Establishment of two quantitative nested qPCR assays targeting the human EPO transgene.

For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.

Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection.

The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient's tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.

Rapid intraoperative tissue expansion for closure of facial defects.

Rapid intraoperative tissue expansion (RITE) has been shown to have definite applicability to reconstruction in the head and neck. However, widespread use of this modality has yet to be adopted by the otolaryngologic community. Believing that RITE might offer some advantages in the reconstruction of soft-tissue facial defects following Mohs micrographic surgery for cutaneous malignant neoplasms, we evaluated 30 consecutive patients referred for cosmetic reconstruction after Mohs micrographic surgery for the use of RITE. Eight patients were selected and underwent reconstruction using a total of 10 tissue expanders. Herein, we describe our experience with this initial series as well as some background and future applications of RITE.

Submental W-plasty for correction of 'turkey gobbler' deformities.

There are many causes and types of submental contour deformities. One such deformity called a turkey gobbler, is caused by an excess of flaccid submental skin and as an isolated defect is seen more commonly in men. We have found that skin excision and a running submental W-plasty for correction of this deformity yields a superior cosmetic result with excellent camouflage of the submental scar. Two cases are illustrated.

Analysis of patient response to preoperative computerized video imaging.

Preoperative computer-assisted video imaging was performed on 50 consecutive rhinoplasty candidates, along with routine preoperative photographs and assessment. Each patient then completed a questionnaire dealing with the imaging process. Patient acceptance of the imaging process was excellent. Responses indicated that most patients felt that video imaging improved communication between patient and surgeon, increased patient confidence in surgery and surgeon, and enhanced the patient-physician relationship. The future use of computer-assisted video imaging in teaching, preoperative planning, and improved post-operative patient satisfaction is discussed.

Local anesthesia for soft-tissue surgery.

The use of local anesthetics in soft-tissue facial surgery can be safe, efficient, economical, and convenient. Understanding these agents and using them with proficiency allows the surgeon to perform a variety of operative procedures with minimal anesthetic morbidity on patients who are relaxed, comfortable, and insensitive to pain.

Scar revision and camouflage.

The successful revision and camouflage of facial scars is as much an art as a science, and the best teacher is continued experience; however, common sense, a broad knowledge of available options, technical ability, and patience are all necessary if one chooses to practice in this challenging field. Patients with facial scars are vulnerable in a way that few others are, and it is the responsibility of the facial plastic surgeon to evaluate these patients properly and realistically, to prepare them for a process that may involve multiple procedures, and to see them through this process with all the expertise and compassion possible. If this is done, the rewards to the patient and to the surgeon can be immeasurable.

Facial plastic surgery in the otolaryngology training program: an update.

In 1985, we demonstrated that, following the institution of a specific curriculum in facial plastic surgery, the number of cosmetic procedures generated and performed by residents rose dramatically. We have now demonstrated that this increase has been sustained in the 5 years since establishing the curriculum. We continue to maintain that the key to a successful curriculum in facial plastic surgery is the presence of at least one full-time staff member whose primary function is the practice and teaching of facial plastic surgery.