One of the most important steps of performing a thesis is “PLANNING” for it. The plan comes after the rationale for the study has been established and existing literature reviewed thoroughly. Executing the plan is easier said than done and requires rigor at every step to accumulate high-quality data. The document containing this “PLAN” is called the “Manual of Operations” (MOP) and represents all the procedural details essential to ensure the smooth performance of each and every step of the study.

While writing a scientific manuscript, it is not only important to report the results of the study but also to enumerate the steps followed to arrive at the results. The latter is referred to as the “Materials and Methods” or simply methods section of the scientific manuscript. It should be described in sufficient detail to allow easy replication by a competent fellow researcher, but the authors should guard against overzealous description and avoid unnecessary technical details. If the MOP is written in great detail, writing the methods section can be a simple exercise and can be considered a precis of the MOP. The MOP/methods section is probably the most important part of a manuscript because it provides vital information on which the quality and validity of the article are judged.

This chapter is aimed at helping one write the methods sections of the resultant scientific manuscript. Though it is not an exhaustive tutorial, it will provide a blueprint to help authors describe what was done, how it was done, how the data was collected, and how it was analyzed. Summarizing the thesis refers to writing the results and we will consider some basics of presenting results at the end of this chapter

Basic understanding of terminologies:

Before learning to write the MOP and methods section, one must understand a few basic terminologies that will be used repeatedly during the study design, execution, and statistical analysis. With respect to the methods section, the two most important terminologies are 1) Variable and 2) Validity.

What is meant by the term Variable?

A scientific study is aimed at establishing “cause and effect relationship” between objects and in scientific literature, these objects are referred to as variables. Everything that is measured (e.g. visual acuity, age, intraocular pressure, etc) in a particular study is a variable. For the purpose of cause-effect relationship studies, variables are classified as independent, dependent, and confounding variables.

• An independent variable is the one, that when manipulated, causes a change in another variable.
• A dependent variable is the one which changes values depending on the manipulations performed on the independent variables.
• A confounding variable is any other variable other than the independent variable which can influence change in the dependent variable.

For example, change in axial length and/or keratometric value results in a change in intraocular lens (IOL) power according to the SRK IOL power calculation formula. Here the axial length and keratometric value are the independent variables and the IOL power is the dependent variable.

Another example, consider a study designed to determine the incidence of pseudophakic cystoid macular edema (CME) in phacoemulsification vs. manual small incision cataract surgery (SICS). Here, the type of surgery is the independent variable, and the occurrence of CME is the dependent variable. The presence of diabetes and coexistent diabetic retinopathy can be considered as a confounding variable as diabetics are known to experience greater pseudophakic CME compared to non – diabetics.

A cause and effect relationship between independent and dependent variables is established by developing of appropriate study design. A study design is simply a strategy to manipulate variables to answer the study question.

What is meant by Validity?

• Internal Validity: Minimizing the influence of confounding variables is key to having a study that actually measures the influence of manipulations of independent variables over the dependent variable and therefore, results in high internal validity.
• External validity refers to the degree to which the results can be applied to the general population of interest. External validity can be improved by reducing selection bias using processes like randomization and masking.

For e.g., in the above-mentioned scenario of pseudophakic CME, either excluding diabetics (omitting the confounder) or measuring the blood glucose/HbA1c and lipid profile (controlling the confounder) of all diabetics will improve the validity of the study. The latter approach will improve the applicability of the study as well as keep the validity high.

Writing Materials and Methods:

This section should provide detailed information on the materials used to conduct the study and the methods used to measure variables in the study. The term “Materials” refers to study participants (e.g. humans, animals, laboratory experiments, etc) and also to various instruments (e.g. applanation tonometry, optical coherence tomography, etc.) and treatments (e.g. drug1 vs. drug2 OR phacoemulsification vs. SICS) used in the study.

The term “Methods” refers to how various procedures were performed on the study participants, how measurements were made, how data was collected and stored, and finally how data was analyzed. In general, it is advisable to use questions like what, when, why, how much, and how often to guide your explanations. The methods should always be written in the past tense.

