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 impairments. This study was a part of a larger one reported previously12. Data for 14 patients recruited in the US in the larger study12 are reported here with permission. An optometrist specialized in vision rehabilitation who routinely evaluate strabismus in clinic performed prism and alternate cover test, following by measurement with the app to prevent bias of the cover test results by the objective app measurement.
1. Prepare the test
NOTE: Testing can be performed in any environment; however, the following controls are likely to aid in successful testing.
<ol>
	<li>Conduct the test in a well-lit environment. Let the patients face in a direction such that corneal reflections from windows and ceiling lights are not located in the center of the eyes. Try to avoid strong background light, such as windows. 
	NOTE: Usually it helps to have a ceiling light right above the head, or a window on one side of the patient. Sometimes, asking the patient to hold their hand above their brow or using a visor can help shield the eyes from strong light sources which create additional reflections on the cornea, as needed. Turning off point light sources such as canister lights or gooseneck lamps will decrease the intensity of extra corneal reflections, making it easier for the software to detect the reflection from the camera flash as intended. If point light source is needed it may be diffused by pointing it at the wall or using a diffuser (i.e., lamp shade).</li>
</ol>
2. Measure tropia (manifest strabismus) with single snapshot&#8722;near fixation
<ol>
	<li>Launch the app and set the mode to No Cover (button at upper right corner).</li>
	<li>Select near fixation (button at lower right corner).</li>
	<li>Hold the phone in landscape orientation about 40 cm from the patient at eye level with the rear-camera facing the patient to be measured. 
	NOTE: The distance does not have to be accurately controlled. The app can automatically compensate for different distances.</li>
	<li>Instruct the patient to fixate on the flash light, which is off at this point. For tests requiring precise accommodation such as when screening for accommodative esotropia, paste a fixation target (such as a letter) on the back of the phone, directly below or above the flashlight.</li>
	<li>While the patient confirms he/she is fixating, press the round button to take a snapshot, which will be analyzed by the app.</li>
	<li>When the analysis is completed, the app will show detected eye features: limbus (iris outer) boundary indicated by a large circle (green), the center of the eye indicated by a cross (green), and the location of the corneal reflection indicated by a small circle (red). Verify these features are detected without obvious errors (such as incorrect limbus fitting, or incorrect location or missing corneal reflection).</li>
	<li>On the same screen, under the captured picture, the app will show the measurements related to the eye alignment. If satisfied with the results, press the save button to save the current test in the phone. Otherwise, press the back-arrow button to retest.</li>
</ol>
3. Measure tropia (manifest strabismus) with snapshot&#8722;far fixation
NOTE: To measure tropia for far fixation, the angle kappa for each eye needs to be measured at least once. The app will automatically choose the latest measurement of angle kappa in the history. If it is not available for either eye, the app will give a reminder to first obtain this measurement (see section 6 for details of angle kappa measurement).
<ol>
	<li>Launch the app and set the mode to No Cover (button at upper right corner).</li>
	<li>Select far fixation mode (button at lower right corner).</li>
	<li>Hold the phone in landscape orientation about 40 cm from the patient at eye level with the rear face of the phone facing the patient. 
	NOTE: The distance does not have to be accurately controlled. The app can automatically compensate for different distances.</li>
	<li>Place the phone slightly below two eyes so that the patient can look above the phone and fixate a target in the distance (typically 5 m away). Make sure the camera is in approximately between the two eyes, not too far to the side of either eye.</li>
	<li>While making sure the patient is fixating properly, press the round button to take a snapshot.</li>
	<li>When the analysis is completed, the app will show detected eye features: limbus boundary indicated by a large circle (green), the center of the eye indicated by a cross (green), and the location of the corneal reflection indicated by a small circle (red). Verify these features are detected without obvious errors (such as incorrect limbus fitting, or incorrect location or missing corneal reflection).</li>
	<li>Under the picture there are measurement outcomes for eye alignment including prism diopters. If satisfied with the results, press the save button to save the test in the phone. Otherwise, press the back-arrow button to retest.</li>
</ol>
