Validation and application of an automated multitarget LC-MS/MS method for drugs of abuse testing using exhaled breath as specimen.

Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel "collectors" that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography - mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.

Proteome profiling of home-sampled dried blood spots reveals proteins of SARS-CoV-2 infections.

BACKGROUND: Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored. METHODS: Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information. RESULTS: Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors. CONCLUSIONS: Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives.

The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases.

Volumetric dried blood spots for determination of phosphatidylethanol: Validation of a liquid chromatography tandem masspectrometry method and clinical application.

Phosphatidylethanol (PEth) measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. Establishment of dried blood spots (DBSs) as a specimen for PEth determination offers several advantages and was the focus of this work. A liquid chromatography tandem mass spectrometry method using a 96-well format for sample preparation was developed and validated. PEth was extracted from DBSs by using isopropanol containing PEth-d5 as internal standard. The blood sampling used a commercial volumetric DBS device having a phosholipase D inhibitor incorporated to stop continuous PEth formation. The method quantified PEth in the range of 0.05-10 µmol/L, with a bias and imprecision of less than 15%. In a clinical study (n = 25) using fingerprick blood, the volumetric device offered more precise quantifications (CV 4.6%) compared with the Whatman 903 Protein Saver card device (CV 16.6%). In another clinical study (n = 48), the use of dried venous and capillary blood, and liquid venous blood was compared under real-life conditions with samples sent by postal service. The capillary and venous DBS samples gave identical results while the liquid blood gave slightly higher values. Calculation of elimination half-life (PEth 16:0/18:1) in 31 cases based on two consecutive samples with 2-9 days in between gave results (mean 6.2 days) that agree with literature but several cases with values over 10 days. In conclusion, this study demonstrates that volumetric DBS is a valid specimen for determination of PEth blood concentrations, offering several advantages.

A multicomponent LC-MS/MS method for drugs of abuse testing using volumetric DBS and a clinical evaluation by comparison with urine.

BACKGROUND: Drug testing commonly use urine as a specimen and immunoassays for screening. The need for supervised urine collection has led to an interest in alternative specimens and a need for using mass spectrometry methods already for screening. In addition, mass spectrometry methods allow for broad multipanel screening which of great value because of the increased number of substances that needs to be covered has increased over time. One alternative specimen of interest for drugs of abuse testing is dried blood spots (DBS) and this work aimed at developing multipanel screening methods based on selected reaction monitoring liquid chromatography - mass spectrometry for both urine and dried finger blood as specimens. MATERIALS AND METHODS: The urine method comprised 37 analytes and utilised salted out liquid/liquid extraction in 96-well format, respectively, and the blood method comprised 35 analytes, a 10 µL volumetric DBS device and a two-step solvent extraction procedure. In both cases stable isotope labelled internal standards were used for almost all analytes. RESULTS: The methods were validated according to forensic standard. The lowest reporting limits were generally set at 100 ng/mL for urine and 1 ng/mL for blood and the accuracy and imprecision were within limits of 15 and 20%. The methods were applied in a clinical study on patients receiving methadone maintenance treatment for opioid dependence. Methadone was detected in all urine and DBS samples, for urine sometimes below the commonly applied screening cutoff limit of 300 ng/mL. In 20 out of 99 cases no other drug was detected in any specimen. The most commonly other detected substances were pregabalin, amphetamine, alprazolam, zopiclone and THCCOOH. Findings in urine and DBS generally agreed well but more positives were detected in DBS. CONCLUSION: Multipanel methods using liquid chromatography - mass spectrometry suitable for clinical drug screening were successfully developed for urine and blood collected by finger-pricking and stored as DBS.

An analytical approach for on-site analysis of breath samples for Δ9-tetrahydrocannabinol (THC).

