Background: At present there is no simple, accurate blood test that may be used to determine the severity of stroke or to predict mortality and morbidity in stroke patients presenting to emergency departments. Methods: Patients with stroke-like symptoms who presented to an emergency department of a university hospital in Hong Kong were recruited for the study. DNA extracted from patients' plasma was analyzed for the ␤-globin gene with a fluorescent-based PCR test. The primary outcome measures were in-hospital and 6-month mortality and morbidity using the post-stroke modified Rankin Score. Results: Among the 88 consecutive patients recruited to the study, 70 (80%) had ischemic stroke, 11 (13%) had intracerebral hemorrhage, and 7 (8%) had transient ischemic attacks. Median plasma DNA concentrations taken within 3 h of symptom onset were higher in patients who died compared with those who survived at discharge (6205 vs 1334 kilogenome-equivalents/L; P ‫؍‬ 0.03). Among patients with NIH Stroke Scale scores >8, median plasma DNA concentrations were higher in patients who died compared with those who survived to 6 months (2273 vs 968 kilogenome-equivalents/L; P ‫؍‬ 0.002). Plasma DNA concentrations correlated with the volume of cerebral hematoma (r ‫؍‬ 0.66; P ‫؍‬ 0.03). Plasma DNA concentrations >1400 kilogenome-equivalents/L had a sensitivity of 100% and a specificity of 74.4% for predicting hospital mortality after stroke, and the area under the ROC curve was 0.89 (95% confidence interval, 0.80 -0.94). The adjusted odds ratio for plasma DNA concentrations predicting 6-month mortality was

Background: Cell-free DNA concentrations increase in the circulation of patients after trauma and may have prognostic potential, but little is know concerning the temporal changes or clearance of the DNA or its relationships with posttraumatic complications. We investigated temporal changes in plasma DNA concentrations in patients after trauma with use of real-time quantitative PCR. Methods: Serial plasma samples were taken from two trauma populations. In the first study, samples were collected every 20 min from 25 patients within the first 3 h of trauma. In the second study, samples were collected every day from 36 other trauma patients admitted to the intensive care unit (ICU). Results: In the first study, plasma DNA was increased within 20 min of injury and was significantly higher in patients with severe injury and in patients who went on to develop organ failure. In patients with less severe injuries, plasma DNA concentrations decreased toward reference values within 3 h. In the second study, plasma DNA concentrations were higher in patients who developed multiple organ dysfunction syndrome between the second and fourth days of admission than in patients who did not develop the syndrome. In patients who remained in the ICU with continuing organ dysfunction, plasma DNA remained higher than in healthy controls even at 28 days after injury. Most survivors with multiple organ dysfunction syndrome showed an initial very high peak followed by a prolonged smaller increase. Conclusions: Plasma DNA concentrations increase early after injury and are higher in patients with severe injuries and in those who develop organ failure. In-

Background: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. Methods: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line ( 0 ) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. Results: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with

Background: As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form. Methods: Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 μm to 0.22 μm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the β-globin gene. Results: The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P <0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 μm filter. In contrast, no significant difference was seen in β-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P <0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-μm-filtered plasma samples and the ultracentrifuged plasma samples (P >0.05). In HCC patients, filtration with a 0.22 μm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P <0.0 5 for filtered plasma samples; P <0.005 for unfiltered plasma samples). Conclusions: A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.

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species is fragmented, cell-free DNA the size of nucleosomal DNA, mostly derived from cells undergoing apoptosis. As discussed earlier, the source of mutated k-ras DNA in the circulation could be apoptotic cells. Thus, mutated k-ras DNA should be enriched in the fragmented, nucleosomal DNA species relative to the large species found near the sample wells of the gel. Therefore, the DNA isolation method that favors isolation of fragmented DNA should produce a better ratio of k-ras-mutated DNA to wild-type DNA. As shown in Table 1, mutated k-ras was readily detected in the DNA isolated by the G/R method but was detected less frequently in DNA isolated by the Qiagen method from the specimen from the same patient, although more DNA was recovered by the Qiagen method. Another possible contributor to this discrepancy in assay results is the nature of the RE-PCR assay. This is a PCR-based assay, and with more wild-type genomic DNA in the assay, the sensitivity of detection of the mutated DNA is reduced because of interference with the PCR.This study clearly demonstrates that the method chosen for isolation of DNA can contribute significantly to the outcome of mutation detection (in this case, k-ras mutations). It has been suggested that malignant, benign, and even preneoplastic cells often proliferate at abnormal rates, accompanied by an increase in apoptotic cell death (9 -11 ), and that this small, fragmented DNA may accumulate in the circulation. These results further suggest that to enhance assay sensitivity for detection of somatic mutations or epigenetic modifications in circulating DNA for cancer detection, monitoring, or prognosis, a method that can preferentially isolate small DNA should be used.

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Use of the phi could improve the accuracy of PCa detection in patients with an elevated PSA level and thus avoid unnecessary prostatic biopsies.