4. Measure intermittent strabismus or phoria with cover test&#8722;near fixation
<ol>
	<li>Launch the app and toggle on the cover test mode (button at upper right corner), and select near fixation (button at lower right corner).</li>
	<li>Hold the phone in landscape orientation about 40 cm from the patient. 
	NOTE: The distance does not have to be accurately controlled. The app can automatically compensate for different distances.</li>
	<li>Instruct the patient to fixate the flash light, which is turned off at this point. For tests requiring precise accommodation, paste a fixation target on the back of the phone, directly below or above the flashlight.</li>
	<li>Use an occluder to cover one of the eyes.</li>
	<li>Press the round button. The app will start to monitor the status of the two eyes (whether one eye is covered).</li>
	<li>While making sure the patient is fixating properly, remove the occluder quickly (i.e., cover-uncover test), or first move the occluder between the two eyes to perform alternate covering a few times and then take the occluder away quickly. The app will automatically take a picture as soon as the occlude is taken away from eyes.</li>
	<li>When the analysis is completed, the app will show detected eye features: iris indicated by a large green circle, the center of the eye indicated by a green cross, and corneal reflection from flash indicated by a small red circle. Verify these features are detected without obvious errors.</li>
	<li>Under the picture there are measurement outcomes for eye alignment in prism diopters. If satisfied with the results, press the save button to save the test in the phone. Otherwise, press the back-arrow button to retest.</li>
</ol>
5. Measure intermittent strabismus or phoria with cover test&#8722;far fixation
NOTE: To measure intermittent ocular misalignment for far fixation, the angle kappa for each eye needs to be measured at least once. The app will automatically choose the latest angle kappa measure. If it is not available for either eye, the app will give a reminder to first obtain this measurement (see section 6 for details of angle kappa measurement).
<ol>
	<li>Launch the app and set the mode to cover test (button at upper right corner).</li>
	<li>Select far fixation (button at lower right corner).</li>
	<li>Hold the phone in landscape orientation about 40 cm from the patient at eye level. 
	NOTE: The distance does not have to be accurately controlled. The app can automatically compensate for different distances. It is best if the flash light/camera is between the eyes. Since the camera and flash are off to one corner in most phone models, this means the phone display itself will be slightly off-center.</li>
	<li>Instruct the patient to look just above the phone and to fixate the target in distance (typically 6 m away).</li>
	<li>Use an occluder to cover an eye.</li>
	<li>Press the round button. The app will start to detect the uncovering of the eye.</li>
	<li>While making sure the patient is fixating properly, remove the occluder quickly (i.e., cover-uncover test), or first move the occluder between the two eyes to perform alternate covering a few times and then take the occluder away quickly. The app will automatically take a picture as soon as the occluder is taken away from eyes.</li>
	<li>When the analysis is completed, the app will show detected eye features: limbus boundary indicated by a large circle (green), the center of the eye indicated by a cross (green), and the location of the corneal reflection indicated by a small circle (red). Verify these features are detected without obvious errors (such as incorrect limbus fitting, or incorrect location or missing corneal reflection).</li>
	<li>Under the picture there are measurement outcomes for eye alignment in prism diopters. If satisfied with the results, press the save button to save the test in the phone. Otherwise, press the back-arrow button to retest.</li>
</ol>
6. Measure angle kappa
<ol>
	<li>Launch the app.</li>
	<li>Select Measure angle kappa.</li>
	<li>Hold the phone in landscape orientation about 40 cm from the patient at eye level. 
	NOTE: The distance does not have to be accurately controlled. The app can automatically compensate for different distances.</li>
	<li>Instruct the patient to use the eye to be tested (either eye) to fixate on the flash light, which is off at this point. Have the other covered by hand or an occluder.</li>
	<li>While making sure the patient is fixating properly, tap the round button to take a snapshot, which will be analyzed by the app.</li>
	<li>When the analysis is completed, the app will show the detected eye features: limbus boundary indicated by a large circle (green), the center of the eye indicated by a cross (green), and the location of the corneal reflection indicated by a small circle (red). Verify these features are detected without obvious errors (such as incorrect limbus fitting, or incorrect location or missing corneal reflection). Under the picture there are measurement outcomes for angle kappa (in degrees).</li>
</ol>

