Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Standardization of vascular assessment of erectile dysfunction: standard operating procedures for duplex ultrasound.

INTRODUCTION: In-office evaluation of erectile dysfunction by color duplex Doppler ultrasound (CDDU) may benefit the decision-making process in regard to choosing the most appropriate therapy. Unfortunately, there is no uniform standardization in performing CDDU resulting in high variability in data expression and interpretation when comparing results among various centers, especially when conducting multicenter trials. Establishing standard operating procedures (SOPs) is a major step that will help minimize such variability. AIM: This SOP describes CDDU procedure with focus on establishing uniformity and normative parameters. MAIN OUTCOME MEASURE: Measure intra-arterial diameter, peak systolic velocity, end-diastolic velocity, and resistive index for each cavernosal artery. METHODS: After initial discussion with the patient about his history and International Index of Erectile Function evaluation describe procedural steps to the patient. Perform the CDDU in a relaxed state, scanning the entire penis (in B-mode image) using a 7.5- to 12-MHz linear array ultrasound probe. An intracorporal injection of a single or combination of vasoactive agents (e.g., prostaglandin E1, phentolamine, and papaverine) is then administered and CDDU performed at various time points, preferably with audiovisual sexual stimulation (AVSS). RESULTS: Monitor penile erection response (tumescence and rigidity) near peak blood flow. Self-stimulation or AVSS leaving the patient alone in room or redosing may be considered to decrease any anxiety and help achieve a maximum rigid erection. CONCLUSION: Considering the complexity and heterogeneity of CDDU evaluation, this communication will help in standardization and establish uniformity in such data interpretation. When indicated, invasive diagnostic testing involving (i) penile angiography and (ii) cavernosography/cavernosometry to establish veno-occlusive dysfunction may be recommended to facilitate further treatment options.