Showing papers in "Clinical Chemistry in 2013"
TL;DR: This review provides an overview of current controversies as well as current recommendations for gestational diabetes care and the recommendations of a number of professional organizations.
Abstract: Background: Gestational diabetes mellitus, defined as diabetes diagnosed during pregnancy that is not clearly overt diabetes, is becoming more common as the epidemic of obesity and type 2 diabetes continues. Newly proposed diagnostic criteria will, if adopted universally, further increase the prevalence of this condition. Much controversy surrounds the diagnosis and management of gestational diabetes.
Content: This review provides information regarding various approaches to the diagnosis of gestational diabetes and the recommendations of a number of professional organizations. The implications of gestational diabetes for both the mother and the offspring are described. Approaches to self-monitoring of blood glucose concentrations and treatment with diet, oral medications, and insulin injections are covered. Management of glucose metabolism during labor and the postpartum period are discussed, and an approach to determining the timing of delivery and the mode of delivery is outlined.
Summary: This review provides an overview of current controversies as well as current recommendations for gestational diabetes care.
1,553 citations
TL;DR: A considerable number of promising CTC-detection techniques have been developed in recent years, but the analytical specificity and clinical utility of these methods must be demonstrated in large prospective multicenter studies to reach the high level of evidence required for their introduction into clinical practice.
Abstract: BACKGROUND: The detection and molecular characterization of circulating tumor cells (CTCs) are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker. The aims of research on CTCs include ( a ) estimation of the risk for metastatic relapse or metastatic progression (prognostic information), ( b ) stratification and real-time monitoring of therapies, ( c ) identification of therapeutic targets and resistance mechanisms, and ( d ) understanding metastasis development in cancer patients.
CONTENT: This review focuses on the technologies used for the enrichment and detection of CTCs. We outline and discuss the current technologies that are based on exploiting the physical and biological properties of CTCs. A number of innovative technologies to improve methods for CTC detection have recently been developed, including CTC microchips, filtration devices, quantitative reverse-transcription PCR assays, and automated microscopy systems. Molecular-characterization studies have indicated, however, that CTCs are very heterogeneous, a finding that underscores the need for multiplex approaches to capture all of the relevant CTC subsets. We therefore emphasize the current challenges of increasing the yield and detection of CTCs that have undergone an epithelial–mesenchymal transition. Increasing assay analytical sensitivity may lead, however, to a decrease in analytical specificity (e.g., through the detection of circulating normal epithelial cells).
SUMMARY: A considerable number of promising CTC-detection techniques have been developed in recent years. The analytical specificity and clinical utility of these methods must be demonstrated in large prospective multicenter studies to reach the high level of evidence required for their introduction into clinical practice.
900 citations
TL;DR: This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation and presents the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines.
Abstract: There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.
686 citations
TL;DR: Evidence suggests recent smoking and/or blood THC concentrations 2-5 ng/mL are associated with substantial driving impairment, particularly in occasional smokers, and future cannabis-and-driving research should emphasize challenging tasks, such as divided attention, and include occasional and chronic daily cannabis smokers.
Abstract: BACKGROUND: Cannabis is the most prevalent illicit drug identified in impaired drivers. The effects of cannabis on driving continue to be debated, making prosecution and legislation difficult. Historically, delays in sample collection, evaluating the inactive Δ9-tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-THC, and polydrug use have complicated epidemiologic evaluations of driver impairment after cannabis use.
CONTENT: We review and evaluate the current literature on cannabis' effects on driving, highlighting the epidemiologic and experimental data. Epidemiologic data show that the risk of involvement in a motor vehicle accident (MVA) increases approximately 2-fold after cannabis smoking. The adjusted risk of driver culpability also increases substantially, particularly with increased blood THC concentrations. Studies that have used urine as the biological matrix have not shown an association between cannabis and crash risk. Experimental data show that drivers attempt to compensate by driving more slowly after smoking cannabis, but control deteriorates with increasing task complexity. Cannabis smoking increases lane weaving and impaired cognitive function. Critical-tracking tests, reaction times, divided-attention tasks, and lane-position variability all show cannabis-induced impairment. Despite purported tolerance in frequent smokers, complex tasks still show impairment. Combining cannabis with alcohol enhances impairment, especially lane weaving.
SUMMARY: Differences in study designs frequently account for inconsistencies in results between studies. Participant-selection bias and confounding factors attenuate ostensible cannabis effects, but the association with MVA often retains significance. Evidence suggests recent smoking and/or blood THC concentrations 2–5 ng/mL are associated with substantial driving impairment, particularly in occasional smokers. Future cannabis-and-driving research should emphasize challenging tasks, such as divided attention, and include occasional and chronic daily cannabis smokers.
