«Novel Biophotonic Imaging Techniques for Assessing Women’s Reproductive Health by Tyler Kaine Drake Department of Biomedical Engineering Duke ...»
The HIV epidemic continues to overwhelm current preventative methods, resulting in approximately 2.5 million infections and 1.7 million deaths each year.4 Although HIV/ AIDS are a worldwide problem, sub- Saharan Africa is the worst affected region, where 1 in every 20 adults is living with HIV.4 Additionally, women account for 58% of people living with HIV in the region, making it necessary for a low- cost, female- controlled method of HIV prevention to be readily available.4 Microbicide gels have been in development to combat sexually transmitted infections, including HIV/AIDS.5- 10 Microbicide gels are designed for women to discreetly protect themselves from HIV infection by inserting the gel into their vaginal canal before sexual intercourse.
They are topical products that act as both a physical barrier for virus migration, as well as delivery vehicles for active pharmaceutical ingredients (APIs).
The APIs either neutralize pathogens before they contact mucosal surfaces or inhibit viral transmission in the mucosa proper of tissue.5- 10 Therefore, a quality microbicide gel is one that has a potent API, loaded into an optimized gel that provides adequate extent and thickness of coating, thus delivering the drug to target tissues and/or fluids.11 2 In 2010, the CAPRISA- 004 trial of a Tenofivir- based microbicide gel showed a 39% reduction in HIV transmission over the control population.12 A follow up trial (VOICE trial) was performed using the same gel, but was stopped early when preliminary data analysis showed no significant reduction in HIV transmission compared to the control population.13 The reason for the disparate trial outcomes is not completely understood.
The two trials did, however, use a different dose regimen for gel application, with CAPRISSA- 004 using a before and after intercourse regimen and VOICE using a once per day regimen.14 It is hypothesized that dose regimen disparities could have affected the outcome of the trials by (1) delivering different amounts of the API (Tenofovir) to the target tissues in the vaginal mucosa at time of HIV exposure, and (2) there may have been differences in user adherence to the dose regimens.
These disparate trial outcomes from an identical microbicide product make it clear that methodologies are needed to monitor microbicide gel distribution in vivo in order to study the effectiveness of products, safely and accurately.
Local details of microbicide gel coating thickness distribution on the tissue surface affect drug delivery.15, 16 Although computational modeling can be used to predict the distribution and resultant drug delivery,15 there is a need to measure local gel coating in vivo.
Such measurement methodologies offer insight to the pharmacokinetics and pharmacodynamics of microbicide products, beyond predictions from modeling.
Remote sensing technologies, such as magnetic resonance imaging (MRI), single- photon 3 emission computed tomography (SPECT), and gamma scintigraphy, have been effective in providing information on in vivo microbicide gel distributions.17- 21 However, these modalities suffer from limitations because they require contrast agents to be added to the gels for imaging.
These contrast agents may diffuse from the gel into tissue and fluids during studies.
Also, with SPECT and gamma scinitgraphy, the contrast agents are radioisotopes, which delivery unnecessary radiation doses to critical radiosensitive organs in the female reproductive tract.
Finally, theses modalities are limited in their spatial resolution, as they often lack the resolution to measure thin coating layers, on the scale of 100 µ;m, that are sufficient for adequate microbicide gel protection.11, 16 Low coherence interferometry (LCI) is a quickly advancing optical measurement technology that provides absolute measurements with high resolution, sensitivity, and speed.22, 23 Fiber optic- based low coherence interferometry can perform measurements that are independent from environmental disturbances, and have been used to measure a wide variety of quantities, such as temperature, pressure, refractive index, and industrial surface metrology with high accuracy.23- 25 LCI utilizes low coherence of broadband light in order to achieve depth- resolve thickness measurements with micrometer resolution, without the need of exogenous contrast agents.
Early studies by Braun et al.
verified the ability of LCI in measuring microbicide gel thickness distributions with high accuracy.26 However, the LCI system had a single scanning point, making scanning of large tissue areas very slow.26 By creating parallel 4 measurement channels, or multiplexing the LCI system, increased scan area could be achieved without requiring a mechanical scanning device.