Increased acceptance of cannabis containing the psychoactive component, Δ9-tetrahydrocannabinol (THC), raises concerns about the potential for impaired drivers and increased highway accidents. In contrast to the "breathalyzer" test, which is generally accepted for determining the alcohol level in a driver, there is no currently accepted roadside test for THC in a motorist. There is a need for an easily collectible biological sample from a potentially impaired driver coupled with an accurate on-site test to measure the presence and quantity of THC in a driver. A novel breath collection device is described, which includes three separate sample collectors for collecting identical A, B, and C breath samples from a subject. A simple one-step ethanol extraction of the "A" breath collector sample can be analyzed by UHPLC/selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS) to provide qualitative and quantitative determination of THC in breath sample in less than 4 min for samples collected up to 6 h after smoking a cannabis cigarette. SIM LC/MS bioanalyses employed d3-THC as the stable isotope internal standard fortified in negative control breath samples for quantitation including replicates of six calibrator standards and three quality control (QC) samples. Subsequent confirmation of the same breath sample in the B collectors was then confirmed by a reference lab by LC/MS/MS analysis. Fit-for-purpose bioanalytical validation consistent with pharmaceutical regulated bioanalyses produced pharmacokinetic (PK) curves for the two volunteer cannabis smokers. These results produced PK curves, which showed a rapid increase of THC in the breath of the subjects in the first hour followed by reduced THC levels in the later time points. A simpler single-point calibration curve procedure with calibrators and QC prepared in ethanol provided similar results. Limitations to this approach include the higher cost and operator skill sets for the instrumentation employed and the inability to actually determine driver impairment.

Δ9-THC and CBD in Plasma, Oral Fluid, Exhaled Breath, and Urine from 23 Patients Administered Sativex.

Background: Detecting the presence of Δ9-THC and CBD is mainly done through venous blood sampling, but other methods are becoming available. Oromucosal administration of Δ9-THC and CBD is less studied than inhalation, but this mode of administration is growing. In this study, we analyze samples obtained through invasive and noninvasive methods in a cohort of patients given oromucosally administered Δ9-THC and CBD to gain understanding in the strengths and weaknesses of the various detection methods. Materials and Methods: Blood, oral fluid (OF), exhaled breath, and urine were collected at several time points from 23 cannabis-naive patients after receiving a single dose of Sativex®; dose ranges: Δ9-THC, 2.7-18.9 mg; CBD 2.5-17.5 mg. Detection of Δ9-THC and CBD was done using liquid chromatography-mass spectrometry methods. Results: Δ9-THC and CBD were present in plasma, OF, and exhaled breath in all 23 patients. The detection time of Δ9-THC and CBD in OF and exhaled breath was longer than in blood. Urine analysis detected the Δ9-THC carboxy metabolite (THC-COOH) up to 7 days after administration, also in a patient who received 8.1/7.5 mg Δ9-THC/CBD. Conclusion: Time to detection of cannabinoids in blood samples was shorter than in exhaled breath and OF. Relative ease of sample collection combined with high sensitivity makes OF and exhaled breath specimens a valuable addition when samples are handled correctly. Δ9-THC metabolites were detected for an unexpected long period of time in urine. EudraCT Number: 2014-005553-39. Date of registration, December 29, 2015.

Determination of lithium concentration in capillary blood using volumetric dried blood spots.

BACKGROUND: Lithium is a cornerstone in the treatment of bipolar disorder and is considered one of the most effective treatments in psychiatry at large. Lithium treatment requires individual dosing with frequent serum concentration measurements due to the narrow therapeutic window and risk of toxicity. There is need for patient-centric methods for lithium monitoring and the use of dried blood spots has recently been proposed for determination of lithium concentration. The purpose of the current study was to assess feasibility of this method by introducing a volumetric technique developed for home-sampling. MATERIALS AND METHODS: Laboratory: Capillary blood was sampled by finger-prick using a volumetric device that collects 10 µL volumes as a dried blood spot. Lithium was measured in the dried blood spots using a validated atomic absorption spectroscopy method. CLINICAL: Thirty-nine lithium-treated patients were recruited, and dried blood spots and venous blood samples were collected. Routine serum analysis was performed for comparison. RESULTS: The range of serum lithium concentrations was 0.41-1.22 mmol/L, and the dried blood spot/serum ratio was 0.78. A strong linear correlation between the two specimens was shown with Pearson's R = 0.95 (r2 = 0.90). Adding hematocrit as a variable only minimally improved prediction. CONCLUSION: Volumetric dried blood spots is a promising technique for measurement of lithium concentrations. This will enable home-sampling and could potentially save resources, improve compliance, and make treatment safer. This may facilitate the use of lithium treatment in regions where monitoring via venous blood sampling remains difficult. However, the usability of dried blood spots for monitoring lithium treatment longitudinally remains to be examined.