500 citations
TL;DR: This study explored the use of shotgun massively parallel sequencing of plasma DNA from cancer patients to scan a cancer genome noninvasively and showed that plasma DNA sequencing is a valuable approach for studying tumoral heterogeneity.
Abstract: BACKGROUND: Tumor-derived DNA can be found in the plasma of cancer patients. In this study, we explored the use of shotgun massively parallel sequencing (MPS) of plasma DNA from cancer patients to scan a cancer genome noninvasively.
METHODS: Four hepatocellular carcinoma patients and a patient with synchronous breast and ovarian cancers were recruited. DNA was extracted from the tumor tissues, and the preoperative and postoperative plasma samples of these patients were analyzed with shotgun MPS.
RESULTS: We achieved the genomewide profiling of copy number aberrations and point mutations in the plasma of the cancer patients. By detecting and quantifying the genomewide aggregated allelic loss and point mutations, we determined the fractional concentrations of tumor-derived DNA in plasma and correlated these values with tumor size and surgical treatment. We also demonstrated the potential utility of this approach for the analysis of complex oncologic scenarios by studying the patient with 2 synchronous cancers. Through the use of multiregional sequencing of tumoral tissues and shotgun sequencing of plasma DNA, we have shown that plasma DNA sequencing is a valuable approach for studying tumoral heterogeneity.
CONCLUSIONS: Shotgun DNA sequencing of plasma is a potentially powerful tool for cancer detection, monitoring, and research.
471 citations
TL;DR: Clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection is demonstrated.
Abstract: BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.
METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.
RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS , 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS -mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS -mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS -mutated DNA. All 5 samples with BRAF mutations were detected.
CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.
448 citations
TL;DR: A better understanding of how the tumor environment affects cancer progression should provide new targets for the isolation and destruction of cancer cells via interference with the complex crosstalk established between cancer cells, host cells, and their surrounding extracellular matrix.
Abstract: BACKGROUND: With the emergence of the tumor microenvironment as an essential ingredient of cancer malignancy, therapies targeting the host compartment of tumors have begun to be designed and applied in the clinic.
CONTENT: The malignant features of cancer cells cannot be manifested without an important interplay between cancer cells and their local environment. The tumor infiltrate composed of immune cells, angiogenic vascular cells, lymphatic endothelial cells, and cancer-associated fibroblastic cells contributes actively to cancer progression. The ability to change these surroundings is an important property by which tumor cells are able to acquire some of the hallmark functions necessary for tumor growth and metastatic dissemination. Thus in the clinical setting the targeting of the tumor microenvironment to encapsulate or destroy cancer cells in their local environment has become mandatory. The variety of stromal cells, the complexity of the molecular components of the tumor stroma, and the similarity with normal tissue present huge challenges for therapies targeting the tumor microenvironment. These issues and their interplay are addressed in this review. After a decade of intensive clinical trials targeting cellular components of the tumor microenvironment, more recent investigations have shed light on the important role in cancer progression played by the noncellular stromal compartment composed of the extracellular matrix.
SUMMARY: A better understanding of how the tumor environment affects cancer progression should provide new targets for the isolation and destruction of cancer cells via interference with the complex crosstalk established between cancer cells, host cells, and their surrounding extracellular matrix.
353 citations
TL;DR: Circulating miRNAs are deregulated in severe obesity and weight loss-induced changes in this profile and the study of in silico targets support this observation and suggest a potential mechanistic relevance.
Abstract: Background: Genomic studies have yielded important insights into the pathogenesis of obesity. Circulating microRNAs (miRNAs) are valuable biomarkers of systemic diseases and potential therapeutic targets. We sought to define the circulating pattern of miRNAs in obesity and examine changes after weight loss.
Methods: We assessed the genome-wide circulating miRNA profile cross-sectionally in 32 men and after surgery-induced weight loss in 6 morbidly obese patients. The most relevant miRNAs were cross-sectionally validated in 80 men and longitudinally in 22 patients (after surgery-induced weight loss). We evaluated the effects of diet-induced weight loss in 9 obese patients. Thirty-six circulating miRNAs were associated with anthropometric variables in the initial sample.
Results: In the validation study, morbidly obese patients showed a marked increase of miR-140-5p, miR-142-3p (both P < 0.0001), and miR-222 ( P = 0.0002) and decreased levels of miR-532–5p, miR-125b, miR-130b, miR-221, miR-15a, miR-423-5p, and miR-520c-3p ( P < 0.0001 for all). Interestingly, in silico targets leukemia inhibitory factor receptor (LIFR) and transforming growth factor receptor (TGFR) of miR-140-5p, miR-142-3p, miR-15a, and miR-520c-3p circulated in association with their corresponding miRNAs. Moreover, a discriminant function of 3 miRNAs (miR-15a, miR-520c-3p, and miR-423-5p) was specific for morbid obesity, with an accuracy of 93.5%. Surgery-induced (but not diet-induced) weight loss led to a marked decrease of miR-140-5p, miR-122, miR-193a-5p, and miR-16-1 and upregulation of miR-221 and miR-199a-3p ( P < 0.0001 for all).
