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Study Design

Study Design. A study design is a careful advance plan of the analytic approach needed to answer the research question under investigation in a scientific way. The basics of study design: A carefully formed research question and a clearly stated outcome measure

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Study Design

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  1. Study Design • A study design is a careful advance plan of the analytic approach needed to answer the research question under investigation in a scientific way. • The basics of study design: • A carefully formed research question and a clearly stated outcome measure • Assessing the feasibility of study objectives and considering alternative research designs • Defining the study population and key concepts in operational terms • Selecting methods of sampling, data collection, and analysis appropriate to the study's objectives • Developing realistic budgets and time schedules for each stage of the research.

  2. Types of Study • Experimental/ Interventional: Investigator controls the assignment of the exposure or of the treatment e.g. randomized controlled trial. • Non-experimental/Observational: The allocation or assignment of factors is not under control of investigator. For example, in a study to see the effect of smoking, it is impossible for an investigator to assign smoking to the subjects. Instead, investigator can study the effect by choosing a control group and find the cause and relation effect. Some examples are- • Cross-sectional study • Cohort study • Case-control study

  3. Study Design • Randomized controlled Trial: Random allocation of different interventions (or treatments) to subjects in which one treatment group is for the purpose of determining the efficacy of the other treatment (s). E.g. placebo or standard medication can be used as a controlled to compare the efficacy of the other (s) treatment (s) • Types of control groups: • Placebo control group: Receive treatment • Active control group: For example a cancer patient can’t be given placebo. Need to use a standard medication in the market. • Types of randomized controlled trials: • Open trial: Investigator and subject know the full details of the treatment. • Single-blind trial: Investigator knows about the treatment but subject does not. • Double-blind: Both investigator and subject do not know about the treatment

  4. Study Design • Cross-sectional study: A descriptive study of the relationship between diseases and other factors at one point of time (usually) in a defined population. This is also known as prevalence study or survey study. • Cohort study: Subjects who presently have a certain condition and/or receive a particular treatment are followed over time and compared with another group who are not affected by the condition under investigation.Cohort analysis attempts to identify cohorts effects.E.g.recruit a group of smokers and a group of non-smokers and follow them for a set period of time and note differences in the incidence of lung cancer between the groups at the end of this time. • Case-control study: A study that compares two groups of people: those with the disease or condition under study (cases) and a very similar group of people who do not have the disease or condition (controls) and look back to see if they had the exposure of interest.E.g. two groups of people (lung cancer group and non-lung cancer group) are selected and compare for an exposure (smoke).

  5. Study Design • A Protocol is a document that describes the background, objective(s), design, methodology, data collection and management, variable assessment, statistical considerations, and organization of the study. • Basically a protocol is a manuscript that describes every step from proposal to completion of the research study.

  6. Basic concepts of clinical trials • A clinical trial is a research study to answer specific questions about vaccines or new therapies or new ways of using known treatments. • Institutional Review Board (IRB): A committee of physicians, statisticians, researchers, and others that ensures that a clinical trial is ethical and that the rights of study participants are protected. • Efficacy is the maximum ability of a drug or treatment to produce a result • Baseline measurement is the measurement taken just before a participant starts to receive the experimental treatment which is being tested • Change from baseline measurement is the difference between baseline and post-baseline measurements. • Percent change from baseline = (Change from baseline / baseline measurement) x 100 • Pharmacokinetics (PK) analysis explores what the body does to the drug. That is, the processes (in a living organism) of absorption, distribution, metabolism, and excretion of a drug or vaccine. It helps to decide the duration of doses. • Pharmacodynamic (PD) analysis detects the effect of drug on the body or microorganisms of the body.

  7. Basic concepts of clinical trials • Classification of clinical trials by their purposes • Treatment trials: Test experimental treatments, new combinations of drugs, or new approaches to surgery or radiation therapy. • Prevention trials: Look for better ways to prevent disease in people who have never had the disease or to prevent a disease from returning. These approaches may include medicines, vitamins, vaccines, minerals, or lifestyle changes. • Diagnostic trials: Conducted to find better tests or procedures for diagnosing a particular disease or condition. • Screening trials: Test the best way to detect certain diseases or health conditions. • Quality of Life: Trials (or Supportive Care trials) explore ways to improve comfort and the quality of life for individuals with a chronic illness.

  8. Study design of a Clinical Trial • Title: Reflects the main research interest. • Background/Rational of study: Importance of the study and previous study results will be explained. • Study Objectives: • Primary objective (s): Focuses on the core research question (s) • Secondary Objectives: Focuses on the secondary/ optional research questions • Investigational Plan: • Variable (parameter) selections to achieve the research objectives. Avoid selection of unnecessary variables • Overall study design and plan description: Brief description of design and assessments.