Microneedle Patch for Painless Intradermal Collection of Interstitial Fluid Enabling Multianalyte Measurement of Small Molecules, SARS-CoV-2 Antibodies, and Protein Profiling.

Blood sampling is a common practice to monitor health, but it entails a series of drawbacks for patients including pain and discomfort. Thus, there is a demand for more convenient ways to obtain samples. Modern analytical techniques enable monitoring of multiple bioanalytes in smaller samples, opening possibilities for new matrices, and microsampling technologies to be adopted. Interstitial fluid (ISF) is an attractive alternative matrix that shows good correlation with plasma concentration dynamics for several analytes and can be sampled in a minimally invasive and painless manner from the skin at the point-of-care. However, there is currently a lack of sampling devices compatible with clinical translation. Here, to tackle state-of-the-art limitations, a cost-effective and compact single-microneedle-based device designed to painlessly collect precisely 1.1 µL of dermal ISF within minutes is presented. The fluid is volume-metered, dried, and stably stored into analytical-grade paper within the microfluidic device. The obtained sample can be mailed to a laboratory, quantitatively analyzed, and provide molecular insights comparable to blood testing. In a human study, the possibility to monitor various classes of molecular analytes is demonstrated in ISF microsamples, including caffeine, hundreds of proteins, and SARS-CoV-2 antibodies, some being detected in ISF for the first time.

Microfluidic Device for Patient-Centric Multiplexed Assays with Readout in Centralized Laboratories.

Patient-centric sampling strategies, where the patient performs self-sampling and ships the sample to a centralized laboratory for readout, are on the verge of widespread adaptation. However, the key to a successful patient-centric workflow is user-friendliness, with few noncritical user interactions, and simple, ideally biohazard-free shipment. Here, we present a capillary-driven microfluidic device designed to perform the critical biomarker capturing step of a multiplexed immunoassay at the time of sample collection. On-chip sample drying enables biohazard-free shipment and allows us to make use of advanced analytics of specialized laboratories that offer the needed analytical sensitivity, reliability, and affordability. Using C-Reactive Protein, MCP1, S100B, IGFBP1, and IL6 as model blood biomarkers, we demonstrate the multiplexing capability and applicability of the device to a patient-centric workflow. The presented quantification of a biomarker panel opens up new possibilities for e-doctor and e-health applications.

Feasibility of using breath sampling of non-volatiles to estimate the prevalence of illicit drug use among nightlife attendees.

The prevalence of drug use among nightlife attendees needs to be accurately estimated to, for example, evaluate preventive interventions. This study tested the feasibility of using a breath-sampling device to estimate the prevalence of drug use among nightlife attendees. The study was conducted at five nightclubs and a large music festival in Stockholm, Sweden. Participants were invited to participate and microparticles in exhaled breath were sampled and analyzed for 47 compounds using a state-of-the-art analytic method that follows forensic standards. In addition, participants' breath alcohol concentration was measured and they were interviewed about demographics, drinking habits, and drug use. Of the people invited, 73.7% (n = 1223) agreed to participate, and breath samples were collected from 1204 participants. Breath sampling was fast and well-accepted by participants. 13 percent of participants tested positive for an illicit drug, but only 4.3% self-reported drug use during the last 48 h. The most common substances detected were cocaine, amphetamine, and MDMA. There was no agreement between self-reported and measured use of any drug. Breath sampling is a convenient method to test illicit drug use among a large number of participants at events, and can be used as an estimate of drug use prevalence.

Increasing prevalence of illicit drug use among employees at Swedish workplaces over a 25-year period.