Conclusions: Circulating miRNAs are deregulated in severe obesity. Weight loss–induced changes in this profile and the study of in silico targets support this observation and suggest a potential mechanistic relevance.
343 citations
TL;DR: Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor.
Abstract: BACKGROUND: Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models.
CONTENT: The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity.
SUMMARY: Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies.
335 citations
TL;DR: Advances in the Human Microbiome Project and human metagenomics research will lead the way toward a greater understanding of the importance and role of the gut microbiome in metabolic disorders such as obesity, metabolic syndrome, and diabetes.
Abstract: Background: Obesity, metabolic syndrome, and type 2 diabetes are major public health challenges. Recently, interest has surged regarding the possible role of the intestinal microbiota as potential novel contributors to the increased prevalence of these 3 disorders.
Content: Recent advances in microbial DNA sequencing technologies have resulted in the widespread application of whole-genome sequencing technologies for metagenomic DNA analysis of complex ecosystems such as the human gut. Current evidence suggests that the gut microbiota affect nutrient acquisition, energy harvest, and a myriad of host metabolic pathways.
Conclusion: Advances in the Human Microbiome Project and human metagenomics research will lead the way toward a greater understanding of the importance and role of the gut microbiome in metabolic disorders such as obesity, metabolic syndrome, and diabetes.
274 citations
TL;DR: A series of experiments were described to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR assays by introducing a panel of clinically relevant inhibitors directly into the PCR reactions, resulting in differences in the resulting inhibition curves.
Abstract: To the Editor:
Real-time quantitative PCR (qPCR)1 is a rapid and sensitive method that forms the foundation for many clinical diagnostic tests. Droplet digital PCR (ddPCR) shares these qualities with qPCR, but owing to reaction partitioning, ddPCR is proposed to exhibit increased tolerance to interfering substances, making it an attractive alternative to qPCR for diagnostic applications (1, 2). The data to support this phenomenon and its mechanism, however, are currently lacking in the literature (3).
Herein, we describe a series of experiments to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR (Bio-Rad Laboratories, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA, and heparin) directly into the PCR reactions (4). Differences in the resulting inhibition curves and the half-maximal inhibitory concentrations (IC50) were then assessed. The laboratory-developed CMV qPCR is a double primer/probe Taqman assay that amplifies and detects the genes UL123 (IE)2 (enhances activation by IE2; interacts with basal transcriptional machinery and cellular transcription factor; disrupts ND10; involved in gene regulation [Human herpesvirus 5]) and UL55 (gB) (type 1 membrane protein; possible membrane fusogen; binds cell surface heparan sulphate; involved in cell entry; involved in cell-to-cell spread [Human herpesvirus 5]) with primers and probes previously described (5). The ddPCR assay uses the same primers and probes, with the dyes HEX replacing 6-FAM on the gB probe and BHQ-1 replacing TAMRA on both probes (Sigma-Aldrich).
SDS, EDTA, and …
TL;DR: Lower 25(OH)D concentrations, by clinical categories or seasonally adjusted quartiles, were associated with higher cumulative incidence of type 2 diabetes and this finding was substantiated in a metaanalysis.
Abstract: BACKGROUND: Vitamin D deficiency has been implicated in decreased insulin secretion and increased insulin resistance, hallmarks of type 2 diabetes mellitus. We tested the hypothesis that low plasma 25-hydroxyvitamin D [25(OH)D] is associated with increased risk of type 2 diabetes in the general population.
METHODS: We measured 25(OH)D in 9841 participants from the general population, of whom 810 developed type 2 diabetes during 29 years of follow-up. Analyses were adjusted for sex, age, smoking status, body mass index, income, physical activity, HDL cholesterol, and calendar month of blood draw.
RESULTS: Lower 25(OH)D concentrations, by clinical categories or seasonally adjusted quartiles, were associated with higher cumulative incidence of type 2 diabetes (trend, P = 2×10−7 and P = 4×10−10). Multivariable adjusted hazard ratios of type 2 diabetes were 1.22 (95% CI 0.85–1.74) for 25(OH)D <5 vs ≥20 μg/L and 1.35 (1.09–1.66) for lowest vs highest quartile. Also, the multivariable adjusted hazard ratio of type 2 diabetes for a 50% lower concentration of 25(OH)D was 1.12 (1.03–1.21); the corresponding hazard ratio for those ≤58 years old was 1.26 (1.15–1.41). Finally, in a metaanalysis of 16 studies, the odds ratio for type 2 diabetes was 1.50 (1.33–1.70) for the bottom vs top quartile of 25(OH)D.
CONCLUSIONS: We observed an association of low plasma 25(OH)D with increased risk of type 2 diabetes. This finding was substantiated in a metaanalysis.
