Name: using an automated hirschberg test app to evaluate ocular alignment
Uploaded: 2020-03-24T15:00:00
Description: Watch this Scientific Journal Video about Using an Automated Hirschberg Test App to Evaluate Ocular Alignment at JoVE.com

This work was supported in part by NIH grant R44EY025902 and by the Mass Eye &#38; Ear Curing Kids Grant.

This study was conducted in accordance with the tenets of the Declaration of Helsinki, at Schepens Eye Research Institute (Boston, MA) and Spaulding Rehabilitation Hospital (Boston, MA). Informed consent was obtained from all the participants. The study was approved by the local institutional review boards of Mass Eye and Ear (Boston, MA).
NOTE: Patient inclusion criteria were prior diagnosis of horizontal strabismus (constant or intermittent exotropia or esotropia) and no other visual impairm...

<ol>
	<li>Kemper, A. R., Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preschool+vision+screening+in+pediatric+practices.">Preschool vision screening in pediatric practices.</a> Clinical Pediatrics. 45 (3), 263-266 (2006).</li><li>Rowe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+profile+of+strabismus+in+stroke+survivors.">The profile of strabismus in stroke survivors.</a> Eye. 24 (4), 682-685 (2010).</li><li>Black, K., McCarus, C., Collins, M. L. Z., Jensen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+Manifestations+of+Autism+in+Ophthalmology.">Ocular Manifestations of Autism in Ophthalmology.</a> Strabismus. 21 (2), 98-102 (2013).</li><li>Taylor, K., Elliott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventions+for+strabismic+amblyopia.">Interventions for strabismic amblyopia.</a> Cochrane Database of Systematic Reviews. 10 (8), (2011).</li><li>Cotter, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+preference+and+visual+acuity+testing+in+a+population-based+cohort+of+preschool+children+with+amblyopia+risk+factors.">Fixation preference and visual acuity testing in a population-based cohort of preschool children with amblyopia risk factors.</a> Ophthalmology. 116 (1), 145-153 (2009).</li><li>Robaei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Associated+with+Childhood+Strabismus:+Findings+from+a+Population-Based+Study.">Factors Associated with Childhood Strabismus: Findings from a Population-Based Study.</a> Ophthalmology. 113 (7), 1146-1153 (2006).</li><li>Tarczy-Hornoch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+decreased+visual+acuity+in+preschool+children:+the+multi-ethnic+pediatric+eye+disease+and+Baltimore+pediatric+eye+disease+studies.">Risk factors for decreased visual acuity in preschool children: the multi-ethnic pediatric eye disease and Baltimore pediatric eye disease studies.</a> Ophthalmology. 118 (11), 2262-2273 (2011).</li><li>Chan, K. W., Deng, L., Weissberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Strabismus+by+Non-Health+Care+Professionals+in+an+Ethnically+Diverse+Set+of+Images.">Detection of Strabismus by Non-Health Care Professionals in an Ethnically Diverse Set of Images.</a> JAMA Ophthalmology. 134 (1), 30-36 (2016).</li><li>Hunter, D. G., Guyton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+location+of+the+corneal+light+reflex+in+strabismus+photography.">Vertical location of the corneal light reflex in strabismus photography.</a> Archives of Ophthalmology. 116 (6), 767-771 (1998).</li><li>Eskridge, J. B., Wick, B., Perrigin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hirschberg+test:+a+double-masked+clinical+evaluation.">The Hirschberg test: a double-masked clinical evaluation.</a> American Journal of Optometry and Physiological Optics. 65 (9), 745-750 (1988).</li><li>Hasebe, S., Ohtsuki, H., Tadokoro, Y., Okano, M., Furuse, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reliability+of+a+video-enhanced+Hirschberg+test+under+clinical+conditions.">The reliability of a video-enhanced Hirschberg test under clinical conditions.</a> Investigative Ophthalmology &amp; Visual Science. 36 (13), 2678-2685 (1995).</li><li>Pundlik, S., Tomasi, M., Liu, R., Houston, K., Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Preliminary+Evaluation+of+a+Smartphone+App+for+Measuring+Eye+Alignment.">Development and Preliminary Evaluation of a Smartphone App for Measuring Eye Alignment.</a> Translational Vision Science &amp; Technology. 8 (1), 19 (2019).</li><li>Cheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EyeTurn+App+for+School+Vision+Screening.">The EyeTurn App for School Vision Screening.</a> American Academy of Optometry Annual Meeting. , (2018).</li><li>Committee on Practice and Ambulatory Medicine and Section on Ophthalmology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Photoscreening+for+Children's+Vision+Screening.">Use of Photoscreening for Children's Vision Screening.</a> Pediatrics. 109 (3), 524-525 (2002).</li><li>Simons, B. D., Siatkowski, R. M., Schiffman, J. C., Berry, B. E., Flynn, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+photoscreening+for+strabismus+and+refractive+errors+in+a+high-risk+population.">Pediatric photoscreening for strabismus and refractive errors in a high-risk population.</a> Ophthalmology. 106 (6), 1073-1080 (1999).</li><li>Kerr, N. C., Somes, G., Enzenauer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+developmentally-at-risk+status+on+the+reliability+of+the+iScreen(R)+photorefractive+device+in+young+children.">The effect of developmentally-at-risk status on the reliability of the iScreen(R) photorefractive device in young children.</a> The American Journal of Orthopedics. 61, 117-123 (2011).</li><li>Arnold, R. W., O'Neil, J. W., Cooper, K. L., Silbert, D. I., Donahue, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+smartphone+photoscreening+app+to+detect+refractive+amblyopia+risk+factors+in+children+aged+1-6+years.">Evaluation of a smartphone photoscreening app to detect refractive amblyopia risk factors in children aged 1-6 years.</a> Clinical Ophthalmology. 12, 1533-1537 (2018).</li></ol>