BACKGROUND: Reports indicate that the proportion of adults using drugs of abuse has been increasing in recent years in Europe. Although there are various indicators of increased drug use in Sweden over time, few studies could demonstrate an increase in the proportion of adults using drugs. To investigate changes in drug use prevalence over time, drug testing at the workplace has been used for a 25-year period. METHODS: The urine samples of employees sent by occupational health services from all over Sweden during a 25-year period were analyzed. The analyzing capacity increased over time (from 3411 in 1994 to 60 315 samples analyzed in 2019), and the majority of the samples was analyzed for the following drugs: cannabis (tetrahydrocannabinol), amphetamine, opiates, cocaine, and benzodiazepines. RESULTS: There was an overall increase in the proportion of samples that tested positive for illicit drugs over a 25-year period. This increase seemed to take place step-wise, with phases of linear increases and plateaus that over time became shorter. About 1.3% of samples tested positive for drugs in 1994, whereas 5.6% tested positive in 2019. Since 2007, the rate of positive samples has increased for cannabis and decreased for benzodiazepines. Although the rate of samples tested positive for opiates had remained relatively stable over the last 20 years, this rate had increased for amphetamine and cocaine between 2013 and 2019. CONCLUSION: The results indicate that the use of illicit drugs among employees at Swedish workplaces has increased during a 25-year period.

Blood cell quantification on dry blood samples: toward patient-centric complete blood counts.

Background: Performing complete blood counts from patients' homes could have a transformative impact on e-based healthcare. Blood microsampling and sample drying are enabling elements for patient-centric healthcare. The aim of this study was to investigate the potential of dry blood samples for image-based cell quantification of red and white blood cells. Methods: A manual sample preparation method was developed and tested for image-based red and white blood cell counting. Results & conclusion: Dry blood samples enable image-based cell counting of red and white blood cells with a good correlation to gold standard hematology analyzer data (average coefficient of variation <6.5%; R2 >0.8) and resolve the basic morphology of white blood cell nuclei. The presented proof-of-principle study is a first step toward patient-centric complete blood counts.

Severe acute respiratory syndrome coronavirus 2 can be detected in exhaled aerosol sampled during a few minutes of breathing or coughing.

BACKGROUND: The knowledge on the concentration of viral particles in exhaled breath is limited. The aim of this study was to explore if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in aerosol from subjects with the coronavirus disease 2019 (COVID-19) during various types of breathing and coughing and how infection with SARS-CoV-2 may influence the number and size of exhaled aerosol particles. METHODS: We counted and collected endogenous particles in exhaled breath in subjects with COVID-19 disease by two different impaction-based methods, during 20 normal breaths, 10 airway opening breaths, and three coughs, respectively. Breath samples were analyzed with reverse transcription real-time polymerase chain reaction (RT-PCR). RESULTS: Detection of RNA in aerosol was possible in 10 out of 25 subjects. Presence of virus RNA in aerosol was mainly found in cough samples (n = 8), but also in airway opening breaths (n = 3) and in normal breaths (n = 4), with no overlap between the methods. No association between viral load in aerosol and number exhaled particles <5 µm was found. Subjects with COVID-19 exhaled less particles than healthy controls during normal breathing and airway opening breaths (all P < 0.05), but not during cough. CONCLUSION: SARS-CoV-2 RNA can be detected in exhaled aerosol, sampled during a limited number of breathing and coughing procedures. Detection in aerosol seemed independent of viral load in the upper airway swab as well as of the exhaled number of particles. The infectious potential of the amount of virus detected in aerosol needs to be further explored.

Clinical effects of a single dose of cannabinoids to patients with chronic lymphocytic leukemia.

This phase II clinical trial investigates a one-time oromucosal dose of tetrahydrocannabinol/cannabidiol (THC/CBD) in 23 patients with indolent leukemic B cell lymphomas. Primary endpoint was a significant reduction in leukemic B cells. Grade 1 - 2 adverse events were seen in 91% of the patients; most common were dry mouth (78%), vertigo (70%), and somnolence (43%). After THC/CBD a significant reduction in leukemic B cells (median, 11%) occurred within two hours (p = .014), and remained for 6 h without induction of apoptosis or proliferation. Normal B cells and T cells were also reduced. CXCR4 expression increased on leukemic cells and T cells. All effects were gone by 24 h. Our results show that a single dose of THC/CBD affects a wide variety of leukocytes and only transiently reduce malignant cells in blood. Based on this study, THC/CBD shows no therapeutic potential for indolent B cell lymphomas (EudraCT trial no. 2014-005553-39).

