Retrospective analysis of reference intervals for dried blood spot based ms/ms newborn screening programs in Chinese preterm neonates: a nationwide study.

OBJECTIVES: Although recent discoveries regarding the biomarkers of newborn screening (NBS) programs by tandem mass spectrometry (MS/MS) highlight the critical need to establish reference intervals (RIs) specifically for preterm infants, no such RIs has been formally published yet. This study addressed the gap by offering a comprehensive set of reference intervals (RIs) for preterm neonates, and illustrating the dynamic changes of each biomarker with age. DESIGN AND METHODS: The NBS data of 199,693 preterm newborns (< 37 weeks of gestation) who met the inclusion and exclusion criteria from the NNSCP database were included in study analysis. The birth weight stratified dynamic trend of each biomarker were captured by their concentrations over age. Reference partitions were determined by the method of Harris and Boyd. RIs, corresponding to the 2.5th and 97.5th percentiles, as well as the 0.5th, 25th, 50th, 75th and 99.5th percentiles were calculated using a non-parametric rank approach. RESULTS: Increasing birth weight is associated with an elevation in the levels of arginine, citrulline, glycine, leucine and isobarics, methionine, ornithine, phenylalanine, and valine, whereas the levels of alanine, proline and tyrosine decrease. Additionally, two short-chain acylcarnitines (butyrylcarnitine + isobutyrylcarnitine and isovalerylcarnitine + methylbutyrylcarnitine) and a median-chain acylcarnitine (octenoylcarnitine) decrease, while four long-chain acylcarnitines (tetradecanoylcarnitine, palmitoylcarnitine, palmitoleylcarnitine and oleoylcarnitine) increase with increasing birth weight. Age impacts the levels of all MS/MS NBS biomarkers, while sex only affects the level of malonylcarnitine + 3-hydroxybutyrylcarnitine (C3-DC + C4-OH) in very low birth weight preterm neonates. CONCLUSION: The current study developed reference intervals (RIs) specific to birth weight, age, and/or sex for 35 MS/MS biomarkers, which can help in the timely evaluation of the health and disease of preterm neonates.

Self-Sampling by Adolescents at Home: Assessment of the Feasibility to Successfully Collect Blood Microsamples by Inexperienced Individuals.

Blood microsampling has increasingly attracted interest in the past decades as a more patient-centric sampling approach, offering the possibility to collect a minimal volume of blood following a finger or arm prick at home. In addition to conventional dried blood spots (DBS), many different devices allowing self-sampling of blood have become available. Obviously, the success of home-sampling can only be assured when (inexperienced) users collect samples of good quality. Therefore, the feasibility of six different microsampling devices to collect capillary blood by inexperienced adolescents at home was evaluated. Participants (n = 95) were randomly assigned to collect blood (dried or liquid) at different time points using four of six different self-sampling devices (i.e., DBS, Mitra volumetric absorptive microsampling (VAMS), Capitainer B, Tasso M20, Minicollect tube and Tasso+ serum separator tube (SST)). The quality of the samples was visually inspected and analytically determined. Moreover, the participants' satisfaction was assessed via questionnaires. Although a majority succeeded based on the visual inspection, the success rate differed largely between the different devices. In general, the lowest success rate was obtained for the Minicollect tubes, although there is an opportunity and need for improvement for the other self-sampling devices as well. Hence, this also emphasizes the importance to assess the quality of samples collected by the target population prior to study initiation. In addition, visual classification by a trained individual was confirmed based on assessment of the analytical variability between replicates. Finally, self-sampling at home was overall (very) positively received by the participants.

Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs.

Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

Exploratory Untargeted Metabolomics of Dried Blood Spot Samples from Newborns with Maple Syrup Urine Disease.

Currently, tandem mass spectrometry-based newborn screening (NBS), which examines targeted biomarkers, is the first approach used for the early detection of maple syrup urine disease (MSUD) in newborns, followed by confirmatory genetic mutation tests. However, these diagnostic approaches have limitations, demanding the development of additional tools for the diagnosis/screening of MUSD. Recently, untargeted metabolomics has been used to explore metabolic profiling and discover the potential biomarkers/pathways of inherited metabolic diseases. Thus, we aimed to discover a distinctive metabolic profile and biomarkers/pathways for MSUD newborns using untargeted metabolomics. Herein, untargeted metabolomics was used to analyze dried blood spot (DBS) samples from 22 MSUD and 22 healthy control newborns. Our data identified 210 altered endogenous metabolites in MSUD newborns and new potential MSUD biomarkers, particularly L-alloisoleucine, methionine, and lysoPI. In addition, the most impacted pathways in MSUD newborns were the ascorbate and aldarate pathways and pentose and glucuronate interconversions, suggesting that oxidative and detoxification events may occur in early life. Our approach leads to the identification of new potential biomarkers/pathways that could be used for the early diagnosis/screening of MSUD newborns but require further validation studies. Our untargeted metabolomics findings have undoubtedly added new insights to our understanding of the pathogenicity of MSUD, which helps us select the appropriate early treatments for better health outcomes.

