Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, ...development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.
•Hematocrit issues still prevent full acceptance of dried blood spot analysis.•Many attempts to overcome this hematocrit bias have been proposed.•Whole spot analysis with volumetric application of ...blood should nullify this bias.•Devices generating dried plasma spots may avoid all hematocrit-related issues.•Strategies to measure the hematocrit of a dried blood spot have been proposed.
Hematocrit-related issues remain a major barrier for (regulatory) acceptance of the classical dried blood spot (DBS) analysis in the bioanalytical and clinical field. Lately, many attempts to cope with these issues have been made. Throughout this review, an overview is provided on new strategies that try to cope with this hematocrit effect (going from avoiding to minimizing), on methods estimating a DBS volume, and on methods estimating or measuring the hematocrit of a DBS. Although many successful strategies have been put forward, a combination of different technologies still provides the most complete solution. Therefore, further efforts and the availability of a straightforward guideline for analytical and clinical method validation should help to overcome the hurdles still associated with DBS sampling.
There is increasing interest in using dried blood spot (DBS) cards to extend the reach of global health and disease surveillance programs to hard-to-reach populations. Conceptually, DBS offers a ...cost-effective solution for multiple use cases by simplifying logistics for collecting, preserving, and transporting blood specimens in settings with minimal infrastructure. This review describes methods to determine both the reliability of DBS-based bioanalysis for a defined use case and the optimal conditions that minimize pre-analytical sources of data variability. Examples by the newborn screening, drug development, and global health communities are provided in this review of published literature. Sources of variability are linked in most cases, emphasizing the importance of field-to-laboratory standard operating procedures that are evidence based and consider both stability and efficiency of recovery for a specified analyte in defining the type of DBS card, accessories, handling procedures, and storage conditions. Also included in this review are reports where DBS was determined to not be feasible because of technology limitations or physiological properties of a targeted analyte.
The sensitivity of dried blood spots (DBS) to identify newborns with congenital cytomegalovirus (cCMV) infection has not been evaluated in screening studies using the current, higher-sensitivity ...methods for DBS processing.
To assess the sensitivity of DBS polymerase chain reaction (PCR) for newborn screening for cCMV infection using saliva as the reference standard for screening, followed by collection of a urine sample for confirmation of congenital infection.
This population-based cohort study took place at 5 newborn nurseries and 3 neonatal intensive care units in the Minneapolis/Saint Paul area in Minnesota from April 2016 to June 2019. Newborns enrolled with parental consent were screened for cCMV using DBS obtained for routine newborn screening and saliva collected 1 to 2 days after birth. Dried blood spots were tested for CMV DNA by PCR at both the University of Minnesota (UMN) and the US Centers for Disease Control and Prevention (CDC). Saliva swabs were tested by CMV DNA PCR at the UMN laboratory only. Newborns who screened positive by saliva or DBS had a diagnostic urine sample obtained by primary care professionals, tested by PCR within 3 weeks of birth. Analysis began July 2019.
Detection of CMV from a saliva swab using a PCR assay.
Number of children with urine-confirmed cCMV and the proportion of them who were CMV positive through DBS screening.
Of 12 554 individuals enrolled through June 2019 (of 25 000 projected enrollment), 56 newborns were confirmed to have cCMV (4.5 per 1000 95% CI, 3.3-5.7). Combined DBS results from either UMN or CDC had a sensitivity of 85.7% (48 of 56; 95% CI, 74.3%-92.6%), specificity of 100.0% (95% CI, 100.0%-100.0%), positive predictive value (PPV) of 98.0% (95% CI, 89.3%-99.6%), and negative predictive value (NPV) of 99.9% (95% CI, 99.9%-100.0%). Dried blood spot results from UMN had a sensitivity of 73.2% (95% CI, 60.4%-83.0%), specificity of 100.0% (100.0%-100.0%), PPV of 100.0% (95% CI, 91.4%-100.0%), and NPV of 99.9% (95% CI, 99.8%-99.9%). Dried blood spot results from CDC had a sensitivity of 76.8% (95% CI, 64.2%-85.9%), specificity of 100.0% (95% CI, 100.0%-100.0%), PPV of 97.7% (95% CI, 88.2%-99.6%), and NPV of 99.9% (95% CI, 99.8%-99.9%). Saliva swab results had a sensitivity of 92.9% (52 of 56; 95% CI, 83.0%-97.2%), specificity of 99.9% (95% CI, 99.9%-100.0%), PPV of 86.7% (95% CI, 75.8%-93.1%), and NPV of 100.0% (95% CI, 99.9%-100.0%).
This study demonstrates relatively high analytical sensitivity for DBS compared with previous studies that performed population-based screening. As more sensitive DNA extraction and PCR methods continue to emerge, DBS-based testing should remain under investigation as a potential low-cost, high-throughput option for cCMV screening.
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.
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.
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.
Display omitted
•Fully autonomous analyses of DBSs collected by hemaPEN® volumetric devices.•Suitable for unmanned quantitative analyses of remote, patient-centric blood samples.•DBS elution, homogenization, and analysis by a single commercial CE instrument.•Sample throughput for the determination of creatinine >12 DBSs per hour.•Excellent repeatability, sensitivity, and transferability; no effects of haematocrit and ageing.
The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed ...by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R2 value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.
Volumetric absorptive microsampling (VAMS) is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and homogeneity issues associated with ...conventional dried blood spot (DBS) sample when a subpunch is taken. The VAMS sampler absorbs a fixed volume of blood (∼10 μL) in 2-4 s with less than 5% volume variation across the hematocrit range of 20-70% with low tip-to-tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.
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.
PDF
