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1.  Optimization of an Enrichment process for Circulating tumor cells from the blood of Head and Neck Cancer patients through depletion of normal cells 
Biotechnology and bioengineering  2009;102(2):521-534.
The optimization of a purely negative depletion, enrichment process for circulating tumor cells, CTC's, in the peripheral blood of Head and Neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 109 to 8.04 × 103 cells/ml and still recover, on average, 2.32 CTC per ml of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per ml of blood, with a range of 282 to 0.53 CTC per ml of blood. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.76 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTC's or other cell type relevant to the specific question (i.e. does the CTC have predominately epithelia or mesenchymal characteristics?).
doi:10.1002/bit.22066
PMCID: PMC3906726  PMID: 18726961
Circulating tumor cells; Immunomagnetic cell separation; Immunocytochemistry; RT-PCR; Squamous cell carcinoma of the head and neck (HNSCC)
2.  Emerging Technologies for CTC Detection Based on Depletion of Normal Cells 
Properly conducted, an enrichment step can improve selectivity, sensitivity, yield, and most importantly, significantly reduce the time needed to isolate rare circulating tumor cells (CTCs). The enrichment process can be broadly categorized as positive selection versus negative depletion, or in some cases, a combination of both. We have developed a negative depletion CTC enrichment strategy that relies on the removal of normal cells using immunomagnetic separation in the blood of cancer patients. This method is based on the combination of magnetic and fluid forces in an axial, laminar flow in long cylinders placed in quadrupole magnets. Using this technology, we have successfully isolated CTCs from patients with breast carcinoma and squamous cell carcinoma of the head and neck. In contrast to a positive selection methodology, this approach provides an unbiased characterization of these cells, including markers associated with epithelial mesenchymal transition.
doi:10.1007/978-3-642-28160-0_9
PMCID: PMC3775349  PMID: 22527498
3.  Effects of Nasal Continuous Positive Airway Pressure and Cannula Use in the Neonatal Intensive Care Unit Setting 
Objective
To investigate the effects of nasal continuous positive airway pressure (CPAP) and cannula use in the neonatal intensive care unit.
Design
Cross-sectional study.
Setting
Tertiary care children’s hospital.
Patients
One hundred patients (200 nasal cavities), younger than 1 year, who received at least 7 days of nasal CPAP (n = 91) or cannula supplementation (n = 9) in the neonatal intensive care unit.
Interventions
External nasal examination and anterior nasal endoscopy with photographic documentation.
Main Outcome Measures
The incidence and characteristics of internal and external nasal findings of patients with nasal CPAP or cannula use.
Results
Nasal complications were seen in 12 of the 91 patients (13.2%) with at least 7 days of nasal CPAP exposure, while no complications were seen in the 9 patients with nasal cannula use alone. The external nasal finding of columellar necrosis, seen in 5 patients (5.5%), occurred as early as 10 days after nasal CPAP use. Incidence of intranasal findings attributed to CPAP use, in the 182 nostrils examined, included ulceration in 6 nasal cavities (3.3%), granulation in 3 nasal cavities (1.6%), and vestibular stenosis in 4 nasal cavities (2.2%). Intranasal complications were seen as early as 8 to 9 days after nasal CPAP administration. Nasal complications from CPAP were associated with lower Apgar scores at 1 (P = .02) and 5 (P = .06) minutes.
Conclusions
External or internal complications of nasal CPAP can be relatively frequent (13.2%) and can occur early, and patients with lower Apgar scores may be at higher risk. Close surveillance for potential complications should be considered during nasal CPAP use.
doi:10.1001/archoto.2010.15
PMCID: PMC3740519  PMID: 20231649
4.  Significance of Circulating Tumor Cells in Patients With Squamous Cell Carcinoma of the Head and Neck 
Objectives
To present and discuss a high-performance negative depletion method for the isolation of circulating tumor cells (CTCs) in the blood of patients with head and neck cancer and to determine the correlation between the presence of CTCs and early clinical outcome in these patients.
Design
Prospective clinical follow-up study of patients with squamous cell carcinoma of the head and neck (SCCHN) undergoing surgical intervention, who had peripheral blood examined for the presence of CTCs.
Patients
The study population comprised 48 patients diagnosed as having SCCHN and undergoing surgical intervention.
Intervention
A negative depletion process to isolate and quantify CTCs from the blood of patients with SCCHN using immunomagnetic separation was developed and validated. Immunostaining for cytokeratin was performed on the enriched samples to determine the number of CTCs extracted from each patient’s blood sample. Correlation of the presence of CTCs, tumor stage, nodal status, clinical characteristics, and outcome was made.
Main Outcome Measure
Disease-free survival.
Results
Our initial data, that have a mean follow-up of 19.0 months, suggest that patients with no detectable CTCs per milliliter of blood had a significantly higher probability of disease-free survival (P=.01). There was no correlation between the presence of CTCs with regard to age, sex, tumor site, stage, or nodal involvement.