Phosphatidylethanol, ethyl glucuronide and ethanol in blood as complementary biomarkers for alcohol consumption.

Alcohol biomarkers can monitor both recent and long-term drinking and provide information about drinking habits as a complement to self-reporting. Ethyl glucuronide (EtG) and phosphatidylethanol (PEth) are the most sensitive available biomarkers for this purpose. The present study aimed to collect data on both PEth and EtG in the same blood sample, in addition to ethanol, in order to evaluate the combined use of these biomarkers. Venous EDTA blood samples (n = 1149) sent to the laboratory as part of a clinical routine service for measuring PEth were investigated. PEth and EtG concentrations were analyzed using liquid chromatography-mass spectrometry methods and ethanol with an enzymatic method. Of the 1149 samples, 95 were positive for ethanol (range 0.11-3.12 g/L), 454 for EtG (1.0-9739 ng/mL), 635 for PEth (0.014-6.0 µmol/L), 534 for PEth ≥ 0.050 µmol/L, and 315 for PEth ≥ 0.30 µmol/L. EtG and PEth concentrations seemed largely independent as the coefficient of determination (r2) between PEth and EtG concentrations was 0.15. However, when the EtG concentrations were evaluated for different subgroups depending on ethanol or PEth concentrations a statistically significant difference between successive higher concentrations was observed. EtG and PEth are independent measures of recent alcohol drinking reflecting different time windows. Their combined measurement in the same blood sample is possible and will provide valuable information regarding recent alcohol consumption as a complement to self-reporting.

Use of Quantitative Dried Blood Spots to Evaluate the Post-Vaccination Level of Neutralizing Antibodies against SARS-CoV-2.

To combat the COVID-19 pandemic, vaccines against SARS-CoV-2 are now given to protect populations worldwide. The level of neutralizing antibodies following the vaccination will evolve with time and vary between individuals. Immunoassays quantifying immunoglobulins against the viral spike (S) protein in serum/plasma have been developed, but the need for venous blood samples could limit the frequency and scale of control in populations. The use of a quantitative dried blood spot (DBS) that can be self-collected would simplify this monitoring. The objective of this study was to determine whether a quantitative DBS device (Capitainer qDBS 10 µL) could be used in combination with an Elecsys anti-SARS-CoV-2 S immunoassay from Roche to follow the development and persistence of anti-S antibodies. This objective was carried out through two clinical studies. The first study investigated 14 volunteers who received two doses of the Comirnaty (Pfizer) vaccine. The levels of anti-S antibodies and the progression over time post-vaccination were studied for three months. The level of produced antibodies varied between subjects, but a similar trend was observed. The anti-S antibodies were highly stimulated by the second dose (×100) and peaked two weeks later. The antibody levels subsequently decreased and three months later were down to 65%. DBS proved to be sufficiently sensitive for use in evaluating the immune status against SARS-CoV-2 over a prolonged time. The second cohort was composed of 200 random patients from a clinical chemistry department in Stockholm. In this cohort, we had no information on previous COVID-19 infections or vaccination. Nevertheless, 87% of the subjects had anti-S immunoglobulins over 0.8 U/mL, and the bias between plasma and DBS proved to be variable, as was also seen in the first vaccination study.

Hydromorphone and codeine concentrations in oral fluid specimens from patients receiving substitution therapy with Substitol™ (morphine sulfate).