TL;DR: A novel, cost-effective, rapid technique was developed to quantify GcfDNA in transplant recipients and embodies a promising, potentially universal biomarker for early detection of rejection, which could enable more effective therapeutic interventions.
Abstract: BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).
METHODS: Plasma samples were collected from stable recipients after liver (LTx, n = 10), kidney (KTx, n = 9), and heart (HTx, n = 8) transplantation as well as from 7 additional patients directly after LTx. Known single-nucleotide polymorphisms were selected for high minor allelic frequencies, of which 41 hydrolysis probe assays were established. Plasma cfDNA was preamplified, followed by conventional real-time PCR to define informative (heterologous) SNPs, which were then used for quantification (percentage) of graft-derived cfDNA (GcfDNA) using ddPCR.
RESULTS: Mean recovery was 94% (SD, 13%) with an imprecision of 4%–14% with the use of controls with 2% minor allele. GcfDNA in stable patients was 60%, whereas in 1 patient with cholestasis no increase was found.
CONCLUSIONS: A novel, cost-effective, rapid technique was developed to quantify GcfDNA in transplant recipients. This technique embodies a promising, potentially universal biomarker for early detection of rejection, which could enable more effective therapeutic interventions.
TL;DR: Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.
Abstract: BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC).
METHODS: We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations.
RESULTS: At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients.
CONCLUSIONS: Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR , KRAS , and PIK3CA , possibly explaining the variable response rates to EGFR inhibition in patients with CRC.
TL;DR: Nontargeted metabolomics investigations led to the following recommendations for the preanalytical phase: test the blood collection tubes, avoid hemolysis, place whole blood immediately in ice water, use EDTA plasma, and preferably use nonrefrozen biobank samples.
Abstract: BACKGROUND: Metabolomics is a powerful tool that is increasingly used in clinical research. Although excellent sample quality is essential, it can easily be compromised by undetected preanalytical errors. We set out to identify critical preanalytical steps and biomarkers that reflect preanalytical inaccuracies.
METHODS: We systematically investigated the effects of preanalytical variables (blood collection tubes, hemolysis, temperature and time before further processing, and number of freeze–thaw cycles) on metabolomics studies of clinical blood and plasma samples using a nontargeted LC-MS approach.
RESULTS: Serum and heparinate blood collection tubes led to chemical noise in the mass spectra. Distinct, significant changes of 64 features in the EDTA-plasma metabolome were detected when blood was exposed to room temperature for 2, 4, 8, and 24 h. The resulting pattern was characterized by increases in hypoxanthine and sphingosine 1-phosphate (800% and 380%, respectively, at 2 h). In contrast, the plasma metabolome was stable for up to 4 h when EDTA blood samples were immediately placed in iced water. Hemolysis also caused numerous changes in the metabolic profile. Unexpectedly, up to 4 freeze–thaw cycles only slightly changed the EDTA-plasma metabolome, but increased the individual variability.
CONCLUSIONS: Nontargeted metabolomics investigations led to the following recommendations for the preanalytical phase: test the blood collection tubes, avoid hemolysis, place whole blood immediately in ice water, use EDTA plasma, and preferably use nonrefrozen biobank samples. To exclude outliers due to preanalytical errors, inspect the biomarker signal intensities reflecting systematic as well as accidental and preanalytical inaccuracies before processing the bioinformatics data.
TL;DR: MPS of maternal plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA, and significant changes in the size profiles of circulating maternal DNA after delivery are observed, but changes in circulating fetalDNA are not observed.
Abstract: BACKGROUND: With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in maternal plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA.
METHODS: Using paired-end MPS, we analyzed serial samples of maternal plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery.
RESULTS: The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA.
CONCLUSIONS: MPS of maternal plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.
TL;DR: A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients, suggesting a particular biology of colorectal cancer affecting CTC detection.
Abstract: BACKGROUND: The incidence and number of circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients are lower than in other cancer types, which may point to a particular biology of colorectal cancer affecting CTC detection.
METHODS: We detected CTCs in the peripheral and mesenteric blood of colorectal cancer patients by use of 2 independent technologies on the basis of different biological properties of colon cancer cells. Seventy-five patients diagnosed with localized (M, n = 60) and metastatic (M1, n = 15) colorectal cancer were included. Peripheral and mesenteric blood samples were collected before tumor resection. We performed CTC enumeration with an EpCAM-independent enrichment method followed by the Epispot assay that detected only viable CK19-releasing CTCs. In parallel, we used the FDA-cleared EpCAM-dependent CellSearch® as the reference method.