A person without professional training can use the EyeTurn app to capture pictures of the eyes and obtain ocular alignment measurements, which might be interpreted by an eye care specialist onsite or remotely. The app only provides magnitude of the misalignment, rather than any interpretation or diagnosis. Eye care professionals such as optometrists or ophthalmologists should determine if the misalignment is significant or not, and make a diagnosis after considering other factors including the conditions under which the ...

Measurement of eye alignment is frequently performed in vision care clinics. Cover test with prism neutralization is the commonly used clinical method for quantifying the degree of eye misalignment (strabismus). This method requires a high degree of training and experience. Accurate measurement becomes more challenging when patients cannot fully engage in the exam such as young children1, individuals with brain injuries or stroke2, or developmental disabilities3. Furthermore, there is a need for ocular alignment testing in school screening, because strabismus develops during childhood in an estimated 5&#8722;8% of the US population4, and is a substantial risk factor for amblyopia with about 30&#8722;40% of cases of amblyopia attributed to strabismus5,6,7. However, school nurses are normally not trained to conduct the standard cover test with prism neutralization for such screening. For non-eye care professionals, an additional challenge in strabismus screening is that intermittent strabismus (misalignment is not always manifested) and smaller magnitudes of misalignment are not visually obvious (&#60;15 prism diopters [&#916;])8.
In an attempt to address the challenges in the detection and measurement of strabismus, we have developed a smartphone app (EyeTurn) that implements and automates the photographic Hirschberg method9 by comparing the displacement of corneal reflections between the eyes. While conventional photographic Hirschberg method has been shown to have good reproducibility in clinics10,11, the cost for dedicated, standalone devices is a barrier for wide adoption. By providing an easy-to-use tool to measure eye alignment with standard smartphones, we hypothesize it will be widely adopted in school vision screening and used by non-eye care professionals. Our previous evaluation studies have shown that the app measurement is consistent with the current clinical standard of prism and alternate cover test12, for strabismus magnitudes of esotropia and exotropia up to 60&#916;. In a pilot school screening study, we also showed that the app can help the school nurse detect children with intermittent exotropia who were missed by standard school vision screening protocols13.
The iOS version of the app is currently available to researchers and clinicians upon request for research purposes. The requesters have thus far included school nurses, pediatric ophthalmologists, optometrists, neuro-ophthalmologists, and strabismus specialists. The purpose of this article is to share the detailed app protocols for using the app to evaluate ocular alignment under different viewing conditions, namely, near and far fixation distance; with and without eye covering to break binocular fusion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EyeTurn</td>
			<td>EyeNexo</td>
			<td></td>
			<td>Smartphone app for measureing eye misalignment</td>
		</tr>
	</tbody>
</table>

using an automated hirschberg test app to evaluate ocular alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

In this work, we describe the protocols to evaluate ocular alignment using a smartphone app that performs the photographic Hirschberg test. The interface of the app is shown in Figure 1. The users can choose to perform cover test or measure a patient with both eyes fixating at a target simultaneously, either at near or far fixation distances. Once the viewing conditions are determined depending on the test purposes, the users can follow the protocols and take...