<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>

All authors have a pending patent application on a method of measuring strabismus using a smartphone camera. The technology is being commercialized by EyeNexo LLC, which was founded by authors GL, PS, MT and KH, under a license from Mass Eye and Ear.

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 was taken.
Taking good quality pictures is essential for the measurement. The camera should be placed at a position between two eyes. Being too far away from the midline can cause a difference in the image size between two eyes, and consequently result in measurement inaccuracy.
The limbus boundary is one of the key features that the app uses for locating eye position. Verifying the limbus boundary fitting (the green circle in results) is a crucial step. If the fitting appears to be inaccurate, the measurement will be subject to errors and the eye care professional will not be able to correctly interpret the test. Usually for patients with larger eye fissures, i.e., iris area being more revealed, the fitting will be robust and accurate. On the other hand, for patients with smaller eye fissures, which have only a small portion of the left and right boundaries revealed, the fitting may be prone to inaccuracies. In this situation, operators can ask the patients to open their eyes widely, or gently lift the eyelid wide open. The current version does not provide measurement of vertical misalignment, which will be implemented in future versions.
In addition to the promise for use in strabismus clinics, another potential application of the app is in vision screening. For prevention of amblyopia, the American Academy of Pediatrics strongly endorsed the development of cost-effective image-based screening as a means to extend screening to all children14. Red reflex method, which compares the brightness of the &#34;red eye&#34; flash artifact with the strabismus eye being a lighter or brighter red color, can detect both refractive error and strabismus, but cannot quantify the magnitude of the strabismus. Devices implementing the red flex method include Photoscreener and Vision Screener15,16. These photoscreeners have not been widely adopted by school districts, likely due to cost. Compared to standalone systems, modern smartphone cameras provide better value, improved accessibility, and rapidly improved and higher resolution cameras. Recently, there is an app that implements the red reflex method, GCK app17. The GCK app has some limitations in that it does not give a quantitative measurement of strabismus and requires more control of ambient lighting than the Hirschberg methods. The app presented in this article can be potentially an alternative or complementary solution for vision screening, because of its ease of use and equivalent accuracy with standard clinical measurement using prisms.

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 a photo of the patient. After image processing, the app will show the analysis results to the users. As an example shown in Figure 2, the limbus boundaries (green circles) of the two eyes as well as the corneal reflection of the flash light (red dots) were detected correctly. This suggests that the ocular alignment measure (18.5&#916;) shown below the image is not subject to image analysis error. In this particular case, the patient had left exotropia, which is obvious from the image as the corneal reflection offset was much larger in the left eye. However, the app does not report which eye is deviated, because in cases of small strabismus angle and unknown angle kappa, it would be unreliable for the app to determine the deviated eye. For comparison, an example without strabismus is shown in Figure 3. Figure 4 shows an example of erroneous limbus detection. While the detection of corneal reflection (small red circle) is correct, the green circle apparently does not match the limbus boundary. The test should be redone.
According to cover test on those patients, the range of strabismus angle was between 25&#916; esotropia to 50&#916; exotropia, with the smallest magnitude of strabismus angle being 6&#916;. There were 10 patients with exotropia and 4 patients with esotropia. As the linear regression analysis showed (slope = 1.02, R2 = 0.94, p &#60; 0.001), the app measurements of strabismus angles were consistent with clinical cover test measurements (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60908/60908fig1.jpg" /> 
Figure 1: User interface of the strabismus testing app. Users can toggle on cover test and fixation distance. Under different conditions, the instructions given to the patient may be different, as described in the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60908/60908fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60908/60908fig2.jpg" /> 
Figure 2: A case of left exptropia. This is the results shown to the users, who should verify the detection of limbus boundary and corneal reflection before reading the strabismus angle. If those image features are not detected correctly, the users should redo the test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60908/60908fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60908/60908fig3.jpg" /> 
Figure 3: An example under near fixation without cover test. The corneal reflection and eye center were aligned well in both eyes. Therefore the horizontal (HOR) ocular misalignment was almost zero, as the app reported. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60908/60908fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60908/60908fig4.jpg" /> 
Figure 4: An example of erroneous limbus detection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60908/60908fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60908/60908fig5.jpg" /> 
Figure 5: Comparison of strabismus angle measurement using the app with clinical measurements done with covert testing (n = 14). Negative values indicate exotropic deviations, positive values indicate esotropic deviations. Overall, measurements with the app were consistent with the clinical measurements of strabismus. This figure has been modified from our previous publication12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60908/60908fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

using-an-automated-hirschberg-test-app-to-evaluate-ocular-alignment

Nanyang Technological University

Research

JoVE Journal

Medicine

11.4K Views.  Harvard Medical School. 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.

Video: Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

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 ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

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 ...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...