High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.

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Clinical Validation of a Volumetric Absorptive Micro-Sampling Device for Pharmacokinetic Studies With Tranexamic Acid

Frontiers in Pharmacology ◽

10.3389/fphar.2021.764379 ◽

2021 ◽

Vol 12 ◽

Author(s):

Stanislas Grassin-Delyle ◽

Elodie Lamy ◽

Michaela Semeraro ◽

Iléana Runge ◽

Jean-Marc Treluyer ◽

...

Keyword(s):

Tranexamic Acid ◽

Whole Blood ◽

Plasma Concentrations ◽

High Sensitivity ◽

Pharmacokinetic Studies ◽

Limits Of Agreement ◽

Sampling Device ◽

Average Bias ◽

Alternative Routes ◽

Matched Samples

We assessed the accuracy of tranexamic acid (TXA) concentrations measured in capillary whole blood using volumetric absorptive micro-sampling (VAMS) devices. Paired venous and VAMS capillary blood samples were collected from 15 healthy volunteers participating in a pharmacokinetic study of alternative routes (oral, IM and IV) of administering TXA. To assess accuracy across a range of concentrations, blood was drawn at different times after TXA administration. We measured TXA concentrations in plasma, whole blood from samples collected by venepuncture and whole blood from venous and capillary samples collected using VAMS devices. TXA was measured using a validated high sensitivity liquid chromatography - mass spectrometry method. We used Bland-Altman plots to describe the agreement between the TXA concentrations obtained with the different methods. In the 42 matched samples, the mean plasma TXA concentration was 14.0 mg/L (range 2.6–36.5 mg/L) whereas the corresponding whole blood TXA concentration was 7.7 mg/L (range 1.6–17.5 mg/L). When comparing TXA concentrations in VAMS samples of venous and capillary whole blood, the average bias was 0.07 mg/L (lower and upper 95% limits of agreement: −2.1 and 2.2 mg/L respectively). When comparing TXA concentrations in venous whole blood and VAMS capillary whole blood, the average bias was 0.7 mg/L (limits of agreement: −2.7 and 4.0 mg/L). Volumetric absorptive micro-sampling devices are sufficiently accurate for use in pharmacokinetic studies of tranexamic acid treatment in the range of plasma concentrations relevant for the assessment of fibrinolysis inhibition.

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Validation of highly specific and sensitive radioimmunoassays for lutropin, follitropin, and free alpha subunit in unextracted urine

Clinical Chemistry ◽

10.1093/clinchem/36.2.340 ◽

1990 ◽

Vol 36 (2) ◽

pp. 340-344 ◽

Cited By ~ 10

Author(s):

H Landy ◽

A L Schneyer ◽

R W Whitcomb ◽

W F Crowley

Keyword(s):

Monoclonal Antibody ◽

Urinary Excretion ◽

Blood Volume ◽

High Sensitivity ◽

High Specificity ◽

Urine Samples ◽

Alpha Subunit ◽

Glycoprotein Hormones ◽

Urinary Ph ◽

Freeze Thaw

Abstract Measurement of the urinary excretion of lutropin (LH) and follitropin (FSH) and their common free alpha subunit (FAS) assists in monitoring the maturation of the hypothalamic-pituitary-gonadal axis and in understanding the physiology of the pituitary glycoprotein hormones. Here we describe sensitive, specific polyclonal radioimmunoassays for LH and FSH and a monoclonal radioimmunoassay for FAS for use with urine--assays unperturbed by alterations in urinary pH or osmolarity within the broad physiological range encountered in urine. Concordance between LH, FSH, and FAS concentrations in extracted and unextracted urine samples was high. Linearity and parallelism with the standard curves was observed with addition of 25 to 200 microL of unextracted urine. No effect on glycoprotein concentration was seen after as many as 10 freeze-thaw cycles. The need for extraction was further obviated by the high sensitivity of each assay, reflected by minimum detectable doses well below the concentrations encountered in patients' samples. Thus we have measured gonadotropins in unextracted urine as precisely as in extracted urine. We also have demonstrated an equally versatile assay for urinary alpha subunit, using a monoclonal antibody of high specificity for this monomer in its free, uncombined form. These radioimmunoassays complement assays of gonadotropins and free alpha subunit in serum and will allow longitudinal investigations otherwise limited by the constraints of the patient's blood volume.