Conclusions
Our enrichment technology, based on the removal of normal cells, has been used on the peripheral blood of patients with head and neck cancer for which follow-up data were collected. If no CTCs were present, a statistically significant improved disease-free survival was observed in SCCHN. A blood test with such a prognostic capability could have important implications in the treatment of patients with head and neck cancer.
doi:10.1001/archoto.2010.223
PMCID: PMC3740520  PMID: 21173379
5.  Mucin-producing Malignant Tumor of Lower Eyelid Presenting in a 14-year-old Patient 
Malignancies of the ocular adnexa are rare, aggressive tumors with significant potential for local recurrence and metastases. Although basal cell carcinoma remains the most common malignancy of the eyelid, encompassing more than 90 percent of all periocular cancers, several other malignant neoplasms have been reported. Malignant ocular adnexal neoplasms are most commonly of sweat gland origin and include hidradenocarcinoma, mucinous eccrine adenocarcinoma, and apocrine adenocarcinoma of the glands of Moll. The different ocular adnexal adenocarcinomas share many of the same characteristics with regard to incidence, primary location, and rates of recurrence and metastases. As a result, these tumors are difficult to distinguish clinically and true diagnosis depends on histological findings. The highest incidence of the reported cases occurred during the sixth decade of life. The head, neck, and trunk are the most frequently reported primary locations. Ocular adnexal adenocarcinomas have a significant rate of metastasis to regional lymph nodes as well as distant sites. Standard treatment of care includes surgical excision of the tumor; however, these malignancies have a tendency to recur locally. The authors present a 14-year-old Caucasian girl with no significant past medical history who presented with a recurrent right lower eyelid tumor. She previously had multiple resections at an outside hospital; however, the margins could not be cleared. The patient was taken to the operating room for Mohs-type resection of the primary site as well as a right parotidectomy with facial nerve preservation and right modified radical neck dissection. The final pathology was consistent with a mucin-producing malignant tumor of ocular adnexa. Due to the great potential for local recurrence and metastases, the prognosis for ocular adnexal adenocarcinoma remains poor even with aggressive surgical management. The use of Mohs surgery for resection has increased as it has been linked to prolonged intervals of metastatic-free disease.
PMCID: PMC3366446  PMID: 22708009
6.  Parker Flex-Tip and Standard-Tip Endotracheal Tubes: A Comparison During Nasotracheal Intubation 
Anesthesia Progress  2010;57(1):18-24.
Abstract
The placement of endotracheal tubes in the airway, particularly through the nose, can cause trauma. Their design might be an important etiologic factor, but they have changed little since their introduction. Recently Parker Medical (Bridgewater, Conn ) introduced the Parker Flex-Tip (PFT) tube, suggesting that it causes less trauma. This study aimed to compare the PFT endotracheal tube to a side-beveled, standard-tip endotracheal tube (ETT) for nasotracheal intubation (Figures 1 and 2). Forty consecutive oral surgery patients requiring nasotracheal intubation were randomized to receive either a standard ETT or the PFT tube. Intubations were recorded using a fiber-optic camera positioned proximal to the Murphy eye of the tube. This allowed visualization of the path and action of the tube tip as it traversed the nasal, pharyngeal, laryngeal, and tracheal airway regions. Video recordings made during intubation and extubation were evaluated for bleeding, trauma, and intubation time. Both bleeding and trauma were recorded using a visual analogue scale (VAS) and by 3 different evaluators. The PFT received significantly better VAS values than the standard tubes from all 3 raters (P < 0.05) in both the extent of trauma and bleeding. Since the intubations were purposefully conducted slowly for photographic reasons, neither tube displayed a time advantage. This study suggests that the PFT tube design may be safer by causing less trauma and bleeding than standard tube designs for nasotracheal intubation.
doi:10.2344/0003-3006-57.1.18
PMCID: PMC2844234  PMID: 20331335
Nasotracheal intubation; Parker Flex-Tip tube; Endotracheal intubation; Endotracheal tube; Fiber-optic intubation
7.  Confocal images of circulating tumor cells obtained using a methodology and technology that removes normal cells 
Molecular pharmaceutics  2009;6(5):1402-1408.
A completely negative enrichment technology was used to detect circulating tumor cells, CTCs, in the peripheral blood of head and neck cancer patients. Of 32 blood samples, 63 percent contained CTCs and the number of CTCs identified per ml of blood collected ranged from 0 to 214. The final purity ranged from 1 CTC in 9 total cells to 1 CTC in 20,000 total cells, the final purity being both a function of the number of CTCs and the performance of the specific enrichment. Consistent with previous reports, CTC were positively identified if: 1) they contained a nucleus based on DAPI stain, 2) stained positive for cytokeratins, and 3) have a high nuclei to cytoplasmic ratio. In addition, for a blood sample to be considered positive for CTCs, the enriched sample must be positive for Epithelial Growth Factor Receptor, EGFR, as measured by RT-PCR. While most of the blood samples were obtained during surgery, a number were taken prior to, and during surgery. In all of the pre- and post- surgery paired samples, significant numbers of CTCs were detected. A number of these enriched samples were observed under confocal microscope in addition to the microscopic observations under traditional wide-field fluorescent microscope. As expected, the FITC stained cytokeratins appeared in the cytoplasm and the average size of these positively stained cells, on the cytospin, was in the range of 8-12 microns. Future studies will involve the investigation if cancer stem cell and mesenchymal markers are present on these CTCs and correlations of patient outcome to the number and type of CTC present.
doi:10.1021/mp9000519
PMCID: PMC2829323  PMID: 19445481
Circulating tumor cells; Immunomagnetic cell separation; Immunocytochemistry; RT-PCR; Squamous cell carcinoma of the head and neck (HNSCC)

Results 1-7 (7)