Watch this Scientific Journal Video about Using an Automated Hirschberg Test App to Evaluate Ocular Alignment at JoVE.com

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

The mobile app that we have developed can be used to measure strabismus for research or for clinical use. This method, facilitates an easy strabismus measurement, which currently requires extensive training and experience to be performed using only a standard phone camera and the app software. In the app interface, users can choose to perform a cover test or to measure a patient with both eyes fixated at a target simultaneously, at either a near or far fixation distance.To measure tropia, with a single snapshot in with a near fixation, launch the app and set the mode to No Cover. Select Near and position the phone in a landscape orientation about 40 centimeters from the patient at eye level, with the rear camera facing the patient. Instruct the patient to fixate on the flashlight, which should be off at this point.For tests requiring a precise accommodation, paste a fixation target on the back of the phone directly below or above the flashlight. When the patient confirms they are fixating, press the round button to take a snapshot to be analyzed by the app. When the analysis is complete, the app will show the detected limbus boundary, the center of the eye, and the location of the corneal reflection.After verifying that these features have been detected without obvious errors, observe the measurements related to the eye alignment. Then, press the Save button to save the current test in the phone, or press the back arrow button to retest. To measure intermittent ocular misalignment for far fixation in the app, set the mode to Cover Test and select Far.Hold the phone in a landscape orientation, about 40 centimeters from the patient at eye level with the flash camera positioned between the patient's eyes. Instruct the patient to look just above the phone, to a target about six meters away. Have the patient cover one eye and press the round button, the app will begin the detection.While making sure that the patient is fixating properly, have them quickly remove the occlusion. The app will automatically take a picture as soon as the occlusion is removed. When the analysis is complete, the app will show the detected limbus boundary, the center of the eye, and the location of the corneal reflection.After verifying that these features are detected without obvious errors, review the measurement outcomes for the eye alignment in prism diopters, before saving the data or repeating the test. For Angle Kappa measurement in the app, select measure Kappa angle. Position the phone in a landscape orientation about 40 centimeters from the patient at eye level, and instruct the patient to use the eye to be tested to fixate on the flashlight while covering the other eye.While making sure the patient is fixating properly, tap the round button to take a snapshot, which will be analyzed by the app. When the analysis is complete, the app will show the detected limbus boundary, the center of the eye, and location of the corneal reflection. After verifying that these features are detected without obvious errors, view the measurement outcomes for Kappa angle in degrees.In this representative analysis, the limbus boundaries of the two eyes and the corneal reflection of the flashlight, were detected correctly, suggesting that the ocular alignment measure is not subject to Image Analysis error. In this particular case, the patient had left exotropia as is obvious from the larger corneal reflection offset in the left eye. The app did not report which eye was deviated however, because in cases of a small strabismus angle and unknown Kappa angle, it would be unreliable for the app to determine the deviated eye.For comparison, here an example without strabismus is shown. In this analysis, erroneous limbus detection can be observed. While the detection of the corneal reflection is correct, the green circle clearly does not match the limbus boundary, indicating that the test should be repeated.In this analysis of 14 subjects, 10 subjects exhibited exotropia and four subjects presented with exotropia. Linear regression analysis, revealed that the eye measurements of the strabismus angles, were consistent with clinical cover test measurements. When using the app, there are two important things to keep in mind.One, set the cover test and the fixation mode correctly for each specific test. Two, check if the eye feature detection is correct. The app only allows strabismus measurement and it doesn't give a diagnosis.Clinicians will need to combine the ocular measurement with other vision measurements to make a diagnosis.

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An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

Preventing Posterior Capsular Opacification Through IOL Rotation in Cataract Surgeries

Rotating the Intraocular Lens to Prevent Posterior Capsular Opacification in Cataract Surgeries

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and migration of lens epithelial cells and can affect long-term visual outcomes significantly. The most effective treatment for PCO is neodymium-doped yttrium aluminum garnet (Nd:YAG) laser capsulotomy; however, this treatment is associated with posterior segment complication and can break the stability of capsular bag, affecting the position and function of trifocal or toric intraocular lenses (IOLs). Advances in surgical procedures, IOL design, and pharmacy have reduced the rate of PCO in recent years, concentrating on the inhibition of proliferative lens epithelial cells (LECs). This protocol aimed to clear LECs more thoroughly during phacoemulsification and IOL implantation. The first several steps, including clear corneal incision, continuous circular capsulorhexis, hydrodissection, hydrodelineation, and phacoemulsification, were completed as conventional procedures. After placing the IOL into the capsular bag, rotation of the IOL by at least 360&#176; was performed using an irrigation/aspiration tip or a hook, with slight stress on the posterior capsule. Some residuals occurred in the originally transparent capsular bag after rotation of the IOLs. Then, these materials and the viscoelastic were cleared completely using an irrigation/aspiration system. A clear posterior capsule was observed after the surgery in patients undergoing this method. This method of rotating IOLs is a simple, effective, and safe way to prevent PCO by clearing residual LECs and can be carried out without extra tools or skills.