This could increase tissue coverage and decrease scan time in measuring microbicide gel thickness distributions.
Work by Henderson et al., demonstrated a probe- based fluorimetry system capable of measuring microbicide gel thicknesses with sufficient depth resolution for imaging thin layers.27, 28 By combining the multiplexed LCI (mLCI) system with the fluorimetry device, simultaneous dual- modality optical measurements are possible.
This would allow the validation of mLCI measurements against the previously verified fluorimetry modality.
However, for this potential to be reached, many technological advancements must be achieved.
The research presented in this dissertation details the theoretical and experimental development of this dual- modality optical imaging instrument.
Along with HIV/AIDS, cervical cancer caused by HPV, is also threat to women’s reproductive health.
It is the second most common female malignancy worldwide and an estimated 500,00 new cases are diagnosed annually, including 12,170 new cases in the U.S.
in 2012.29 Prophylactic vaccines against HPV can significantly reduce cervical cancer risk, however these vaccines have a high cost which has limited widespread use in developing countries.
Also, older women are typically not vaccinated and the vaccines do not cover all oncogenic HPV subtypes, so many women remain at high risk for cervical cancer.
5 Cancers typically develop slowly over time, beginning with just a small group of abnormal cells that proliferate.
Detecting these early structural changes in tissue offers the greatest opportunity for therapeutic intervention and prevention of invasive cancers.
However, detecting precancerous development is a challenge for current cervical screening techniques because of limited sensitivity and specificity, as well as interobserver reproducibility.30- 33 The current screening technique for detecting cervical cancer is the Papaniclolaou test (Pap test), also known as cervical cytology.
A speculum is used to open the vaginal canal and cells are collected from the outer opening of the cervix, the ectocervix, and the endocervix.
The cells are then examined visually under a microscope to find abnormalities that indicate pre- cancerous changes.
Pap tests are prone to under sampling, interpretation errors, and lack of regular screening.3 If a Pap test is positive, typically a colposcopy- directed biopsy with histopathologic assessment will follow.
Biopsy samples are removed from abnormal tissue areas and then are fixed, stained, and examined by a pathologist.
However, as in the Pap test, colposcopy- directed biopsy is limited in that it must be subjectively interpreted by a pathologist.
It is often difficult distinguishing dysplastic tissue from atypical metaplastic tissue in colposcopy, since cervical cancer often arises in the metaplastic transformation zone between the ectocervix and endocervix.
There is also a delay of 1- 2 weeks between the time a biopsy 6 is taken and a diagnosis is made, and like the Pap test, coverage of the cervix is very limited.
Improved screening and diagnostic technologies are needed to overcome these limitations of the current process of detecting cervical cancer.
Angle- resolved low coherence interferometry (a/LCI) has shown success in detecting dysplasia in a variety of epithelial tissues in both humans and animals.34- 40 a/LCI is an elastic light scattering technique that provides depth- resolved nuclear morphology measurements from sites within epithelial tissues.41, 42 It combines LCI with angle- resolved light scattering to probe subcellular structure in subsurface tissues.
a/LCI examines scattered light and compares it to Mie Theory models to determine the average size distribution of scatterers in the sample.
The depth- resolving power of LCI allows a/LCI to isolate light from different layers in the sample, such as light from the basal layer of epithelium, where early signs of dysplasia typically occur.40, 43 a/LCI has great potential to be applied as tool for screening and diagnosis of cervical pre- cancers and to guide physical biopsies.
However, pilot studies must first be performed to show the feasibility of a/LCI in detecting dysplasia in the cervical epithelium.
1.2 Project overview The overall goal of the work described in this dissertation was to (1) develop the dual- modality optical imaging technology from a simple proof- of- principle instrument into an integrated system capable of in vivo clinical measurements, and (2) apply the 7 clinical a/LCI device in a pilot study to determine the feasibility of a/LCI in identifying cervical dysplasia (CIN).