• Ethical considerations
• Study period, location and type
• Describe the Subjects in detail
• Describe how the subjects were prepared
• Describe the study design and protocol
• Explain how measurements were made and how calculations were performed
• Describe statistical tests in sufficient detail

It is important to maintain a systematic and logical chronology in the writing so that it is easily comprehended by reviewers and readers alike. The following chronology can be followed in most instances:

We will consider the salient points of each of the above to make the Materials and Methods section of the manuscript succinct yet comprehensive.

Ethical considerations

When the study involves manipulations on human or animal subjects, it is essential that the methods section begins with a statement stating that the study was approved by the institutional review board, informed consent was obtained from all the participants and the study protocol adhered to the tenets of the declaration of Helsinki. For animal experiments, including a statement regarding approval by the institutional animal care committee or appropriate body.

Declaration of Helsinki

• The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data.
• It has 37 guiding principles under various classifications such as Scientific Requirements of Research Protocols, Research Ethics Committees, patient Privacy and Confidentiality, informed consent process and use of placebo.

Study period, location and type

Specify the duration of the study with the start and end dates (month/year) followed by the location of the study. If the journal does not allow disclosure of the institute’s name to maintain anonymity during the review process, then mention whether it’s a primary, secondary, or tertiary care eye center and from which part of the country (south, north, etc.). It is essential to mention the type of study such as a randomized controlled trial, prospective/retrospective cohort study, case?control study, or cross?sectional survey. The following points can be considered before choosing a prospective or retrospective study design:

• Can the study question be answered by a retrospective study?
• Are medical records maintained meticulously by MRD staff?
• How good is documentation of findings in the case sheets?
• How common is the disease?

If findings are routinely documented well and case records are preserved properly, most studies can be done both prospectively and retrospectively.

If your study does not fit a particular definition you may describe the key design components, e.g. an interventional and/or observational study and whether data were collected longitudinally or cross?sectionally.

Subjects

It is of utmost importance to explicitly describe who the study participants were and how they were chosen for the study. It is useful to use words such as volunteers, patients, controls, lab animals, etc. “Inclusion and exclusion criteria” should be described in great detail as these will help in establishing the limits set by the authors and thereby help in generalizing the study results to the population (i.e. to whom the study results will be applicable). For the purpose of the study, it is important to “define the disease” in measurable terms. For e.g. for a glaucoma study, how glaucoma was defined should be explained using credible references. In case of controls, explain how they were selected and how they relate to the study group, e.g. matching by age, gender, ethnicity, clinical condition, etc. Also, describe in brief the sampling methods used. Randomization and masking should be described in detail in case the manuscript is describing a randomized controlled trial. The randomization/masking details should be highly descriptive in the MOP but lesser so in the methods section.

Preparation of Subjects

In studies involving human subjects, steps taken to prepare the participants prior to the actual study should be described briefly. For e.g., how dilatation of pupils was achieved or what type of anesthesia was used prior to the actual surgical procedure or how OCT (optical coherence tomography) was performed prior to injecting intravitreal anti VEGF agents etc. In animal experiments and mechanical models, the setting up of the animals and models is very important and should be described explicitly.

Study design and Protocol

The research protocol is the exact sequence of manipulations and measurements performed to answer the study question. It should be described in precisely the same chronology as the steps performed in the study. Typically, this first involves a description of baseline measurements, followed by the sequence of events to manipulate the independent variable and the subsequent measurement of changes in the dependent variable. It is advisable to provide a flow plan especially in case of prospective studies to help readers understand the protocol easily.

Measurement of variables

All the tools and instruments used to manipulate the independent variable and measure changes in the dependent variable should be mentioned including the name and location of the manufacturer. For e.g., the central foveal thickness was measured using the spectral-domain OCT (Spectralis, Heidelberg Engineering GmbH, Heidelberg, Germany) or IOP was measured using Goldmann applanation tonometry. Additionally, describe in detail any modifications made to the equipment or any equipment that was constructed specifically for the study and, if necessary, provide illustrations or photographs of the modifications.

It is important to remember that any new method employed for measuring a variable will require additional validation either in the form of a previous publication that can be referenced or by providing proof of the concept of the new measurement technique used. The former is the better approach. This is especially true in studies where questionnaires are used. The questionnaires must be developed in a systematic manner and should have been used previously to prove its validity.

