microfluidic-platform-for-measuring-neutrophil-chemotaxis-from

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Massachusetts General Hospital

Harvard University School of Engineering and Applied Sciences

Index of suspicion.

Aicardi syndrome is a rare congenital disorder with a classic triad of infantile spasms, chorioretinal lacunae, and agenesis of the corpus callosum. We report the use of fluorescein angiography and spectral domain optical coherence tomography to examine the posterior segment structures in an 8-month-old girl with Aicardi syndrome. Most of the observed features correlated with previously published histopathological findings, but inner nuclear layer cysts have not been previously described. To our knowledge, this is the first study of the tomographic and angiographic chorioretinal features in vivo.

Chorioretinal architecture in Aicardi syndrome: an optical coherence tomography and fluorescein angiography study.

A 65-year-old man with unilateral exudative age-related macular degeneration presented initially with hypotony and a shallow choroidal detachment 5 days after an intravitreal ranibizumab injection. This was followed by an acute increase in intraocular pressure (IOP) to 60 mm Hg despite open angles, no evidence of intraocular inflammation, and no corticosteroid use. Ultrasound biomicroscopy showed a focal atrophic area in the ciliary body from the 7- to 9-o'clock positions, corresponding to the site of intravitreal injection. The IOP elevation was successfully treated with aqueous suppressants and resolved spontaneously after 48 hours. This report describes a patient with a rare case of labile IOP secondary to angle changes following ranibizumab injection. Injury to angle structures following intravitreal ranibizumab injection is a rare but potentially severe complication. Ultrasound biomicroscopy may be helpful in elucidating angle changes in the setting of labile IOP following intravitreal ranibizumab injection.

Severe intraocular pressure fluctuation after intravitreal anti-vascular endothelial growth factor injection.

This is a report of the use, efficacy, and theoretic safety of negative-pressure wound therapy over ocular structures as a part of surgical treatment for necrotizing fasciitis. We treated a 65-year-old man with facial necrotizing fasciitis requiring serial debridement and closure of extensive periorbital and nasal wounds with skin grafts. Negative-pressure wound therapy was first used as a bridge to allow temporary closure and to encourage granulation tissue development. It was then used as a bolster dressing to stabilize skin grafts in the complex wound, not amenable to tie-over dressings. Excellent functional and cosmetic reconstruction of the periorbital and nasal regions was achieved. After treatment, the patient's corrected vision was 20/20. To our knowledge, the use of negative-pressure wound therapy directly over ocular structures has not been previously documented. In this case, it was safely used over the eyes with no sequelae to the patient's vision.

Safety of negative-pressure wound therapy over ocular structures.

To describe the corticosteroid-sparing success in controlling chronic uveitis in patients treated with TNFα inhibitors.

Infliximab and adalimumab for uveitis.

This report details a comprehensive study of inertial focusing dynamics and particle behavior in low aspect ratio (h/w ∼ 1/1 to 1/8) spiral microchannels. A continuum of particle streak behavior is shown with longitudinal, cross-sectional, and velocity resolution, yielding a large analyzed parameter space. The dataset is then summarized and compared to prior results from both straight microchannels and other low aspect ratio spiral microchannel designs. Breakdown of focusing into a primary and secondary fluorescent streak is observed in the lowest aspect ratio channels at high average downstream velocities. Streak movement away from the theoretically predicted near inner wall equilibrium position towards the center of the channel at high average downstream velocities is also detailed as a precursor to breakdown. State diagrams detail the overall performance of each device including values of the required channel lengths and the range of velocities over which quality focusing can be achieved.

Inertial focusing dynamics in spiral microchannels.

The approach used by the authors for managing a patient with a schizoaffective disorder and advanced basal cell carcinoma involving the eyelids, orbit, and face is presented. Complexities included the advanced nature of the disease, neglect of the patient's condition due to schizoaffective disorder, the difficulty of obtaining informed consent, the required aggressive surgical intervention, reconstruction, and the necessary management during the postsurgical period. A multidisciplinary team approach with psychiatry, ophthalmology, ear, nose, and throat, plastic surgery, radiation oncology, oncology, legal, and bioethics specialties is required in patients with cognitive disabilities. Curative treatment requires complete excision, reconstruction, and proper postoperative care, which can be prohibitive in a schizophrenic patient from a surgical and ethical perspective. Staging of this condition after proper informed consent with biopsy, computed tomography, and magnetic resonance imaging is presented. The options for management are discussed, including surgical intervention and palliative care.

Advanced Basal cell carcinoma in a patient with schizoaffective disorder: constraints and management.

Massive submacular hemorrhage can cause disastrous visual complications. Prompt displacement away from the fovea is desirable in some patients. We describe a novel surgical technique involving subretinal air as a therapeutic adjuvant for massive submacular hemorrhage displacement.

Subretinal pneumatic displacement of subretinal hemorrhage.