In order to achieve these goals, several key milestones were reached.
The first milestone was the expansion of a single channel LCI instrument into a fully characterized, multiplexed LCI benchtop device.
To achieve this milestone, the device was built, and an investigation of the optical performance of the device was completed.
Once the benchtop mLCI device was fully characterized, the clinical dual- modality optical imaging instrument was built.
This was done by combining the mLCI instrument with a fluorimetry modality, into a single endoscopic device which allowed simultaneous imaging of the two modalities.
The optical performance of this device was again investigated and a proof- of- concept study was done with microbicide gel thickness measurements from a single in vivo human dataset.
The next milestone was a larger in vivo human study in which microbicide thickness measurements from 15 study sessions were obtained with the dual- modality instrument.
This data was then used to verify that mLCI was capable of measuring in vivo gel thicknesses with high depth resolution (10 µ;m) by comparing mLCI coating distributions to those of fluorimetry.
A dilution study was then performed in which measurements obtained with the dual- modality optical imaging instruments were used to calculate the extent of microbicide gel dilution.
This milestone was completed by measuring thickness data of serial dilutions of a placebo microbicide gel in a calibration socket to create a calibration curve.
8 This curve can be used to calculate the extent of dilution of microbicide gels from in vivo data, and an example calculation of the methodology is presented.
The final milestone was to use a clinical a/LCI device in a pilot study to detect cervical dysplasia on ex vivo tissues.
The study scanned 23 biopsy sites on 20 tissue samples and showed the feasibility of a/LCI in detecting cervical dysplasia with high sensitivity (100%) and specificity (90%).
1.3 Document organization The dissertation document is organized in the following manner.
Chapter 2 begins with background information about microbicide gels and how they function to prevent sexually transmitted infections such as HIV/AIDS.
The importance of accurately measuring in vivo microbicide coating is discussed, as well as the effects of dilution on coating distribution and gel rheology.
Other in vivo microbicide imaging techniques are summarized to give the reader context of where the dual- modality optical imaging instrument fits in field.
Early studies using a common path LCI, and then full Michelson geometry LCI are presented to show the ability of LCI in measuring microbicide gel thicknesses.
Background information on cervical intraepithelial neoplasia (CIN) and a/LCI is then discussed in Section 2.3.
The current common screening method for CIN is explained, and risk factors for CIN, such as human papillomavirus (HPV) and HIV, are discussed.
Again, a survey of other optical screening techniques is presented, followed 9 by the advantages of a/LCI in detecting CIN.
Finally, a recap of previous a/LCI studies is offered in order to support the use of a/LCI for in vivo detection of CIN.
Chapter3 then explores further into the instrumentation that was designed and built for both microbicide gel imaging.
The chapter begins with describing the optical design and performance of the benchtop mLCI instrument.
The integration process of this mLCI instrument with a fluorimetry system, and growth into a viable, dual- modality clinical instrument is described.
Example calibration and in vivo data are presented to characterize the dual- modality clinical instrument.
An additional update is provided, which was performed to minimize both the footprint of the instrument and its clinical scan time.
The focus of Chapter 4 is an in vivo clinical study, in which the dual- modality optical imaging instrument was used to measure microbicide gel thickness distributions in the human vagina.
The chapter describes the design, data analysis, and results from the study in which gel distributions from mLCI were compared with those measured with fluorimetry to assess the ability and accuracy of mLCI in measuring gel thicknesses.
Chapter5 provides details on a study completed in order to measure dilution of microbicide gels with the dual- modality instrument.
The chapter describes the application of the dual- modality device to measuring diluted gels, and the data processing techniques that are necessary in measuring the extent of dilution.
Chapter 5 10 also provides an example dilution calculation performed on data from the aforementioned in vivo microbicide gel thickness clinical study to illustrate the method.
A pilot study performed to determine the feasibility of a/LCI in detecting cervical dysplasia is the emphasis of Chapter 6.
The design, application, and results from the ex vivo cervical tissue study are discussed in detail.