Improving LC-MS/MS measurements of steroids with differential mobility spectrometry.

Introduction: Steroid measurements are important for diagnosis and monitoring of many conditions and treatment regiments; however, due to structural and chemical similarities amongst steroids, these analyses are challenging, even for highly specific techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential mobility spectrometry (DMS) has the potential to improve these analyses by providing an orthogonal and complementary separation technique. Methods: Initially, the potential for DMS to improve signal-to-noise ratio (S/N) and reduce interference was tested by comparing chromatograms acquired with and without DMS when performing measurements of six different steroids. Subsequently, a full clinical validation of cortisol and cortisone in urine was performed with the LC-DMS-MS/MS method. Results and Discussion: DMS significantly reduced interferences observed in the chromatograms and boosted S/N by between 1.6 and 13.8 times. Additionally, DMS improved the agreement between quantifier/qualifier fragment ion results for cortisol and cortisone as indicated by the increase in R2 from approximately 0.81 to 0.98. All validation studies met acceptance criteria and we observed exceptional analytical performance in terms of precision, with % CVs less than 8%. Conclusions: DMS improved the specificity of the steroid measurements by reducing interferences and improving S/N. The validation studies prove that these benefits did not come at the expense of other aspects of analytical performance. This study indicates that DMS has the potential to benefit not just clinical measurements of challenging analytes, but many clinical LC-MS/MS analyses.

Identification, Measurement, and Assessment of the Clinical Utility of Human Pancreatic Polypeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Human pancreatic polypeptide (HPP) is a 36 amino acid peptide hormone that plays a role in the bidirectional communication between the digestive system and the brain. HPP measurements are used to assess vagal nerve function following sham feeding and to detect gastroenteropancreatic-neuroendocrine tumors. These tests have historically been conducted by radioimmunoassays, but liquid chromatography-tandem mass spectrometry (LC-MS/MS) has several advantages such as improved specificity and elimination of radioactive molecules. Here, we present our LC-MS/MS method. Initially, samples were immunopurified and subjected to LC-high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) to identify circulating forms of the peptide in human plasma. We identified 23 forms of HPP, including several glycosylated forms. The most abundant peptides then were used for targeted LC-MS/MS measurements. LC-MS/MS performance for precision, accuracy, linearity, recovery, limit of detection, and carryover met our acceptance criteria based on CLIA regulations. Additionally, we observed the expected physiological rise in HPP in response to sham feeding. Our results indicate that HPP measurement by LC-MS/MS produces clinically equivalent results to our established immunoassay when several peptides are monitored, making it a suitable replacement. The measurement of peptide fragments, including modified species, might have additional clinical value.

Overcoming the chromatographic challenges when performing LC-MS/MS measurements of pyridoxal-5'-phosphate.

Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, is required for numerous enzymatic reactions. Vitamin B6 deficiency or exceptionally high levels of PLP have negative implications, making measurements of PLP imperative for diagnoses and monitoring in many clinical scenarios. Traditional assays are enzymatic, ELISA based, or employ HPLC with various detection modalities; all of these are prone to interferences and crossreactivity with other compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to overcome these issues, but the high polarity of PLP raises chromatographic challenges. Using ion pairing reagents in the mobile phases is a possible solution, but these reagents often have deleterious effects on instrumentation. An alternative strategy is the addition of an ion pairing reagent after extraction, but prior to injection. To prove this, we used 1-octanesulfonic acid (OSA) without changing the LC method or column. With this technique, we observed a 2-4 fold increase in signal-to-noise ratio. Intraday and interday precision of replicate measurements also improved drastically compared to analyses without OSA, while also yielding a dramatic improvement in column life compared to our previous approach and to this point no deleterious effects on instrument hardware commonly associated with traditional ion pairing reagent techniques have been observed.

Development of a multiomics model for identification of predictive biomarkers for COVID-19 severity: a retrospective cohort study.