Ocular trauma is recognized as the leading cause of unilateral blindness. However, few studies to date have focused on the clinical features of hospital-based ocular emergencies. Effectiveness of trauma centers in treating ocular emergencies was compared with treatment in traditional community hospital emergency departments. Demographics, causes, and nature of ocular emergencies, as well as visual outcome in community hospitals emergency departments and trauma centers, were also examined.

Hospital-based ocular emergencies: epidemiology, treatment, and visual outcomes.

The current paradigm of unidirectional migration of neutrophils from circulation to sites of injury in tissues has been recently challenged by observations in zebrafish showing that neutrophils can return from tissues back into the circulation. However, the relevance of these observations to human neutrophils remains unclear, the forward and reverse migration of neutrophils is difficult to quantify, and the precise conditions modulating the reverse migration cannot be isolated. Here, we designed a microfluidic platform inside which we observed human neutrophil migration in response to chemoattractant sources inside channels, simulating the biochemical and mechanical confinement conditions at sites of injury in tissues. We observed that, after initially following the direction of chemoattractant gradients, more than 90% of human neutrophils can reverse their direction and migrate persistently and for distances longer than one thousand micrometers away from chemoattractant sources (retrotaxis). Retrotaxis is enhanced in the presence of lipoxin A4 (LXA4), a well-established mediator of inflammation resolution, or Tempol, a standard antioxidant. Retrotaxis stops after neutrophils encounter targets which they phagocytise or on surfaces presenting high concentrations of fibronectin. Our microfluidic model suggests a new paradigm for neutrophil accumulation at sites of inflammation, which depends on the balance of three simultaneous processes: chemotaxis along diffusion gradients, retrotaxis following mechanical guides, and stopping triggered by phagocytosis.

Retrotaxis of human neutrophils during mechanical confinement inside microfluidic channels.

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 10⁷ cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2-5 d.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples.

To demonstrate the difficulties of diagnosing a patient with Tolosa-Hunt syndrome (THS) due to its complicated presentation and extensive diagnostic testing, and how to manage the treatment of a patient in an emergent setting.

Tolosa-Hunt syndrome masquerading as a carotid artery dissection.

Neutrophil chemotaxis is critical for defense against infections and its alterations could lead to chronic inflammation and tissue injury. The central role that transient alterations of neutrophil chemotaxis could have on patient outcomes calls for its quantification in the clinic. However, current methods for measuring neutrophil chemotaxis require large volumes of blood and are time consuming. To address the need for rapid and robust assays, we designed a microfluidic device that measures neutrophil chemotaxis directly from a single droplet of blood. We validated the assay by comparing neutrophil chemotaxis from finger prick, venous blood and purified neutrophil samples. We found consistent average velocity of (19 ± 6 μm/min) and directionality (91.1%) between the three sources. We quantified the variability in neutrophil chemotaxis between healthy donors and found no significant changes over time. We also validated the device in the clinic and documented temporary chemotaxis deficiencies after burn injuries.

Article	Year
Index of suspicion. Pediatrics in review / American Academy of Pediatrics\| PubMed ID: 17012490	2006
Chorioretinal architecture in Aicardi syndrome: an optical coherence tomography and fluorescein angiography study. Journal of AAPOS : the official publication of the American Association for Pediatric Ophthalmology and Strabismus / American Association for Pediatric Ophthalmology and Strabismus\| PubMed ID: 21777802	2011
Severe intraocular pressure fluctuation after intravitreal anti-vascular endothelial growth factor injection. Ophthalmic surgery, lasers & imaging : the official journal of the International Society for Imaging in the Eye\| PubMed ID: 21986163	2011
Safety of negative-pressure wound therapy over ocular structures. Ophthalmic plastic and reconstructive surgery\| PubMed ID: 22186986	2012
Infliximab and adalimumab for uveitis. Ocular immunology and inflammation\| PubMed ID: 22324897	2012
Inertial focusing dynamics in spiral microchannels. Physics of fluids (Woodbury, N.Y. : 1994)\| PubMed ID: 22454556	2012
Advanced Basal cell carcinoma in a patient with schizoaffective disorder: constraints and management. Ophthalmic plastic and reconstructive surgery\| PubMed ID: 23235512	2013
Subretinal pneumatic displacement of subretinal hemorrhage. JAMA ophthalmology\| PubMed ID: 24337559	2013
Hospital-based ocular emergencies: epidemiology, treatment, and visual outcomes. The American journal of emergency medicine\| PubMed ID: 24418441	2014
Retrotaxis of human neutrophils during mechanical confinement inside microfluidic channels. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 24419464	2014
Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nature protocols\| PubMed ID: 24577360	2014
Tolosa-Hunt syndrome masquerading as a carotid artery dissection. Clinical ophthalmology (Auckland, N.Z.)\| PubMed ID: 24741291	2014
Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood. Technology\| PubMed ID: 24809064	2013

Joseph Martel

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