  9. Study design of a Clinical Trial • Selection of Study Population: • Inclusion criteria: A set of conditions to include a subject in the study. E.g. a adult study will include subjects only of age 18 or more. • Exclusion criteria: A set of conditions under which a subject (met inclusion criteria) will be excluded from the study. E.g. protocol violations, Non-compliance of the treatment etc. • Sample size calculation: Based on the effect size and the statistical power needed to test of main research question. Here are some useful websites for power and sample size calculation- • compare means, compare proportions, population survey http://department.obg.cuhk.edu.hk/researchsupport/Sample_size_EstMean.asp • Experimental Design, survival analysis http://hedwig.mgh.harvard.edu/sample_size/size.html • Regression/multiple regression http://www.danielsoper.com/statcalc/calc01.aspx

  10. Study design of a Clinical Trial • Description of the treatment groups/ treatment administration/ treatment period • Randomization of the treatment to the subjects • Detailed descriptions of assessment/collection of all parameters/variables including sign and symptoms (adverse events) • Statistical Methods: Detailed descriptions of the statistical analyses of all variables in the study. A typical clinical trial may include- • Hypothesis and Decision rules • Rules for handling missing values • Interim/Final analysis • Subjects Disposition/summaries including the summary of the reasons of early termination

  11. Study design of a Clinical Trial • Statistical Methods (continued) • Disease Diagnosis/History: Usually descriptive statistics is enough • Summary of Medical/Surgical history • Demographics (age, sex, race, BMI, height, weight, etc.) and baseline characteristics : Usually descriptive statistics are enough but these variables are often used as covariates in efficacy analysis. • Efficacy analysis: Needs some reasonable statistical analysis to justify the research goal. Researchers sometimes perform analysis on the change from baseline and percent change from baseline values instead of the observed values.

  12. Study design of a Clinical Trial • Statistical Methods (continued) • Safety analysis (if subjects receives medication): Vital signs (temperature, blood pressures, respiration, pulse etc.), ECG/MRI results, Laboratory parameters, Physical exams, Adverse Events, pregnancy tests etc.)- Usually summary of the observed values and change from baseline values are provided. • Prior/concomitant medications: Summary of the all medications taken during the study or just immediate prior to study ( usually not more than one month) are provided. • Quality of life measurements: Both summary statistics and reasonable statistical analysis are required. • Pharmacokinetic (PK) & pharmacodynamic (PD) parameters: Summary statistics is enough for most cases.

  13. Study design of a Clinical Trial • Ethics: • Independent Ethics committee (IEC) or Institutional Review Board (IRB) • Ethical conduct of the study: Guidelines of Food and Drug Administration (FDA) and International Conference on Harmonization (ICH) for good clinical practices and maintaining the quality of research. • Patient information and consent: A document that describes the rights and risks of the study participants, and includes details about the study, such as its purpose, duration, required procedures, and key contacts. The participant then decides whether or not to sign the document. • Data collection and management • Storage security • Protection from data loss • Checking inconsistency of the data

  14. Experimental Design for Microarray Experiments Suzanne McCahan, Ph.D. Molecular Biologist

  15. Microarray Research • Should be Hypothesis Driven • Test a specific statement • Ask a specific question • Involves data mining • Often generates new hypotheses

  16. Microarrays can measure… • Gene Expression • Chromatin Structure • Methylation of Cytosine • Histone Binding • Array Comparative Genomic Hybridization (aCGH) • Amplification of Chromosomal Regions • Deletion of Chromosomal Regions

  17. General Background • The application and type of array to be used determine what should be considered when designing microarray experiments. • A general understanding of microarrays is needed.

  18. Image courtesy of Affymetrix. General Characteristics of Microarrays • Microarrays are small. • This is an picture of an Affymetrix GeneChip.

  19. Microarrays are comprised of DNA probes • Probes are attached to (or synthesized on) a surface. • Oligos - 25 bp • Oligos – 50-70 bp • Cloned or Amplified DNA • PCR – 500 bp • BAC (Bacterial Artificial Chromosome) – 300kb Image courtesy of Affymetrix.

  20. C : G T : A A : T A : T G : C A : T G : C C : G Hybridization • Strands represent • A probe on an array • Labeled DNA or RNA from a sample (This is also referred to as the ‘target’.) G A T T C T C G C T A A G A G C Image courtesy of Affymetrix.

  21. Hybridization No fluorescence where labeled DNA (or RNA) does NOT hybridize to probe. Fluorescence where labeled DNA (or RNA) hybridizes to probe. Images courtesy of Affymetrix.

  22. DNA Microarrays • There are many probes on a single microarray. • Amount of target is relative to the intensity of fluorescent signal. Image courtesy of Affymetrix.