While describing the measurement of multiple variables in the same paper, it is advisable to have subheadings for each. For e.g., in a paper studying ASOCT characteristics of eyes with angle closure, it may be worthwhile having separate subheadings on the clinical examination, biometry (axial length, AC depth, pachymetry, etc.) and ASOCT details to segregate the methods and provide clarity. The description of the primary outcome variable is a good way to clarify exactly how it was recorded. For e.g., IOP of <18mmHg may be considered as a success for a study on trabeculectomy for glaucoma or central foveal thickness less than 275 microns may be considered as a successful resolution for diabetic macular edema. These should be defined with precision to make interpretations and future comparisons easy.

Statistical tests and Data Analysis

A statistician should be involved in the study from the beginning and his/her help can be sought to write this section. This section describes how the data will be presented in the results section (e.g., mean vs. median), which statistical tests will used for the inferential data, and what p value is deemed to be a statistically significant difference between groups. While using regression analysis, it is important to mention a rationale for the covariates used in the multivariable models. For survival analysis, one must explain how the event and time to the event was calculated. While using specialized or unusual statistical methods (e.g. negative binominal regression), one must provide a reference. Also, it is good practice to mention the name and version of the statistical package used for the analysis (for e.g. Stata13 I/C, College Station, TX).

Summarizing the thesis

Once the methods are written and the experiment is performed, the results should be presented in a particular order as follows:

• Mention sample size over the study period (months)
• Demographics of the study population can be presented as a table and mention the important ones in words. For comparative study, mention whether the 2 groups were comparable in terms of the parameters studied before the intervention.
• Results of the primary outcome measure
• Results of the secondary outcome measures
• Other results. E.g., Complications of a procedure

It is advisable to describe results from the primary outcome measure before the other results. Additionally, it is important to first plan which results answer the questions that have been posed in the research and which results may be left out.

Using Tables to summarize results:

Summarizing complex data with absolute values (not percentages) in the form of tables is a good practice. The tables should be labeled appropriately and should be stand – alone such that readers will be able to interpret it without having to reference the text repeatedly. The tables should be cited in the manuscript, the tabular data should agree to those mentioned in the manuscript text, and information in tables should never be duplicated in text and graphs

Using graphs and figures to summarize results:

One may consider using graphs when there is a clear relation between variables or when pronounced trends are seen in the results. Choosing the appropriate graph type is essential to best convey the results. Box and whisker plots display continuous variables with 25^th and 75^th percentile and outlier values and can be used to show distribution of the variable. If there is a relationship between two numerical variables (for e.g. IOP and ocular perfusion pressure, both in mmHg), using a scatter diagram with a LOWESS curve is most appropriate. Pie diagrams help in displaying proportions and bar diagrams (with standard error or 95% confidence intervals) can be used to display numerical data as well as comparative proportions. Labeling the graph and citing it in the text is also extremely important. Design, provide legends and label the graphs carefully so that they are clear and easy to understand. If the study warrants clinical illustrations of the human or animal study such as clinical photographs or microscopic structures, these should be prepared according to the requirements of the journal of choice. Commonly, color illustrations require 300 dpi resolution and CMYK color format for most journals.

Summarizing the results section of the thesis is a difficult task but one that is fulfilling. The ability to effectively communicate ones own findings to the scientific community is critical to get your manuscript accepted by a reputed journal.

Writing the manual of operations, executing the thesis protocol well, and converting it into the methods and results in sections of a scientific manuscript are probably the most important tasks that lead to eventual acceptance and publication of the manuscript. Writing these can be a daunting task, especially for the novice. However, following a stepwise approach as mentioned in this chapter and breaking down the task into smaller steps goes a long way in alleviating the fear associated with this process. We hope that the guidelines provided herein will help future residents in ophthalmology to plan, perform and execute their thesis in a precise and orderly fashion as well as write it up in a clear and lucid manner.

The author isthe director of Sengupta's Research Academy and offers online lectures and clinical research training that had garnered more than 1100 enrollments from more than 70 countries. Click here to visit his online lecture series