RESULTS: The enumeration of CK19-releasing cells by the CK19-Epispot assay revealed viable CTCs in 27 of 41 (65.9%) and 41 of 74 (55.4%) ( P = 0.04) patients in mesenteric and peripheral blood, respectively, whereas CellSearch detected CTCs in 19 of 34 (55.9%) and 20 of 69 (29.0%) ( P = 0.0046) patients. In mesenteric blood, medians of 4 (range 0–247) and 2.7 CTCs (range 0–286) were found with Epispot and CellSearch ( P = 0.2), respectively, whereas in peripheral blood, Epispot and CellSearch detected a median of 1.2 (range 0–92) and 0 CTCs (range 0–147) ( P = 0.002).
CONCLUSIONS: A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients.
TL;DR: For potential use in epidemiologic studies, the majority of plasma metabolites had low CVs and were reproducible over a 24-h processing delay and within individuals over 1-2 years.
Abstract: BACKGROUND: Rigorous studies are necessary to demonstrate suitability of metabolomics platforms to profile metabolites in archived plasma within epidemiologic studies of human disease, for which attenuation of effect estimates due to measurement error is a key concern.
METHODS: Using a liquid chromatography–tandem mass spectrometry platform, we quantified 257 metabolites from archived plasma to evaluate metabolite interassay reproducibility, reproducibility with delayed processing, and within-person reproducibility over time. Interassay reproducibility was assessed with CVs from 60 duplicate plasma samples donated by participants in the Nurses' Health Study and Health Professionals Follow-up Study, and 20 QC pool plasma replicates. Metabolite reproducibility over a 24- to 48-h processing delay (n = 48 samples) and within-person reproducibility over 1–2 years (n = 80 samples) were assessed using Spearman and intraclass correlation coefficients (ICCs).
RESULTS: CVs were <20% for 92% of metabolites and generally were similar by plasma anticoagulant type (heparin or EDTA) and fasting time. Approximately 75% of metabolites were reproducible over delays in processing of blood samples (Spearman correlation or ICC ≥0.75, comparing immediate and 24-h delayed processing). Carbohydrates and purine/pyrimidine derivatives were most adversely affected by the processing delay. Ninety percent of metabolites were reproducible over 1–2 years within individuals (Spearman correlation or ICC ≥0.4).
CONCLUSIONS: For potential use in epidemiologic studies, the majority of plasma metabolites had low CVs and were reproducible over a 24-h processing delay and within individuals over 1–2 years. Certain metabolites, such as carbohydrates and purine/pyrimidine derivatives, may be challenging to evaluate if samples have delayed processing.
TL;DR: The findings indicate that serum concentrations of deregulated microRNAs may be linked to a particular biology of breast carcinomas favoring progression and metastatic spread.
Abstract: BACKGROUND: MicroRNAs (miRs) are small, noncoding RNAs that target genes involved in tumor development and progression. In the current study, we investigated the use of circulating miR concentrations as biomarkers in the serum of breast cancer patients.
METHODS: We analyzed serum samples from 120 patients with primary breast cancer after surgery and before chemotherapy (M0, classified into 3 subgroups of 40 patients with progesterone/estrogen-positive, HER2-positive, and triple-negative cancer), 32 patients with overt metastasis (M1), and 40 healthy women. Using quantitative TaqMan MicroRNA PCR, we measured the relative concentrations of 6 circulating microRNAs (miR-10b, -17, -34a, -93, -155, and -373) known to be relevant for tumor development and progression. The data were correlated with clinicopathologic risk factors, with particular reference to HER2 and hormone receptor status of the primary tumor and the presence of metastases.
RESULTS: The relative serum concentrations of circulating miR-34a [ P = 0.013, area under the curve (AUC) 0.636], miR-93 ( P = 0.001, AUC 0.699), and miR-373 ( P = 0.0001, AUC 0.879) were significantly different between M0 breast cancer patients and healthy women, whereas miR-17 ( P = 0.002, AUC 0.679) and miR-155 ( P = 0.0001, AUC 0.781) were differently expressed between M0 and M1 patients. Increased concentrations of miR-373 were associated with negative HER2 status of the primary tumor ( P = 0.0001). Deregulated concentrations of miR-17 ( P = 0.019) and miR-34a ( P = 0.029) were detected in patients with progesterone/estrogen receptor–positive and –negative status, respectively.
CONCLUSIONS: Our findings indicate that serum concentrations of deregulated microRNAs may be linked to a particular biology of breast carcinomas favoring progression and metastatic spread.
TL;DR: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated, and this finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior inqPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.
Abstract: Background: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility.
Methods: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield.
Results: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3’ end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased.
Conclusions: Single mismatches located >5 bp from the 3’ end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for >3 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.
TL;DR: The affinity-assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies and has acceptable performance characteristics for use in clinical diagnostic applications.
Abstract: Background: Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAbs), which can interfere with immunoassays (IAs) and cause false-negative results.