BACKGROUND: COVID-19 is a multi-system disorder with high variability in clinical outcomes among patients who are admitted to hospital. Although some cytokines such as interleukin (IL)-6 are believed to be associated with severity, there are no early biomarkers that can reliably predict patients who are more likely to have adverse outcomes. Thus, it is crucial to discover predictive markers of serious complications. METHODS: In this retrospective cohort study, we analysed samples from 455 participants with COVID-19 who had had a positive SARS-CoV-2 RT-PCR result between April 14, 2020, and Dec 1, 2020 and who had visited one of three Mayo Clinic sites in the USA (Minnesota, Arizona, or Florida) in the same period. These participants were assigned to three subgroups depending on disease severity as defined by the WHO ordinal scale of clinical improvement (outpatient, severe, or critical). Our control cohort comprised of 182 anonymised age-matched and sex-matched plasma samples that were available from the Mayo Clinic Biorepository and banked before the COVID-19 pandemic. We did a deep profiling of circulatory cytokines and other proteins, lipids, and metabolites from both cohorts. Most patient samples were collected before, or around the time of, hospital admission, representing ideal samples for predictive biomarker discovery. We used proximity extension assays to quantify cytokines and circulatory proteins and tandem mass spectrometry to measure lipids and metabolites. Biomarker discovery was done by applying an AutoGluon-tabular classifier to a multiomics dataset, producing a stacked ensemble of cutting-edge machine learning algorithms. Global proteomics and glycoproteomics on a subset of patient samples with matched pre-COVID-19 plasma samples was also done. FINDINGS: We quantified 1463 cytokines and circulatory proteins, along with 902 lipids and 1018 metabolites. By developing a machine-learning-based prediction model, a set of 102 biomarkers, which predicted severe and clinical COVID-19 outcomes better than the traditional set of cytokines, were discovered. These predictive biomarkers included several novel cytokines and other proteins, lipids, and metabolites. For example, altered amounts of C-type lectin domain family 6 member A (CLEC6A), ether phosphatidylethanolamine (P-18:1/18:1), and 2-hydroxydecanoate, as reported here, have not previously been associated with severity in COVID-19. Patient samples with matched pre-COVID-19 plasma samples showed similar trends in muti-omics signatures along with differences in glycoproteomics profile. INTERPRETATION: A multiomic molecular signature in the plasma of patients with COVID-19 before being admitted to hospital can be exploited to predict a more severe course of disease. Machine learning approaches can be applied to highly complex and multidimensional profiling data to reveal novel signatures of clinical use. The absence of validation in an independent cohort remains a major limitation of the study. FUNDING: Eric and Wendy Schmidt.

Machine Learning-Based Fragment Selection Improves the Performance of Qualitative PRM Assays.

Targeted mass spectrometry-based platforms have become a valuable tool for the sensitive and specific detection of protein biomarkers in clinical and research settings. Traditionally, developing a targeted assay for peptide quantification has involved manually preselecting several fragment ions and establishing a limit of detection (LOD) and a lower limit of quantitation (LLOQ) for confident detection of the target. Established thresholds such as LOD and LLOQ, however, inherently sacrifice sensitivity to afford specificity. Here, we demonstrate that machine learning can be applied to qualitative PRM assays to discriminate positive from negative samples more effectively than a traditional approach utilizing conventional methods. To demonstrate the utility of this method, we trained an ensemble machine learning model using 282 SARS-CoV-2 positive and 994 SARS-CoV-2 negative nasopharyngeal swabs (NP swab) analyzed using a targeted PRM method. This model was then validated using an independent set of 200 positive and 150 negative samples and achieved a sensitivity of 92% relative to results obtained by RT-PCR, which was superior to a traditional approach that resulted in 86.5% sensitivity when analyzing the same data. These results demonstrate that machine learning can be applied to qualitative PRM assays and results in superior performance relative to traditional methods.

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay-based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe several COVID-19 cases where we were unable to detect the expected peptide targets from clinical nasopharyngeal swabs. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Minor modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants could be detected by performing the bottom-up proteomic analysis of whole viral genome-sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.

A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples.

SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.

Isotopic Distribution Calibration for Mass Spectrometry.

Mass spectrometry (MS) is widely used in science and industry. It allows accurate, specific, sensitive, and reproducible detection and quantification of a huge range of analytes. Across MS applications, quantification by MS has grown most dramatically, with >50 million experiments/year in the USA alone. However, quantification performance varies between instruments, compounds, different samples, and within- and across runs, necessitating normalization with analyte-similar internal standards (IS) and use of IS-corrected multipoint external calibration curves for each analyte, a complicated and resource-intensive approach, which is particularly ill-suited for multi-analyte measurements. We have developed an internal calibration method that utilizes the natural isotope distribution of an IS for a given analyte to provide internal multipoint calibration. Multiple isotope distribution calibrators for different targets in the same sample facilitate multiplex quantification, while the emerging random-access automated MS platforms should also greatly benefit from this approach. Finally, isotope distribution calibration allows mathematical correction for suboptimal experimental conditions. This might also enable quantification of hitherto difficult, or impossible to quantify, targets, if the distribution is adjusted in silico to mimic the analyte. The approach works well for high resolution, accurate mass MS for analytes with at least a modest-sized isotopic envelope. As shown herein, the approach can also be applied to lower molecular weight analytes, but the reduction in calibration points does reduce quantification performance.

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Analytical Sensitivity and Specificity of Four Point of Care Rapid Antigen Diagnostic Tests for SARS-CoV-2 Using Real-Time Quantitative PCR, Quantitative Droplet Digital PCR, and a Mass Spectrometric Antigen Assay as Comparator Methods.

