Wayne L. Ryan

New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.

Whole Blood Glucose Standard is Key to Accurate Insulin Dosages

Introduction: There is no available glucose standard for whole blood. None of the various glucose controls can be used as standards (calibrators) for glucose meters or test strips. This article describes the need for a whole blood glucose standard for areas such as manufacturing or proficiency evaluations. Furthermore, it describes the performance of the standard developed by Streck. Implementation of a whole blood glucose reference standard may allow the manufacturers of products for the diabetes health care industry to reduce the level of systematic difference between true blood glucose values and those obtained by point-of-care (POC) glucose meters. Methods: Glucose agreement data were collected across four prominent POC glucose meters representing >97% of all meters reporting data in the 2006 College of American Pathologists survey. Glucose concentrations of whole blood were adjusted to replicate the concentrations contained in the blood glucose standard developed by Streck. Commercial glucose controls, provided by the respective strip manufacturer, remained unaltered and were tested in accordance with the manufacturers recommendations. Results: Only slight variations in hematocrit, surface tension, and viscosity were measured over a period of 90 days with the Streck Blood Glucose Standard compared to fresh whole blood at time zero. Glucose measurements on whole blood and the blood glucose standard were in agreement for all four commonly used glucose meters. In contrast, there is a lack of agreement between a manufacturers set of recommended aqueous-based glucose controls in measurements taken on their POC meter relative to the YSI. Finally, the blood glucose standard demonstrated stability in 35-day open- and 110-day closed-vial assessments. Conclusions: Results of our experiments illustrate the ability of the blood glucose standard to closely mimic whole blood results. In addition, the blood glucose standard shows good open-vial and closed-vial stability at 6°C.

Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays

BACKGROUND Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. METHODS Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. RESULTS The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. CONCLUSION Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.

Evaluation of the Performance and Stability of a Novel A1c-Cellular Control

Research shows lifestyle changes and regular monitoring of diabetes indices are the most effective means to manage diabetes.1,2 Measuring hemoglobin A1c (A1C) is an accepted means of diagnosis and a helpful indicator when monitoring diabetes status. Hemoglobin A1c analyzers are usually based on three analytical principles: high-performance liquid chromatography, immunoturbidimetry, and boronate affinity. The A1C values obtained from different analyzers often vary because of the differences in analytical principles and matrix effects.3 Thus a quality control that consistently provides similar A1C values within a relevant range across various analyzers would be helpful when correlating results from various methodologies. Clinical and Laboratory Standard Institute regulations state that control materials should be treated in the same manner as a patient specimen.4 A whole blood control would be ideal; however, the instability of whole blood and the unavailability of large volumes of diabetic blood, as well as ethical concerns, are major limitations. Streck A1c-Cellular®, a ready-to-use bi-level liquid control, resembles whole blood, as it contains intact red cells and is used in the same manner as a patient sample. In this letter, we describe the performance of this control across multiple analyzers and report the stability of the recovered A1C values and the stability of the intact red cells that constitute the control. Table 1 shows that the mean A1C values of 10 consecutive runs of the control across different analyzers are within their respective ±2 standard deviation (SD) ranges [A1C values are expressed in National Glycohemoglobin Standardization Program (NGSP) percentage units]. The imprecision values [percentage coefficient of variation (CV)] for all instruments are within 3.0%, a traceability criteria approved by the NGSP.5 The interinstrument mean values for two levels are 5.8% and 11.6%. The interinstrument ±2 SD ranges are 4.5–7.1% and 10.5–12.8%, respectively. Therefore, the mean A1C values of all instruments are within an interinstrument ±2 SD range for both levels of the control. These results indicate that the A1c-Cellular can be used as a quality control for multiple analyzers. The variation between the mean values for each instrument may be the result of matrix effects. Table 1 Reproducibility of Bi-Level A1c-Cellular across Multiple Analyzersa The control is stable for 6 months in closed vial and 30 days in open vial when stored at 2–10 °C. We measured the A1C values of both levels of the control using the Tosoh G8 A1c analyzer over 6 months and found that the A1C values are within the ±2 SD range of the mean. The control also maintains the integrity of intact red cells throughout its stability claim. The presence of intact red cells in A1c-Cellular can detect errors in lysing steps that would be bypassed by lysate-based controls. In conclusion, A1C values for A1c-Cellular control, obtained from various analyzers, are reproducible, and the impression values are within the NGSP acceptable range. The mean A1C values for each instrument are within the interinstrument ±2 SD ranges. The control resembles whole blood by containing stable intact red cells that could also be useful to indicate malfunctions in the lysing step.