Quality considerations and major pitfalls for high throughput DNA-based newborn screening for severe combined immunodeficiency and spinal muscular atrophy.

BACKGROUND: Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD). METHODS: This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined. RESULTS: Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS. CONCLUSIONS: Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per µl may not be reliable and should therefore be avoided.

Increased glucosylsphingosine levels and Gaucher disease in GBA1-associated Parkinson's disease.

INTRODUCTION: Gaucher's disease (GD) is caused by biallelic mutations in the GBA1 gene, leading to reduced glucocerebrosidase (GCase) activity and substrate (glucosylceramide and glucosylsphingosine, GlcSph) accumulation. GBA1 variant carriers are at risk of Parkinson's disease (PD), but only those with biallelic mutations cross the threshold of GCase reduction, leading to substrate accumulation and GD. The link between GBA1 mutations, GD and PD is not fully understood. Here we aimed at reporting the results of a large PD population screening with dried blood spot tests for GD. METHODS: We measured GCase activity and GlcSph levels in 1344 PD patients with dried blood spot tests, and performed GBA1 genetic sequencing. RESULTS: While the GCase activity was reduced in GBA1-PD carriers compared to wild type PD, GlcSph was increased in GBA1-PD compared to GBA1-controls, regardless of the underlying type of GBA1 variant. 13.6 % and 0.4 % of PD patients had mono- or biallelic GBA1 mutations respectively. GCase deficiency, lipid accumulation and clinical manifestations of GD was detected in five PD patients with biallelic GBA1 mutations, of whom four had a risk combined with a GD causing variant. CONCLUSIONS: GlcSph appearing higher in PD may represent a reliable biomarker of the disease and deserves to be further investigated. This study highlights the importance of screening PD patients for possible underlying GD, which is a treatable condition that should not be missed. We diagnosed GD cases carrying a "risk" variant in one allele, which is an unprecedented finding deserving further investigation.

Newborn Dried Blood Spot Folate in Relation to Maternal Self-reported Folic Acid Intake, Autism Spectrum Disorder, and Developmental Delay.

BACKGROUND: Maternal folic acid intake has been associated with decreased risk for neurodevelopmental disorders including autism spectrum disorder (ASD). Genetic differences in folate metabolism could explain some inconsistencies. To our knowledge, newborn folate concentrations remain unexamined. METHODS: We measured folate in archived newborn dried blood spots of children from the CHARGE (Childhood Autism Risks from Genetics and the Environment) case-control study who were clinically confirmed at 24-60 months to have ASD (n = 380), developmental delay (n = 128), or typical development (n = 247). We quantified monthly folic acid intake from maternally-reported supplements and cereals consumed during pregnancy and 3 months prior. We assessed associations of newborn folate with maternal folic acid intake and with ASD or developmental delay using regression. We stratified estimates across maternal and child MTHFR genotypes. RESULTS: Among typically developing children, maternal folic acid intake in prepregnancy and each pregnancy month and prepregnancy prenatal vitamin intake were positively associated with newborn folate. Among children with ASD, prenatal vitamin intake in pregnancy months 2-9 was positively associated with newborn folate. Among children with developmental delay, maternal folic acid and prenatal vitamins during the first pregnancy month were positively associated with neonatal folate. Associations differed by MTHFR genotype. Overall, neonatal folate was not associated with ASD or developmental delay, though we observed associations with ASD in children with the MTHFR 677 TT genotype (odds ratio: 1.76, 95% CI = 1.19, 2.62; P for interaction = 0.08). CONCLUSION: Maternal prenatal folic acid intake was associated with neonatal folate at different times across neurodevelopmental groups. Neonatal folate was not associated with reduced ASD risk. MTHFR genotypes modulated these relationships.