This study aimed to determine whether hydromorphone and codeine can be detected in oral fluid specimens following administration of Substitol™, a slow-release formulation of morphine. This is of interest for those monitoring treatment compliance using drug testing. Oral fluid specimens collected for compliance assessment in routine clinical practice or as part of a clinical trial were subjected to quantitative analysis of hydromorphone, morphine, codeine, and 6-acetylmorphine using highly sensitive mass spectrometric methods. Oral fluid was collected using a Greiner Bio-One saliva collection system. Patients undergoing substitution treatment with Substitol™, methadone, or buprenorphine were included, together with patients undergoing pain treatment with hydromorphone. Hydromorphone was detected in 642 of the 663 (97%) samples from substitol-treated patients. Concentrations were not higher in methadone- and buprenorphine-treated patients who relapsed into heroin use, or in patients on hydromorphone therapy. Codeine was detected in 29% of the samples. These concentrations were lower than those in patients who had relapsed to heroin use. Clinical administration of morphine can lead to detectable concentrations of both hydromorphone and codeine in oral fluids. This should be taken into consideration when using drug testing in oral fluid samples for compliance assessment in this patient group.

Multianalyte serology in home-sampled blood enables an unbiased assessment of the immune response against SARS-CoV-2.

Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG+IgM+ against several SARS-CoV-2 proteins, as well as 2.1% being IgG-IgM+ and 5.0% being IgG+IgM- for the virus' S protein. Subjects classified as IgG+ for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG+ for only the S protein (OR = 6.1; p < 0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus.

Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood-importance of inhibition of post-sampling formation from ethanol.

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption by action of the enzyme phospholipase D (PLD). PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. However, in blood specimens containing ethanol, formation of PEth may continue after sampling leading to falsely elevated concentrations. This study evaluated the use of dried blood spot (DBS) and microsampling specimens to avoid post-sampling formation of PEth. Filter paper cards and three commercial devices for volumetric microsampling of finger-pricked blood were assessed, using PEth-negative and PEth-positive whole blood fortified with 2 g/L ethanol. PEth (16:0/18:1) was measured by LC-MS/MS. Post-sampling formation of PEth occurred in wet blood and in the volumetric devices, but not filter paper cards, when stored at room temperature for 48 h. Addition of an inhibitor of PLD, sodium metavanadate (NaVO3), eliminated post-sampling formation during storage and drying. In conclusion, the present study confirmed previous observations that PEth can be formed in blood samples after collection, if the specimen contains ethanol. The results further demonstrated that post-sampling formation of PEth from ethanol also occurred with commercial devices for volumetric dried blood microsampling. In order for a PEth result not to be questioned, it is recommended to use a PLD inhibitor, whether venous blood is collected in a vacutainer tube or finger-pricked blood is obtained using devices for dried blood microsampling.

Peanuts in the air - clinical and experimental studies.

BACKGROUND: Allergic reactions to food allergens usually occur after ingestion. However, fear of reactions to airborne peanut is a common concern for people with peanut allergy. There are no scientific reports on severe reactions with airborne peanut allergen. OBJECTIVE: To investigate the occurrence of allergic reactions in peanut-allergic children undergoing airborne peanut challenge and to determine levels of airborne peanut protein in a separate experimental evaluation. METHODS: Eighty-four children with peanut allergy underwent an airborne peanut challenge, 0.5 m from a bowl of peanuts for 30 min under controlled conditions. In a separate experiment, airborne peanut proteins from roasted and dry-roasted peanuts were collected at varying distances and at varying times with an electret SensAbues filter connected to an air pump. Collected airborne peanut proteins were extracted, dissolved and detected by ELISA. Basophil activation test was used to confirm biological activity. RESULTS: No moderate/severe allergic reactions to airborne peanut allergens were observed. Two children (2%) had mild rhino-conjunctivitis which required no treatment. The IgE-antibodies to peanut or Ara h 2 did not predict a reaction. In the experimental set-up, biological active peanut proteins were detected, in a very low amount, in median 166 ng/ml for dry-roasted and 33 ng/ml for roasted peanuts and decreased dramatically when the collection occurred at a greater distance (0.5-2 m) from the peanut source. Increased exposure time did affect the amount of collected peanut protein at 0 m, and the highest median was obtained after 60 min (p = .012); for time trend p = .0006. CONCLUSIONS AND CLINICAL RELEVANCE: Allergic reactions to airborne peanut proteins are rare and cannot be predicted by high levels of IgE-antibodies to peanut or Ara h 2. Only small amounts of biologically active peanut proteins were detected in the air and seem unlikely to trigger moderate/severe allergic reactions.