  23. Numbers of Probes on Microarrays • Gene Expression • Affymentrix Rat GeneChip has ~300,00 probes representing ~15,000 genes • Chromatin Structure • Agilent Mouse CpG Island Array has ~100,000 probes • aCGH (Amplification/Deletion) • NimbleGen Human X Chromosome Tiling Array ~385,000 probes

  24. Keep comparisons simple • Two well defined groups is best, although more can be used. • Normal vs control • Untreated vs treated (one drug) • Less complex samples are better than complex ones. • Cell lines are the least complex • Blood • RBC should be removed, hemoglobin mRNA and protein can interfere • Mononuclear cells are better than total white cells • Solid tissue • Tumor only, no contaminating normal tissue • Muscle only, no contaminating fat

  25. Decrease Variability • Samples should be as much the same as possible • If from patients • Exact same tissue • Strict criteria for diagnosis • Only meds to be studied • Same pubertal stage • Handled in a similar manner (immediately on ice) • Same quality of starting material (RNA or DNA) • Hybridization, washing and scanning should be done by a single person at a single location.

  26. How many samples should be included in a study? • Many ‘tests’ are done on a single sample. • Each hybridization is expensive. This usually limits the number of samples that can be included in an experiment. • With the usual budget, it is not feasible to use standard statistical tools to determine the number of samples to be included in a study and analyze the data.

  27. How many samples should be included in a study? • The best way to determine sample size is to do a pilot study to obtain data from a particular experimental system and do a power analysis taking the challenges of microarrays into consideration. • A few publications assess sample size with public gene expression microarray data sets. The results vary with dataset. One estimation for sample size was 10-12 per group. • When a pilot study is not feasible, a general rule of thumb is 5 – 10 samples per group.

  28. 1-Color Hybridizations • Common format for gene expression arrays • RNA or DNA from each sample is hybridized to a single array • If there are two groups (control and treated) with 10 samples each, then • A total of 20 samples will be used • A total of 20 arrays will be used

  29. 2-Color Hybridizations • Format for some gene expression, some aCGH, and all chromatin structure arrays • RNA or DNA from two samples simultaneously hybridized to a single array • One sample is experimental • Other sample is control • Each of the two samples is labeled with a different fluorochrome. • One array is needed for each pair of samples

  30. aCGH Arrays – Detection of Amplification/Deletion • Most platforms for aCGH require 2-color hybridizations • Tumor Studies • Hybridize labeled DNA from tumor and normal tissue from a single subject (patient/animal) together. • Genetic Studies • Hybridize labeled DNA from control subject with DNA from diseased subject • The same control should be used with all diseased subjects. • The control could be DNA from a single individual or from a single pool of individuals

  31. Confirmation of results is necessary • Since there is such disparity in the number of samples examined and the number of tests performed (e.g. level of transcripts measured) microarray experiments results must be confirmed.

  32. Methods for confirmation of results • Additional samples that were not examined on the microarray should be used. • Quantitative PCR methods are often performed. • Confirmation usually involves a small number of genes (or chromosomal regions, depending on the type of array that was used). • The combination of a larger sample size and small number of tests allows standard statistical methods to be used.

  33. Example of a microarry experiment • Modeled after: Insight into Pathogenesis of Antibiotic-Resistant Lyme Arthritis through Gene Expression Profiling • AnneMare Brescia, MD • Principal Investigator

  34. Hypothesis There are differences in gene expression of synovial fibroblasts from individuals with acute Lyme synovitis and chronic Lyme synovitis and these differences allow for the perpetuation of inflammation in chronic Lyme synovitis.

  35. Biological material – Cell lines • Collect synovial fluid • Site of disease activity • Prospectively – don’t know whether the case is acute or chronic Lyme disease at time of collection • Culture cells from fluid • Primary cells • Adherent cells are selected • Consistent phenotype - no monocytes • Harvest while cells are dividing • At same passage • Before reaching confluence

  36. Synovial fluid from 3 groups • Control • Injured joints • Acute Lyme Disease • Lyme synovitis resolved within 2 months of initiation of antibiotic therapy • Chronic Lyme Disease • Lyme synovitis persisted for six or more months despite antibiotic therapy

  37. Experimental Details • Uniform samples • Cell line probably representing single cell type • Affymetrix GeneChip • Single color hybridization • Human gene expression arrays – 15 chips • Biological replicates • 5 samples per group

  38. Overview of Analysis • Determine differential expression • Confirm results with real-time RT-PCR • Determine functional categories that are represented by differentially expressed genes • Replication? • Recruitment and/or activation of immune system? • Unexpected results can generate additional hypotheses

  39. The Future New technologies may replace microarrays • High through-put sequencing is on the horizon • Less expensive • Faster • Short sequences (25 nt – 400 nt) • Presents new computing challenges • Experimental design will need to be adjusted

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