Development and performance of an analytical method for polychlorinated naphthalenes and other persistent organic pollutants in dried blood spots.

BACKGROUND: Dried blood spots (DBSs) collected and archived in newborn screening programs (NSP) represent a potentially valuable resource for assessing exposure to a range of organic and inorganic chemicals in newborns. This study develops and optimizes a method to measure polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides (OCPs) in DBS using the isotope dilution technique, ultrasonic-assisted liquid-liquid extraction, simple cleanup, triple quadrupole GC-MS/MS analysis, and background correction. RESULTS: We minimize the number of extraction repetitions and the volume of solvent, which helps increase throughput while minimizing the potential for contamination. We obtained high recovery and precision for most compounds, and method detection limits (MDLs) were sufficiently low to detect the more prevalent compounds based on representative sample of the US population. MDLs averaged 0.020 ng/mL (recovery: 107 %, precision: 4 %) for PCNs, 0.021 ng/mL (recovery: 97 %, precision: 4 %) for PCBs, 0.021 ng/mL (recovery: 117 %, precision: 2 %) for OCPs, and 0.021 ng/mL (recovery: 96 %, precision: 3 %) for PBDEs. SIGNIFICANCE AND NOVELTY: To our knowledge, this is the first study presenting an analytical method and for PCNs in DBS, and one of the few studies providing an assessment of method performance for persistent organic pollutants in DBS. The optimized method can be applied to a wide range of applications, including exposure assessment, environmental epidemiology, forensics, environmental surveillance, and ecological monitoring.

Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors.

Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.

Newborn Screening for High-Risk Congenital Heart Disease by Dried Blood Spot Biomarker Analysis.

Importance: Congenital heart disease (CHD) is the most common human organ malformation, affecting approximately 1 of 125 newborns globally. Objectives: Assessing the performance of 2 diagnostic tests using minimal amounts of dried blood spots (DBS) to identify high-risk CHD compared with controls in a Swedish cohort of neonates. Design, Setting, and Participants: This diagnostic study took place in Sweden between 2019 and 2023 and enrolled full-term babies born between 2005 and 2023. All cases were identified through centralized pediatric cardiothoracic surgical services in Lund and Gothenburg, Sweden. Controls were followed up for 1 year to ensure no late presentations of high-risk CHD occurred. Cases were verified through surgical records and echocardiography. Exposure: High-risk CHD, defined as cases requiring cardiac surgical management during infancy due to evolving signs of heart failure or types in which the postnatal circulation depends on patency of the arterial duct. Using 3-µL DBS samples, automated quantitative tests for NT-proBNP and interleukin 1 receptor-like 1 (IL-1 RL1; formerly known as soluble ST2) were compared against established CHD screening methods. Main Outcomes and Measures: Performance of DBS tests to detect high-risk CHD using receiver operating characteristic curves; Bland-Altman and Pearson correlation analyses to compare IL-1 RL1 DBS with plasma blood levels. Results: A total of 313 newborns were included (mean [SD] gestational age, 39.4 [1.3] weeks; 181 [57.8%] male). Mean (SD) birthweight was 3495 (483) grams. Analyzed DBS samples included 217 CHD cases and 96 controls. Among the CHD cases, 188 participants (89.3%) were high-risk types, of which 73 (38.8%) were suspected prenatally. Of the 188 high-risk cases, 94 (50.0%) passed pulse oximetry screening and 36 (19.1%) were initially discharged after birth without diagnoses. Combining NT-proBNP and IL-1 RL1 tests performed well in comparison with existing screening methods and enabled additional identification of asymptomatic babies with receiver operating characteristic area under the curve 0.95 (95% CI, 0.93-0.98). Conclusions and relevance: In this diagnostic study, NT-proBNP and IL-1 RL1 DBS assays identified high-risk CHD in a timely manner, including in asymptomatic newborns, and improved overall screening performance in this cohort from Sweden. Prospective evaluation of this novel approach is warranted.

Determination of remifentanil in neonatal dried blood spots by liquid chromatography-tandem mass spectrometry.