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

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction.

BACKGROUND AIMS: Viral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct. METHODS: Assay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations. RESULTS: The limit of detection was 10 copies/µL, whereas negative samples showed <1.3 copies/µL. Within and between assay imprecision coefficients of variation across the reportable range (10-10 000 copies/µL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R2 >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations). CONCLUSIONS: DDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.

Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum.

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

Center of Mass Calculation in Combination with MS/MS Allows Robust Identification of Single Amino Acid Polymorphisms in Clinical Measurements of Insulin-Like Growth Factor-1.

Insulin-like growth factor-1 (IGF-1) measurement by high-resolution accurate mass-mass spectrometry (HRAM-MS) is replacing IGF-1 immunoassays and allows for identification of single amino acid variants; by contrast, both normal and deleterious sequence variants might be missed by immunoassays or non-HRAM-MS methods. We have developed an intact molecule HRAM-MS method to identify IGF-1 variants, distinguishing them by a center of mass (COM) calculation, followed by various tandem-MS activation techniques (HCD, ETD, ETciD, EThcD, UVPD). We found single amino acid variants in 841 of 146 620 patient samples (0.57%). Most were benign (A67T, A70T). We also observed a pathogenic variant (V44M), likely pathogenic variants (A38V, V17M), and a likely benign variant (A67V). For 207 samples from unique patients with residual serum, the MS variant results were confirmed by cell-free DNA sequencing. Our approach allows accurate quantitative reporting of functional IGF-1 in the presence of single amino acid variants. The COM approach potentially enables omission of tandem-MS for known, common variants, while the combination of COM and tandem-MS allows accurate identification in all cases we encountered. This approach should be applicable to qualitative and quantitative analyses of other peptides/proteins in clinical and research settings and might lend itself to the characterization of other protein variations.

Monozygotic twins discordant for congenital adrenal hyperplasia due to mosaicism.

INTRODUCTION: Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. CASE DESCRIPTION: A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5'-CYP21A1P/CYP21A2-3' hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. CONCLUSIONS: The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

Adrenal Cortical Carcinoma Associated With Lynch Syndrome: A Case Report and Review of Literature.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis. ACC was reported in 3.2% patients with Lynch syndrome (LS), however no particular case-detection strategies have been recommended. PARTICIPANTS: We report a case of a 65-year-old woman who was incidentally discovered with a large adrenal mass during work-up of postmenopausal uterine bleeding. She was recently diagnosed with MSH6 germline mutation after her sister presented with uterine carcinoma in the setting of LS. RESULTS: Whereas the patient was asymptomatic for overt hormonal excess, biochemical work-up confirmed glucocorticoid autonomy and androgen and estrogen excess. Urine steroid profiling was suggestive of ACC. Adrenalectomy confirmed an oncocytic ACC with focal extracapsular extension into the periadrenal adipose tissue with a Ki-67 of 15% and a peak mitotic count of 40/50 high-power fields. CONCLUSION: ACC can be the only manifestation of LS. A best case-detection approach for ACC in the asymptomatic patient with LS is unclear, however urine steroid profiling could be considered.

Compound heterozygosity for a whole gene deletion and p.R124C mutation in CYP21A2 causing nonclassic congenital adrenal hyperplasia.

We present a family with 2 members who received long-term steroid treatment for presumed classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, until molecular testing revealed nonclassic CAH, not necessarily requiring treatment. A 17-year-old male presented to our clinic on glucocorticoid and mineralocorticoid treatment for classic CAH. He was diagnosed at 4 years of age based on mild-moderate elevations of 17-hydroxyprogesterone (17-OHP) and adrenocorticotropic hormone (ACTH), but without evidence of precocious adrenarche/puberty. Due to his diagnosis, his clinically asymptomatic 3-year-old sister was tested and also found to have elevated ACTH and 17-OHP levels and was started on glucocorticoids for classic CAH. Family history revealed a healthy sibling who had no biochemical evidence of CAH and consanguineous healthy parents. We questioned the diagnosis of classic CAH and performed an ACTH1-24 stimulation test, which showed a level of 17-OHP in the borderline range between classic and nonclassic CAH. Molecular testing, using sequencing and multiplex ligation-dependent probe amplification analysis of CYP21A2, revealed that both affected siblings were compound heterozygotes for a whole-gene deletion and a, likely pathogenic (nonclassical), sequence variant, p.R124C. The asymptomatic father had the same genotype, while the mother showed one deleted copy and 2 active copies, making her an asymptomatic carrier. Our report demonstrates the importance of molecular testing in atypical cases of CAH, as well as the importance of both sequencing and deletion analysis. The results of molecular testing should be interpreted in clinical context, and treatment should be prescribed according to guidelines when available.

Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing.

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.