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Measurement of blood O2 and CO2 concentrations using PO2 and PCO2 electrodes

Journal of Applied Physiology ◽

10.1152/jappl.1978.44.5.818 ◽

1978 ◽

Vol 44 (5) ◽

pp. 818-820 ◽

Cited By ~ 4

Author(s):

A. L. Harabin ◽

L. E. Farhi

Keyword(s):

Carbon Monoxide ◽

Blood Volume ◽

Whole Blood ◽

Co2 Concentration ◽

Saturated Solution ◽

Additional Step ◽

Co2 Concentrations ◽

O2 Concentration ◽

Co2 Electrode ◽

Small Blood Volume

Accurate dilution of a small blood volume with a carbon monoxide-saturated solution allows measurement of the whole blood O2 concentration as an increase in O2 tension in the solution. We have improved the method by simplifying both equipment and procedure. We also suggest an additional step in which the mixture is acidified, thereby allowing the measurement of CO2 concentration in the same solution with a CO2 electrode. The accuracy of both the O2 and CO2 determinations compares favorably with that obtained with other micromethods.

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BIODOSIMETRY ON SMALL BLOOD VOLUME USING GENE EXPRESSION ASSAY

Health Physics ◽

10.1097/01.hp.0000346706.44253.5c ◽

2010 ◽

Vol 98 (2) ◽

pp. 179-185 ◽

Cited By ~ 51

Author(s):

Muriel Brengues ◽

Brigitte Paap ◽

Michael Bittner ◽

Sally Amundson ◽

Bruce Seligmann ◽

...

Keyword(s):

Gene Expression ◽

Blood Volume ◽

Gene Expression Assay ◽

Small Blood Volume

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Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method

Radiation Research ◽

10.1667/rr13897.1 ◽

2015 ◽

Vol 184 (1) ◽

pp. 95-104 ◽

Cited By ~ 6

Author(s):

Maria Wojewodzka ◽

Sylwester Sommer ◽

Marcin Kruszewski ◽

Katarzyna Sikorska ◽

Maciej Lewicki ◽

...

Keyword(s):

Blood Volume ◽

Processing Parameters ◽

Optimal Detection ◽

Blood Processing ◽

Volume Method ◽

Small Blood Volume

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Development of small blood volume assays for the measurement of oxidative stress markers in mammals

PLoS ONE ◽

10.1371/journal.pone.0209802 ◽

2018 ◽

Vol 13 (12) ◽

pp. e0209802 ◽

Cited By ~ 2

Author(s):

Evan Langille ◽

Vincent Lemieux ◽

Dany Garant ◽

Patrick Bergeron

Keyword(s):

Oxidative Stress ◽

Blood Volume ◽

Oxidative Stress Markers ◽

Stress Markers ◽

Small Blood Volume

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Diagnostic qPCR Assay to Detect Fusarium brasiliense, a Causal Agent of Soybean Sudden Death Syndrome and Root Rot of Dry Bean

Plant Disease ◽

10.1094/pdis-01-19-0016-re ◽

2020 ◽

Vol 104 (1) ◽

pp. 246-254

Author(s):

Mitchell G. Roth ◽

Kjersten A. Oudman ◽

Amanda Griffin ◽

Janette L. Jacobs ◽

Hyunkyu Sang ◽

...