Methods: We enriched Tg from serum samples using rabbit polyclonal anti-Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg-specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2-dimensional LC-MS/MS. Instrument cycle time was 6.5 min per sample.
Results: The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL dimer). Total imprecision of triplicate measurements in serum samples over 5 days was <10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n = 73) showed Deming regression, IA = 1.00 * LC-MS/MS − 2.35, r = 0.982, S y|x = 9.52. In a set of Tg-Aab–positive samples that tested negative for Tg using IA (n = 71), concentrations determined by LC-MS/MS were ≥0.5 ng/mL in 23% of samples (median 1.2, range 0.7–11 ng/mL).
Conclusions: The introduced method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between methods was observed in Tg-Aab–positive samples with concentrations <2 ng/mL (determined with LC-MS/MS). The affinity-assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies.
TL;DR: This study establishes comprehensive pediatric reference intervals for several common endocrine and immunochemical biomarkers obtained in a large cohort of healthy children, and ensures appropriate interpretation of pediatric disease biomarkers.
Abstract: Background: Reference intervals are indispensable in evaluating laboratory test results; however, appropriately partitioned pediatric reference values are not readily available. The CALIPER (Canadian Laboratory Initiative for Pediatric Reference Intervals) program is aimed at establishing the influence of age, sex, ethnicity, and body mass index on biochemical markers and developing a comprehensive database of pediatric reference intervals using an a posteriori approach.
Methods: A total of 1482 samples were collected from ethnically diverse healthy children ages 2 days to 18 years and analyzed on the Abbott ARCHITECT i2000. Following the CLSI C28-A3 guidelines, age- and sex-specific partitioning was determined for each analyte. Nonparametric and robust methods were used to establish the 2.5th and 97.5th percentiles for the reference intervals as well as the 90% CIs.
Results: New pediatric reference intervals were generated for 14 biomarkers, including α-fetoprotein, cobalamin (vitamin B12), folate, homocysteine, ferritin, cortisol, troponin I, 25(OH)-vitamin D [25(OH)D], intact parathyroid hormone (iPTH), thyroid-stimulating hormone, total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine. The influence of ethnicity on reference values was also examined, and statistically significant differences were found between ethnic groups for FT4, TT3, TT4, cobalamin, ferritin, iPTH, and 25(OH)D.
Conclusions: This study establishes comprehensive pediatric reference intervals for several common endocrine and immunochemical biomarkers obtained in a large cohort of healthy children. The new database will be of global benefit, ensuring appropriate interpretation of pediatric disease biomarkers, but will need further validation for specific immunoassay platforms and in local populations as recommended by the CLSI.
TL;DR: Measurements of CSF Aβ(1-42), t-τ, and p-τ(181) with analytically qualified immunoassays reliably reflect the neuropathologic hallmarks of AD in patients at the early predementia stage of the disease and even in presymptomatic patients.
Abstract: BACKGROUND: Over the past 2 decades, clinical studies have provided evidence that cerebrospinal fluid (CSF) amyloid β1–42 (Aβ1–42), total τ (t-τ), and τ phosphorylated at Thr181 (p-τ181) are reliable biochemical markers of Alzheimer disease (AD) neuropathology.
CONTENT: In this review, we summarize the clinical performance and describe the major challenges for the analytical performance of the most widely used immunoassay platforms [based on ELISA or microbead-based multianalyte profiling (xMAP) technology] for the measurement of CSF AD biomarkers (Aβ1–42, t-τ, and p-τ181). With foundational immunoassay data providing the diagnostic and prognostic values of CSF AD biomarkers, the newly revised criteria for the diagnosis of AD include CSF AD biomarkers for use in research settings. In addition, it has been suggested that the selection of AD patients at the predementia stage by use of CSF AD biomarkers can improve the statistical power of clinical trial design. Owing to the lack of a replenishable and commutable human CSF-based standardized reference material (SRM) and significant differences across different immunoassay platforms, the diagnostic–prognostic cutpoints of CSF AD biomarker concentrations are not universal at this time. These challenges can be effectively met in the future, however, through collaborative ongoing standardization efforts to minimize the sources of analytical variability and to develop reference methods and SRMs.
SUMMARY: Measurements of CSF Aβ1–42, t-τ, and p-τ181 with analytically qualified immunoassays reliably reflect the neuropathologic hallmarks of AD in patients at the early predementia stage of the disease and even in presymptomatic patients. Thus these CSF biomarker tests are useful for early diagnosis of AD, prediction of disease progression, and efficient design of drug intervention clinical trials.
TL;DR: The fetal methylation profile deduced from maternal plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site, which offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.
Abstract: BACKGROUND: Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in maternal plasma and can be analyzed noninvasively. METHODS: We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of maternal plasma DNA at single-nucleotide resolution. The first approach used maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of maternal blood cells and the fractional fetal DNA concentration in maternal plasma to deduce the placental methylomic profile from maternal plasma DNA-sequencing data. RESULTS: Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and maternal plasma with maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from maternal plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the maternal plasma data. We demonstrated one potential clinical application of maternal plasma bisulfite sequencing with the successful detection of fetal trisomy 21. CONCLUSIONS: We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.