Remifentanil is an ultra-short-acting synthetic opioid-class analgesic which might be increasingly used "off-label" as pain management during labour. Side effects in parturients during labour, and in the infant at birth are of particular concern, especially respiratory depression which is concentration-dependent, and can occur at levels as low as 3-5 ng mL-1. The safety of such use, particularly in newborns due to remifentanil placental transfer, has not been fully demonstrated yet, partly due to the lack of a suitable non-invasive analytical method. The aim of our work was to develop a sensitive method to monitor the levels of remifentanil in neonates by a non-invasive sampling of umbi lical cord blood to support efficacy and safety trials. The presented LC-MS method is sensitive enough to reliably quantify remifentanil in just 20 µL of blood at only 0.3 ng mL-1. The dried blood spot sample preparation included solvent extraction with subsequent solid-phase extraction. The method was validated in terms of accuracy, precision, recovery, matrix effect, and stability, and was successfully applied to a small pilot study. The estimated arterial blood concentrations at the time of delivery ranged from 0.2 to 0.3, and up to 0.9 ng mL-1 in neonatal, and maternal samples, respectively.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

CalScope: methodology and lessons learned for conducting a remote statewide SARS-CoV-2 seroprevalence study in California using an at-home dried blood spot collection kit and online survey.

BACKGROUND: To describe the methodology for conducting the CalScope study, a remote, population-based survey launched by the California Department of Public Health (CDPH) to estimate SARS-CoV-2 seroprevalence and understand COVID-19 disease burden in California. METHODS: Between April 2021 and August 2022, 666,857 randomly selected households were invited by mail to complete an online survey and at-home test kit for up to one adult and one child. A gift card was given for each completed survey and test kit. Multiple customized REDCap databases were used to create a data system which provided task automation and scalable data management through API integrations. Support infrastructure was developed to manage follow-up for participant questions and a communications plan was used for outreach through local partners. RESULTS: Across 3 waves, 32,671 out of 666,857 (4.9%) households registered, 6.3% by phone using an interactive voice response (IVR) system and 95.7% in English. Overall, 25,488 (78.0%) households completed surveys, while 23,396 (71.6%) households returned blood samples for testing. Support requests (n = 5,807) received through the web-based form (36.3%), by email (34.1%), and voicemail (29.7%) were mostly concerned with the test kit (31.6%), test result (26.8%), and gift card (21.3%). CONCLUSIONS: Ensuring a well-integrated and scalable data system, responsive support infrastructure for participant follow-up, and appropriate academic and local health department partnerships for study management and communication allowed for successful rollout of a large population-based survey. Remote data collection utilizing online surveys and at-home test kits can complement routine surveillance data for a state health department.

Automated analyses of dried blood spots collected by volumetric microsampling devices.

BACKGROUND: Dried blood spot (DBS) sampling on cellulose cards suffers from varying blood haematocrit levels and from chromatographic effects, which have a direct impact on quantitative DBS analyses. Commercial volumetric microsampling devices were, therefore, introduced to mitigate these effects, however, these devices are not compatible with automated DBS processing systems and must be processed manually. RESULTS: Capillary electrophoresis (CE) instruments use fused-silica (FS) capillaries for precise and accurate liquid handling as well as for injection, separation, and quantitative analyses of liquid samples. These inherent features of an Agilent 7100 CE instrument were employed for the automated processing (elution and homogenization) of DBSs collected by hemaPEN® volumetric devices (2.74 µL of capillary blood per spot). The hemaPEN® samples were processed directly in CE vials by consecutive transfers of 56 µL of methanol and 14 µL of deionized water through the FS capillary in a sequence of 39 DBSs with repeatability of the liquid transfers better than 1.4 %. The resulting DBS eluates were homogenized by a quick air flush through the capillary and analyzed by the same capillary and CE instrument. Creatinine was selected as a clinically relevant model analyte and its endogenous concentrations in DBSs were determined by CE with capacitively coupled contactless conductivity detection (CE-C4D) in a background electrolyte solution consisting of 50 mM acetic acid and 0.1 % (v/v) Tween 20 (pH 3.0). The overall repeatability of the automated DBS processing and CE-C4D analyses of 39 DBSs was ≤7.1 % (peak areas) and ≤0.6 % (migration times), the calibration curve was linear in the 25-500 µM range (R2 = 0.9993) and covered all endogenous blood creatinine levels, the limit of detection was 5.0 µM, and sample throughput was >12 DBSs per hour. DBS ageing for 60 days and varying blood haematocrit levels (20-70 %) did not affect creatinine quantitative results (≤6.9 % for peak areas). Inter-capillary and inter-instrument repeatability was ≤7.7 % (peak areas) and ≤3.4 % (migration times) and demonstrated an excellent transferability of the proposed analytical concept among laboratories. SIGNIFICANCE AND NOVELTY: This contribution is the first-ever report on the use of a single off-the-shelf analytical instrument for fully automated analyses of DBSs collected by commercial volumetric microsampling devices and holds great promise for future unmanned quantitative DBS analyses.

Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease.

BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term. In this review, the focus is on advancements in the field made from 2017 up to 2023. In the first part reviews concerning the subject are discussed. After this, advancements made for clinical purposes are highlighted. Both targeted protein analyses, with and without the use of affinity extractions, as well as untargeted, global proteomic approaches are discussed. In the last part, both methodological advancements are being reviewed as well as the possibility to integrate sample preparation steps during the sample handling. The focus, of this so-called smart sampling, is on the incorporation of cell separation, proteolysis, and antibody-based affinity capture.

A validated method for simultaneous quantification of four antiretrovirals in dried blood spot and plasma using LC-MS/MS: Application to efavirenz therapeutic drug monitoring in pregnant patients.

INTRODUCTION: The specific physiological background induced by pregnancy leads to significant changes in maternal pharmacokinetics, suggesting potential variability in plasma concentrations of antiretrovirals. Pregnant HIV patients exposed to subtherapeutic doses, particularly in the last trimester of the pregnancy, have higher chances to transmit the infection to their children. Therefore, the therapeutic drug monitoring of antiretrovirals in HIV pregnant patients would be of great value. OBJECTIVES: This study aimed to develop and validate a sensitive liquid chromatograph tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of efavirenz, raltegravir, atazanavir, and ritonavir in dried blood spots (DBS) and plasma. DESIGN AND METHODS: The analytes were extracted from the DBS punch and plasma with a mixture of methanol:zinc sulfate 200 mM (50:50, v/v) and 100 % methanol, respectively. For the chromatographic separation a Shim-pack® C18, 4.6 mm × 150 mm, 5 µm column was used. Detection was performed in a 3200-QTRAP® mass spectrometer, with a run time of 6 min. RESULTS: The assay was linear in the range of 15-1,000 ng/mL for raltegravir, 50-10,000 ng/mL for both atazanavir and ritonavir, 50-5,000 ng/mL for efavirenz. Precision and accuracy at these concentrations were less than 15 % for all analytes. Raltegravir, atazanavir, and ritonavir were stable for seven days at 23 °C and 40 °C, whereas efavirenz was stable for twenty-four hours at the same conditions. CONCLUSIONS: The method was successfully applied to quantify efavirenz in DBS samples obtained from HIV-1 infected pregnant volunteers under antiretroviral therapy. The concentrations of efavirenz in DBS and plasma were comparable according to Passing-Bablok regression and Bland-Altman analysis.

Microsampling Techniques Suitable for Therapeutic Drug Monitoring of Antipsychotics.

BACKGROUND: Therapeutic drug monitoring (TDM) of antipsychotics for dose titration or detection of noncompliance is not uncommon in daily practice. Normally, TDM implies measuring a drug concentration in venous blood samples. This technique is invasive and requires trained assistants and patients normally need to go to an outpatient clinic. Over the past decades, sensitivity of analytical equipment has improved leading to a growing interest in microsampling techniques. These techniques are minimally invasive, require a small volume (<100 µL), usually result in stable samples, and can be collected by the patient or a caregiver at home. Before a microsampling technique can be used in daily routine, proper method development and a clinical validation study should be performed. METHOD: For this review, the databases of PubMed and Embase were systematically searched. Currently available microsampling techniques for antipsychotics in blood, serum, or plasma are summarized. Subsequently, it has also been assessed whether these techniques are sufficiently validated for TDM monitoring in daily practice. RESULTS: Several microsampling techniques are available today, for example, dried blood spot sampling, dried plasma extraction cards, and volumetric absorptive microsampling. Eighteen studies were identified in which a microsampling technique for 1 or a few antipsychotics was chemically analytically and clinically validated. However, the majority of these studies have relevant shortcomings that mean its usefulness for different antipsychotics is not yet well established. CONCLUSIONS: Microsampling for TDM can be recommended for patients using clozapine. For TDM of other antipsychotics, it is a very promising development.

Establishing Personalized Blood Protein Reference Ranges Using Noninvasive Microsampling and Targeted Proteomics: Implications for Antidoping Strategies.

To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling with highly multiplexed MRM assays is the best-suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome "fingerprint" showcasing that the targeted proteome is unique to an individual and likely comparable to a DNA fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.