Keyword(s):

Sudden Death ◽

Root Rot ◽

Management Strategies ◽

High Sensitivity ◽

High Specificity ◽

Sudden Death Syndrome ◽

Qpcr Assay ◽

Taqman Probe ◽

Dry Bean ◽

Accurate Detection

Species within clade 2 of the Fusarium solani species complex (FSSC) are significant pathogens of dry bean (Phaseolus vulgaris) and soybean (Glycine max), causing root rot and/or sudden death syndrome (SDS). These species are morphologically difficult to distinguish and often require molecular tools for proper diagnosis to a species level. Here, a TaqMan probe-based quantitative PCR (qPCR) assay was developed to distinguish Fusarium brasiliense from other closely related species within clade 2 of the FSSC. The assay displays high specificity against close relatives and high sensitivity, with a detection limit of 100 fg. This assay was able to detect F. brasiliense from purified mycelia, infected dry bean roots, and soil samples throughout Michigan. When multiplexed with an existing qPCR assay specific to Fusarium virguliforme, accurate quantification of both F. brasiliense and F. virguliforme was obtained, which can facilitate accurate diagnoses and identify coinfections with a single reaction. The assay is compatible with multiple qPCR thermal cycling platforms and will be helpful in providing accurate detection of F. brasiliense. Management of root rot and SDS pathogens in clade 2 of the FSSC is challenging and must be done proactively, because no midseason management strategies currently exist. However, accurate detection can facilitate management decisions for subsequent growing seasons to successfully manage these pathogens.

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Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress

Human & Experimental Toxicology ◽

10.1177/0960327106070463 ◽

2007 ◽

Vol 26 (4) ◽

pp. 273-282 ◽

Cited By ~ 18

Author(s):

Galina F Makhaeva ◽

Vladimir V Malygin ◽

Nadezhda N Strakhova ◽

Larisa V Sigolaeva ◽

Lidia G Sokolovskaya ◽

...

Keyword(s):

Whole Blood ◽

Colorimetric Assay ◽

High Sensitivity ◽

Epidemiological Studies ◽

Neuropathy Target Esterase ◽

Carbon Paste ◽

New Approach ◽

Ongoing Work ◽

Hydrolysis Of ◽

Small Blood Volume

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 μL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area. Human & Experimental Toxicology (2007) 26, 273-282

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Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2018-1298 ◽

2020 ◽

Vol 58 (5) ◽

pp. 701-708 ◽

Cited By ~ 2

Author(s):

Elodie Lamy ◽

Fanta Fall ◽

Lisa Boigne ◽

Kirill Gromov ◽

Nicolas Fabresse ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Plasma ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

High Sensitivity ◽

Clinical Samples ◽

Pharmacokinetic Studies ◽

Plasma Fraction ◽

Drug Pharmacokinetic ◽

Liquid Chromatography Tandem Mass

AbstractBackgroundRopivacaine is a widely used local anaesthetic drug, highly bound to plasma proteins with a free plasma fraction of about 5%. Therefore, the monitoring of free drug concentration is most relevant to perform pharmacokinetic studies and to understand the drug pharmacokinetic/pharmacodynamic (PK/PD) relationship.MethodsA high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using reverse-phase LC and electrospray ionisation mass spectrometry with multiple reaction monitoring (MRM) is described for the quantitation of both free and total ropivacaine in human plasma. Ropivacaine-d7 was used as an internal standard (IS).ResultsThe method was validated in the range 0.5–3000 ng/mL, with five levels of QC samples and according to the European Medicine Agency and Food and Drug Administration guidelines. The performance of the method was excellent with a precision in the range 6.2%–14.7%, an accuracy between 93.6% and 113.7% and a coefficient of variation (CV) of the IS-normalised matrix factor below 15%. This suitability of the method for the quantification of free and total ropivacaine in clinical samples was demonstrated with the analysis of samples from patients undergoing knee arthroplasty and receiving a local ropivacaine infiltration.ConclusionsA method was developed and validated for the quantification of free and total ropivacaine in human plasma and was shown suitable for the analysis of clinical samples.

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