Author Spotlight: Enhancing Visual Outcomes in Cataract Surgery: A Novel Technique to Prevent Posterior Capsular Opacification Through IOL Rotation 

The present protocol describes removing residual epithelial cells by rotating the intraocular lens in extracapsular cataract surgery without extra tools for preventing posterior capsular opacification.

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and ...

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production 

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

Manufacturing Chimeric Antigen Receptor T-Cells Using an Automated Cell Processor

Begin by turning on the instrument and selecting the T-cell transduction process from the touchscreen interface. Click run to initiate the procedure and follow the onscreen instructions provided. On the perimeter input screen input the operator initials tubing set, lot number, and expiration date.Next, select full process on the process setup screen. Now, choose two vials of selection reagent for the CD4, CD8 selection method. Install the tubing set, ensuring that all lure connections are tightly set and complete the upper and lower integrity tests by following the instructions on the touchscreen.Now, attach the medium and processing buffer bags per the onscreen guidance and begin the automatic priming of the tubing set. Once the transfer cell product screen appears, transfer the thawed T-cell product to a 150 milliliter transfer bag and sterile weld it to the application bag of the tubing set. Next, use a quality control pouch to obtain a sample from the application bag and perform a cell count using a hematology analyzer.Connect the CD4 and CD8 reagent vials to the tubing set and start the selection process for T-cell enrichment. After enrichment, remove a sample of the selected cells from the reapplication bags quality control pouch. Enter the desired cell concentration and starting number.Now, attach a vial of the activation reagent according to onscreen instructions. Then, set the carbon dioxide concentration to 5%and the culture chamber temperature to 39 degrees Celsius. Establish the activity matrix using the enhanced feeding protocol as a starting point and modify individual steps to your specific optimized protocol.In this case, set the transduction activity time to 24 hours after seeding and the culture wash to 48 hours after transduction. Set the activate shaker time to begin 30 minutes after starting the culture wash. Delete any medium bag exchange and waste bag exchange activities as they are no longer required.Then, press okay on the screen to initiate cultivation. Allow the instrument to automatically pump the required volume from the reapplication bag into the culture chamber. Thaw and prepare the calculated vector volume in a 20 milliliter reagent bag with a cold medium to reach a final volume of 10 milliliters.Adjust the activity matrix to set the transduction time to two minutes into the future and press okay to initiate the transduction activity. Sterile weld the vector bag to the tubing set following the onscreen instructions. Modify the activity matrix according to the actual transduction start time with the culture wash scheduled 48 hours after transduction.Schedule the time of activate shaker to 30 minutes after the culture wash. Finally, ensure the media exchange occurs in the afternoon at 2:00 PM on day six. Press the sample button and follow the onscreen instructions to acquire a quality control sample from the active culture.Divide the sample into three one milliliter aliquots for various analyses. Prepare the final formulation buffer, saving a portion for the cryoprotectant preparation later. Next, adjust the activity matrix, setting the end of culture to two minutes in the future.Attach the final formulation buffer to the tubing set and begin harvest. Seal off and remove the target cell bag. This bag contains the CAR T-cells ready for infusion or cryo-preservation.RT runs of the clinical trial was 46%This indicates that the TCT process effectively promoted cell expansion. The cell viability of the final product was between 88 to 94%and the CD3 T-cell purity was 89 to 93%Endotoxin, mycoplasma, sterility, replication competent lentivirus, and residual leukemic cells were not detectable.

Establishment of an Ulcerative Coloproctitis-Colorectal Cancer (UC-CRC) Model to Evaluate the Therapeutic Effect of Chinese Medicine

To begin, gently position the mouse with its belly up and the head slightly down. Grasp the back skin to secure the abdominal area and insert a one milliliter syringe needle, one centimeter to the right of the midline of the abdomen at the intersection of the line, connecting both thighs. Gradually insert the one milliliter syringe needle to a depth of three to five millimeters under the skin.Insert the needle approximately 0.3 to 0.5 millimeters into the abdominal cavity at a 45 degree angle. Once the needle tip passes through the abdominal muscle, notice a sudden decrease in resistance. Slowly inject 0.1 milliliters of the azoxymethane injection solution per 10 grams of body weight into the mice.Subsequently, retract the syringe gently. Then add 500 milliliters of sterile distilled water to 10 grams of DSS. To prepare a 2%DSS solution.Mix the solution with a vortex mixer and store at four degrees Celsius until use, administer the prepared LJZD and five amino salicylic acid solution daily at a consistent time via gastric gavage to the respective groups at week seven and week 15.