TL;DR: It is too early to expect that the new technological advances will catalyze the anticipated biomarker revolution any time soon, but the journey of a protein biomarker from the bench to the clinic is long and challenging.
Abstract: BACKGROUND: Protein cancer biomarkers serve multiple clinical purposes, both early and late, during disease progression. The search for new and better biomarkers has become an integral component of contemporary cancer research. However, the number of new biomarkers cleared by the US Food and Drug Administration has declined substantially over the last 10 years, raising concerns regarding the efficiency of the biomarker-development pipeline.
CONTENT: We describe different clinical uses of cancer biomarkers and their performance requirements. We also present examples of protein cancer biomarkers currently in clinical use and their limitations. The major barriers that candidate biomarkers need to overcome to reach the clinic are addressed. Finally, the long and arduous journey of a protein cancer biomarker from the bench to the clinic is outlined with an example.
SUMMARY: The journey of a protein biomarker from the bench to the clinic is long and challenging. Every step needs to be meticulously planned and executed to succeed. The history of clinically useful biomarkers suggests that at least a decade is required for the transition of a marker from the bench to the bedside. Therefore, it may be too early to expect that the new technological advances will catalyze the anticipated biomarker revolution any time soon.
TL;DR: High-sensitivity cardiac troponin (hs-cTnI) is remarkably sensitive in the general population, and there are important sex and age differences among healthy reference individuals, which have important implications for defining hs-c TnI reference values and identifying disease.
Abstract: Background: High-sensitivity cardiac troponin (hs-cTn) assays are now available that can detect measurable troponin in significantly more individuals in the general population than conventional assays. The clinical use of these hs-cTn assays depends on the development of proper reference values. Therefore, our objective was to define hs-cTnI reference values and determinants in the general community, in a healthy reference cohort, and in subsets with diseases.
Materials and Methods: A well-characterized community-based cohort of 2042 study participants underwent clinical assessment and echocardiographic evaluation. Baseline hs-cTnI measurements were obtained in 1843 individuals. A healthy reference cohort (n = 565) without cardiac, renal, or echocardiographic abnormalities was identified.
Results: Measurable hs-cTnI was identified in 1716 (93%) of the community-based study cohort and 499 (88%) of the healthy reference cohort. Parameters that significantly contributed to higher hs-cTnI concentrations in the healthy reference cohort included age, male sex, systolic blood pressure, and left ventricular mass. Glomerular filtration rate and body mass index were not independently associated with hs-cTnI in the healthy reference cohort. Individuals with diastolic and systolic dysfunction, hypertension, and coronary artery disease (but not impaired renal function) had significantly higher hs-cTnI values than the healthy reference cohort.
Conclusions: We assessed an hs-cTnI assay with the aid of echocardiographic imaging in a large, well-characterized community-based cohort. hs-cTnI is remarkably sensitive in the general population, and there are important sex and age differences among healthy reference individuals. These results have important implications for defining hs-cTnI reference values and identifying disease.
TL;DR: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.
Abstract: Background: 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored.
Methods: We developed an online trapping/capillary hydrophilic-interaction liquid chromatography ( c HILIC)/ in-source fragmentation/tandem mass spectrometry system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues. A polymer-based hydrophilic monolithic column was prepared and used for the separation of 12 nucleosides by c HILIC coupled with an online trapping system. Limits of detection and quantification, recovery, and imprecision of the method were determined.
Results: Limits of detection for 5-mC and 5-hmC were 0.06 and 0.19 fmol, respectively. The imprecision and recovery of the method were determined, with the relative SDs and relative errors being <14.9% and 15.8%, respectively. HCC tumor tissues had a 4- to 5-fold lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated with tumor stage (tumor-nodes-metastasis, P = 0.0002; Barcelona Clinic liver cancer, P = 0.0003).
Conclusions: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.
TL;DR: The findings indicate a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor.
Abstract: INTRODUCTION: Detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in the peripheral blood of patients with solid tumors has been widely studied for the early detection of metastatic spread. We evaluated whether there was an association between the origin of cfDNA and CTCs. We investigated whether SRY (sex determining region Y)-box 17 ( SOX17 ) promoter methylation in CTCs was associated with the methylation pattern of this gene in matched cfDNA isolated from plasma of patients with breast cancer.
METHODS: We examined SOX17 methylation in 79 primary breast tumors, in 114 paired samples of DNA isolated from CTCs and cfDNA, and in 60 healthy individuals. Isolated DNA was modified by sodium bisulfite and subjected to methylation specific PCR.
