared to other analytes (144h). Conclusions: Data demonstrated variability and similarities in analyte concentrations and stability, respectively in both tubes

Independent and combined effects of biotin and hemolysis on high-sensitivity cardiac troponin assays

Objectives This study compared the independent and combined effects of hemolysis and biotin on cardiac troponin measurements across nine high-sensitivity cardiac troponin (hs-cTn) assays. Methods Parallel cTn measurements were made in pooled lithium heparin plasma spiked with hemolysate and/or biotin using nine hs-cTn assays: Abbott Alinity, Abbott ARCHITECT i2000, Beckman Access 2, Ortho VITROS XT 7600, Siemens Atellica, Siemens Centaur, Siemens Dimension EXL cTnI, and two Roche Cobas e 411 Elecsys Troponin T-hs cTnT assays (outside US versions, with and without increased biotin tolerance). Absolute and percent cTn recovery relative to two baseline concentrations were determined in spiked samples and compared to manufacturer’s claims. Results All assays except the Ortho VITROS XT 7600 showed hemolysis and biotin interference thresholds equivalent to or greater than manufacturer’s claims. While imprecision confounded analysis of Ortho VITROS XT 7600 data, evidence of biotin interference was lacking. Increasing biotin concentration led to decreasing cTn recovery in three assays, specifically both Roche Cobas e 411 Elecsys Troponin T-hs assays and the Siemens Dimension EXL. While one of the Roche assays was the most susceptible to biotin among the nine studied, a new version showed reduced biotin interference by approximately 100-fold compared to its predecessor. Increasing hemolysis also generally led to decreasing cTn recovery for susceptible assays, specifically the Beckman Access 2, Ortho VITROS XT 7600, and both Roche Cobas e 411 Elecsys assays. Equivalent biotin and hemolysis interference thresholds were observed at the two cTn concentrations considered for all but two assays (Beckman Access 2 and Ortho VITROS XT 7600). When biotin and hemolysis were present in combination, biotin interference thresholds decreased with increasing hemolysis for two susceptible assays (Roche Cobas e 411 Elecsys and Siemens Dimension EXL). Conclusions Both Roche Cobas e 411 Elecsys as well as Ortho VITROS XT assays were susceptible to interference from in vitro hemolysis at levels routinely encountered in clinical laboratory samples (0–3 g/L free hemoglobin), leading to falsely low cTn recovery up to 3 ng/L or 13%. While most assays are not susceptible to biotin at levels expected with over-the-counter supplementation, severely reduced cTn recovery is possible at biotin levels of 10–2000 ng/mL (41–8,180 nmol/L) for some assays. Due to potential additive effects, analytical interferences should not be considered in isolation.

Abstract MP11: Circulating Plasma Biomarkers Associated With Brain Arteriovenous Malformations

Objective: Circulating plasma protein profiling may aid in the identification of cerebrovascular disease signatures. This study aimed to identify circulating angiogenic and inflammatory biomarkers that may serve as biomarkers to differentiate sporadic brain arteriovenous malformation (bAVM) patients from other conditions with brain AVMs, including hereditary hemorrhagic telangiectasia (HHT) patients. Methods: The Quantibody Human Angiogenesis Array 1000 (Raybiotech) is an ELISA multiplex panel that was used to assess the levels of 60 proteins related to angiogenesis and inflammation in heparin plasma samples from 13 sporadic unruptured bAVM patients (69% male, mean age 51 years) and 37 patients with HHT (40% male, mean age 47 years, n=19 (51%) with bAVM). The Quantibody Q-Analyzer tool was used to calculate biomarker concentrations based on the standard curve for each marker and log-transformed marker levels were evaluated for associations between disease states using a multivariable interval regression model adjusted for age, sex, ethnicity and collection site. Statistical significance was based on Bonferroni correction for multiple testing of 60 biomarkers (P< 8.3x10 - 4 ). Results: Circulating levels of two plasma proteins differed significantly between sporadic bAVM and HHT patients: PDGF-BB (P=2.6x10 -4 , PI= 3.37, 95% CI:1.76-6.46) and CCL5 (P=6.0x10 -6 , PI=3.50, 95% CI=2.04-6.03). When considering markers with a nominal p-value of less than 0.01, MMP1 and angiostatin levels also differed between patients with sporadic bAVM and HHT. Markers with nominal p-values less than 0.05 when comparing sporadic brain AVM and HHT patients also included angiostatin, IL2, VEGF, GRO, CXCL16, ITAC, and TGFB3. Among HHT patients, the circulating levels of UPAR and IL6 were elevated in patients with documented bAVMs when considering markers with nominal p-values less than 0.05. Conclusions: This study identified differential expression of two promising plasma biomarkers that differentiate sporadic bAVMs from patients with HHT. Furthermore, this study allowed us to evaluate markers that are associated with the presence of bAVMs in HHT patients, which may offer insight into mechanisms underlying bAVM pathophysiology.