RESULTS: The SOX17 promoter was methylated in 68 (86.0%) of 79 of primary breast tumors. In CTCs, SOX17 was methylated in 19 (34.5%) of 55 patients with early breast cancer, 27 (45.8%) of 59 patients with metastatic cancer, and 1 (4.3%) of 23 healthy individuals, whereas in matched cfDNA SOX17 was methylated in 19 (34.5%) of 55, 24 (40.7%) of 59, and 1 (2.0%) of 49 of these same groups, respectively. There was a significant correlation between SOX17 methylation in cfDNA and CTCs in patients with early breast cancer ( P = 0.008), but not in patients with verified metastasis ( P = 0.283).
CONCLUSIONS: The SOX17 promoter is highly methylated in primary breast tumors, in CTCs isolated from patients with breast cancer, and in corresponding cfDNA samples. Our findings indicate a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor.
TL;DR: The results of this multicenter study show that Phi, compared with tPSA or %fPSA, demonstrated superior clinical performance in detecting PCa at t PSA 1.6-8.0 μg/L and is better able to detect aggressive PCa.
Abstract: BACKGROUND: Total prostate-specific antigen (tPSA) is flawed for prostate cancer (PCa) detection. [−2]proprostate-specific antigen (p2PSA), a molecular isoform of free PSA (fPSA), shows higher specificity compared with tPSA or percentage of free PSA (%fPSA). The prostate health index (Phi), a measure based on p2PSA and calculated as p2PSA/fPSA × √tPSA, was evaluated in a multicenter study for detecting PCa.
METHODS: A total of 1362 patients from 4 different study sites who had tPSA values of 1.6–8.0 μg/L (668 patients with PCa, 694 without PCa) underwent ≥10 core biopsies. Serum concentrations of tPSA, fPSA (both calibrated against a WHO reference material), and p2PSA were measured on Access2 or DxI800 analyzers (Beckman Coulter).
RESULTS: The percentage ratio of p2PSA to fPSA (%p2PSA) and Phi were significantly higher in all PCa subcohorts (positive initial or repeat biopsy result or negative digital rectal examination) ( P < 0.0001) compared with patients without PCa. Phi had the largest area under the ROC curve (AUC) (AUC = 0.74) and provided significantly better clinical performance for predicting PCa compared with %p2PSA (AUC = 0.72, P = 0.018), p2PSA (AUC = 0.63, P < 0.0001), %fPSA (AUC = 0.61) or tPSA (AUC = 0.56). Significantly higher median values of Phi were observed for patients with a Gleason score ≥7 (Phi = 60) compared with a Gleason score <7 (Phi = 53; P = 0.0018). The proportion of aggressive PCa (Gleason score ≥7) increased with the Phi score.
CONCLUSIONS: The results of this multicenter study show that Phi, compared with tPSA or %fPSA, demonstrated superior clinical performance in detecting PCa at tPSA 1.6–8.0 μg/L (i.e., approximately 2–10 μg/L in traditional calibration) and is better able to detect aggressive PCa.
TL;DR: Cannabinoids can be detected in blood of chronic daily cannabis smokers during a month of sustained abstinence, consistent with the time course of persisting neurocognitive impairment reported in recent studies.
Abstract: BACKGROUND: Cannabis is the illicit drug most frequently reported with impaired driving and motor vehicle accidents. Some “per se” laws make it illegal to drive with any amount of drug in the body, while others establish blood, saliva, or urine concentrations above which it is illegal to drive. The persistence of Δ9-tetrahydrocannabinol (THC) in chronic daily cannabis smokers' blood is unknown.
METHODS: Thirty male chronic daily cannabis smokers resided on a secure research unit for up to 33 days, with daily blood collection. Samples were processed in an ice bath during sample preparation to minimize cannabinoid adsorption onto precipitant material. We quantified THC by 2-dimensional GC-MS.
RESULTS: Of the 30 participants, 27 were THC-positive on admission, with a median (range) concentration of 1.4 μg/L (0.3–6.3). THC decreased gradually; only 1 of 11 participants was negative at 26 days, 2 of 5 remained THC-positive (0.3 μg/L) for 30 days, and 5.0% of participants had THC ≥1.0 μg/L for 12 days. Median 11-hydroxy-THC concentrations were 1.1 μg/L on admission, with no results ≥1.0 μg/L 24 h later. 11-Nor-9-carboxy-THC (THCCOOH) detection rates were 96.7% on admission, decreasing slowly to 95.7% and 85.7% on days 8 and 22, respectively; 4 of 5 participants remained THCCOOH positive (0.6–2.7 μg/L) after 30 days, and 1 remained positive on discharge at 33 days.
CONCLUSIONS: Cannabinoids can be detected in blood of chronic daily cannabis smokers during a month of sustained abstinence. This is consistent with the time course of persisting neurocognitive impairment reported in recent studies.